JP2014062067A - Proliferation acceleration and/or mobilization agent for hematopoietic stem cell and/or hemopoietic precursor cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide proliferation acceleration and/or mobilization agents for hematopoietic stem cells and/or hemopoietic precursor cells, containing a sterol compound or its pharmacologically acceptable salt as an active ingredient.SOLUTION: This invention provides proliferation acceleration and/or mobilization agents for hematopoietic stem cells and/or hemopoietic precursor cells, containing a sterol compound represented by general formula (I) or (A) or its pharmacologically acceptable salt as an active ingredient.

Description

  The present invention relates to a growth promoting and / or mobilizing agent for hematopoietic stem cells and / or hematopoietic progenitor cells containing a sterol compound or a pharmaceutically acceptable salt thereof as an active ingredient.

Techniques for proliferating stem cells and / or inducing differentiation of human tissues and cells from stem cells include heart failure, liver failure, renal failure, neurodegenerative diseases (such as Parkinson's disease and Alzheimer's disease), and cardiovascular disorders that do not currently have effective root treatment From the viewpoint of providing new treatments for cerebrovascular disorders, spinal cord injury, arthropathy, osteoporosis, diabetes, and the like.
Stem cells are cells having both self-replicating ability and multipotency capable of differentiating into various tissues. Since they have self-replicating ability, they can be applied to mass production of cells. Stem cells can be roughly classified into five types: embryonic stem cells (ES cells), embryonic stem cells, adult stem cells, umbilical cord blood stem cells and placental stem cells.

  Among these, an adult stem cell is a stem cell present in various tissues, is proliferated and maintained by self-replication ability, differentiates into a progenitor cell by differentiation ability, and further differentiates into a functional cell. For example, hematopoietic stem cells are present mainly in bone marrow tissue in adults, differentiate into hematopoietic progenitor cells, and then mature into circulating blood cells such as red blood cells, white blood cells, platelets and lymphocytes. Thus, the production of blood cells begins with hematopoietic stem cells and is maintained. Similarly, cells responsible for the functions of the respective tissues of the adult, such as skeletal muscle cells, smooth muscle cells, cardiomyocytes, blood cells, vascular endothelial cells, adipocytes, osteoblasts, chondrocytes, fibroblasts, kidney glomeruli As for cells, renal tubular cells, nerve cells, hepatocytes, pancreatic cells, tracheal epithelial cells, etc., stem cells and progenitor cells of the respective tissues exist and maintain the tissues.

As drugs that mobilize hematopoietic stem cells and / or hematopoietic progenitor cells to peripheral blood, chemokines and analogs thereof, various antibodies, cytokines, growth factors, and the like are known (Exp Hematology, 34, p.996-1009, 2006). For example, granulocyte colony stimulating factor (G-CSF) preparations are used as hematopoietic stem cell mobilizers (Blood, 89 (7), p.2233-2258, 1997) for the collection of stem cells. Is used to treat leukemia, lymphoma, and other life-threatening diseases. In addition, G-CSF preparations are used as leukocyte-increasing agents (Japanese Patent No. 2718426), for example, for the treatment of infections in immunocompromised patients, leukopenia upon administration of anticancer agents, aplastic anemia, and the like. In addition, it has been reported that AMD3100, a CXCR4 antagonist, enhances the effect of mobilizing hematopoietic stem cells by G-CSF (Transfusion, 45, p.295-300, 2005). Furthermore, in mice and monkeys, IL-8 has been reported to mobilize hematopoietic stem cells and / or hematopoietic progenitor cells from the bone marrow (Blood, 85, p.2269-2275, 2005). In addition, as a substance related to the mobilization of hematopoietic stem cells and / or hematopoietic progenitor cells, one of chemokines, SDF-1 (Stromal cell derived factor-1) (Blood, 97, p. 3354-3360, 2001; Blood , 99, p.44-51, 2002), Vascular endothelial cell growth factor (VEGF), Angiopoietin-1 (J Exp Med, 193, p.1005-1014, 2001), and stromal cells VCAM-1 (Vascular cell adhesion molecule-1), an antibody that neutralizes VLA-4 (Very late antigen-1) expressed in human hematopoietic stem cells and / or hematopoietic progenitor cells (Proc. Natl. Acad. Sci. USA 92 , p.9647-9651, 1995), oligonucleotide with CpG motif characteristic of bacterial DNA (CpG-ODN) (Journal of cellular physiology, 204, p.889-895, 2005), prostaglandin I _Two derivatives (Japanese Patent Laid-Open No. 2007-269784) are known.

  GSK-3β inhibitor (Nature Medicine, 12, p.89-98, 2006), an agent for the treatment of osteoporosis, is an agent that has the activity of promoting the proliferation of hematopoietic stem cells and / or hematopoietic progenitor cells. Thyroid hormone (Patent No. 512897; Nature, 425, p.841-846, 2003), prostaglandin E2 derivative (Nature, 447, p.1007-1011, 2007) and the like are known. In addition, compounds that amplify hematopoietic stem cells and / or hematopoietic progenitor cells in vitro (Japanese Patent Laid-Open No. 2012-65644), compounds for increasing the hematopoietic stem cell population (Special Table 2012-507554), and the like are also known.

  On the other hand, compounds (see Patent Document 1) and sterol derivatives (see Patent Document 2) produced by Penicillium sp. CND1007 strain (FERM BP-10917) are known as compounds having a neural stem cell proliferation promoting action. ing.

International Publication No. 2009/096445 Pamphlet International Publication No. 2011/010682 Pamphlet

  An object of the present invention is to provide an agent for promoting proliferation and / or mobilizing hematopoietic stem cells and / or hematopoietic progenitor cells.

The present invention relates to the following (1) to (10).
(1) Growth-promoting and / or mobilizing agent for hematopoietic stem cells and / or hematopoietic progenitor cells containing, as an active ingredient, a sterol compound represented by the following general formula (I) or (A) or a pharmaceutically acceptable salt thereof: .

[Where:
Y represents lower alkyl optionally having substituent (s) or lower alkenyl optionally having substituent (s);
X ^a and X ^b are the same or different and are a bond or —NR ^a — (wherein R ^a is a hydrogen atom, optionally substituted lower alkyl, optionally substituted) Cycloalkyl, lower alkanoyl which may have a substituent or aroyl which may have a substituent)
R ¹ , R ² , R ³ , R ⁴ , R ⁷ and R ⁸ are the same or different and each represents a hydrogen atom or hydroxy, or
R ¹ and R ² , R ³ and R ⁴ , or R ⁷ and R ⁸ together represent ═O,
R ⁵ and R ⁶ are the same or different and each is a hydrogen atom, halogen, azide, hydroxy, optionally substituted lower alkoxy, optionally substituted cycloalkyloxy, or optionally substituted. Lower alkenyloxy which may have a substituent, lower alkynyloxy which may have a substituent, aryloxy which may have a substituent, aromatic heterocyclic oxy which may have a substituent, substituted Aliphatic heterocyclic oxy which may have a group, lower alkanoyloxy which may have a substituent, aroyloxy which may have a substituent, aromatic heterocyclic which may have a substituent Ring carbonyloxy or —NR ^b R ^c (wherein R ^b and R ^c are the same or different and are a hydrogen atom, optionally substituted lower alkyl, optionally substituted lower Alkenyl, substituted Cycloalkyl which may have a group, aryl which may have a substituent, aromatic heterocyclic group which may have a substituent, aliphatic heterocyclic ring which may have a substituent Group, lower alkanoyl optionally having substituent, aroyl optionally having substituent, lower alkoxycarbonyl optionally having substituent, aryloxycarbonyl optionally having substituent , Optionally substituted aromatic heterocyclic oxycarbonyl, optionally substituted lower alkyl carbamoyl, optionally substituted di-lower alkyl carbamoyl, substituted Arylcarbamoyl which may be substituted, aromatic heterocyclic carbamoyl which may have a substituent, arylsulfonyl which may have a substituent or lower alkylsulfonyl which may have a substituent Or represents represents a sulfonyl), or, O = R ⁵ and R ⁶ together, R ^d ON = (wherein, R ^d represents a hydrogen atom, an optionally substituted lower alkyl, substituted Cycloalkyl which may have a group, aryl which may have a substituent, aromatic heterocyclic group which may have a substituent or aliphatic heterocyclic ring which may have a substituent Or R ^e R ^f C = (wherein R ^e and R ^f are the same or different and have a hydrogen atom, halogen, optionally substituted lower alkyl, or substituent) Lower alkenyl which may have, lower alkynyl which may have a substituent, cycloalkyl which may have a substituent, aryl which may have a substituent, which may have a substituent Good aromatic heterocyclic group, optionally substituted aliphatic heterocyclic group, optionally substituted lower alkyl Xyl, optionally substituted aryloxy, optionally substituted aromatic heterocyclic oxy, optionally substituted lower alkanoyl, optionally substituted Aroyl, lower alkanoyloxy optionally having substituent (s) or aroyloxy optionally having substituent (s))
R ⁹ has a hydrogen atom, optionally substituted lower alkyl, optionally substituted lower alkenyl, optionally substituted lower alkynyl, or optionally substituted. Represents a good cycloalkyl, or
R ¹ and R ³ , R ³ and R ⁵ (only when X ^a is a bond), R ⁵ and R ⁷ (but only when X ^b is a bond) or R ⁷ and R ⁹ together Represents a bond or an oxygen atom,
R ¹⁰ , R ¹¹ and R ¹² represent a bond or an oxygen atom when R ¹⁰ and R ¹¹ are taken together; R ¹² represents a hydrogen atom or a bond or oxygen taken together with R ¹⁰ and R ¹² Represents an atom, and R ¹¹ represents a hydrogen atom]
(2) The agent according to (1), wherein the sterol compound is a sterol compound represented by the formula (I).
(3) The agent according to (2), wherein R ¹⁰ and R ¹¹ together represent a bond, and R ¹² is a hydrogen atom.
(4) The agent according to (2) or (3), wherein X ^a and X ^b are a bond.
(5) The agent according to (1), wherein the sterol compound is a compound represented by any of the following formulas (A), (110), (18), (49), (81) and (83).

(6) The agent according to (1), wherein the sterol compound is a compound represented by any of the following formulas (110), (18), (49), (81) and (83).

(7) The agent according to any one of (1) to (6), which is a hematopoietic stem cell proliferation promoter.
(8) The agent according to any one of (1) to (6), which is an agent for promoting mobilization of hematopoietic stem cells.
(9) The agent according to any one of (1) to (6), which is a proliferation promoter for hematopoietic progenitor cells.
(10) The agent according to any one of (1) to (6), which is a mobilization promoter for hematopoietic progenitor cells.

  The present invention provides an agent for promoting proliferation and / or mobilization of hematopoietic stem cells and / or hematopoietic progenitor cells containing a sterol compound or a pharmaceutically acceptable salt thereof as an active ingredient.

In the present invention, “proliferation promotion” is used in the sense of enhancing the ability of a cell to grow or accelerating the speed of cell proliferation, not limited to the type of cell. For example, “hematopoietic stem cells and / or progenitor cells promote proliferation” means that the proliferation rate of hematopoietic stem cells and / or progenitor cells is accelerated and the number of hematopoietic stem cells and / or progenitor cells increases.
In the present invention, “mobilization” is used in the sense that cells accumulate not only from the type of cells but also from other sites. For example, “hematopoietic stem cells and / or progenitor cells mobilize” means that hematopoietic stem cells and / or progenitor cells accumulate from the bone marrow into peripheral blood. The agent for mobilizing hematopoietic stem cells and / or progenitor cells in the present invention is, in other words, an agent for mobilizing hematopoietic stem cells and / or progenitor cells from bone marrow to peripheral blood.

The agent for promoting proliferation and / or mobilizing hematopoietic stem cells and / or progenitor cells of the present invention contains a sterol compound represented by formula (I) or (A) or a pharmaceutically acceptable salt thereof as an active ingredient.
Hereinafter, the compound represented by the general formula (I) is referred to as Compound I. The same applies to the compounds of other formula numbers.

In the definition of each group of general formula (I):
Lower alkyl and lower alkoxy, lower alkanoyl, lower alkoxycarbonyl, lower alkanoyloxy, lower alkylcarbamoyl, dilower alkylcarbamoyl and lower alkylsulfonyl include, for example, linear or branched alkyl having 1 to 10 carbon atoms. More specifically, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl and the like can be mentioned. The two lower alkyls in the di-lower alkylcarbamoyl may be the same or different.

Examples of lower alkenyl and lower alkenyl moiety of lower alkenyloxy include linear or branched alkenyl having 2 to 10 carbon atoms, and more specifically vinyl, allyl, 1-propenyl, 1-butenyl, 2- Examples include butenyl, 3-butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl and the like.
Examples of the lower alkynyl moiety of lower alkynyl and lower alkynyloxy include linear or branched alkynyl having 2 to 10 carbon atoms, and more specifically, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, Noninyl, decynyl and the like can be mentioned.

Examples of the cycloalkyl part of cycloalkyl and cycloalkyloxy include cycloalkyl having 3 to 8 carbon atoms, and more specifically, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.
Examples of aryl and aryl moiety of aryloxy, aroyl, aroyloxy, aryloxycarbonyl, arylcarbamoyl and arylsulfonyl include aryl having 6 to 14 carbon atoms, and more specifically, phenyl, naphthyl, azulenyl, anthryl and the like. can give.

  Examples of the aliphatic heterocyclic group and the aliphatic heterocyclic group portion of the aliphatic heterocyclic oxy include, for example, a 5-membered or 6-membered monocyclic group including at least one atom selected from a nitrogen atom, an oxygen atom, and a sulfur atom. An aliphatic heterocyclic group, a bicyclic or tricyclic condensed 3- to 8-membered ring containing at least one atom selected from a nitrogen atom, an oxygen atom and a sulfur atom, etc. More specifically, aziridinyl, azetidinyl, pyrrolidinyl, piperidino, piperidinyl, azepanyl, 1,2,5,6-tetrahydropyridyl, imidazolidinyl, pyrazolidinyl, piperazinyl, homopiperazinyl, pyrazolinyl, oxiranyl, tetrahydrofuranyl, tetrahydro-2H- Pyranyl, 5,6-dihydro-2H-pyranyl, oxazolidinyl, morpholino, morpholinyl, thioxazolid Thiomorpholinyl, 2H-oxazolyl, 2H-thioxazolyl, dihydroindolyl, dihydroisoindolyl, dihydrobenzofuranyl, benzimidazolidinyl, dihydrobenzoxazolyl, dihydrobenzothioxazolyl, tetrahydroquinolyl, tetrahydroiso Examples include quinolyl, dihydro-2H-chromanyl, dihydro-1H-chromanyl, dihydro-2H-thiochromanyl, dihydro-1H-thiochromanyl, tetrahydroquinoxalinyl, tetrahydroquinazolinyl, dihydrobenzodioxanyl and the like.

  Aromatic heterocyclic groups and aromatic heterocyclic groups in aromatic heterocyclic oxy, aromatic heterocyclic carbonyloxy, aromatic heterocyclic oxycarbonyl and aromatic heterocyclic carbamoyl include, for example, nitrogen atom, oxygen atom and sulfur 5- or 6-membered monocyclic aromatic heterocyclic group containing at least one atom selected from atoms, bicyclic or tricyclic condensed 3- to 8-membered ring, nitrogen atom, oxygen atom and sulfur atom A condensed aromatic heterocyclic group containing at least one atom selected from the group consisting of furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, Triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl Benzofuranyl, benzothiophenyl, benzoxazolyl, benzothiazolyl, isoindolyl, indolyl, indazolyl, benzoimidazolyl, benzotriazolyl, oxazolopyrimidinyl, thiazolopyrimidinyl, pyrrolopyridinyl, pyrrolopyrimidinyl, imidazopyridinyl, purinyl, quinolinyl, isoquinolinyl, Cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl and the like.

Halogen means each atom of fluorine, chlorine, bromine and iodine.
Lower alkyl which may have a substituent, Lower alkenyl which may have a substituent, Lower alkynyl which may have a substituent, Lower alkoxy which may have a substituent, Substituent Lower alkenyloxy which may have a substituent, lower alkynyloxy which may have a substituent, lower alkanoyloxy which may have a substituent, lower alkanoyl which may have a substituent, substituted Lower alkoxycarbonyl which may have a group, Lower alkylcarbamoyl which may have a substituent, Dilower alkylcarbamoyl which may have a substituent and Lower alkyl which may have a substituent Examples of the substituent for sulfonyl are the same or different, for example, having 1 to 3 substituents.
Halo; azide; cyano; carboxy; carbamoyl; formyl; C _3-8 cycloalkyl;
C _6-14 which may have 1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, nitro, carboxy, C _1-10 alkoxycarbonyl, C _1-10 alkoxy and trifluoromethyl Aryl;
Aliphatic heterocyclic group; aromatic heterocyclic group;
1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, carboxy, C _1-10 alkoxy, C _1-10 alkylamino, di-C _1-10 alkylamino and C _1-10 alkoxycarbonyl _Optionally C _1-10 alkoxy;
C _2-10 alkenyloxy;
C _3-8 cycloalkoxy;
C _6-14 which may have 1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, nitro, carboxy, C _1-10 alkoxycarbonyl, C _1-10 alkoxy and trifluoromethyl Aryloxy;
C _7-16 optionally having 1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, nitro, carboxy, C _1-10 alkoxycarbonyl, C _1-10 alkoxy and trifluoromethyl Aralkyloxy;
C _2-11 alkanoyloxy; C _7-15 aroyloxy; C _1-10 alkylsulfonyloxy; trifluoromethanesulfonyloxy; C _6-14 arylsulfonyloxy; p-toluenesulfonyloxy;
C _1-10 alkylsulfanyl; C _6-14 arylsulfanyl;
-NR ^Xa R ^Ya (wherein R ^Xa and R ^Ya are the same or different, hydrogen atom; formyl;
1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, carboxy, C _1-10 alkoxy, C _1-10 alkylamino, di-C _1-10 alkylamino and C _1-10 alkoxycarbonyl _{Optionally substituted} C _1-10 alkyl;
C _3-8 cycloalkyl;
C _6-14 which may have 1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, nitro, carboxy, C _1-10 alkoxycarbonyl, C _1-10 alkoxy and trifluoromethyl Aryl;
An aromatic heterocyclic group;
C _7-16 optionally having 1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, nitro, carboxy, C _1-10 alkoxycarbonyl, C _1-10 alkoxy and trifluoromethyl Aralkyl;
1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, carboxy, C _1-10 alkoxy, C _1-10 alkylamino, di-C _1-10 alkylamino and C _1-10 alkoxycarbonyl _Optionally C _2-11 alkanoyl;
C _7-15 aroyl; C _1-10 alkoxycarbonyl; C _7-16 aralkyloxycarbonyl;
C _1-10 alkylcarbamoyl, diC _1-10 alkylcarbamoyl, C _6-14 arylcarbamoyl, C _1-10 alkylsulfonyl; trifluoromethanesulfonyl; C _6-14 arylsulfonyl or p-toluenesulfonyl);
C _2-11 alkanoyl; C _3-8 cycloalkylcarbonyl; C _7-15 aroyl, aliphatic heterocyclic carbonyl; aromatic heterocyclic carbonyl; C _1-10 alkoxycarbonyl; C _6-14 aryloxycarbonyl; C _{7- 16} Aralkyloxycarbonyl; from the group consisting of hydroxy, halogen, C _1-10 alkoxy, amino, C _1-10 alkylamino, di-C _1-10 alkylamino, C _2-11 alkanoylamino and C _1-10 alkoxycarbonylamino C _1-10 alkylcarbamoyl optionally having 1 to 3 substituents selected; hydroxy, halogen, C _1-10 alkoxy, amino, C _1-10 alkylamino, di-C _1-10 alkylamino, C _2-11 alkanoylamino and C _1-10 alkoxy which may have 1 to 3 substituents selected from the group consisting carbonylamino di C _1-10 alkylcarbamoyl and C _6-14 aryl Such substituent selected from the group consisting of Rubamoiru the like.

Aryl which may have a substituent, aryloxy which may have a substituent, aroyl which may have a substituent, aroyloxy which may have a substituent, having a substituent An optionally substituted aryloxycarbonyl, an optionally substituted arylcarbamoyl, an optionally substituted arylsulfonyl, an optionally substituted aromatic heterocyclic group, and a substituent. Aromatic heterocyclic oxy which may have, aromatic heterocyclic carbonyloxy which may have a substituent, aromatic heterocyclic oxycarbonyl which may have a substituent and a substituent As the substituents in the aromatic heterocyclic carbamoyl which may be the same or different, for example, having 1 to 5 substituents,
Halo; cyano; carboxy; carbamoyl; C _1-10 alkyl; trifluoromethyl; C _3-8 cycloalkyl;
C _6-14 which may have 1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, nitro, carboxy, C _1-10 alkoxycarbonyl, C _1-10 alkoxy and trifluoromethyl Aryl;
Aliphatic heterocyclic group; aromatic heterocyclic group;
1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, carboxy, C _1-10 alkoxy, C _1-10 alkylamino, di-C _1-10 alkylamino and C _1-10 alkoxycarbonyl _Optionally C _1-10 alkoxy;
C _3-8 cycloalkoxy;
C _6-14 which may have 1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, nitro, carboxy, C _1-10 alkoxycarbonyl, C _1-10 alkoxy and trifluoromethyl Aryloxy;
C _7-16 optionally having 1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, nitro, carboxy, C _1-10 alkoxycarbonyl, C _1-10 alkoxy and trifluoromethyl Aralkyloxy;
C _2-11 alkanoyloxy; C _7-15 aroyloxy; C _1-10 alkylsulfonyloxy; trifluoromethanesulfonyloxy; C _6-14 arylsulfonyloxy; p-toluenesulfonyloxy;
C _1-10 alkylsulfanyl; C _6-14 arylsulfanyl;
-NR ^Xb R ^Yb (wherein R ^Xb and R ^Yb are the same or different, a hydrogen atom; formyl;
1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, carboxy, C _1-10 alkoxy, C _1-10 alkylamino, di-C _1-10 alkylamino and C _1-10 alkoxycarbonyl _{Optionally substituted} C _1-10 alkyl;
C _3-8 cycloalkyl;
C _6-14 which may have 1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, nitro, carboxy, C _1-10 alkoxycarbonyl, C _1-10 alkoxy and trifluoromethyl Aryl;
An aromatic heterocyclic group;
C _7-16 optionally having 1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, nitro, carboxy, C _1-10 alkoxycarbonyl, C _1-10 alkoxy and trifluoromethyl Aralkyl;
1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, carboxy, C _1-10 alkoxy, C _1-10 alkylamino, di-C _1-10 alkylamino and C _1-10 alkoxycarbonyl _Optionally C _2-11 alkanoyl;
C _7-15 aroyl; C _1-10 alkoxycarbonyl; C _7-16 aralkyloxycarbonyl; C _1-10 alkylsulfonyl; trifluoromethanesulfonyl; C _6-14 arylsulfonyl or p-toluenesulfonyl)
C _2-11 alkanoyl; C _3-8 cycloalkylcarbonyl; C _7-15 aroyl, aliphatic heterocyclic carbonyl; aromatic heterocyclic carbonyl; C _1-10 alkoxycarbonyl; C _6-14 aryloxycarbonyl; C _{7- 16} aralkyloxycarbonyl; C _1-10 alkylcarbamoyl; di-C _1-10 alkylcarbamoyl; C _6-14 arylcarbamoyl;
And substituents selected from the group consisting of C _1-10 alkylsulfonyl and C _6-14 arylsulfonyl.

Cycloalkyl optionally having substituent, cycloalkyloxy optionally having substituent, aliphatic heterocyclic group optionally having substituent and aliphatic optionally having substituent The substituents in the group heterocyclic oxy are the same or different, for example, having 1 to 5 substituents.
Oxo; halogen; hydroxy; sulfanyl; nitro; cyano; carboxy; carbamoyl; 1-3 hydroxy optionally substituted C _1-10 alkyl; trifluoromethyl; C _3-8 cycloalkyl;
C _6-14 which may have 1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, nitro, carboxy, C _1-10 alkoxycarbonyl, C _1-10 alkoxy and trifluoromethyl Aryl;
Aliphatic heterocyclic group; aromatic heterocyclic group;
1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, carboxy, C _1-10 alkoxy, C _1-10 alkylamino, di-C _1-10 alkylamino and C _1-10 alkoxycarbonyl _Optionally C _1-10 alkoxy;
C _3-8 cycloalkoxy;
C _6-14 which may have 1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, nitro, carboxy, C _1-10 alkoxycarbonyl, C _1-10 alkoxy and trifluoromethyl Aryloxy;
C _7-16 optionally having 1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, nitro, carboxy, C _1-10 alkoxycarbonyl, C _1-10 alkoxy and trifluoromethyl Aralkyloxy;
C _2-11 alkanoyloxy; C _7-15 aroyloxy; C _1-10 alkylsulfonyloxy; trifluoromethanesulfonyloxy; C _6-14 arylsulfonyloxy; p-toluenesulfonyloxy;
C _1-10 alkylsulfanyl; C _6-14 arylsulfanyl;
-NR ^Xc R ^Yc (wherein R ^Xc and R ^Yc are the same or different and represent a hydrogen atom; formyl;
1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, carboxy, C _1-10 alkoxy, C _1-10 alkylamino, di-C _1-10 alkylamino and C _1-10 alkoxycarbonyl _{Optionally substituted} C _1-10 alkyl;
C _3-8 cycloalkyl;
C _6-14 which may have 1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, nitro, carboxy, C _1-10 alkoxycarbonyl, C _1-10 alkoxy and trifluoromethyl Aryl;
An aromatic heterocyclic group;
C _7-16 optionally having 1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, nitro, carboxy, C _1-10 alkoxycarbonyl, C _1-10 alkoxy and trifluoromethyl Aralkyl;
1 to 3 substituents selected from the group consisting of halogen, hydroxy, amino, carboxy, C _1-10 alkoxy, C _1-10 alkylamino, di-C _1-10 alkylamino and C _1-10 alkoxycarbonyl _Optionally C _2-11 alkanoyl;
C _7-15 aroyl; C _1-10 alkoxycarbonyl; C _7-16 aralkyloxycarbonyl; C _1-10 alkylsulfonyl; trifluoromethanesulfonyl; C _6-14 arylsulfonyl or p-toluenesulfonyl)
C _2-11 alkanoyl; C _3-8 cycloalkylcarbonyl; C _7-15 aroyl, aliphatic heterocyclic carbonyl; aromatic heterocyclic carbonyl; C _1-10 alkoxycarbonyl; C _6-14 aryloxycarbonyl; C _{7- 16} aralkyloxycarbonyl; C _1-10 alkylcarbamoyl; di-C _1-10 alkylcarbamoyl; C _6-14 arylcarbamoyl;
And substituents selected from the group consisting of C _1-10 alkylsulfonyl and C _6-14 arylsulfonyl.

C _1-10 alkyl and C _1-10 alkoxy, C _2-11 alkanoyloxy, C _1-10 alkylsulfanyl, C _1-10 alkylamino, di-C _1-10 alkylamino, C _2-11 alkanoyl shown here Amino, C _1-10 alkoxycarbonylamino, C _2-11 alkanoyl, C _1-10 alkoxycarbonyl, C _1-10 alkylcarbamoyl, di-C _1-10 alkylcarbamoyl, C _1-10 alkylsulfonyl and C _1-10 alkyl Examples of the C _1-10 alkyl moiety of sulfonyloxy include the groups exemplified above for the lower alkyl. Two C _1-10 alkyl in di C _1-10 alkylamino and di C _1-10 alkylcarbamoyl may be the same or different.

Examples of the C _2-10 alkenyl moiety of C _2-10 alkenyloxy include the groups exemplified above for the lower alkenyl.
The C _3-8 cycloalkyl moiety of the C _3-8 cycloalkyl and C _3-8 cycloalkoxy and C _3-8 cycloalkylcarbonyl, for example groups mentioned for illustrative said cycloalkyl is exemplified.

C _6-14 aryl and C _6-14 aryloxy, C _7-15 aroyloxy, C _6-14 arylsulfanyl, C _6-14 arylsulfonyl, C _7-15 aroyl, C _6-14 aryloxycarbonyl, C _6- Examples of the C _6-14 aryl moiety of ₁₄ arylcarbamoyl, C _6-14 arylsulfonyl, and C _6-14 arylsulfonyloxy include the groups exemplified in the aforementioned aryl.

Examples of the aryl moiety of C _7-16 aralkyloxy, C _7-16 aralkyl and C _7-16 aralkyloxycarbonyl include the groups exemplified in the above examples of aryl, and examples of the alkyl moiety include, for example, C _1-10 Examples thereof include alkylene, and more specifically, groups obtained by removing one hydrogen atom from the groups exemplified in the lower alkyl.
Examples of the aliphatic heterocyclic group and the aliphatic heterocyclic group part of the aliphatic heterocyclic carbonyl, the aromatic heterocyclic group, the aromatic heterocyclic group part of the aromatic heterocyclic carbonyl, and the halogen include, for example, the above aliphatic heterocyclic group, respectively. And the groups exemplified in the examples of the aromatic heterocyclic group and the halogen.

  Specific examples of compound I include, for example, the following compounds.

  Examples of the sterol compound contained as an active ingredient in the promoter for proliferation and / or mobilization of hematopoietic stem cells and / or hematopoietic progenitor cells of the present invention include the compound A as well as the compounds exemplified above. preferable.

Among these, compounds 110, 18, 49, 81, 83 and the like are more preferable, and compound 110 and the like are even more preferable.
Pharmaceutically acceptable salts of Compound I or A include, for example, pharmaceutically acceptable acid addition salts, metal salts, ammonium salts, organic amine addition salts, amino acid addition salts, and the like. Examples of the pharmaceutically acceptable acid addition salt of Compound I or A include inorganic acid salts such as hydrochloride, hydrobromide, nitrate, sulfate, phosphate, acetate, oxalate, and maleic acid. Organic salts such as salts, fumarate, citrate, benzoate, methanesulfonate and the like, and pharmaceutically acceptable metal salts include, for example, alkali metal salts such as sodium salt and potassium salt , Alkaline earth metal salts such as magnesium salts and calcium salts, aluminum salts, zinc salts and the like. Examples of pharmaceutically acceptable ammonium salts include salts such as ammonium and tetramethylammonium. Examples of acceptable organic amine addition salts include addition salts such as morpholine and piperidine, and pharmaceutically acceptable amino acid addition salts include, for example, Jin, glycine, phenylalanine, aspartic acid, addition salts, such as glutamic acid.

In addition, compounds I and A and pharmaceutically acceptable salts thereof may exist in the form of adducts with water or various solvents, and these adducts are also present in the hematopoietic stem cells and / or hematopoietics of the present invention. It can be used for progenitor cell growth promotion and / or mobilization agents.
Some compounds I may have stereoisomers such as geometric isomers and optical isomers, tautomers, etc., but the proliferation of hematopoietic stem cells and / or hematopoietic progenitor cells of the present invention and / or Alternatively, all possible isomers and mixtures thereof can be used for the mobilizing agent.

Compounds I and A and their pharmaceutically acceptable salts are not particularly limited in their origins of origin, and may be those extracted and purified from nature, or those synthesized by chemical techniques. For example, it can be produced by the method described in WO2009 / 096445 or WO2011 / 010682.
As the agent for promoting proliferation and / or mobilizing hematopoietic stem cells and / or hematopoietic progenitor cells of the present invention, Compound I or A or a pharmaceutically acceptable salt thereof can be used alone as it is. It can also be used as a pharmaceutical preparation.

  In the use of the hematopoietic stem cell and / or hematopoietic progenitor cell growth promoting and / or mobilizing agent of the present invention, compound I or A or a pharmaceutically acceptable salt thereof can be used alone or as various pharmaceutical preparations in human form. Or it can implement by the method of administering with respect to an animal. Further, in the use of the hematopoietic stem cell and / or hematopoietic progenitor cell proliferation promoter of the present invention, compound I or A or a pharmaceutically acceptable salt thereof can be used alone or as various pharmaceutical preparations in the living body or umbilical cord. Can be carried out by a method such as in vitro contact with hematopoietic stem cells and / or hematopoietic progenitor cells obtained from the above.

  The pharmaceutical preparation according to the present invention may contain Compound I or A or a pharmaceutically acceptable salt thereof as an active ingredient alone or as a mixture with any other active ingredient for treatment. These pharmaceutical preparations are well known in the technical field of pharmaceutics by mixing the active ingredient with one or more pharmaceutically acceptable carriers (eg, diluents, solvents, excipients, etc.). Manufactured by any method.

As the administration route for administration to humans or animals, it is desirable to use the most effective route for carrying out the present invention, and examples thereof include oral or parenteral such as intravenous.
Examples of the dosage form include tablets and injections.
For example, tablets suitable for oral administration can be produced using excipients such as lactose, disintegrants such as starch, lubricants such as magnesium stearate, binders such as hydroxypropylcellulose, and the like.

For example, an injection suitable for parenteral administration can be produced using a diluent or a solvent such as a salt solution, a glucose solution or a mixed solution of a saline solution and a glucose solution.
The agent for promoting proliferation and / or mobilization of hematopoietic stem cells and / or hematopoietic progenitor cells of the present invention promotes proliferation of hematopoietic stem cells and / or hematopoietic progenitor cells in the bone marrow and / or spleen by being administered to humans or animals. The cells can be increased in a short period of time. In addition, mobilization of hematopoietic stem cells and / or hematopoietic progenitor cells in the bone marrow into the peripheral blood can be promoted. Further, by contacting compound I or A or a pharmaceutically acceptable salt thereof with hematopoietic stem cells and / or hematopoietic progenitor cells fractionated from human or animal bone marrow, umbilical cord blood and / or peripheral blood, Cells can be grown in a short time.

  Therefore, the agent for promoting the proliferation and / or mobilization of hematopoietic stem cells and / or hematopoietic progenitor cells of the present invention can be used for the treatment of patients with impaired hematopoietic function, patients requiring transplantation of hematopoietic stem cells and / or hematopoietic progenitor cells (for example, , Acute myeloid leukemia, chronic myelogenous leukemia, malignant lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma and other hematopoietic tumors, breast cancer, ovarian cancer, lung cancer, renal cell cancer, pancreatic cancer, colon cancer, It can be used for treatment of solid tumors such as neuroblastoma, brain tumor, osteosarcoma, germ cell tumor, Ewing sarcoma, myelodysplastic syndrome, aplastic anemia, autoimmune disease).

  Specifically, for example, hematopoietic stem cells and / or bone marrow cells containing hematopoietic progenitor cells collected from a donor (provider), peripheral blood, umbilical cord blood and the like are transplanted into a recipient (a patient receiving a transplant), and the hematopoietic of the present invention is performed. Administration of stem cell and / or hematopoietic progenitor cell proliferation promoting and / or mobilizing agent according to the above increases hematopoietic stem cells and / or hematopoietic progenitor cells transplanted into the recipient, and engrafts the recipient with peripheral blood. Mobilization is promoted, and as a result, blood cell recovery can be promoted.

  In addition, by administering the hematopoietic stem cells and / or hematopoietic progenitor cells of the present invention to the donor and increasing the number of hematopoietic stem cells and / or hematopoietic progenitor cells in the bone marrow of the donor, The mobilization of the cells can also be promoted to increase the yield of hematopoietic stem cells and / or hematopoietic progenitor cells that can be collected from donor bone marrow and / or peripheral blood. Thereby, a sufficient amount of hematopoietic stem cells and / or hematopoietic progenitor cells necessary for transplantation can be secured.

  When hematopoietic stem cells and / or hematopoietic progenitor cells are mobilized from bone marrow to peripheral blood, a blood sample can be separated to obtain the cells. These cells can be transplanted immediately, but can be further enriched for cells having the characteristics of “hematopoietic stem cells and / or hematopoietic progenitor cells” from isolated blood samples. The enrichment method includes, for example, a step of removing hematopoietic stem cells and / or hematopoietic progenitor cells from a sample that is also rich in more differentiated progeny cells. Samples containing hematopoietic stem cells and / or hematopoietic progenitor cells can be enriched by selecting with CD34 positive cells. The step of removing hematopoietic stem cells and / or hematopoietic progenitor cells can be achieved using, for example, commercially available magnetic anti-CD34 beads (Dynal, Lake Success, NY).

  Furthermore, a cell population rich in hematopoietic stem cells and / or hematopoietic progenitor cells is isolated from bone marrow cells, peripheral blood, umbilical cord blood and the like collected from a donor, and this is used to promote the growth of hematopoietic stem cells and / or hematopoietic progenitor cells of the present invention and / or Alternatively, by culturing with the addition of a mobilizing agent, the proliferation of hematopoietic stem cells and / or hematopoietic progenitor cells can be promoted and the number of cells can be increased, and hematopoietic stem cells and / or hematopoietic progenitor cells necessary for transplantation can be sufficiently obtained. The amount can be secured.

  There are various techniques for analyzing the number of hematopoietic stem cells and / or hematopoietic progenitor cells, including culture methods, flow cytometry methods, transplantation methods, and histochemical analysis methods. In the culture method, hematopoietic stem cells and / or hematopoietic progenitor cells and / or cells containing hematopoietic stem cells and / or hematopoietic progenitor cells are cultured in a liquid medium and the increase in the number of cells is quantified. Colony-forming cell (CFC) assay, which measures the number of colonies formed, long-term culture-initiating cells (LTC-IC) ) There are assay methods, etc. (Presentation of hematopoietic stem cells and development of medical treatment Yodosha p.125-142). In addition, the number of hematopoietic stem cells and / or hematopoietic progenitor cells can be quantified by using a fluorescently labeled antibody and a flow cytometry instrument. For example, in the case of mouse hematopoietic stem cells and / or hematopoietic progenitor cells, c-kit positive, Sca-1 positive and cell lineage marker (CD3, Gr-1, Mac-1, B220, ter-119) negative The cells are frequently present in the cell population. In the case of the number of human hematopoietic stem cells and / or hematopoietic progenitor cells, the cells are frequently present in the cell population of CD34 and / or CD133 positive cells.

  Also, hematopoietic stem cells and / or hematopoietic progenitor cells and / or cell populations containing hematopoietic stem cells and / or hematopoietic progenitor cells are transplanted into mice whose immune system is suppressed by X-ray or the like, and for a certain period, for example, 2 weeks to 3 months There is a method to quantify the abundance of transplanted cell-derived blood cells, hematopoietic stem cells and / or hematopoietic progenitor cells by combining the above methods (Norio Nakajo “Stem Cell / Clone Research Protocol” p.117-124, 138- 144, 2001).

By such a technique, hematopoietic stem cells and / or hematopoietic progenitor cells present in bone marrow tissue, peripheral blood, spleen and other various tissues can be evaluated.
Hematopoietic stem cells and / or hematopoietic progenitor cells are usually present in the bone marrow, and are considered to maintain their biological hematopoietic system by repeating proliferation and differentiation. The proliferation promoting activity can also be evaluated by quantitative evaluation of the number of hematopoietic stem cells and / or hematopoietic progenitor cells in bone marrow tissue. Further, by quantifying the number of hematopoietic stem cells and / or hematopoietic progenitor cells in peripheral blood and spleen, the mobilization activity from bone marrow tissue to peripheral blood or spleen can be evaluated.

By the above method and evaluation method or a method similar to the above method, the proliferation promotion of the hematopoietic stem cells and / or hematopoietic progenitor cells of the present invention and / or the effect of the mobilizing agent can be verified.
The agent for promoting proliferation and / or mobilization of hematopoietic stem cells and / or hematopoietic progenitor cells of the present invention inhibits, for example, an agent that proliferates or mobilizes other hematopoietic stem cells and / or hematopoietic progenitor cells, epidermal growth factor receptor (EGFR) signal (Nature Medicine, 16, p.1141-1146. 2010), immunosuppressive agents and the like. Specifically, for example, plerixafor, G-CSF preparations [for example, filgrastim, Nartograstim, Lenograstim, etc.], erlotinib, antithymocytes Globulin (ATG), cyclosporine and the like.

  The dose and frequency of administration when administering Compound I or A or a pharmaceutically acceptable salt thereof vary depending on the dosage form, patient age, body weight, nature or severity of symptoms to be treated, etc. In the case of oral administration, the dose is 0.01 to 10000 mg, preferably 0.05 to 100 mg per adult, once to several times a day. In the case of parenteral administration such as intravenous administration, 0.001 to 10,000 mg, preferably 0.01 to 100 mg, is usually administered once or several times a day for each adult. However, the dose and the number of doses vary depending on the various conditions described above.

  When compound I or A or a pharmaceutically acceptable salt thereof is added when culturing hematopoietic stem cells and / or hematopoietic progenitor cells collected, compound I or A or a pharmaceutically acceptable salt thereof is, for example, dimethyl sulfoxide. After being dissolved in a suitable solvent such as, the concentration of Compound I in the medium is 10 nmol / L to 10 mmol / L, preferably 100 nmol / L to 1 mmol / L, more preferably 1 μmol / L to 100 What is necessary is just to add in a culture medium so that it may become micromol / L.

Next, the effect of the growth promotion and / or mobilization agent of the hematopoietic stem cells and / or hematopoietic progenitor cells of the present invention will be specifically described with reference to test examples.
Each test was performed by the following method unless otherwise specified.
Compound A, 110, 18, 49, 81 and 83 used for each test compound were obtained by the method described in WO2009 / 096445 or WO2011 / 010682. Recombinant human G-CSF was prepared by Kyowa Hakko Kirin Co., Ltd. (Narutograstim; J Pharmacol Exp Ther, 273, p.1114-1122, 1995) in phosphate buffer (PBS; Gibco). It dissolved and used so that it might become a microg / mL density | concentration.
For experimental animals of Experimental Animal Test Example 2, 8-week-old male DBA / 2 mice (Japan SLC) were used. The mice were bred for 7 to 14 days for both quarantine and habituation, and administration was started.

The experimental animals of Examples 3 to 5 were males of C57BL / 6-CD45.2 mice male (Japan SLC) and C57BL / 6-CD45.1 mice (Sankyo Lab Service), 8 weeks old. The mice were bred for 7 days for both quarantine and habituation, and then the experiment was started.
Method of administration When administered from the abdominal cavity, the test compound is dissolved in N, N-dimethylacetamide (DMA; Nacalai Tesque), and then Cremophor EL (Wako Pure Chemical Industries) is added and mixed and stirred. Then, it was used as a solution prepared by adding physiological saline (Otsuka Pharmaceutical), mixing and stirring. At this time, the final concentration was 10 vol% for DMA, 15 vol% for Cremophor EL, and 75 vol% for physiological saline. In addition, a solvent consisting of 10 vol% DMA, 15 vol% Cremophor EL, and 75 vol% physiological saline, which does not contain a test compound, is hereinafter referred to as DMA solvent, and the group administered with the DMA solvent is negative control (Negative Control; NC ).

When administered orally, the test compound is pulverized uniformly in a mortar, then the administration medium (0.5 w / v% methylcellulose; Wako Pure Chemical Industries, Ltd.) so that the test compound concentration is 25 mg / mL Suspended with, stirred well and used as a suspension.
Bone marrow cell collection Mice were dislocated from the cervical vertebrae, and the whole body was disinfected with 70% ethanol, and then the femurs of one or both feet were collected using scissors and tweezers. Muscles and periosteum adhering to the femur were removed, and the epiphysis was cut off. A syringe with 21 G needle (Terumo) was inserted into the bone marrow, and the bone marrow was pushed out and collected by the flow pressure of PBS. Using a syringe with a 21 G needle, suction and elution were repeated to loosen the bone marrow, and the bone marrow cells were suspended. After adding PBS to the resulting cell suspension to a total volume of 5 mL, bone marrow cells used in the test are passed through a 40 μm cell strainer (Becton Dickinson) to remove bone fragments and cell aggregates. Was obtained as a cell suspension. The number of viable cells in the cell suspension was measured by mixing with trypan blue (Gibco) or Turku stain (Nacalai Tesque).
Collection of peripheral blood cells Using a Pasteur with several μL of heparin (Shimizu Pharmaceutical) from the fundus of mice anesthetized with diethyl ether , collect several tens of μL of peripheral blood and mix with 500 μL of 0.5 vol% heparin in PBS. did. To separate mononuclear cells, this mixture was layered on Nycoprep ^™ 1.077A (Daiichi Kagaku), centrifuged at 2000 rpm for 30 minutes at room temperature, and the supernatant (plasma) and precipitate (red blood cells) The intermediate layer (mononuclear cell) was recovered. Next, add 10 mL of Dulbecco's Modified Eagle's Medium (DMEM) medium (Gibco) containing 10 vol% fetal bovine serum (FBS; Gibco) to the collected mononuclear cells and mix at 4 ° C. And centrifuged at 1700 rpm for 8 minutes to discard the supernatant. This operation was performed twice to wash the cells. To the cell pellet after centrifugation, 300 μL of DMEM medium containing 10 vol% FBS was added and suspended to obtain peripheral blood cells used in the test. The number of cells was measured by mixing with trypan blue (Gibco) or Turku stain (Nacalai Tesque).
Statistical analysis The results of Test Examples 1-4 below were expressed as mean ± standard deviation. For statistical analysis, statistical processing file ystat2000 (version 2000, published by medical books) was used. Comparison between the two groups was tested using an unpaired t-test, and a risk rate of less than 5% was considered significant.

Statistical analysis SAS release (9.1.3) was used for the statistical analysis of the survival rate (%) of the mice shown in Test Example 5. Comparison between the two groups was tested using Fisher's exact probability method, and a risk rate of less than 5% was considered significant.
Test Example 1 Colony-forming cell (CFC) assay Hematopoietic stem cells and / or hematopoietic progenitor cells repeatedly proliferate in a semi-solid medium such as methylcellulose to form blood cell colonies. Since this blood cell colony is formed from the level of one cell, the number of blood cell colony forming cells [Colony-forming cells (CFC)] having colony forming ability is quantified by counting the number of blood cell colonies by the following method. be able to.

For the peripheral blood CFC assay, a sample of 300,000 peripheral blood mononuclear cells suspended in 300 μL of DMEM medium containing 10 vol% FBS is used.For the bone marrow CFC assay, 18,000 cells of bone marrow cells are added with 10 vol% FBS. A sample suspended in 300 μL of the DMEM medium containing was used. Add 300 μL of these cell suspensions to four different cytokines (Stem Cell Factor, IL-3, IL-6 and Erytropoietin) in methylcellulose medium MethoCult GF ^a M3434 (Stem Cell Technology; 2.7 mL). Suspended. In the case of the peripheral blood CFC assay, 30 μL (1/100 volume) of a streptomycin (10,000 μg / mL) / penicillin (10,000 units / mL) solution (Gibco) was added to a cytokine-added methylcellulose medium MethoCult GF ^a M3434. Using a 1 mL microsyringe, 1 mL of the resulting suspension was blotted and seeded on a 3.5 cm culture dish. Two culture dishes were set per mouse and cultured in a 37 ° C. CO ₂ incubator. The number of blood cell colonies that appeared after 7 to 10 days was counted under a microscope (TE2000-U; Nikon), and the average number of colonies in the two culture dishes was calculated. In the case of the peripheral blood CFC assay, the number of CFCs per 100,000 cells of peripheral blood mononuclear cells was shown, and in the case of the bone marrow CFC assay, the number of CFCs per 6,000 cells of bone marrow was shown.
(1) Bone marrow CFC growth-promoting activity of Compound I by intraperitoneal administration Compound A (100 mg / kg) or Compound 110 (100 mg / kg) is intraperitoneally injected into DBA / 2 mice once a day for 5 days Repeated administration. The number of bone marrow CFCs in the negative control (NC group) was 47 ± 7, which was significantly increased to 58 ± 6 in the group administered Compound A (N = 5; bone marrow CFC increase rate relative to NC group: 1.23 times ( p <0.05)). In the group administered Compound 110, the number was 68 ± 16, which was significantly increased compared to 43 ± 2 in the NC group (N = 4; bone marrow CFC increase rate relative to the NC group: 1.58 times (p <0.05). )). From this result, it was shown that Compound I has an activity of promoting the growth of hematopoietic stem cells and / or progenitor cells in bone marrow.
(2) Peripheral blood CFC mobilization activity of Compound I by intraperitoneal administration Compound A (100 mg / kg) or Compound 110 (100 mg / kg) was intraperitoneally injected into DBA / 2 mice once a day for 5 days. Repeated administration (N = 4). The number of CFCs per 10 ⁵ peripheral blood cells was 65 ± 17 in the group administered with Compound A, which was significantly increased compared to 12 ± 4 in the NC group (5.4 times; p <0.05). In addition, the group administered Compound 110 also had a significant increase in the number of CFCs compared to the NC group (8.7 times; p <0.05). From this result, it was shown that Compound I has an activity of mobilizing hematopoietic stem cells and / or progenitor cells from bone marrow to peripheral blood.
(3) Evaluation of bone marrow CFC growth promoting activity of compounds 110, 18, 49, 81 and 83 by oral administration 100 mg / kg of each of compounds 110, 18, 49, 81 and 83 once a day in DBA / 2 mice, Orally administered for 5 consecutive days, bone marrow cells were collected 3 or 6 hours after the final administration, and the number of bone marrow CFCs was quantified (N = 5). The results are shown in the table.

Test Example 2 Combination Use of Compound 110 and G-CSF Test compound 110 (100 mg / kg) was orally administered to DBA / 2 mice once a day for 10 days (1 to 10 days). As the NC group, methylcellulose solvent was similarly administered. G-CSF (100 μg / kg) was subcutaneously administered once a day for 5 days from the 6th day to the 10th day. In the case of administration of Compound 110 and G-CSF on the same day, Compound 110 was administered first, and G-CSF was administered about 1 hour later. On the 5th, 9th or 11th day, peripheral blood was collected from the fundus for both the compound 110 + G-CSF group and the NC + G-CSF group.

The collected peripheral blood (60 μL) was suspended in hemolyzed BD Pharm Lyse ^™ lysing solution (Becton Dickinson; 15 mL) and allowed to stand at room temperature for 5 to 15 minutes. After confirming that the cell suspension is clear, centrifuge at 1700 rpm for 8 minutes at room temperature, and add 270 μL of DMEM medium containing 10 vol% FBS to the cell pellet from which the supernatant has been removed. did.
The cell suspension (270 μL) was added to 2.7 mL of methylcellulose medium (MethoCult GF ^a M3434) containing cytokines (Stem Cell Factor, IL-3, IL-6 and Erythropoietin) and well suspended. 30 μL (1/100 volume) of a solution of streptomycin (10,000 μg / mL) / penicillin (10,000 units / mL) was added to the suspension and well suspended. Using a 1 mL microsyringe, 1 mL of the obtained suspension was sucked and seeded on a 3.5 cm culture dish. Two culture dishes were set per animal and cultured in a CO ₂ incubator at 37 ° C. Blood cell colonies that appeared after 7 to 10 days were counted under a microscope (TE2000-U; Nikon), and the average value of the number of colonies in the two culture dishes was calculated and indicated as the number of CFCs per 20 μL of peripheral blood.

On day 9 of administration, the peripheral blood CFC of compound 110 group was significantly increased (p <0.05) to 1.6 times that of NC group.
Test example 3 in vivo transplantation experiment
(1) Serial Transplantation Competitive Reconstitution Assay (Serial Transplantation)
Compound 110 (100 mg / kg) is orally administered once daily for 5 consecutive days, and bone marrow cells collected 6 hours after the final dose are used as donors to measure the degree of competition for bone marrow reconstitution at the time of continuous transplantation A transplantation bone marrow reconstruction method (Serial Transplantation Competitive Reconstitution Assay) was performed according to the following method.

Using an X-ray irradiation device (MBR-1505R; Hitachi Medical), the recipient mouse (C57BL / 6-CD45.2) was irradiated with whole body at a dose of 9.5 Gy. Bone marrow cells prepared for transplantation were transplanted intravenously to recipient mice 4-8 hours after irradiation. The preparation of bone marrow cells used for transplantation and the method of continuous transplantation are as follows, referring to the literature (Current Protocols in Immunology. 22B.2.1-31, 2008; Cell Stem Cell. 1 (1), 101-12, 2007). As detailed above.
Primary transplantation experiment: Bone marrow cells (4 x 10 ⁵ per mouse) derived from C57BL / 6-CD45.1 mice treated with methylcellulose solvent (NC) or compound 110, and C57BL / 6-CD45 as competitor cells (competitors) .2 Mouse-derived bone marrow cells (4 × 10 ⁵ per mouse) were mixed and each was transplanted into a recipient mouse (C57BL / 6-CD45.2) (primary transplanted mouse). Bone marrow cells are collected from the primary transplanted mice 10 weeks after transplantation, and CD45 marker-positive whole bone marrow cells and hematopoietic stem cells and / or hematopoietic progenitor cells [c-kit positive and Sca-1 positive and cell line markers] (CD3, Mac-1, Gr-1, Ter-119 and B220) Negative cells (KSL cells)] The ratio of CD45.1 positive cells and CD45.2 positive cells present in the cells was analyzed by flow cytometry . The chimerism was calculated by the formula of CD45.1 positive cell number / (CD45.1 positive cell number + CD45.2 positive cell number).
Secondary transplantation experiment: Bone marrow cells derived from primary transplanted mice (6 x 10 ⁵ per mouse) were transplanted into secondary recipient mice (C57BL / 6-CD45.2) (secondary transplanted mice). Bone marrow cells were collected from secondary transplanted mice 12 weeks after transplantation, and CD45.1 positive cells present in CD45 marker-positive whole bone marrow cells and hematopoietic stem cells and / or hematopoietic progenitor cells (KSL cells) The proportion of CD45.2 positive cells was analyzed, and the chimerism of the donor (CD45.1) was calculated as described above.
Third transplantation experiment: Bone marrow cells (6 × 10 ⁵ per mouse) derived from the second transplanted mouse were transplanted into the third recipient mouse (C57BL / 6-CD45.2) (third transplanted mouse). Bone marrow cells were collected from the third transplanted mice 10 weeks after transplantation, and CD45.1 positive cells present in CD45 marker positive whole bone marrow cells and hematopoietic stem cells and / or hematopoietic progenitor cells (KSL cells) were analyzed by flow site. The proportion of CD45.2 positive cells was analyzed, and the chimerism of the donor (CD45.1) was calculated as described above.

The analysis by flow cytometry was performed by the following method.
Chimerism was analyzed by analyzing the proportion of bone marrow cells and hematopoietic stem cells and / or hematopoietic progenitor cells in the transplanted mice, respectively, of CD45.1 positive cells and CD45.2 positive cells. That is, bone marrow cells were collected from transplanted mice, bone marrow cells 10 ⁷ per, and addition of an anti-CD16 / 32 antibody 10 [mu] L for Fc receptor blocking was allowed to react for 10 minutes at 4 ° C.. Next, FITC-labeled anti-mouse CD45.2 (Ly5.2) antibody, PE-labeled anti-mouse CD45.1 (Ly5.1) antibody, biotin-labeled anti-mouse cell line marker (CD3, Mac-1, Gr-1, Ter -119, B220) antibody, PE-Cy7-labeled anti-mouse c-kit antibody and APC-labeled anti-mouse Sca-1 antibody were added in an amount of 10 μL each and allowed to react at 4 ° C. for 30 minutes. PBS containing 5 vol% fetal bovine serum (FBS; Gibco) (5% FBS / PBS) was added, centrifuged at 1700 rpm for 8 minutes at 4 ° C, supernatant was removed, and bone marrow cells were recovered (This operation is hereinafter referred to as cell washing operation). After the cells were suspended in 5% FBS / PBS, the PerCP-Cy5.5-labeled avidin was added 30 [mu] L per myeloid cells ¹⁰⁷ and reacted for 15 minutes at 4 ° C.. After hemolysis with the addition of sodium chloride hemolyzing agent (Becton Dickinson; 1 mL), using a flow cytometer FACSCantoII or FACSAria (Becton Dickinson), CD45.1 positive cells / CD45.2 positive cells in bone marrow cells The ratio and the ratio of CD45.1 positive cells / CD45.2 positive cells were analyzed by gating the area of KSL cells.

  Table 15 shows CD45-positive whole bone marrow cells and KSL cells (c-kit positive / Sca-1 positive / cell) meaning hematopoietic stem cells and / or hematopoietic progenitor cells collected from primary, secondary and tertiary transplanted mice. The ratio (chimerism) of CD45.1 positive donor cells in (liner marker negative cells) was shown. Higher chimerism means that donor cells competed predominantly. In the group transplanted with bone marrow cells derived from mice pre-administered with compound 110 as a donor, the chimerism of CD45.1 positive donor cells of whole bone marrow cells and hematopoietic stem cells and / or hematopoietic progenitor cells was higher than that of the group administered NC. Significantly higher.

(2) Limiting Dilution Competitive Repopulating Unit Assay (LDA)
Compound 110 (100 mg / kg) was orally administered to C57BL / 6-CD45.1 mice once daily for 5 consecutive days, and bone marrow cells collected 6 hours after the final administration were used as donors to prepare serially diluted cells. An assay for calculating the number of functional hematopoietic stem cells (CRU: Competitive Repopulating Unit) from the non-engraftment rate at the time of transplantation was performed by the following method. Methylcellulose solvent was used as a negative control (NC).

Using an X-ray irradiation device (MBR-1505R; Hitachi Medical), a recipient mouse (C57BL / 6-CD45.2) was irradiated with a whole body dose of 9.5 Gy. Marrow cells for transplantation were transplanted intravenously into mice 4 to 8 hours after irradiation. Preparation of bone marrow cells used for transplantation and the method of continuous transplantation were performed as follows with reference to literature (Proc. Natl. Acad. Sci. USA, 87, p.8736-8740, 1990).
That is, bone marrow cells derived from C57BL / 6-CD45.1 mice administered with methylcellulose solvent (NC) or compound 110 (9 x 10 ⁴ , 3 x 10 ⁴ , 1 x 10 ⁴ , 3 x 10 ³ and 1 per mouse) 5 groups of x 10 ³ ) and bone marrow cells derived from C57BL / 6-CD45.2 mice (1 x 10 ⁵ per mouse) as competing cells, each of which is mixed with recipient mice (C57BL / 6-CD45.2) ). Peripheral blood was collected from transplanted mice 18 weeks after transplantation, and T cells (CD3 positive cells), B cells (B220 positive cells) and myeloid cells (Mac-1 positive cells and Gr-1 positives) were obtained by flow cytometry. The ratio of CD45.1 positive cells and CD45.2 positive cells present in the cells was analyzed.

Individuals whose CD45.1 positive cells had a chimerism of 1% or less in each cell population of T cells, B cells and myeloid cells were determined to be non-engrafting. L-Calc (Stem Cell Technology) statistical software for limiting dilution competitive bone marrow reconstruction based on the percentage of non-engrafted individuals and the number of transplanted cells derived from C57BL / 6-CD45.1 mice Using to calculate CRU Frequency.
The analysis by flow cytometry was performed by the following method.

  CD45.1-positive and CD45.2-positive T cells (CD3 positive cells), B cells (B220 positive cells) and myeloid cells (Mac-1 positive cells and Gr-1 positive cells) in the peripheral blood of transplanted mice Chimerism was analyzed by analyzing the proportion of cells. About 60 μl of peripheral blood was collected from the transplanted mice, 5 μL of anti-CD16 / 32 antibody was added for blocking Fc receptor, and reacted at 4 ° C. for 10 minutes. Next, 5 μL each of FITC-labeled anti-mouse CD45.2 (Ly5.2) antibody and PE-labeled anti-mouse CD45.1 (Ly5.1) antibody were added. Divide the sample into three parts: (a) sample added with 5 μL of biotin-labeled anti-mouse CD3 antibody, (b) sample added with 5 μL of biotin-labeled anti-mouse B220 antibody, and (c) biotin-labeled anti-mouse Mac-1 Samples were prepared by adding 5 μL of antibody and 5 μL of biotin-labeled anti-mouse Gr-1 antibody, and each sample was reacted at 4 ° C. for 30 minutes. After cell washing with 5% FBS / PBS, 5 μL of APC-labeled avidin was added and reacted at 4 ° C. for 15 minutes. Sodium chloride hemolyzing agent (Becton Dickinson; 1 mL) was added to lyse, and then peripheral blood cells (a) T cells (CD3 positive) using a flow cytometer FACSCanto II (Becton Dickinson) or FACSAria (Becton Dickinson) Cell), (b) B cell (B220 positive cell) and (c) myeloid cell (Mac-1 positive cell and Gr-1 positive cell) CD45.1 positive cell and CD45.2 positive cell ratio, respectively Analyzed. All antibody-related reagents were from Becton Dickinson.

As a result of calculating CRU Frequency, the CRU frequency of NC mouse-derived bone marrow cells was 1 / 15,261. In contrast, the CRU frequency of bone marrow cells derived from compound 110-administered mice was 1 / 8,116.
Test Example 4 Ex vivo expansion experiment
Compound 110 (1 nmol / L) or dimethyl sulfoxide (DMSO; NC) at the same concentration is added to Stemspan SFEM medium containing 4 types of cytokines (mouse SCF, mouse TPO, mouse IGF-II and human FGF-1). KSL cells were cultured ex vivo. The cultured cells were collected, and the CFC assay and transplantation experiment were carried out by the following method to evaluate the growth promoting effect of Compound 110 on CFC or CRU in the in vitro amplification method.
(1) Separation of cell lineage marker negative cells To enrich mouse hematopoietic stem cells and / or hematopoietic progenitor cells, Becton Dickinson's magnetic cell separator BD IMagnet ^TM and Mouse Hematopoietic Progenitor (Stem) Cell Enrichment Set Cell line marker (CD3e, CD11b, CD45R / B220, Ly-6G and Ly6C and TER-119) positive cells were removed by BD IMag ^™ Cell Separation System using -DM, and cell line marker negative cells were obtained.

That is, anti-CD16 / 32 antibody (Becton Dickinson; 10 μL) was added per 10 ⁷ bone marrow cells for Fc receptor blocking, and reacted at 4 ° C. for 10 minutes. Next, after the biotinylated murine cell line depletion cocktail antibody (Biotin Mouse Lineage Depletion Cocktail) (kit) was added 50μl per myeloid cells ^107, allowed to react for 15 minutes at 4 ℃, 5% FBS / Dulbecco's phosphate Cell washing operation was performed by adding 10 mL of acid buffer (D-PBS). The cell pellet was added 5% FBS / D-PBS to 2 mL, the cells were well suspended, magnetic bead-labeled streptavidin; a (Imag Streptavidin kit) was added 50μL per myeloid cells ^107, were mixed at 4 ° C. The reaction was allowed to proceed for 15 minutes, and then cell washing was performed by adding 10 mL of 5% FBS / D-PBS. 2 mL of 5% FBS / D-PBS was added to the cell pellet, and the cells were well suspended. The cell suspension was set in BD IMagnet ^™ and allowed to stand for 8 minutes, and then the cell suspension (cell line marker negative cells) was collected with a Pasteur. This recovery operation was performed twice. The collected cell line marker negative cells were subjected to cell washing twice and suspended in 5% FBS / D-PBS for use in the test. When hematopoietic stem cells and / or hematopoietic progenitor cells were separated by a sorting flow cytometer, the cells were suspended in 5% FBS / D-PBS, reacted with a fluorescently labeled antibody, and used for the test.
(2) Separation of mouse hematopoietic stem cells and / or hematopoietic progenitor cells by FACS To isolate hematopoietic stem cells and / or hematopoietic progenitor cells (KSL cells) from mouse cell line marker negative cells, use FACSAria (Becton Dickinson). Using.

That is, per 10 ^{7 of} cell line marker negative cells, PE labeled anti-mouse Sca-1 antibody, biotin labeled anti mouse cell line marker (CD3e, CD11b, CD45R / B220, Ly-6G and Ly6C and TER-119) antibody and APC label 10 μL of anti-mouse c-kit antibody was added and reacted at 4 ° C. for 30 minutes. After cell washing, suspended in 5% FBS / D-PBS, the PE-Cy7-labeled avidin was added 10 [mu] L per myeloid cells ^107, was reacted at 4 ° C. 20 min. After cell washing, propidium iodide (Invitrogen) was added to the cell suspension to a final concentration of 1 μg / mL. Using FACSAria, KSL cells in propidium iodide negative cells, which are living cells, were separated by gating.
(3) Cell culture in in vitro expansion method (Ex vivo expansion) Separated KSL cells are cultured ex vivo in a serum-free medium, Stemspan SFEM (Stem Cell Technology) supplemented with cytokines and compound 110, followed by CFC assay and transplantation Experiments were conducted to evaluate the growth promoting effect of Compound 110 on CFC or CRU in ex vivo expansion.

Ex vivo culture was performed on Stemspan SFEM using 10 ng / mL mouse SCF (# 455-MC; R & D Systems), 20 ng / mL mouse TPO (# 488-TO; R & D Systems), 20 ng / mL mouse IGF-II (# 792-MG; R & D Systems) and 10 ng / mL human FGF-1 (# PHG0014; Invitrogen), respectively, were prepared and used at the time of use. A 96-well plate was seeded with 2 × 10 ³ cells / 100 μL / well of KSL cells, and a test compound was added after 1 hour.

CFC assay was performed after 3 days of culture, and transplantation experiments were performed by collecting cells cultured ex vivo after 4 days of culture.
(4) CFC assay of ex vivo cultured cells
KSL cells after 3 days of ex vivo culture were collected and subjected to CFC assay by culturing on a methylcellulose medium containing cytokines. That is, the cultured cells were collected by pipetting from a 96-well plate cultured ex vivo with the addition of a test compound or DMSO solvent. Dulbecco's Modified Eagle's Medium (DMEM) medium (Gibco; 300 μL) containing 10 vol% fetal bovine serum (FBS; Gibco), respectively, for 1/10, 1/30, and 1/50 volumes of the collected cell suspension It was suspended in. A methylcellulose medium (MethoCult GF ^a M3434; Stem Cell Technology; 2.7 mL) containing cytokines (Stem Cell Factor, IL-3, IL-6 and Erythropoietin) was added thereto and well suspended. Using a 1 mL microsyringe, 1 mL of 1/3 of the obtained suspension was sucked out, seeded on two 3.5 cm culture dishes, and cultured in a CO ₂ incubator at 37 ° C. Blood cell colonies that appeared after 7 to 10 days were counted under a microscope (TE2000-U; Nikon), the average value of the number of colonies in the two culture dishes was calculated, and the total number of CFCs in the cell suspension was converted.

As a result of the test, by culturing 3,000 KSL cells for 3 days under the condition where Compound 110 was added, the number of KSL cells was significantly increased to 4640 ± 262 versus 3600 ± 375 in the NC addition group ( Increase rate of CFC with respect to NC addition group: 1.29 times (P <0.05, N = 3)).
(5) Ex vivo cultured cell transplantation experiment
3,000 KSL cells derived from C57BL / 6-CD45.1 mice were cultured for 4 days under Compound 110 1 nmol / L or DMSO addition conditions (NC) at the same concentration. As competing cells, 3 × 10 ⁵ bone marrow cells derived from C57BL / 6-CD45.2 mice were transplanted into X-irradiated recipient mice (C57BL / 6-CD45.2). Mouse peripheral blood was collected 8 weeks after transplantation, and the ratio of CD45.1 positive cells and CD45.2 positive cells in blood cells was analyzed by flow cytometry, and chimerism was calculated. As a result, the chimerism tended to be high in mice transplanted with cells cultured in the medium supplemented with Compound 110.

From this result, it was shown that Compound 110 increases hematopoietic stem cells and / or hematopoietic progenitor cells having engraftment ability even ex vivo.
Test Example 5 Evaluation of engraftment of hematopoietic stem cells and / or hematopoietic progenitor cells using bone marrow transplantation model mice According to the following, using hematopoietic stem cells and / or hematopoietic progenitor cells using X-irradiated bone marrow transplantation model mice An engraftment evaluation experiment was conducted to evaluate the arrival by viability of mice.

Using an X-ray irradiation apparatus (MBR-1505R; Hitachi Medical), a recipient mouse (C57BL / 6-CD45.2) was irradiated with a dose of 10 Gy whole body (Day 0). On the next day (Day 1), bone marrow mononuclear cells (1.6 x 10 ⁵ cells / mouse) were intravenously administered and transplanted. From the next day (Day 2), compound 110 (50 mg / kg) or solvent control (methylcellulose solvent administration) ) Was orally administered once a day for 14 consecutive days. The survival of mice was evaluated for 30 days after X-ray irradiation.

As a result, administration of Compound 110 promoted the engraftment of hematopoietic stem cells and / or hematopoietic progenitor cells, and confirmed a significant improvement in the survival rate of transplanted mice from day 7 to 9 (survival on day 30 of transplantation). (The rate was 10% in the methylcellulose solvent-administered group compared to 45% in the compound 110 group (N = 20; p <0.05).)
As a result of the above tests, Compound I or A or a pharmaceutically acceptable salt thereof, particularly Compound A, 110, 18, 49, 81, 83 is used as an agent for promoting proliferation and / or mobilizing hematopoietic stem cells and / or hematopoietic progenitor cells. It has been shown to be useful.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the scope of the present invention is not limited to these examples.

Tablet (Compound 110)
A tablet having the following composition is prepared by a conventional method. Compound 110, 40 g, lactose 286.8 g and potato starch 60 g are mixed, and 10% aqueous solution of hydroxypropylcellulose 120 g is added thereto. The obtained mixture is kneaded by a conventional method, granulated and dried, and then sized to obtain granules for tableting. To this was added 1.2 g of magnesium stearate, mixed, and tableted with a tableting machine (RT-15 type, manufactured by Kikusui Co., Ltd.) with a 8 mm diameter punch, and tablets (containing 20 mg of active ingredient per tablet) )
Formula Compound 110 20 mg
Lactose 143.4 mg
Potato starch 30 mg
Hydroxypropylcellulose 6 mg
Magnesium stearate 0.6 mg
200 mg

Injection (Compound 110)
An injection having the following composition is prepared by a conventional method. Compound 110, 1 g is added to and mixed with distilled water for injection. Further, hydrochloric acid and aqueous sodium hydroxide solution are added to adjust the pH to 7, and then the total volume is made up to 1000 mL with distilled water for injection. The obtained mixed solution is aseptically filled into glass vials by 2 mL to obtain an injection (containing 2 mg of active ingredient per vial).
Formula Compound 110 2 mg
Hydrochloric acid appropriate amount Sodium hydroxide aqueous solution appropriate amount
Distilled water for injection
2.00 mL

A DMSO solution (0.1 mmol / L) of compound 18 is prepared by a conventional method, and a proliferation promoter for hematopoietic stem cells and / or hematopoietic progenitor cells containing compound 18 is obtained.

According to the present invention, an agent for promoting proliferation and / or mobilizing hematopoietic stem cells and / or hematopoietic progenitor cells can be provided.

Claims (10)

An agent for promoting and / or mobilizing proliferation of hematopoietic stem cells and / or hematopoietic progenitor cells, which contains a sterol compound represented by the following general formula (I) or (A) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Where:
Y represents lower alkyl optionally having substituent (s) or lower alkenyl optionally having substituent (s);
X ^a and X ^b are the same or different and are a bond or —NR ^a — (wherein R ^a is a hydrogen atom, optionally substituted lower alkyl, optionally substituted) Cycloalkyl, lower alkanoyl which may have a substituent or aroyl which may have a substituent)
R ¹ , R ² , R ³ , R ⁴ , R ⁷ and R ⁸ are the same or different and each represents a hydrogen atom or hydroxy, or
R ¹ and R ² , R ³ and R ⁴ , or R ⁷ and R ⁸ together represent ═O,
R ⁵ and R ⁶ are the same or different and each is a hydrogen atom, halogen, azide, hydroxy, optionally substituted lower alkoxy, optionally substituted cycloalkyloxy, or optionally substituted. Lower alkenyloxy which may have a substituent, lower alkynyloxy which may have a substituent, aryloxy which may have a substituent, aromatic heterocyclic oxy which may have a substituent, substituted Aliphatic heterocyclic oxy which may have a group, lower alkanoyloxy which may have a substituent, aroyloxy which may have a substituent, aromatic heterocyclic which may have a substituent Ring carbonyloxy or —NR ^b R ^c (wherein R ^b and R ^c are the same or different and are a hydrogen atom, optionally substituted lower alkyl, optionally substituted lower Alkenyl, substituted Cycloalkyl which may have a group, aryl which may have a substituent, aromatic heterocyclic group which may have a substituent, aliphatic heterocyclic ring which may have a substituent Group, lower alkanoyl optionally having substituent, aroyl optionally having substituent, lower alkoxycarbonyl optionally having substituent, aryloxycarbonyl optionally having substituent , Optionally substituted aromatic heterocyclic oxycarbonyl, optionally substituted lower alkyl carbamoyl, optionally substituted di-lower alkyl carbamoyl, substituted Arylcarbamoyl which may be substituted, aromatic heterocyclic carbamoyl which may have a substituent, arylsulfonyl which may have a substituent or lower alkylsulfonyl which may have a substituent Or represents represents a sulfonyl), or, O = R ⁵ and R ⁶ together, R ^d ON = (wherein, R ^d represents a hydrogen atom, an optionally substituted lower alkyl, substituted Cycloalkyl which may have a group, aryl which may have a substituent, aromatic heterocyclic group which may have a substituent or aliphatic heterocyclic ring which may have a substituent Or R ^e R ^f C = (wherein R ^e and R ^f are the same or different and have a hydrogen atom, halogen, optionally substituted lower alkyl, or substituent) Lower alkenyl which may have, lower alkynyl which may have a substituent, cycloalkyl which may have a substituent, aryl which may have a substituent, which may have a substituent Good aromatic heterocyclic group, optionally substituted aliphatic heterocyclic group, optionally substituted lower alkyl Xyl, optionally substituted aryloxy, optionally substituted aromatic heterocyclic oxy, optionally substituted lower alkanoyl, optionally substituted Aroyl, lower alkanoyloxy optionally having substituent (s) or aroyloxy optionally having substituent (s))
R ⁹ has a hydrogen atom, optionally substituted lower alkyl, optionally substituted lower alkenyl, optionally substituted lower alkynyl, or optionally substituted. Represents a good cycloalkyl, or
R ¹ and R ³ , R ³ and R ⁵ (only when X ^a is a bond), R ⁵ and R ⁷ (but only when X ^b is a bond) or R ⁷ and R ⁹ together Represents a bond or an oxygen atom,
R ¹⁰ , R ¹¹ and R ¹² represent a bond or an oxygen atom when R ¹⁰ and R ¹¹ are taken together; R ¹² represents a hydrogen atom or a bond or oxygen taken together with R ¹⁰ and R ¹² Represents an atom, and R ¹¹ represents a hydrogen atom] The agent according to claim 1, wherein the sterol compound is a sterol compound represented by the formula (I). 3. The agent according to claim 2, wherein R ¹⁰ and R ¹¹ together represent a bond, and R ¹² is a hydrogen atom. 4. The agent according to claim 2, wherein ^Xa and ^Xb are a bond. 2. The agent according to claim 1, wherein the sterol compound is a compound represented by any one of the following formulas (A), (110), (18), (49), (81) and (83).

2. The agent according to claim 1, wherein the sterol compound is a compound represented by any of the following formulas (110), (18), (49), (81) and (83).

The agent according to any one of claims 1 to 6, which is a hematopoietic stem cell proliferation promoter. The agent according to any one of claims 1 to 6, which is an agent for promoting mobilization of hematopoietic stem cells. The agent according to any one of claims 1 to 6, which is a hematopoietic progenitor cell proliferation promoter. The agent according to any one of claims 1 to 6, which is an agent for promoting mobilization of hematopoietic progenitor cells.