JP2006180763A - Tissue stem cell growing agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a tissue stem cell growing agent, to provide a culturing method using the same, to provide a culture medium given by using the same, and to provide a cell cultured and prepared by using the tissue stem cell growing agent. <P>SOLUTION: This tissue stem cell growing agent contains a low-molecular weight compound or its salt as an active ingredient. The tissue stem cell growing agent has an effect of growing the tissue stem cell in an undifferentiated state. The cell for medicinal use and a tissue for the same are cultured by using the tissue stem cell which is cultured by using the tissue stem cell growing agent, respectively. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a tissue stem cell proliferating agent comprising a low molecular weight compound, particularly a bicyclic compound as an active ingredient, a tissue stem cell culture method using the same, a medium, and a tissue stem cell line produced using the same.

  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, regeneration of tissue and organs using stem cells that exist in embryos or adult organs and tissues and that are thought to have the ability to differentiate in one or more directions to compensate for defective tissues Medical care has attracted attention as a treatment method 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.

  Tissue stem cells (including organ stem cells) present in each organ have been identified in many fields such as bone marrow, blood vessels, nerves, blood, cartilage, bone, liver, pancreas, and the main stem cells include bone marrow and peripheral blood. Hematopoietic stem cells present in umbilical cord blood, neural stem cells present in brain hippocampus, skin epidermal stem cells present in epithelial tissues, hair follicle stem cells, corneal stem cells and gastrointestinal epithelial stem cells, liver stem cells present in liver, skeletal muscle In addition, skeletal muscle stem cells present in the body and mesenchymal stem cells present in the bone marrow and umbilical cord blood are known. For example, the bone marrow includes mesenchymal stem cells that play a useful role in the regeneration of mesenchymal tissue systems such as bone, cartilage, muscle, ligament, tendon, fat and stroma, and are undifferentiated It has been shown to retain pluripotency that can grow as cells and differentiate into, for example, chondrocytes or osteoblasts. Furthermore, mesenchymal stem cells can be easily isolated from bone marrow and umbilical cord blood, and retain the monolayer properties in in vitro culture (Non-patent Document 1). It is proposed to induce differentiation of cartilage tissue or bone tissue from the undifferentiated cells obtained by culturing and use them for transplantation treatment. However, in the conventional culture method, the proliferation of stem cells is extremely slow, and a very long culture period is required to obtain a sufficient number of mesenchymal stem cells, or the proliferation stops on the way. There is. Furthermore, there is a problem that the ability to differentiate into chondrocytes or osteoblasts is lost. However, a method for efficiently proliferating these tissue stem cells in vitro has not been known so far. Fibroblast growth factor (FGF) has been reported as a growth factor for mesenchymal stem cells (Patent Document 1). However, since this factor is a protein, there are problems such as high cost and storage stability, and mass culture. Not suitable for. Furthermore, the fibroblast growth factor does not show an effect of increasing the proliferation rate of mesenchymal stem cells, and is not suitable for culturing that requires a large amount of mesenchymal stem cells in a short period of time, such as therapeutic use. That is, a method for culturing a large amount of tissue stem cells such as mesenchymal stem cells in a short period of time has not been known so far.

Pittenger M.F., et al., Science 284,143-147, 1999 International Publication WO2002 / 022788

  An object of the present invention is to provide a tissue stem cell proliferating agent that can proliferate tissue stem cells in a short period of time. Another object of the present invention is to provide a method for growing tissue stem cells using such a tissue stem cell proliferating agent, to provide a medium containing such a tissue stem cell proliferating agent, and such tissue stem cell proliferation. An object of the present invention is to provide a cell line produced by culturing using an agent.

  As a result of intensive studies on compounds having a tissue stem cell proliferation promoting activity, the present inventors have found that the bicyclic compounds represented by the following general formulas (1) to (6) have a tissue stem cell proliferation promoting action. The present invention has been completed.

  The present invention has been made to achieve the above-described object, and is a tissue stem cell proliferating agent capable of proliferating tissue stem cells, a method for culturing tissue stem cells using the same, a medium containing a tissue stem cell proliferating agent, and such The present invention relates to a cell line prepared by culturing using a tissue stem cell proliferating agent.

［式中、R¹、R²、R³、及びR⁴は、それぞれ同一または異なっていてもよく、電子吸引基、電子供与基または水素原子を表す。Ａ環は、少なくとも１つのヘテロ原子を環内に含む５〜８員環を表す。Ｘは、主鎖の原子数が０〜１０のアルキレン基を表す。原子数０のアルキレン基とは、単結合を表す。該アルキレン基を構成する１つ以上のエチレンが、−Ｃ＝Ｃ−基及び／又は−Ｎ＝Ｎ−基及び／又は−ＣＯＮＨ−基で置き換わっていてもよい。また、環Ａと結合するボンドが二重結合となる基であってもよい。さらに、該アルキレン基は、置換基として、電子吸引基、電子供与基または水素原子を１つ以上有してもよい。Ｇは、電子吸引基、電子供与基または水素原子を有してもよい芳香族基を表す。該Ａ環は、−ＸＧ基以外の置換基として電子吸引基及び／又は電子供与基を１つ以上有してもよい。］
で表される化合物である、（１）に記載の組織幹細胞増殖剤。 That is, the present invention has the following configuration.
(1) A tissue stem cell proliferating agent containing a low molecular compound or a salt thereof as an active ingredient.
(2) The low molecular weight compound is represented by the general formula (1)

[Wherein R ¹ , R ² , R ³ , and R ⁴ may be the same or different and each represents an electron-withdrawing group, an electron-donating group, or a hydrogen atom. The A ring represents a 5- to 8-membered ring containing at least one hetero atom in the ring. 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 -CONH- group. Moreover, the group which becomes a double bond may be sufficient as the bond couple | bonded with the ring A. Furthermore, the alkylene group may have one or more electron-withdrawing groups, electron-donating groups, or hydrogen atoms as a substituent. G represents an aromatic group which may have an electron withdrawing group, an electron donating group or a hydrogen atom. The A ring may have one or more electron withdrawing groups and / or electron donating groups as a substituent other than the —XG group. ]
The tissue stem cell proliferating agent according to (1), which is a compound represented by:

(3) The tissue stem cell proliferating agent according to (2), wherein the A ring is a 5- or 6-membered ring containing at least one heteroatom selected from the group consisting of a nitrogen atom, an oxygen atom and a sulfur atom.
(4) The tissue stem cell proliferating agent according to (2), wherein the A ring is a 5- or 6-membered ring containing one nitrogen atom in the ring.
(5) The alkylene group represented by X is selected from the group consisting of an alkyl group, an acyl group, an alkoxy group, a nitro group, a hydroxycarbonyl group, an alkoxycarbonyl group, an aminocarbonyl group, a nitrile group, and a halogen atom as a substituent. The tissue stem cell proliferating agent according to any one of (2) to (4), which has one or more groups and / or atoms.

［式中、R¹、R²、R³、R⁴、Ｘ及びＧは、前記（２）中の定義と同義である。R⁵、R⁶、R⁷、R⁸、及びR⁹は、それぞれ同一または異なっていてもよく、電子吸引基、電子供与基または水素原子を表す。］
で表される化合物である、（２）に記載の組織幹細胞増殖剤。 (6) The low molecular weight compound is represented by the general formula (2)

[Wherein, R ¹ , R ² , R ³ , R ⁴ , X and G are as defined in the above (2). R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ may be the same or different and each represents an electron-withdrawing group, an electron-donating group or a hydrogen atom. ]
The tissue stem cell proliferating agent according to (2), which is a compound represented by:

［式中、R¹、R²、R³、R⁴、Ｘ及びＧは、前記（２）中の定義と同義である。R⁵及びR⁶は、それぞれ同一または異なっていてもよく、電子吸引基、電子供与基または水素原子を表す。］
で表される化合物である、（２）に記載の組織幹細胞増殖剤。 (7) The low molecular weight compound is represented by the general formula (3)

[Wherein, R ¹ , R ² , R ³ , R ⁴ , X and G are as defined in the above (2). 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 tissue stem cell proliferating agent according to (2), which is a compound represented by:

［式中、R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、及びR⁹は、前記（６）中の定義と同義である。R¹⁰、R¹¹、R¹²、及びR¹³は、それぞれ同一または異なっていてもよく、電子吸引基、電子供与基または水素原子を表す。二重片側破線は単結合または二重結合を表す。二重片側破線が二重結合を表す場合、波線部に関して幾何異性体が存在する。これらの幾何異性体の配置は特に限定されず、それぞれ独立に、Ｅ体又はＺ体のいずれであってもよい。］
で表される化合物である、（６）に記載の組織幹細胞増殖剤。 (8) The low molecular weight compound is represented by the general formula (4)

[Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , and R ⁹ are as defined in (6) above. R ¹⁰ , R ¹¹ , R ¹² and R ¹³ may be the same or different and each represents an electron withdrawing group, an electron donating group or a hydrogen atom. A double broken line on a single side represents a single bond or a double bond. When the double dashed line represents a double bond, there is a geometric isomer with respect to the wavy line. The arrangement of these geometric isomers is not particularly limited, and may be either E-form or Z-form independently. ]
The tissue stem cell proliferating agent according to (6), which is a compound represented by:

(9) R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , and R ¹³ are each independently an alkyl group Or a tissue stem cell according to (8), which is a group or atom selected from the group consisting of acyl group, alkoxy group, nitro group, hydroxycarbonyl group, nitrile group, alkoxycarbonyl group, aminocarbonyl group, halogen atom and hydrogen atom Proliferator.

ただし、Aは水素原子もしくは

を表す。
［式中、R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、及びR⁹は、前記（６）中の定義と同義である。R¹⁰、R¹¹、R¹²は、それぞれ同一または異なっていてもよく、電子吸引基、電子供与基または水素原子を表す。］
で表される化合物である、（６）に記載の組織幹細胞増殖剤。 (10) The low molecular weight compound is represented by the general formula (5)

Where A is a hydrogen atom or

Represents.
[Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , and R ⁹ are as defined in (6) above. R ¹⁰ , R ¹¹ and R ¹² may be the same or different and each represents an electron withdrawing group, an electron donating group or a hydrogen atom. ]
The tissue stem cell proliferating agent according to (6), which is a compound represented by:

(11) R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , and R ¹² are each independently an alkyl group or an acyl group The tissue stem cell proliferating agent according to (10), which is a group or an atom selected from the group consisting of an alkoxy group, a nitro group, a hydroxycarbonyl group, a nitrile group, an alkoxycarbonyl group, an aminocarbonyl group, a halogen atom and a hydrogen atom.

［式中、R¹、R²、R³、R⁴、R⁵、及びR⁶は前記（７）中の定義と同義である。R⁷、R⁸、及びR⁹は、それぞれ同一または異なっていてもよく、電子吸引基、電子供与基または水素原子を表す。］
で表される化合物である、（７）に記載の組織幹細胞増殖剤。 (12) The low molecular compound is represented by the general formula (6)

[Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , and R ⁶ have the same definitions as in (7) above. R ⁷ , R ⁸ and R ⁹ may be the same or different and each represents an electron withdrawing group, an electron donating group or a hydrogen atom. ]
The tissue stem cell proliferating agent according to (7), which is a compound represented by:

(13) R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , and R ⁹ are each independently an alkyl group, an alkoxy group, a hydroxyl group, a nitro group, a nitrile group, From acetoxy group, acetoxyalkyl group, cyclic alkylaminoalkyl group which may contain oxygen atom, dialkylaminoalkyl group, dialkylaminovinyl group, hydroxyalkylaminoalkyl group, arylaminovinyl group, alkoxycarbonyl group, halogen atom and hydrogen atom The tissue stem cell proliferating agent according to (12), which is a group or atom selected from the group consisting of

(14) R ¹ , R ² , R ³ , R ⁴ , R ⁸ , R ⁹ and R ¹⁰ are hydrogen atoms, R ⁵ is a hydrogen atom, a lower acyl group, or a lower alkyl group, and R ⁶ and R ⁷ is the same or different lower alkyl group, R ¹¹ is a hydrogen atom, halogen atom, nitro group, lower alkyl group, lower acyl group or lower alkoxy group, and R ¹² is hydrogen An atom, a lower alkyl group, a lower acyl group, or a lower alkoxy group, and R ¹³ is a hydroxycarbonyl group, a nitrile group, a carbamoyl group, a mono-lower alkylaminocarbonyl group, a di-lower alkylaminocarbonyl group, a secondary aminocarbonyl group The tissue stem cell proliferating agent according to (8), which is a tertiary aminocarbonyl group or a lower alkoxycarbonyl group.

(15) R ¹ , R ² , R ³ , R ⁴ , R ⁸ , and R ⁹ are hydrogen atoms, R ⁵ is a hydrogen atom, a lower acyl group, or a lower alkyl group, and R ⁶ and R ⁷ are A lower alkyl group which may be the same or different, R ¹⁰ is a hydrogen atom or a lower alkyl group, R ¹¹ is a hydrogen atom or a nitro group, and R ¹² is a hydrogen atom or a lower alkyl group. The tissue stem cell proliferating agent according to (10), which is a group, a lower acyl group, a lower alkoxycarbonyl group, or a lower alkoxy group.

(16) R ¹ and R ² are hydrogen atoms, R ³ is a hydroxyl group or an acetoxy group, R ⁴ is an acetoxyalkyl group, a cyclic alkylaminoalkyl group which may contain an oxygen atom, di-lower alkylamino lower alkyl Group, a hydroxy lower alkylamino lower alkyl group, or a hydrogen atom, R ⁵ is a lower alkyl group, a di-lower alkylaminovinyl group, or an arylaminovinyl group, R ⁶ is a nitro group, R ⁷ , R The tissue stem cell proliferating agent according to (12), wherein ⁸ and R ⁹ are the same or different lower alkyl group, lower alkoxy group, or hydrogen atom.

(17) The tissue stem cell proliferating agent according to any one of (1) to (16), wherein the tissue stem cells are mesenchymal stem cells.
(18) The tissue stem cell proliferating agent according to any one of (1) to (17), wherein the tissue stem cells are human tissue stem cells.

(19) A tissue stem cell culture method, wherein tissue stem cells are grown using the tissue stem cell proliferating agent according to any one of (1) to (18).
(20) A medium containing the tissue stem cell proliferating agent according to any one of (1) to (18) as an active ingredient.
(21) The medium according to (20), wherein the tissue stem cell proliferating agent has a concentration of 10 ng / ml to 100 μg / ml.

(22) A tissue stem cell cultured using the tissue stem cell proliferating agent according to any one of (1) to (18).
(23) A cell or tissue obtained by differentiating a tissue stem cell cultured using the tissue stem cell proliferating agent according to any one of (1) to (18).
(24) The cell or tissue according to (22) or (23), wherein the cell or tissue is for transplantation into a living body.
(25) A therapeutic method of transplanting the cell or / and tissue according to any one of (22) to (24) into a living body.

  The tissue stem cell proliferating agent of the present invention has an effect of proliferating tissue stem cells in an undifferentiated state. Medical cells and tissues can be cultured using tissue stem cells cultured using the tissue stem cell proliferating agent of the present invention.

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. .
Tissue stem cells: Tissue stem cells are pluripotent stem cells present in the body, such as hematopoietic stem cells, neural stem cells, skin epidermal stem cells, hair follicle stem cells, corneal stem cells, gastrointestinal epithelial stem cells, liver stem cells, skeletal muscle stem cells, Examples include mesenchymal stem cells.
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 stem cell is a hematopoietic stem cell, which can differentiate into various blood cell types such as lymphocytes and erythrocytes.

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.

  The present invention provides a tissue stem cell proliferating agent capable of proliferating tissue stem cells, a culture method using the same, a medium using the same, and a cell line cultured and produced using the same. The tissue stem cell proliferating agent, culture method and medium provided in the present invention can proliferate tissue stem cells more simply and safely than before. Cell culture methods comprising the tissue stem cell proliferating agent of the present invention can also be used to screen for specific differentiation inducers and useful combinations of differentiation inducers. The ability to grow tissue stem cells using the tissue stem cell proliferating agents and culture methods of the present invention is important, including the ability to produce tissue stem cells with single or multiple genetic modifications that have important therapeutic applications. Provide benefits.

  The tissue stem cell proliferating agent 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 tissue stem cells in an undifferentiated state. Examples thereof include a low molecular compound represented by 1).

In the formula, R ¹ , R ² , R ³ , and R ⁴ 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. It is also possible to define σ <0 as an electron-donating group and σ> 0 as an electron-withdrawing group using Hammett's substituent constant σ (Basic Organic Reaction Theory, written by Shizunobu Hashimoto, Sankyo Publishing, 1997) .

The A ring represents a 5- to 8-membered ring containing at least one hetero atom in the ring.
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 -CONH- group. Moreover, the group which becomes a double bond may be sufficient as the bond couple | bonded with the ring A. Furthermore, the alkylene group may have one or more electron-withdrawing groups, electron-donating groups, or hydrogen atoms as a substituent.

  G represents an aromatic group which may have an electron withdrawing group, an electron donating group or a hydrogen atom. The A ring may have one or more electron withdrawing groups and / or electron donating groups as a substituent other than the —XG group.

Among these, the A ring is preferably a 5- or 6-membered ring containing at least one heteroatom selected from the group consisting of a nitrogen atom, an oxygen atom and a sulfur atom.
It is also desirable when the A ring is a 5- or 6-membered ring containing one nitrogen atom in the ring. In this case, the 5-membered ring is preferably unsaturated, and the 6-membered ring is preferably saturated.

  Further, the alkylene group represented by X is a group selected from the group consisting of an alkyl group, an acyl group, an alkoxy group, a nitro group, a hydroxycarbonyl group, an alkoxycarbonyl group, an aminocarbonyl group, a nitrile group, and a halogen atom as a substituent. And / or having one or more atoms.

For example, examples of the general formula (1) include a tetrahydroisoquinoline derivative represented by the formula (2) and an indole derivative represented by the general formula (3).

In the formula, R ¹ , R ² , R ³ , R ⁴ , X and G are as defined above. R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ 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 , X and G are as defined above. 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.

Specific examples of the tetrahydroisoquinoline derivative represented by the general formula (2) include compounds and salts having a structure represented by the formula (4) or the formula (5).

を表す。 Where A is a hydrogen atom or

Represents.

In the formula, R ^{1 to} R ¹³ may be the same or different and each represents an electron-withdrawing group, an electron-donating group, or a hydrogen atom.
More preferably, R ^{1 to} R ¹³ are groups selected from the group consisting of alkyl groups, acyl groups, alkoxy groups, nitro groups, hydroxycarbonyl groups, nitrile groups, alkoxycarbonyl groups, aminocarbonyl groups, halogen atoms and hydrogen atoms. Or atoms.

In the case of the general formula (4), more preferably, R ¹ , R ² , R ³ , R ⁴ , R ⁸ , R ⁹ and R ¹⁰ are hydrogen atoms, R ⁵ is a hydrogen atom, a lower acyl group, Or a lower alkyl group, R ⁶ and R ⁷ may be the same or different lower alkyl group, and R ¹¹ is a hydrogen atom, a halogen atom, a nitro group, a lower alkyl group, a lower acyl group or A lower alkoxy group, R ¹² is a hydrogen atom, a lower alkyl group, a lower acyl group, or a lower alkoxy group, and R ¹³ is a hydroxycarbonyl group, a nitrile group, a carbamoyl group, a mono-lower alkylaminocarbonyl group, a di- Examples thereof include a lower alkylaminocarbonyl group, a secondary aminocarbonyl group, a tertiary aminocarbonyl group, and a lower alkoxycarbonyl group.

In the case of the general formula (5), more preferably, R ¹ , R ² , R ³ , R ⁴ , R ⁸ , and R ⁹ are hydrogen atoms, and R ⁵ is a hydrogen atom, a lower acyl group, or a lower alkyl group. R ⁶ and R ⁷ are the same or different lower alkyl group, R ¹⁰ is a hydrogen atom or a lower alkyl group, R ¹¹ is a hydrogen atom or a nitro group, R ¹² is a hydrogen atom, a lower alkyl group, a lower acyl group, a lower alkoxycarbonyl group, or a lower alkoxy group.

A specific example of the general formula (4) is a compound represented by the general formula (7) in which the double one-side broken line is a double bond and R ¹³ is an aminocarbonyl group in the formula (4).

Moreover, the following are illustrated as a more specific compound contained in the said General formula (4) which is a tetrahydroisone quinoline derivative.
2- (4-Acetyl-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide
2- (3-Acetyl-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide
2- (4-Bromo-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide
2- (3-Bromo-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide
2- (4-Chloro-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide
2- (3-Chloro-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide
2- (3,3-Dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -2-m-tolylazo-acetamide
2- (3,3-Dimethyl-3,4-dihydro-2H-isoquinoline-1-ylidene) -2-p-tolylazo-acetamide
2- (3,3-Dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -2- (4-methoxy-phenylazo) -acetamide
2- (3,3-Dimethyl-3,4-dihydro-2H-isoquinoline-1-ylidene) -2- (3-methoxy-phenylazo) -acetamide

2- (3,3-Dimethyl-3,4-dihydro-2H-isoquinoline-1-ylidene) -2- (4-nitro-phenylazo) -acetamide
2- (3,3-Dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -2- (3-nitro-phenylazo) -acetamide
2- (3,3-Dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -2- (4-sulfamoyl-phenylazo) -acetamide
2- (3,3-Dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -2- (3-sulfamoyl-phenylazo) -acetamide
2- (4-Acetylamino-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide
2- (3-Acetylamino-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide
2- (2-Acetyl-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide
2- (3,3-Dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -2-phenylazo-acetamide
(4-Acetyl-phenylazo)-(3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetic acid ethyl ester
2- (4-Acetyl-phenylazo) -2- (3,3-dimethyl-1,2,3,4-tetrahydro-isoquinolin-1-yl) -acetamide

2- (4-Acetyl-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -N-methyl-acetamide
2- (4-Acetyl-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -N-phenyl-acetamide
2- (4-Acetyl-phenylazo) -2- (2,3,3-trimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide
(4-Acetyl-phenylazo)-(3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetonitrile
2- (4-Acetyl-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene)-N, N-dimethyl-acetamide
(4-Acetyl-phenylazo)-(3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetic acid
2- (2-Acetyl-3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -2- (4-acetyl-phenylazo) -acetamide

Moreover, the compound represented by General formula (8) shown below as a specific example of General formula (5) is mention | raise | lifted.

Moreover, the following are illustrated as a compound contained in General formula (5).
2-Cyano-2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -Np-tolyl-acetamide
2-Cyano-2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -Nm-tolyl-acetamide
2-Cyano-2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -No-tolyl-acetamide
2-Cyano-2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -N- (4-methoxy-phenyl) -acetamide
2-Cyano-2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -N- (3-methoxy-phenyl) -acetamide
2-Cyano-2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -N- (4-nitro-phenyl) -acetamide
2-Cyano-2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -N- (3-nitro-phenyl) -acetamide
4- [2-Cyano-2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetylamino] -benzoic acid ethyl ester
3- [2-Cyano-2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetylamino] -benzoic acid ethyl ester
2-Cyano-2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -N-phenyl-acetamide
2-Cyano-2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -N- (2,4-dimethyl-phenyl) -acetamide
2-Cyano-2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide

Examples of the indole derivative represented by the general formula (3) include compounds represented by the following formula (6).

In the formula, R ^{1 to} R ⁹ represent the same or different electron-withdrawing groups, electron-donating groups, or hydrogen atoms. Among these, preferably, R ^{1 to} R ⁹ are an alkyl group, an alkoxy group, a hydroxyl group, a nitro group, a nitrile group, an acetoxy group, an acetoxyalkyl group, a cyclic alkylaminoalkyl group that may contain an oxygen atom, or a dialkylaminoalkyl group. A group or an atom selected from the group consisting of a dialkylaminovinyl group, a hydroxyalkylaminoalkyl group, an arylaminovinyl group, an alkoxycarbonyl group, a halogen atom and a hydrogen atom. Furthermore, R ¹ and R ² are hydrogen atoms, R ³ is a hydroxyl group or an acetoxy group, R ⁴ is an acetoxyalkyl group, a cyclic alkylaminoalkyl group which may contain an oxygen atom, di-lower alkylamino lower alkyl Group, a hydroxy lower alkylamino lower alkyl group, or a hydrogen atom, R ⁵ is a lower alkyl group, a di-lower alkylaminovinyl group or an arylaminovinyl group, R ⁶ is a nitro group, R ⁷ , R ⁸ , And R ⁹ may be the same or different lower alkyl group, lower alkoxy group, or hydrogen atom.

Moreover, the following are illustrated as a compound contained in General formula (6).
2-Methyl-3-nitro-1-phenyl-1H-indole-6-ol
1- (4-Methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol
2-Methyl-3-nitro-1-p-tolyl-1H-indole-6-ol
2- [2- (4-Methoxy-phenylamino) -vinyl] -3-nitro-1-p-tolyl-1H-indole-6-ol
1- (2-Methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol
7-Dimethylaminomethyl-2- (2-dimethylamino-vinyl) -3-nitro-1-p-tolyl-1H-indole-6-ol
1- (4-Methoxy-phenyl) -2-methyl-3-nitro-7-piperidin-1-ylmethyl-1H-indole-6-ol hydrochloride
2- (2-Dimethylamino-vinyl) -1- (4-methoxy-phenyl) -7-morpholin-4-ylmethyl-3-nitro-1H-indole-6-ol
7-[(3-Hydroxy-propylamino) -methyl] -1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol hydrochloride
7-Dimethylaminomethyl-2- (2-dimethylamino-vinyl) -1- (4-methoxy-phenyl) -3-nitro-1H-indole-6-ol

7-Diethylaminomethyl-1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol
7-Dimethylaminomethyl-2-methyl-3-nitro-1-p-tolyl-1H-indole-6-ol
1- (4-Methoxy-phenyl) -2-methyl-3-nitro-7-piperidin-1-ylmethyl-1H-indole-6-olacetic acid 7-acetoxymethyl-2-methyl-3-nitro-1-p -Tolyl-1H-indol-6-yl ester
2- (2-Dimethylamino-vinyl) -1- (4-methoxy-phenyl) -3-nitro-7-piperidin-1-ylmethyl-1H-indole-6-ol
7-Dimethylaminomethyl-2-methyl-3-nitro-1-phenyl-1H-indole-6-ol
7-dimethylaminomethyl-1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-olacetic acid 6-acetoxy-1- (4-methoxy-phenyl) -2-methyl- 3-Nitro-1H-indole-7-ylmethyl ester
2- (2-Dimethylamino-vinyl) -3-nitro-1-p-tolyl-1H-indole-6-ol
2- (2-Dimethylamino-vinyl) -3-nitro-1-phenyl-1H-indole-6-ol

Acetic acid 6-acetoxy-2- (2-dimethylamino-vinyl) -1- (4-methoxy-phenyl) -3-nitro-1H-indol-7-ylmethyl ester
1- (4-Chloro-phenyl) -2-methyl-3-nitro-1H-indole-6-olacetic acid 2- (2-dimethylamino-vinyl) -6-hydroxy-1- (4-methoxy-phenyl) -3-Nitro-1H-indole-7-ylmethyl ester
5-hydroxy-2-methyl-4,6-dinitro-1-phenyl-1H-indole-3-carboxylic acid ethyl ester
7-[[Bis- (2-hydroxy-ethyl) -amino] -methyl] -1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol
7-[[Bis- (2-hydroxy-ethyl) -amino] -methyl] -2-methyl-3-nitro-1-p-tolyl-1H-indole-6-ol
7-Dimethylaminomethyl-2- (2-dimethylamino-vinyl) -3-nitro-1-phenyl-1H-indole-6-ol
2- (6-Hydroxy-3-nitro-1-phenyl-1H-indol-2-ylmethyl) -isothioureaacetic acid 2- (N, N'-diphenyl-carbamimidoylsulfanylmethyl) -1- (4- Methoxy-phenyl) -3-nitro-1H-indol-6-yl ester acetic acid 6-acetoxy-1- (4-acetoxy-phenyl) -2- (2-dimethylamino-vinyl) -3-nitro-1H-indole -7-ylmethyl ester

2- (2-Dimethylamino-5-hydroxy-benzofuran-3-yl) -1- (4-methoxy-phenyl) -3-nitro-1H-indole-6-ol
2- (2-Dimethylamino-vinyl) -1- (4-methoxy-phenyl) -3-nitro-1H-indole-6-ol
5-Bromo-1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-olacetic acid 1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H- Indole-6-yl ester
7-Dimethylaminomethyl-6-hydroxy-2-methyl-1-phenyl-1H-indole-3-carbonitrile
7-Diethylaminomethyl-6-hydroxy-2-methyl-1-phenyl-1H-indole-3-carbonitrile
2- (2-Dimethylamino-5-hydroxy-benzofuran-3-yl) -6-methoxy-1-phenyl-1H-indole-3-carbonitrile
6-Methoxy-2-methyl-1-phenyl-1H-indole-3-carbonitrile
2- (2-Dimethylamino-vinyl) -6-methoxy-1-phenyl-1H-indole-3-carbonitrile
5-Bromo-6-hydroxy-1- (4-methoxy-phenyl) -2-methyl-1H-indole-3-carbonitrile

5,7-Dibromo-6-hydroxy-1- (4-methoxy-phenyl) -2-methyl-1H-indole-3-carbonitrile
6-Hydroxy-1- (4-methoxy-phenyl) -2-methyl-1H-indole-3-carbonitrile
6-Hydroxy-2-methyl-1-p-tolyl-1H-indole-3-carbonitrile
6-Hydroxy-2-methyl-1-phenyl-1H-indole-3-carbonitrile
1H-furo [2,3-g] indole-3-acetic acid, 5-hydroxy-1- (4-methoxyphenyl) -2,8-dimethyl-, ethyl ester
5-Bromo-7-dimethylaminomethyl-6-hydroxy-1-phenyl-2-phenylsulfanylmethyl-1H-indole-3-acetic acid ethyl ester
6-Hydroxy-2-methyl-1-phenyl-1H-indole-3-acetic acid
5-Bromo-6-hydroxy-1-phenyl-2-phenylsulfanylmethyl-1H-indole-3-acetic acid ethyl ester
6-acetoxy-5-bromo-2-methyl-1-phenyl-1H-indole-3-acetic acid ethyl ester
5-Bromo-7-dimethylaminomethyl-6-hydroxy-1-phenyl-2-phenylsulfanylmethyl-1H-indole-3-acetic acid ethyl ester

6-acetoxy-5-bromo-2-bromomethyl-1-phenyl-1H-indole-3-acetic acid ethyl ester
6-acetoxy-2-bromomethyl-1-phenyl-1H-indole-3-acetic acid ethyl ester
6-acetoxy-2-methyl-1-phenyl-1H-indole-3-acetic acid ethyl ester pyrrolo [2,3-f] [1,3] benzoxazine-3-acetic acid, 8-ethyl-1,7,8, 9-Tetrahydro-2-methyl-1- (4-nitrophenyl)-, ethyl ester
6-Acetoxy-2-methyl-1- (2-trifluoromethyl-phenyl) -1H-indole-3-acetic acid ethyl ester
1- (2,4-Dimethoxy-phenyl) -6-hydroxy-2-methyl-1H-indole-3-acetic acid ethyl ester
6-hydroxy-1- (4-methoxy-phenyl) -2-methyl-1H-indole-3-acetic acid ethyl ester
1- (4-Ethoxycarbonyl-phenyl) -6-hydroxy-2-methyl-1H-indole-3-acetic acid ethyl ester
1- (4-Cyano-phenyl) -6-hydroxy-2-methyl-1H-indole-3-acetic acid ethyl ester
6-hydroxy-2-methyl-1- (2-trifluoromethyl-phenyl) -1H-indole-3-acetic acid ethyl ester

6-Hydroxy-2-methyl-1- (4-trifluoromethyl-phenyl) -1H-indole-3-acetic acid ethyl ester
6-Hydroxy-2-methyl-1- (4-nitro-phenyl) -1H-indole-3-acetic acid ethyl ester
1- (4-Bromo-phenyl) -6-hydroxy-2-methyl-1H-indole-3-acetic acid ethyl ester
1- (4-Fluoro-phenyl) -6-hydroxy-2-methyl-1H-indole-3-acetic acid ethyl ester
6-Hydroxy-2-methyl-1- (4-nitro-phenyl) -5,7-bis-piperidin-1-ylmethyl-1H-indole-3-acetic acid ethyl ester
5,7-Bis-dimethylaminomethyl-6-hydroxy-2-methyl-1- (4-nitro-phenyl) -1H-indole-3-acetic acid ethyl ester
6-Hydroxy-2-methyl-1- (4-nitro-phenyl) -1H-indole-3-acetic acid ethyl ester
7-Dimethylaminomethyl-6-hydroxy-2-methyl-1- (4-nitro-phenyl) -1H-indole-3-acetic acid ethyl ester
6-acetoxy-1- (4-chloro-phenyl) -2-methyl-1H-indole-3-acetic acid ethyl ester
7-Dimethylaminomethyl-6-hydroxy-2-methyl-1-p-tolyl-1H-indole-3-acetic acid ethyl ester

7-Dimethylaminomethyl-6-hydroxy-2-methyl-1-phenyl-1H-indole-3-acetic acid ethyl ester
6-hydroxy-2-methyl-1-p-tolyl-1H-indole-3-acetic acid ethyl ester
6-Hydroxy-2-methyl-1-phenyl-1H-indole-3-acetic acid ethyl ester
1- (4-Chloro-phenyl) -7-dimethylaminomethyl-6-hydroxy-2-methyl-1H-indole-3-acetic acid ethyl ester
1- (4-Dimethylamino-phenyl) -6-hydroxy-2-methyl-1H-indole-3-acetic acid ethyl ester
1- (2-Chloro-phenyl) -6-methoxy-2-methyl-1H-indole-3-acetic acid ethyl ester
1- (2-Chloro-phenyl) -6-hydroxy-2-methyl-1H-indole-3-acetic acid ethyl ester
6-acetoxy-1- (4-chloro-phenyl) -2-methyl-5,7-dinitro-1H-indole-3-acetic acid ethyl ester
1- (4-Chloro-phenyl) -6-hydroxy-2-methyl-5,7-dinitro-1H-indole-3-acetic acid ethyl ester
6-acetoxy-5,7-dibromo-1- (4-chloro-phenyl) -2-methyl-1H-indole-3-acetic acid ethyl ester

5,7-Dibromo-1- (4-chloro-phenyl) -6-hydroxy-2-methyl-1H-indole-3-acetic acid ethyl ester
6-acetoxy-5-bromo-1- (4-chloro-phenyl) -2-methyl-1H-indole-3-acetic acid ethyl ester
5-Bromo-1- (4-chloro-phenyl) -6-methoxy-2-methyl-1H-indole-3-acetic acid ethyl ester
1- (4-Chloro-phenyl) -6-methoxy-2-methyl-1H-indole-3-acetic acid
1- (4-Chloro-phenyl) -6-methoxy-2-methyl-1H-indole-3-acetic acid ethyl ester
1- (4-Chloro-phenyl) -6-hydroxy-2-methyl-1H-indole-3-acetic acid ethyl ester

Examples of the lower alkyl group represented by R include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 1-methylpropyl, Examples include n-hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 3,3-dimethylpropyl, 2-ethylbutyl and the like. Preferably, it is methyl.
Examples of the lower alkoxy group represented by R 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 represented by R include fluorine, chlorine, bromine, iodine and the like, preferably chlorine or bromine.
Examples of the lower acyl group represented by R include formyl, acetyl, propionyl, butyryl and the like, preferably acetyl.
Examples of the lower alkyl group that may form a cyclic structure represented by R include cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like, and cyclopentyl, cyclohexyl, and cycloheptyl are preferred.

Examples of lower alkoxycarbonyl represented by R include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl and the like, and methoxycarbonyl or ethoxycarbonyl is preferred.
Examples of the aminocarbonyl represented by R 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). Can be mentioned.

Examples of the secondary aminocarbonyl group represented by R include -CONHR (representing 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 the aminoalkyl group represented by R, for example, — (CH ₂ ) _n —NR ₂ (n is 1 to 8, preferably 1, but R is the same or different hydrogen atom, A lower alkyl group, a lower alkyl group that may form a cyclic structure (the cyclic structure may contain 1 to 3 heteroatoms such as nitrogen or oxygen), and a phenyl group that may have a substituent ) Etc.
Examples of the acetoxyalkyl represented by R include — (CH ₂ ) _n —OAc (n represents 1 to 8), and preferably n is 1.

  Moreover, the double one-sided broken line in Formula (4) represents a single bond or a double bond. Furthermore, in the compound represented by the formula (4), when the double one-sided broken line represents a double bond, geometric isomers exist with respect to the wavy line (two places). The arrangement of these geometric isomers is not particularly limited, and may be independently either E-form or Z-form, and the compound of the present invention is a pure form of geometric isomer based on these geometric isomers. Any mixture may be used. Furthermore, tautomers may be included in any ratio.

  As the salt of the compound represented by the general formulas (2) and (3), 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.

In addition, ester compounds of the compounds represented by the general formulas (4) and (5) are also within the scope of the present invention, and examples thereof include carboxylic acid esters, sulfonic acid esters, and inorganic acid esters.
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 used in the present invention can be produced by carrying out the methods described below, methods analogous thereto, or known methods. A method for producing the compound represented by the formula (9) is shown below.

  A suitable solvent that does not adversely affect the reaction between the equivalent amount of the thioether derivative (I) and the phenylazoacetamide derivative (II) (for example, amides such as dimethylformamide and dimethylacetamide, ethers such as tetrahydrofuran, benzene, toluene and the like It can be produced by dissolving in aromatic hydrocarbons) and refluxing until there are no more raw materials. The derivative (I) can be purchased by a known method (for example, described in Khim. Geterotsikl. Soedin., No. 7, p995 (1990)) or from a commercial product. Specifically, it is as shown in Table 1 below.

The phenylazoacetamide derivative (II) is prepared by a known method (Materialy Ural'sk. Soveshch. Po Spektroskopii, 4th, Sverdlovsk 1963, p205 (1965), Bulletin de l'Academie Polonaise des Sciences, Serie des Sciences Chimiques, 14 (1 ), p29 (1966), Am. Chem. Soc., Div. Org. Coatings, Plastics Chem. Preprints, 23 (2), p486 (1963), Zhurnal Obshchei Khimii, 35 (3), p559 (1965), Zhurnal Obshchei Khimii, 32, p526 (1962) etc.).
The compound represented by the formula (9) can also be synthesized by the following scheme.

  The compound (IV) obtained by reacting a benzylcarbinol derivative with a 2-cyanoacetamide derivative in the presence of sulfuric acid is reacted with a diazonium salt (V) in an alcohol aqueous solution that does not adversely affect the reaction in the presence of hydrochloric acid. Can be obtained. Various derivatives of benzyl carbinol derivatives can be purchased from commercial products, and further can be obtained by known methods (for example, J. Gen. Chem. USSR, No. 6, p1263 (1936). Diazonium salts are commercially available. The aminobenzene derivative can be derived from hydrochloric acid or an aqueous sodium nitrite solution by a known method.

  Among the compounds represented by the formula (9), 2- (4-acetyl-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide (Asinex, Russia), 2- (3-Chloro-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide (Asinex, Russia), 2- (3,3 -Dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -2- (3-methoxy-phenylazo) -acetamide (Asinex, Russia), 2- (3,3-dimethyl-3,4-dihydro- 2H-isoquinoline-1-ylidene) -2- (3-nitro-phenylazo) -acetamide (PHARMEKS, Russia), 2- (3,3-dimethyl-3,4-dihydro-2H-isoquinoline-1-ylidene)- 2- (4-Methoxy-phenylazo) -acetamide (PHARMEKS, Russia), 2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -2-m-tolylazo-acetamide (IBS , Russia) is a commercial product and supplier It can be obtained from.

A production method in the case where R ⁵ is a methyl group among the compounds represented by the formula (9) is shown below. A compound represented by the formula (4) (R5 = -H) in an appropriate solvent that does not adversely influence the reaction (for example, nitriles of acetonitrile, ethers such as tetrahydrofuran, aromatic hydrocarbons such as benzene and toluene) And an appropriate solvent that does not adversely affect the reaction (for example, amides such as dimethylformamide and dimethylacetamide, ethers such as tetrahydrofuran, aromatic hydrocarbons such as benzene and toluene) Can be produced by slowly dropping and dissolving the product dissolved in

A production method in the case where R ⁵ is an acetyl group among the compounds represented by the formula (9) is shown below. It can be produced by dissolving the compound represented by formula (9) (R ⁵ = -H) and dimethylaminopyridine in pyridine in an ice bath, adding acetic anhydride, and stirring.

A method for producing the compound represented by the formula (11) is shown below.

  The compound represented by the formula (9) is dissolved in an appropriate solvent that does not adversely influence the reaction (for example, amides such as dimethylformamide and dimethylacetamide), and thionyl chloride is dissolved in an appropriate solvent that does not adversely influence the reaction (for example, , Dimethylformamide, amides such as dimethylacetamide) and the like can be prepared by dropping them in an ice bath.

A method for producing the compound represented by the formula (12) is shown below.

  It can be produced by adding potassium hydroxide and a compound represented by the formula (9) to ethanol and refluxing.

A method for producing the compound represented by the formula (13) is shown below.

  It can be produced by suspending the compound represented by the formula (12) in ethanol, slowly adding dropwise thionyl chloride while cooling, and stirring at room temperature.

A method for producing the compound represented by the formula (14) is shown below.

  The compound represented by the formula (12), the corresponding primary or secondary amine, HOBt (Advanced ChemTech, USA) and a suitable solvent that does not adversely influence the reaction (for example, amides such as dimethylformamide and dimethylacetamide) HBTU (Advanced ChemTech, USA) is dissolved in an appropriate solvent that does not adversely affect the reaction (for example, amides such as dimethylformamide and dimethylacetamide, and halogen solvents such as dichloromethane). I added what I did. It can be produced by stirring at room temperature.

A method for producing the compound represented by the formula (10) is shown below.

  A compound represented by the formulas (9) and (11) to (14) is dissolved in an appropriate solvent that does not adversely influence the reaction (for example, nitrile of acetonitrile), and triphenyltin hydride (Aldrich, USA) Is dissolved in an appropriate solvent that does not adversely affect the reaction (for example, aromatic hydrocarbons such as benzene, toluene, xylene, etc.) and added to the solvent, followed by refluxing.

The compound represented by the general formula (4) can be dissolved in an appropriate solvent, but is present in the solvent as it is, in a reduced form represented by the following formula (15), or as a mixture thereof. Also good.

  The compound used in the present invention or a salt thereof thus obtained can be isolated and purified by a usual separation means such as recrystallization, distillation, chromatography and the like. Thus, when the compound used in 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. On the other hand, when it is obtained as 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 concentration of the low molecular weight compound which is an active ingredient in the tissue stem cell proliferating agent of the present invention is desirably used in the range of 0.1 ng / ml to 1 mg / ml, preferably 10 ng / ml to 100 μg / ml, more preferably Is used in the range of 100 ng / ml to 10 μg / ml.

  The tissue stem cell proliferating agent 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 (above Invitrogen Invitrogen, USA). In one embodiment, the cell culture medium is Dulbecco's Modified Eagle Medium (DMEM). Furthermore, proteins relating to cell growth and differentiation control such as serum or serum substitutes, various growth factors and cytokines can be added to these basal media for use. Moreover, you may add arbitrary compounds. The serum can be any serum or serum-based solution that provides nutrients that are effective in maintaining tissue stem cell proliferation and viability. 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 Vitamin etc. 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 10%. In another embodiment, the serum replacement is knockout serum replacement: KSR (Invitrogen, USA). Cell growth factors that can be added include hepatocyte growth factor (HGF), epithelial cell growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), nerve growth factor (NGF), platelets Examples include, but are not limited to, derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), bone morphogenetic factor (BMP), stem cell factor (SCF), Wnt, and the like. Examples of cytokines that can be added include, but are not limited to, interleukin (IL) and granulocyte-macrophage colony stimulating factor (GM-CSF). Moreover, differentiation control factors, such as LIF and a notch ligand, can also be added. The compound to be added is not particularly limited, but may be an agonist or antagonist for any protein, and may be any phosphorylation inhibitor. Moreover, the bullet kit MSCGM set (made by CAMBREX) which is a commercially available culture kit can also be used.

  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 tissue stem cell proliferating agent and its active ingredient of the present invention 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, as natural polymer substances, mica (mica), asbestos (asbestos), graphite (graphite), saccharides and gelatin represented by diamond, starch, cellulose, alginic acid, etc., and gelatin, fibronectin, fibrinogen, laminin, collagen, etc. And the like. 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 preferred, proteins and peptides, and organic synthetic polymer materials are more preferred, and a suitable example is Matrigel (BD Biosciences).

  By using the culture substrate on which the tissue stem cell proliferating agent of the present invention is fixed as a cell capturing material, a method and apparatus capable of separating and culturing tissue stem cells from a population consisting of a plurality of different cells can be provided. That is, a cell-containing solution containing tissue 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 tissue stem cell proliferating agent of the present invention is fixed. A tissue stem cell culturing method characterized in that a tissue stem cell is captured by a capture material, and the cells to be removed are led out of the container and then cultured together, and the culture substrate on which the tissue stem cell proliferating agent of the present invention is immobilized A cell culture device in which a container is filled with a cell capturing material comprising: the cell capturing material can be used as a carrier for cell culture, and the container can be used for cell culture. A cell culture device. The cells to be removed refer to all cells other than tissue stem cells. This also includes cells that have differentiated from tissue stem cells and have lost pluripotency. The cell-containing liquid to be introduced into the cell trapping material may be any cell liquid containing tissue stem cells, and examples thereof include blood, bone marrow, debris tissue liquid, tissue stem cell culture liquid, and the like.

  The present invention relates to a method for growing tissue stem cells. A culture of tissue stem cells grown using the tissue stem cell proliferating agent of the present invention can be provided.

  The cells cultured using the tissue stem cell proliferating agent of the present invention include all tissue stem cells that can be obtained using known methods and materials. Examples of tissue stem cells include cells that can be obtained by the following known methods.

  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, Gage et al., Science, 287, p1433-1438, 2000), 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., Nature Medicine, 6, p1282-1286, 2000. Pittenger et al., Science, 284, p143. -147, 1999), skin stem cells / epidermal stem cells (Murota Seiya, edited by “Regenerative Medicine / Regenerative Therapy”, Modern Chemistry 41, Tokyo Chemical Doujin, Fuchs et al., Nature Reviews Genetics, 3, p199-209, 2002, Tumbar Science, 303, p359-363, 2004), pigment stem cells (Nishimura et al., Nature, 416, p854-860, 2002), hair follicle stem cells (Oshima et al., Cell, 104, p233-245, 2001) Nishimura et al., Nature, 416 p854-860, 2002 years, Tumbar et al., Science, 303, p359-363, 2004 years).

  The tissue stem cell proliferating agent provided by the present invention can be used for an already isolated tissue stem cell, or the tissue stem cell can be isolated and cultured using the tissue stem cell proliferating agent of the present invention. The degree of undifferentiation of tissue stem cells cultured using the tissue stem cell proliferating agent of the present invention can be confirmed by detecting a specific marker present on the cell membrane of each stem cell. For identification of mesenchymal stem cells, CD105 antigen, CD166 antigen, CD29 antigen, CD44 antigen (positive), CD45 antigen, CD14 antigen, CD34 antigen (negative) can be used. Tuj1 antigen, GFAP antigen, O4 antigen and the like can be used for identification of neural stem cells. For identification of hematopoietic stem cells, CD34 antigen (negative or weak positive), c-Kit antigen, Sca-1 antigen (more positive), lineage marker (negative), etc. can be used (Nakagata et al., “Stem Cell / Clone Research Protocol” "Yodosha). In one embodiment, the surface antigen is incubated with a specific antibody (primary antibody) that recognizes the antigen and further incubated with a second antibody (secondary antibody) coupled to a reporter such as a fluorescent label. Can be labeled. 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 targeted differentiation inhibitor. 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). The number of cells from which the desired surface antigen is expressed 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.

  A stem cell-specific marker or a differentiated cell-specific marker can also be confirmed by measuring the expression level of the marker gene. 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 marker 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 a marker gene, primers are designed using commercially available gene analysis software based on the sequence of the marker gene 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 marker gene can be determined for cells exposed to the tissue stem cell proliferating agent of the present invention. Compared to the expression level of a marker gene in a control cell that has not been exposed to the proliferating agent, that is, induced to differentiate from a tissue stem cell, the expression level of a stem cell-specific marker gene is significantly maintained, or a differentiation cell-specific marker gene It is considered that a compound having an activity capable of suppressing the expression of is a proliferating agent capable of proliferating while maintaining undifferentiation of tissue stem cells. In a specific embodiment, ISOGEN (Nippon Gene Co., Ltd., Japan) is used from human mesenchymal stem cells cultured by the method described in Example 1 (3) “Human mesenchymal stem cell proliferation assay”, and the attached method Extract total RNA according to That is, the medium is removed from the dish after culturing, washed twice with 10 ml of PBS, and dissolved in 1 ml of ISOGENE. Allow to stand at room temperature for 5 minutes, then collect in a 1.5 ml Eppendorf tube. 0.2 ml of chloroform (Wako Pure Chemical Industries) was added, shaken for 15 seconds, and allowed to stand at room temperature for 2 to 3 minutes. Centrifuge at 4 ° C. and 10,000 rpm for 15 minutes in a microcentrifuge (Tomy Seiko). Transfer 400 μl of the supernatant to a new 1.5 ml Eppendorf tube, add 500 μl of isopropanol (Wako Pure Chemical Industries, Ltd.), allow to stand at room temperature for 10 minutes, and then centrifuge for 10 minutes at 4 ° C. and 10,000 rpm in a microcentrifuge. After removing the supernatant, add 1 ml of 70% ethanol aqueous solution, shake, and centrifuge in a microcentrifuge at 4 ° C, 10,000 rpm for about 5 minutes. After removing the supernatant and drying the precipitate, the precipitate is dissolved in 30 μl of distilled water to obtain a total RNA solution.

  Using 2 μg of the total RNA thus obtained as a template, Deoxyribonuclease I (Amplification Grade) (Invitrogen), Oligo (dT) 12-18 primer (Invitrogen 18418-012), Omniscript Reverse Transcriptase (QIAGEN) Synthesize cDNA according to the attached protocol. That is, 1 μl of 10 × DNase I Reaction Buffer® 1 μl of 10 × DNase I (manufactured by Invitrogen) and distilled water are added to 2 μg of total RNA to prepare a reaction solution, which is then incubated at room temperature for 10 minutes. Add 1 μl of 25 mM EDTA solution, heat at 65 ° C. for 10 minutes, return to room temperature, then 2 μl 10 × Buffer RT, 2 μl 5 mM dNTP Mix, 2 μl Oligo (dT) 12-18 primer, 0.25 μl RNaseOUT (manufactured by Invitrogen, 10777-019), 1 μl of Omniscript Reverse Transscriptase is added, adjusted to a total volume of 20 μl with RNase-free purified water, and then incubated at 37 ° C. for 60 minutes to obtain a cDNA solution. A portion of the synthetic cDNA thus obtained is diluted 5-fold with distilled water, and 2 μl of the diluted cDNA is used as a template and a light cycler first start DNA master SYBR Green I kit (manufactured by Roche Diagnostics) is attached. Perform PCR according to the protocol. The expression level of the marker gene and the glyceraldehyde triphosphate dehydrogenase (GAPDH) gene as an internal standard are measured. The reaction solution composition and reaction conditions are shown below.

  Thereby, the expression level of the marker gene of the tissue stem cell cultured in the presence of the tissue stem cell proliferating agent of the present invention and the tissue stem cell cultured in a medium not containing the proliferating agent can be compared.

  The tissue stem cell proliferating agent, the culture method and medium using the proliferating agent provided by the present invention are expected to be applied to all techniques in which tissue stem cells are useful.

  The tissue stem cell proliferating agent of the present invention, the culture method using the proliferating agent, and the cells produced using the medium are differentiated and used for cell transplantation or used for artificial tissue construction together with the use of artificial support tissue And can be transplanted into a living body or used as an artificial organ. The use of tissue stem cells for cell transplantation and tissue engineering can solve the problems associated with conventional transplantation treatments including autologous transplantation, such as tissue defects 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.

  Tissue stem cell proliferating agent of the present invention, culture method using the same, unmodified and modified tissue stem cells obtained by a medium containing the same, and cultures thereof are substances that improve monitoring of tissue stem cells or collection of tissue stem cells Used to screen for. For example, a putative tissue stem cell differentiation inducer can be added to a cell culture grown using the method described above. A substance capable of inducing differentiation is identified as a tissue stem cell differentiation inducing factor as compared to a control cell culture lacking a putative tissue stem cell differentiation inducing substance.

  The tissue stem cell proliferating agent of the present invention has an excellent ability to maintain undifferentiated tissue stem cells and proliferate, and therefore can be used as a therapeutic or preventive agent for tissues or organs damaged by aging, disease 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 tissue stem cell proliferating agent 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, and is usually a tablet. Oral administration as a solid preparation such as a capsule, granule or powder, or parenteral administration as an injection, transdermal agent, suppository, 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 In general, 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 1 to 4 times a day. Divide into two doses. 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 tissue stem cell proliferating agent of the present invention in the pharmaceutical composition 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.

  To the tissue stem cell proliferating agent of the present invention, a suspending agent, a solubilizing agent, a stabilizing agent, a tonicity agent, a preservative, etc. may be added to obtain a intravenous, subcutaneous, or intramuscular injection by a known method. it can. At that time, if necessary, a lyophilized product can be obtained by a known method. When the tissue stem cell proliferating agent of the present invention is administered to, for example, humans, it is mixed with itself or an appropriate pharmacologically acceptable carrier, excipient, diluent, and orally or parenterally as a pharmaceutical composition. Can be administered safely. 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 tissue stem cell proliferating agent of the present invention includes a dispersing agent (eg, Tween 80 (manufactured by Atlas Powder, USA, HCO 60 (manufactured by Nikko Chemicals) polyethylene glycol, carboxymethylcellulose, sodium alginate, etc.), a preservative (eg, methylparaben). , Propylparaben, benzyl alcohol, etc.), isotonic agents (eg, sodium chloride, mannitol, sorbitol, glucose, etc.) together with aqueous injections, or olive oil, sesame oil, cottonseed oil, corn oil and other vegetable oils, propylene glycol, etc. It can be dissolved, suspended or emulsified and molded into an oily injection to obtain an injection.In order to obtain an oral preparation, the tissue stem cell proliferating agent of the present invention is treated with, for example, an excipient (eg , Lactose, sucrose, starch, etc.), disintegrants (eg, starch, carbonated carbonate) ), Binder (eg, starch, gum arabic, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose, etc.) or lubricant (eg, talc, magnesium stearate, polyethylene glycol 6000, etc.) Then, if necessary, it can be made into an orally-administered preparation by coating by a method known per se for the purpose of taste masking, enteric or sustained, and examples thereof include hydroxypropylmethylcellulose, ethylcellulose and the like. , Hydroxymethylcellulose, hydroxypropylcellulose, polyoxyethylene glycol, Tween 80, Pluronic F68, cellulose acetate phthalate, hydroxypropylmethylcellulose Tallate, hydroxymethylcellulose acetate succinate, Eudragit (Rohm, Germany, methacrylic acid / acrylic acid copolymer) and pigments (eg, Bengala, titanium dioxide, etc.) are used. An intermediate phase may be provided between the 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 tissue stem cell proliferating agent of the present invention can be made into a solid, semi-solid or liquid external preparation according to a known method. 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.

  Next, examples embodying the present invention will be shown. This example is provided to assist one of ordinary skill in practicing the present invention. These examples in no way limit the scope of the invention in any way. Moreover, you may change in the range which does not deviate from the range of this invention. “Room temperature” described in the following examples represents 0 to 30 ° C. “%” Means weight percent unless otherwise specified.

Synthesis example: Compound production method
[1] Synthesis of 3,3-dimethyl-1,2,3,4-tetrahydroisoquinolinidene-1-acetamide

2-Cyanoacetamide (Aldrich, USA) is added to benzene and concentrated sulfuric acid is slowly added dropwise while stirring so as not to rise to 10 ° C. After returning to room temperature, dimethylbenzyl carbinol (Lancaster, UK) is added. Reflux for about 30 minutes, allow to cool to room temperature, and pour the reaction mixture into ice water. The extracted aqueous phase is neutralized and the resulting precipitate is filtered off. After drying, the title compound is obtained by recrystallization from isopropanol.
MS (m / z) = 217 (M + 1)

[2] Synthesis of 2- (4-acetyl-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide

  p-Aminoacetophenone (Aldrich, USA) is dissolved in benzene, bubbled with hydrochloric acid gas, the resulting precipitate is filtered off and recrystallized from isopropanol to obtain p-aminoacetophenone hydrochloride. The obtained p-aminoacetophenone hydrochloride is dissolved in a 20% aqueous ethanol solution and acidified with concentrated hydrochloric acid in an ice bath. A diazonium salt solution is obtained by dropping an aqueous sodium nitrite solution and treating with urea.

3,3-Dimethyl-1,2,3,4-tetrahydroisoquinolinidene-1-acetamide is dissolved in 20% aqueous ethanol and concentrated hydrochloric acid is added. Adjust the temperature so that it does not exceed 10 ° C and add the diazonium salt solution. The reaction changes and a saturated aqueous sodium acetate solution is added with stirring. The resulting precipitate is filtered off and recrystallized from isopropanol to give the title compound.
MS (m / z) = 363 (M + 1)

[3] Synthesis of 2- (3-acetyl-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide

  p-Aminoacetophenone hydrochloride is obtained by dissolving m-aminoacetophenone (Aldrich, USA) in benzene, bubbling hydrochloric acid gas, filtering the resulting precipitate and recrystallizing with isopropanol. The obtained p-aminoacetophenone hydrochloride is dissolved in a 20% aqueous ethanol solution and acidified with concentrated hydrochloric acid in an ice bath. A diazonium salt solution is obtained by dropping an aqueous sodium nitrite solution and treating with urea.

3,3-Dimethyl-1,2,3,4-tetrahydroisoquinolinidene-1-acetamide is dissolved in 20% aqueous ethanol and concentrated hydrochloric acid is added. Adjust the temperature so that it does not exceed 10 ° C and add the diazonium salt solution. The reaction changes and a saturated aqueous sodium acetate solution is added with stirring. The resulting precipitate is filtered off and recrystallized from isopropanol to give the title compound.
MS (m / z) = 363 (M + 1)

[4] Synthesis of 2- (4-acetyl-phenylazo) -2- (2,3,3-trimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide

To acetonitrile was added 2- (4-acetyl-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide (Asinex, Russia) and sodium carbonate. A solution of methyl chloride in acetonitrile is slowly added dropwise and refluxed. The refluxed product is allowed to cool to room temperature, and then the solvent is distilled off under reduced pressure. The residue is dissolved in dichloromethane and washed with distilled water. Thereafter, the organic layer is dried over anhydrous sodium sulfate, filtered, and the solvent is distilled off under reduced pressure. The residue containing the reaction product is purified by column chromatography to give the title compound.
MS (m / z) = 377 (M + 1)

[5] Synthesis of 2- (2-acetyl-3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -2- (4-acetyl-phenylazo) -acetamide

In an ice bath, 2- (4-acetyl-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide (Asinex, Russia) and dimethylaminopyridine Dissolve in pyridine and add acetic anhydride. After stopping, the solvent after the reaction is poured into ice water, and the reaction product is extracted with dichloromethane. The reaction product is dried over anhydrous sodium sulfate, filtered, and the solvent is distilled off under reduced pressure. The residue containing the reaction product is purified by column chromatography to give the title compound.
MS (m / z) = 405 (M + 1)

[6] Synthesis of (4-acetyl-phenylazo)-(3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetonitrile

2- (4-acetyl-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide (Asinex, Russia), thionyl chloride and DMF in an ice bath And stir slowly. Excess thionyl chloride is distilled off from the mixed solvent under reduced pressure, and the residue containing the reaction product is purified by column chromatography to give the title compound.
MS (m / z) = 345 (M + 1)

[7] Synthesis of (4-acetyl-phenylazo)-(3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetic acid

Add potassium hydroxide and 2- (4-acetyl-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide (Asinex, Russia) to ethanol, Heat to reflux. The refluxed product is allowed to cool to room temperature, distilled water is added thereto, and hydrochloric acid is added to neutralize the reaction product. Then, the reaction product is extracted with ether. The reaction product is dried over anhydrous sodium sulfate, filtered, and the solvent is distilled off under reduced pressure. The residue containing the reaction product is purified by column chromatography to give the title compound.
MS (m / z) = 364 (M + 1)

[8] Synthesis of (4-acetyl-phenylazo)-(3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetic acid ethyl ester

(4-Acetyl-phenylazo)-(3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetic acid is suspended in ethanol and stirred. While cooling, thionyl chloride is slowly added dropwise and then stirred at room temperature. The solvent is distilled off from the stirred mixed solvent under reduced pressure and washed with water. The reaction product is dried over anhydrous sodium sulfate, filtered, and the solvent is distilled off under reduced pressure. The residue containing the reaction product is purified by column chromatography to give the title compound.
MS (m / z) = 392 (M + 1)

[9] Synthesis of 2- (4-acetyl-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -N-methyl-acetamide

Dissolve (4-acetyl-phenylazo)-(3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetic acid, methylamine, HOBt (Advanced ChemTech, USA) and triethylamine in DMF. HBTU (Advanced ChemTech, USA) in dichloromethane is added and then stirred at room temperature. The stirred mixed solvent is washed with a saturated aqueous sodium chloride solution and a 5% aqueous sodium hydrogen carbonate solution. The reaction product is dried over anhydrous sodium sulfate, filtered, and the solvent is distilled off under reduced pressure. The residue containing the reaction product is purified by column chromatography to give the title compound.
MS (m / z) = 377 (M + 1)

[10] Synthesis of 2- (4-acetyl-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -N, N-dimethyl-acetamide

Dissolve (4-acetyl-phenylazo)-(3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetic acid, dimethylamine, HOBt (Advanced ChemTech, USA) and triethylamine in DMF. HBTU (Advanced ChemTech, USA) in dichloromethane is added and then stirred at room temperature. The stirred mixed solvent is washed with a saturated aqueous sodium chloride solution and a 5% aqueous sodium hydrogen carbonate solution. The reaction product is dried over anhydrous sodium sulfate, filtered, and the solvent is distilled off under reduced pressure. The residue containing the reaction product is purified by column chromatography to give the title compound.
MS (m / z) = 391 (M + 1)

[11] Synthesis of 2- (4-acetyl-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -N-phenyl-acetamide

Dissolve (4-acetyl-phenylazo)-(3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetic acid, phenylamine, HOBt (Advanced ChemTech, USA) and triethylamine in DMF. HBTU (Advanced ChemTech, USA) in dichloromethane is added and then stirred at room temperature. The stirred mixed solvent is washed with a saturated aqueous sodium chloride solution and a 5% aqueous sodium hydrogen carbonate solution. The reaction product is dried over anhydrous sodium sulfate, filtered, and the solvent is distilled off under reduced pressure. The residue containing the reaction product is purified by column chromatography to give the title compound.
MS (m / z) = 439 (M + 1)

[12] Synthesis of 2- (4-acetyl-phenylazo) -2- (3,3-dimethyl-1,2,3,4-tetrahydro-isoquinolin-1-yl) -acetamide

2- (4-Acetyl-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide (Asinex, Russia) was dissolved in acetonitrile under a nitrogen stream. To this is added xylene in which triphenyltin hydride (Aldrich, USA) is dissolved and refluxed. The reflux is allowed to cool to room temperature and the insoluble material is filtered off. The solvent is removed from the filtrate under reduced pressure, and the residue containing the reaction product is purified by column chromatography to give the title compound.
MS (m / z) = 365 (M + 1)

Example 1
(1) Culture of human mesenchymal stem cells
Mesenchymal Stem Cell Basal Medium (MSCBM) (manufactured by CAMBREX) is supplemented with Mesenchymal Cell Growth Supplement (MCGS), glutamine, penicillin and streptomycin (manufactured by CAMBREX) according to the attached protocol, and human mesenchymal stem cell culture medium Was prepared.

Human mesenchymal stem cells (purchased from CAMBREX) were thawed and cultured according to the attached protocol. That is, a human mesenchymal stem cell frozen vial is thawed at 37 ° C., and becomes 5 to 6 × 10 ³ cells / cm ² using a medium that has been kept warm at 37 ° C. in a 5% CO ₂ incubator for 30 minutes or more. The cell culture dish was seeded after dilution. In a 75 cm ² square flask (Costar), about 4 × 10 ⁵ cells are cultured using 37.5 ml of medium. The medium was changed every 3 to 4 days and cultured until confluent.

(2) Preparation of human mesenchymal stem cells After washing human mesenchymal stem cells cultured according to the method described in "Culture of human mesenchymal stem cells" in (1) above using PBS containing no Ca and Mg Add trypsin-EDTA solution (Cosmo Bio), incubate at 37 ° C for 5 minutes, add 5 ml of mesenchymal stem cell medium, disperse the cells using a pipette, transfer to a 15 ml sterile tube, The mixture was pelleted by centrifugation at 1000 rpm for about 5 minutes in a tabletop centrifuge (Tomy Seiko). The supernatant was removed and the cells were added with 5 ml of fresh mesenchymal stem cell medium to obtain a cell suspension.

(3) Mesenchymal stem cell proliferation assay Human mesenchymal stem cells prepared according to the method described in (2) Preparation of human mesenchymal stem cells are placed in a 6 cm ² cell culture dish (FALCON, USA), 3 × 10 ⁴ to 1 × 10 ⁵ cells were seeded by suspending in 5.5 ml of medium. A sample obtained by diluting the tissue stem cell proliferating agent of the present invention described below dissolved in dimethyl sulfoxide (DMSO), water, or a mixture thereof with a human mesenchymal stem cell medium to 0.4 to 100 μg / ml. , 0.5 ml each was added to a human mesenchymal stem cell culture dish and cultured in a 37 ° C., 5% CO ₂ incubator. DMSO was brought into the medium at a final concentration of 0.1% or less. For control, DMSO alone was added to a final concentration of 0.1%. Mesenchymal stem cells cultured for 7 to 14 days were collected according to the method described in “Preparation of human mesenchymal stem cells” in (2) above, and the number of cells was measured using a hemocytometer. The mesenchymal stem cells cultured with the tissue stem cell proliferating agent of the invention added significantly proliferated compared to the cells cultured in a medium not containing the proliferating agent (FIG. 1).

Tissue stem cell proliferating agent A used in Examples: 2-cyano-2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -Np-tolyl-acetamide B: 2- (4 -Acetyl-phenylazo) -2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide C: 2- (3-chloro-phenylazo) -2- (3,3- Dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -acetamide D: 2- (3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -2- (3-methoxy -Phenylazo) -acetamide E: 2- (3,3-Dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -2- (3-nitro-phenylazo) -acetamide F: 2- (3,3 -Dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -2- (4-tolylazo-phenylazo) -acetamide G: 2- (3-acetyl-phenylazo) -2- (3,3-dimethyl- 3,4-Dihydro-2H-isoquinoline-1-ylidene) -acetamide H 2- (3,3-Dimethyl-3,4-dihydro-2H-isoquinolin-1-ylidene) -2-m-tolylazo-acetamide a: 2-methyl-3-nitro-1-phenyl-1H-indole-6 -Ol b: 1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol c: 2-methyl-3-nitro-1-p-tolyl-1H-indole-6 -All d: 2- [2- (4-methoxy-phenylamino) -vinyl] -3-nitro-1-p-tolyl-1H-indole-6-ol e: 1- (2-methoxy-phenyl)- 2-Methyl-3-nitro-1H-indole-6-ol f: 7-dimethylaminomethyl-2- (2-dimethylamino-vinyl) -3-nitro-1-p-tolyl-1H-indole-6- All g: 1- (4-methoxy-phenyl) -2-methyl-3-nitro-7-piperidin-1-ylmethyl-1H-indole-6-ol h: 2- (2-dimethylamino-vinyl) -1 -(4-Methoxy-phenyl) -7-morpholin-4-ylmethyl-3-nitro-1H-indole-6-ol i: 7-[(3 -Hydroxy-propylamino) -methyl] -1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol j: 7-dimethylaminomethyl-2- (2-dimethylamino) -Vinyl) -1- (4-methoxy-phenyl) -3-nitro-1H-indole-6-ol k: 7-diethylaminomethyl-1- (4-methoxy-phenyl) -2-methyl-3-nitro- 1H-indole-6-ol l: 7-dimethylaminomethyl-2-methyl-3-nitro-1-p-tolyl-1H-indole-6-ol m: 1- (4-methoxy-phenyl) -2- Methyl-3-nitro-7-piperidin-1-ylmethyl-1H-indole-6-ol n: 7-acetoxymethyl-2-methyl-3-nitro-1-p-tolyl-1H-indol-6-yl acetate Ester o: 2- (2-dimethylamino-vinyl) -1- (4-methoxy-phenyl) -3-nitro-7-piperidin-1-ylmethyl-1H-indole-6-ol p: 7-dimethylaminomethyl -2-methyl-3-nitro-1-fe Ru-1H-indole-6-ol q: 7-dimethylaminomethyl-1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol r: acetic acid 6-acetoxy-1 -(4-Methoxy-phenyl) -2-methyl-3-nitro-1H-indol-7-ylmethyl ester s: 2- (2-dimethylamino-vinyl) -3-nitro-1-p-tolyl-1H- Indole-6-ol t: 2- (2-dimethylamino-vinyl) -3-nitro-1-phenyl-1H-indole-6-ol u: acetic acid 6-acetoxy-2- (2-dimethylamino-vinyl) -1- (4-Methoxy-phenyl) -3-nitro-1H-indol-7-ylmethyl ester v: 1- (4-chloro-phenyl) -2-methyl-3-nitro-1H-indole-6-ol w: Acetic acid 2- (2-dimethylamino-vinyl) -6-hydroxy-1- (4-methoxy-phenyl) -3-nitro-1H-indol-7-ylmethyl ester x: 5-hydroxy-2-methyl- 4,6-Dinitro-1-phenyl-1H-India Le-3-carboxylic acid ethyl ester

(4) Expression study of marker protein of human mesenchymal stem cell Human mesenchymal stem cell marker of human mesenchymal stem cell obtained by culturing according to the method described in the “human mesenchymal stem cell proliferation assay” of (3) above Protein expression was confirmed. That is, a dish in which human mesenchymal stem cells were cultured was washed with PBS, 0.05% trypsin-EDTA solution (manufactured by Cosmo Bio) was added, and incubated at 37 ° C. for 5 minutes. 5 ml of mesenchymal stem cell medium was added, the cells were dispersed using a pipette, transferred to a 15 ml sterile tube, and pelleted by centrifugation at 1000 rpm for about 5 minutes in a tabletop centrifuge (Tomy Seiko). After washing with Hanks balanced salt solution containing 2% FBS (HBSS, manufactured by Invitrogen), HBSS containing 2% FBS was added so that the number of cells was 1-6 × 10 ⁵ . Antibodies against human mesenchymal stem cell surface antigens, CD105, CD29, CD44, CD14, and CD34, ie, anti-CD105-PE antibody (Ancell Corporation), anti-CD-29-PE antibody (Coulter Corporation), anti-CD-44- FITC antibody (IMMUNOTECH), anti-CD-14-PE antibody (Coulter Corporation), and anti-CD-34-PE antibody (Coulter Corporation) are each diluted with HBSS according to the attached protocol, and added for 30 to 50 minutes on ice. I put it. After washing twice with HBSS (1 ml), the sample was redispersed in HBSS (1 ml) to obtain a sample used for flow cytometer measurement. The sample used for the flow cytometer measurement was measured after passing through a nylon mesh having a pore diameter of 100 μm for the purpose of removing aggregates. The flow cytometer is FACSCalibur (BECTON DICKINSON, USA), and the measurement condition data collection and analysis software is CELLQuest (BECTON DICKINSON, USA). Human mesenchymal stem cells cultured with the tissue stem cell proliferating agent of the present invention are positive for CD105, CD29 and CD44, and negative for CD14 and CD34. (Fig. 2). That is, it was confirmed that the tissue stem cell proliferating agent of the present invention can proliferate human mesenchymal stem cells.

  According to the present invention, tissue stem cells can be proliferated in large quantities in a single period. The tissue stem cell proliferating agent provided by the present invention, the culture method using the same, and the culture medium can be applied to produce tissue stem cells as a cell source used in cell transplantation applications. In addition to the many benefits obtained by utilizing tissue stem cells, the tissue stem cell proliferating agent provided by the present invention and the culture method using the same produce tissue stem cells having one or many genetic modifications. Can be applied for. Examples of such applications include, but are not limited to, the development of cell-based models for disease as well as the development of tissues that are specific for transplantation to treat genetic diseases.

Furthermore, since the tissue stem cell proliferating agent of the present invention has an excellent ability to maintain undifferentiated tissue stem cells and proliferate, it can be used as a therapeutic or prophylactic agent for tissues or organs damaged by aging, illness or trauma. Can do. In particular, it is applied to alveolar bone damage due to periodontal disease / alveolar pus leakage, and male pattern baldness, and can be used as an oral or parenteral agent as a prophylactic and / or therapeutic agent.
As described above, the tissue stem cell proliferating agent of the present invention can proliferate tissue stem cells in an undifferentiated state, and can be suitably used in the field of regenerative medicine.

FIG. 1 shows the results of the mesenchymal stem cell proliferation assay of the present invention. FIG. 2 shows the expression study of the marker protein of the mesenchymal stem cell of the present invention.

Claims (25)

A tissue stem cell proliferating agent comprising a low molecular weight compound or a salt thereof as an active ingredient. The low molecular weight compound is represented by the general formula (1)

[Wherein R ¹ , R ² , R ³ , and R ⁴ may be the same or different and each represents an electron-withdrawing group, an electron-donating group, or a hydrogen atom. The A ring represents a 5- to 8-membered ring containing at least one hetero atom in the ring. 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 -CONH- group. Moreover, the group which becomes a double bond may be sufficient as the bond couple | bonded with the ring A. Furthermore, the alkylene group may have one or more electron-withdrawing groups, electron-donating groups, or hydrogen atoms as a substituent. G represents an aromatic group which may have an electron withdrawing group, an electron donating group or a hydrogen atom. The A ring may have one or more electron withdrawing groups and / or electron donating groups as a substituent other than the —XG group. ]
The tissue stem cell proliferating agent according to claim 1, which is a compound represented by: The tissue stem cell proliferating agent according to claim 2, wherein the A ring is a 5- or 6-membered ring containing at least one heteroatom selected from the group consisting of a nitrogen atom, an oxygen atom and a sulfur atom in the ring. The tissue stem cell proliferating agent according to claim 2, wherein the A ring is a 5- or 6-membered ring containing one nitrogen atom in the ring. An alkylene group represented by X is a group selected from the group consisting of an alkyl group, an acyl group, an alkoxy group, a nitro group, a hydroxycarbonyl group, an alkoxycarbonyl group, an aminocarbonyl group, a nitrile group and a halogen atom as a substituent; Or the tissue stem cell proliferation agent in any one of Claims 2-4 which has one or more atoms. The low molecular weight compound is represented by the general formula (2)

[Wherein R ¹ , R ² , R ³ , R ⁴ , X and G have the same definitions as in claim 2. R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ may be the same or different and each represents an electron-withdrawing group, an electron-donating group or a hydrogen atom. ]
The tissue stem cell proliferating agent according to claim 2, which is a compound represented by: The low molecular weight compound is represented by the general formula (3)

[Wherein R ¹ , R ² , R ³ , R ⁴ , X and G have the same definitions as in claim 2. 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 tissue stem cell proliferating agent according to claim 2, which is a compound represented by: The low molecular weight compound is represented by the general formula (4)

[Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , and R ⁹ have the same definitions as in claim 6. R ¹⁰ , R ¹¹ , R ¹² and R ¹³ may be the same or different and each represents an electron withdrawing group, an electron donating group or a hydrogen atom. A double broken line on a single side represents a single bond or a double bond. When the double dashed line represents a double bond, there is a geometric isomer with respect to the wavy line. The arrangement of these geometric isomers is not particularly limited, and may be either E-form or Z-form independently. ]
The tissue stem cell proliferating agent according to claim 6, which is a compound represented by: R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , and R ¹³ are each independently an alkyl group or an acyl group The tissue stem cell proliferating agent according to claim 8, which is a group or an atom selected from the group consisting of an alkoxy group, a nitro group, a hydroxycarbonyl group, a nitrile group, an alkoxycarbonyl group, an aminocarbonyl group, a halogen atom and a hydrogen atom. The low molecular weight compound is represented by the general formula (5)

Where A is a hydrogen atom or

Represents.
[Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , and R ⁹ have the same definitions as in claim 6. R ¹⁰ , R ¹¹ and R ¹² may be the same or different and each represents an electron withdrawing group, an electron donating group or a hydrogen atom. ]
The tissue stem cell proliferating agent according to claim 6, which is a compound represented by: R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , and R ¹² are each independently an alkyl group, an acyl group, or an alkoxy group. The tissue stem cell proliferating agent according to claim 10, which is a group or atom selected from the group consisting of nitro group, hydroxycarbonyl group, nitrile group, alkoxycarbonyl group, aminocarbonyl group, halogen atom and hydrogen atom. The low molecular weight compound is represented by the general formula (6)

[Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , and R ⁶ have the same definitions as in claim 7. R ⁷ , R ⁸ and R ⁹ may be the same or different and each represents an electron withdrawing group, an electron donating group or a hydrogen atom. ]
The tissue stem cell proliferating agent according to claim 7, which is a compound represented by: R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , and R ⁹ are each independently an alkyl group, an alkoxy group, a hydroxyl group, a nitro group, a nitrile group, an acetoxy group, An acetoxyalkyl group, a cyclic alkylaminoalkyl group which may contain an oxygen atom, a dialkylaminoalkyl group, a dialkylaminovinyl group, a hydroxyalkylaminoalkyl group, an arylaminovinyl group, an alkoxycarbonyl group, a halogen atom and a hydrogen atom; The tissue stem cell proliferating agent according to claim 12, which is a selected group or atom. R ¹ , R ² , R ³ , R ⁴ , R ⁸ , R ⁹ and R ¹⁰ are hydrogen atoms, R ⁵ is a hydrogen atom, a lower acyl group, or a lower alkyl group, and R ⁶ and R ⁷ are A lower alkyl group which may be the same or different, R ¹¹ is a hydrogen atom, a halogen atom, a nitro group, a lower alkyl group, a lower acyl group or a lower alkoxy group, and R ¹² is a hydrogen atom, An alkyl group, a lower acyl group, or a lower alkoxy group, and R ¹³ is a hydroxycarbonyl group, a nitrile group, a carbamoyl group, a mono-lower alkylaminocarbonyl group, a di-lower alkylaminocarbonyl group, a secondary aminocarbonyl group, a tertiary The tissue stem cell proliferating agent according to claim 8, which is an aminocarbonyl group or a lower alkoxycarbonyl group. R ¹ , R ² , R ³ , R ⁴ , R ⁸ , and R ⁹ are hydrogen atoms, R ⁵ is a hydrogen atom, a lower acyl group, or a lower alkyl group, and R ⁶ and R ⁷ are the same A lower alkyl group which may be different or different, R ¹⁰ is a hydrogen atom or a lower alkyl group, R ¹¹ is a hydrogen atom or a nitro group, and R ¹² is a hydrogen atom, a lower alkyl group, a lower group. The tissue stem cell proliferating agent according to claim 10, which is an acyl group, a lower alkoxycarbonyl group, or a lower alkoxy group. R ¹ and R ² are hydrogen atoms, R ³ is a hydroxyl group or an acetoxy group, R ⁴ is an acetoxyalkyl group, a cyclic alkylaminoalkyl group which may contain an oxygen atom, a di-lower alkylamino lower alkyl group, a hydroxy group A lower alkylamino lower alkyl group, or a hydrogen atom, R ⁵ is a lower alkyl group, a di-lower alkylaminovinyl group, or an arylaminovinyl group, R ⁶ is a nitro group, R ⁷ , R ⁸ , and The tissue stem cell proliferating agent according to claim 12, wherein R ⁹ is the same or different lower alkyl group, lower alkoxy group, or hydrogen atom. The tissue stem cell proliferating agent according to any one of claims 1 to 16, wherein the tissue stem cells are mesenchymal stem cells. The tissue stem cell proliferating agent according to any one of claims 1 to 17, wherein the tissue stem cells are human tissue stem cells. A tissue stem cell culturing method comprising growing a tissue stem cell using the tissue stem cell proliferating agent according to any one of claims 1 to 18. A medium containing the tissue stem cell proliferating agent according to any one of claims 1 to 18 as an active ingredient. The medium according to claim 20, wherein the concentration of the tissue stem cell proliferating agent is 10 ng / ml to 100 µg / ml. Tissue stem cells cultured using the tissue stem cell proliferating agent according to any one of claims 1 to 18. A cell or tissue obtained by differentiating a tissue stem cell cultured using the tissue stem cell proliferating agent according to any one of claims 1 to 18. The cell or tissue according to claim 22 or 23, wherein the cell or tissue is for transplantation into a living body. The treatment method which transplants the cell or / and tissue in any one of Claims 22-24 in a biological body.

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