WO2019117262A1 - Composition for promoting cell adhesion and method for culturing cells using same - Google Patents

Abstract

The present invention provides a composition for promoting cell adhesion containing a compound represented by general formula (I) or a salt thereof. {In the formula, each symbol is as defined in the description.}

Description

Composition for promoting cell adhesion and method of culturing cells using the same

The present invention relates to a composition and the like for promoting cell adhesion of adherent cells, and in particular, to a composition for promoting cell adhesion and a method for culturing cells characterized by using the composition. .

In recent years, techniques for growing, differentiating or maintaining various cells in an animal body in vitro have been developed. The present technology has become essential to study in detail the function and structure of cells, to evaluate the efficacy and toxicity of drugs and the like, and to carry out regenerative medicine. Due to their properties, animal-derived cells are roughly divided into floating cells and adherent cells. Suspended cells are cells that do not require a scaffold for growth and proliferation, and adherent cells are cells that require a scaffold for growth and proliferation, but most of the cells that make up the organism have the latter adhesiveness. It is a cell. Here, although it is first necessary to adhere the adherent cells to the container when culturing, problems such as low cell adhesion ratio or long time to cell adhesion may occur. When such a problem arises, studies using cells can not be conducted quickly and efficiently. Therefore, a method for promoting adhesion of cells to a culture vessel is required, and, for example, a method for promoting cell adhesion using an extracellular matrix has been reported (Non-patent Document 1). In addition, methods for promoting cell adhesion using chemically synthesized low molecular weight compounds and polymers have been reported (Patent Documents 1 and 2).

International Publication No. 2009/154201 JP-A-11-322614

An object of the present invention is to provide a composition for adding a medium capable of promoting adhesion of adherent cells to a culture vessel.

As a result of intensive studies for the above problems, the present inventors have found that by adding a specific compound to a culture medium, adhesion of adherent cells to a culture vessel can be promoted extremely well, The present invention has been completed by conducting further research based on it. That is, the present invention is as follows.

[1] A composition for promoting cell adhesion, comprising a compound represented by the following general formula (I), or a salt thereof:

Wherein X is a single bond, -CH ₂ COO-, -CONH-, or -NHCO-, R ₁ is an optionally substituted alkyl group having 1 to 10 carbon atoms, substituted An aryl group which may have a group or —Y—NH—Z—Ar, wherein Y and Z each have a single bond or a carbon number of 1 to 6 which may have a substituent Ar is an aryl group which may have a substituent), R ₂ is an alkyl group having 1 to 6 carbon atoms which may have a substituent, R ₃ Is a hydrogen atom or a hydroxyl group. }.
[2] The composition according to [1], wherein X is -NHCO-.
[3] The composition according to [1] or [2], wherein R ₂ is a methyl or ethyl group which may have a substituent, and R ₃ is a hydrogen atom.
[4] The composition according to any one of [1] to [3], wherein R ₁ is —Y—NH—Z—Ar.
[5] The composition according to [4], wherein Ar is a phenyl group which may be substituted with a halogen atom, a hydroxyl group or a methyl group.
[6] The composition according to [4] or [5], wherein Y is a methylene group, an ethylidene group or a propylidene group.
[7] The composition according to any one of [1] to [6], wherein the compound is a compound selected from the group consisting of: or a salt thereof.

and,

[8] The composition according to any one of [1] to [5], wherein the compound is a compound shown below or a salt thereof.

and,

[9] The composition according to any one of [1] to [8], wherein the cells are selected from the group consisting of normal cell lines, cancer cell lines and stem cells.
[10] The composition according to [9], wherein the normal cell line is HUVEC, C3H10T1 / 2 or HEK293.
[11] The composition according to [9], wherein the cancer cell line is HeLa cells, A431, HCT116, or MCF7.
[12] The composition of [9], wherein the stem cells are human mesenchymal stem cells (hMSCs) or human induced pluripotent stem cells (hiPSCs).
[13] A medium for culturing cells, comprising the composition according to any one of [1] to [8].
[14] The medium according to [13], wherein the cells are selected from the group consisting of normal cell lines, cancer cell lines and stem cells.
[15] The medium according to [14], wherein the normal cell line is HUVEC, C3H10T1 / 2 or HEK293.
[16] The medium according to [14], wherein the cancer cell line is HeLa cells, A431, HCT116, or MCF7.
[17] The medium according to [14], wherein the stem cells are human mesenchymal stem cells (hMSCs) or human induced pluripotent stem cells (hiPSCs).
[18] A method for promoting adhesion of cells to a culture vessel, comprising adding the composition according to any one of [1] to [8] to a culture medium.
[19] The method according to [18], wherein the cells are selected from the group consisting of normal cell lines, cancer cell lines and stem cells.
[20] The method according to [19], wherein the normal cell line is HUVEC, C3H10T1 / 2 or HEK293.
[21] The method according to [19], wherein the cancer cell line is HeLa cells, A431, HCT116, or MCF7.
[22] The method according to [19], wherein the stem cells are human mesenchymal stem cells (hMSCs) or human induced pluripotent stem cells (hiPSCs).

According to the present invention, it is possible to promote the adhesion of adherent cells to a culture vessel, in which adhesion to a substrate or the like is essential in cell growth. This makes it possible to adhere the cells efficiently, and to carry out studies using the cells more rapidly and efficiently than in the past. In the case where the adherent cells cultured using the present invention are stem cells (eg, induced pluripotent stem cells), the present invention promotes adhesion of the stem cells to a culture vessel while the cells are being cultured. An undifferentiated state of stem cells can be suitably maintained.

FIG. 1 shows that when the composition of the present invention is added to the culture medium at stepwise concentrations, cell adhesion to the culture vessel of the HEK 293 cell line is promoted in a concentration dependent manner.

The terms used in the present specification are defined below.

The "halogen atom" is a fluorine atom, a chlorine atom, a bromine atom or an iodine atom. The "halogeno group" is fluoro, chloro, bromo or iodo.

The “alkyl group” means a linear or branched alkyl group, and specifically, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n- Groups such as pentyl, isopentyl, tert-pentyl, neopentyl, 2-pentyl, 3-pentyl, n-hexyl, 2-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like can be mentioned.

"Aryl" includes monocyclic, bicyclic, tricyclic and tetracyclic carbocyclic groups in which at least one ring is aromatic and each ring has 5 to 8 ring atoms. Specifically, examples include phenyl, indenyl, naphthyl, fluorenyl and the like. In particular, the aryl group may be C _6-10 aromatic phenyl, indenyl, naphthyl.

The "alkylene group" means a linear or branched alkylene group, and specifically, methylene, ethylene, n-propylene, isopropylene, propylidene, n-butylene, sec-butylene, tert-butylene, n -Groups such as pentylene, sec-pentylene, tert-pentylene, n-hexylene, sec-hexylene, tert-hexylene and the like can be mentioned.

The "alkyl group", the "aryl group" and the "alkylene group" may have a substituent, and examples of such a substituent include the following.
(1) halogeno group,
(2) hydroxy group,
(3) cyano group,
(4) nitro group,
(5) Carboxyl group,
(6) Alkenyl group (C _2-10 alkenyl group; for example, vinyl, allyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, butadienyl, hexatrienyl, and each isomer thereof),
(7) alkynyl group (C _2-10 alkynyl group; for example, ethynyl, propynyl, butynyl, pentynyl, hexynyl and each isomer thereof),
(8) a halogenoalkyl group (eg, monofluoromethyl, difluoromethyl, trifluoromethyl, monofluoroethyl, difluoroethyl, trifluoroethyl, chloromethyl, chloroethyl, dichloroethyl and their respective isomers),
(9) Cyclic alkyl group (may contain a hetero atom in the ring) (eg, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, tetrahydrofuran, tetrahydrofuranyl, tetrahydropyranyl, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl),
(10) aryl group (eg, phenyl, naphthyl),
(11) Heteroaryl groups (eg, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, furyl, thiophenyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl (eg, 1,2,3-triazolyl, 1,2,4-triazolyl), tetrazolyl, Oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl (eg, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl), thiadiazolyl (eg, 1,2,3-thiadiazolyl, 1 , 2,4-thiadiazolyl, 1,3,4-thiadiazolyl), benzofuryl, benzothiophenyl, indolyl, isoindolyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, indazolyl, benzisoxazolyl, benziso Thiazolyl, benzoxadiazolyl, benzothiadiazolyl, purinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, pteridinyl, imidazooxazolyl, imidazothiazolyl, imidazoimidazolyl),
(12) Alkoxy groups (eg, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, tert-pentyloxy, neopentyloxy , 2-pentyloxy, 3-pentyloxy, n-hexyloxy, 2-hexyloxy),
(13) alkylthio group (eg, methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, sec-butylthio, tert-butylthio, n-pentylthio, isopentylthio, tert-pentylthio, neopentylthio, 2-pentylthio, 3-pentylthio, n-hexylthio, 2-hexylthio),
(14) an alkoxy group (defined in the above (12)) substituted with an aryl group (defined in the above (10)),
(15) an alkylthio group (defined in the above (13)) substituted with an aryl group (defined in the above (10)),
(16) an alkoxy group (defined in the above (12)) substituted with a heteroaryl group (defined in the above (11)),
(17) an alkylthio group (as defined above in (13)) substituted with a heteroaryl group (defined as above in (11)),
(18) Cyclic alkyl (which may contain a hetero atom in the ring) oxy group (eg, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, tetrahydrofuranyl, tetrahydropyranyloxy, aziridinyloxy, azetylene Dinyloxy, pyrrolidinyloxy, piperidinyloxy, morpholinyloxy),
(19) an aryloxy group (eg, a group in which an aryl group (as defined in (10) above) is bonded to an oxygen atom),
(20) heteroaryloxy group (eg, a group in which a heteroaryl group (as defined in the above (11)) is bonded to an oxygen atom),
(21) a halogenoalkoxy group (eg, a group in which a halogenoalkyl group (as defined in (8) above) is bonded to an oxygen atom),
(22) a halogenoalkylthio group (eg, a group in which a halogenoalkyl group (as defined in (8) above) is bonded to a sulfur atom),
(23) an alkoxy group substituted with a hydroxy group (as defined in the above (12)),
(24) an alkoxy group (defined in the above (12)) substituted with an alkoxy group (defined in the above (12)),
(25) amino group,
(26) an amino group mono- or di-substituted with an alkyl group,
Here, the “alkyl group” includes a C _1-6 alkyl group, and specifically, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n -Pentyl, isopentyl, tert-pentyl, neopentyl, 2-pentyl, 3-pentyl, n-hexyl, 2-hexyl and the like.
(27) carbamoyl group,
(28) a carbamoyl group mono- or di-substituted with an alkyl group (as defined for the “alkyl group” in the above (26)) (eg, methylcarbamoyl, ethylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl, ethylmethylcarbamoyl)
(29) sulfamoyl group,
(30) A sulfamoyl group mono- or di-substituted with an alkyl group (as defined for the "alkyl group" in the above (26)) (eg, methylsulfamoyl, ethylsulfamoyl, dimethylsulfamoyl, diethylsulfamoyl, Ethyl methyl sulfamoyl),
(31) an alkanoyl group (eg, a hydrogen atom or a carbonyl group having an alkyl group (as defined for the “alkyl group” in the above (26)) bonded to a carbon atom)
(32) an aroyl group (eg, a carbonyl group in which an aryl group (as defined in (10) above) is bonded to a carbon atom),
(33) an alkylsulfonylamino group (eg, a sulfonylamino group substituted with an alkyl group (as defined as the “alkyl group” in the above (26))
(34) an arylsulfonylamino group (eg, a sulfonylamino group substituted with an aryl group (as defined in the above (10))),
(35) Heteroarylsulfonylamino group (eg, sulfonylamino group substituted with heteroaryl group (as defined in the above (11))),
(36) an acylamino group (eg, an amino group substituted with an acyl group),
Here, the “acyl group” is an acyl group having a C _1-6 alkyl group or a C _6-10 aryl group. Here, the “C _1-6 alkyl group” is one having 1 to 6 carbon atoms among the above “alkyl groups”, and the “C _6-10 aryl group” is one of the above “aryl groups”. Among them, those having 6 to 10 carbon atoms. Specific examples of the acyl group include acetyl group, propionyl group, butyroyl group, isobutyroyl group, valeroyl group, isovaleroyl group, pivaloyl group, hexanoyl group, acryloyl group, methacryloyl group, crotonoyl group, isocrotonoyl group, benzoyl group and naphthoyl group. Groups, etc.,
(37) an alkoxycarbonylamino group (eg, a carbonylamino group substituted with an alkoxy group (as defined in the above (12)),
(38) an alkylsulfonyl group (eg, a sulfonyl group substituted with an alkyl group (as defined as the “alkyl group” in the above (26)),
(39) an alkylsulfinyl group (eg, a sulfinyl group substituted with an alkyl group (as defined as the “alkyl group” in the above (26)),
(40) alkoxycarbonyl group (eg, methoxycarbonyl group, ethoxycarbonyl group) and the like.
When two or more substituents are present, they may be the same or different.

The compounds of formula (I) may be in the form of salts. Examples of the salt of the compound represented by the formula (I) include salts with inorganic acids such as hydrochloric acid and hydrobromic acid, as well as acetic acid, propionic acid, tartaric acid, fumaric acid, maleic acid, malic acid, oxalic acid, And salts with organic acids such as succinic acid, citric acid and benzoic acid.

[Synthesis of a Compound Composed of a Combination of Ketone and Hydrazide]
In general, as represented by the following formula, one equivalent each of ketone and hydrazide is used, and the solvent is 100 ° C. or more in a solvent such as toluene, 1,4-dioxane, N, N-dimethylformamide, dimethyl sulfoxide, etc. The reaction is preferably carried out in the temperature range of 1 hour to 3 days.

[Reaction Formula 1]

After completion of the reaction, the reaction mixture can be precipitated by adding distilled water, or when not precipitated, it can be subjected to a usual post treatment such as extraction after extraction with an organic solvent to obtain the desired compound of the present invention. In addition, when the need for purification arises, it can be separated and purified by any purification method such as recrystallization, column chromatograph, thin layer chromatograph, liquid chromatograph or the like.

[Synthesis of k-1 derivative]
Generally, using 2 ', 4'-dihydroxy-3'-methylpropiophenone (k-1) and a desired primary amine or a salt thereof, one equivalent each of each, toluene, 1, 4 The reaction is preferably carried out in a solvent such as dioxane, N, N-dimethylformamide, dimethylsulfoxide or the like at a temperature range of 100 ° C. or more for 1 hour to 24 hours. As a salt of amine, hydrochloride, p-toluenesulfonate, trifluoroacetate and the like can be used.

Further, when the above method is not used, 2 ', 4'-dihydroxy-3'-methylpropiophenone (k-1) is dissolved in a methanol solution of ammonia, and stirred while injecting ammonia gas. Thus, imine (k-1 ′) can be synthesized and then reacted with the corresponding isocyanate.

After completion of the reaction, the reaction mixture can be subjected to usual post treatments such as extraction with solvent and concentration after addition of distilled water to obtain the desired compound of the present invention. In addition, when the need for purification arises, it can be separated and purified by any purification method such as recrystallization, column chromatograph, thin layer chromatograph, liquid chromatograph or the like.

1. Composition for promoting cell adhesion The present invention provides a composition for promoting cell adhesion (hereinafter sometimes referred to as "the composition of the present invention"), which contains the following compound. The composition of the present invention can promote cell adhesion of cells (particularly adherent cells) to a culture vessel by adding to a culture medium for cell culture.

The compounds included in the composition of the present invention have the general formula (I):

Wherein X is a single bond, -CH ₂ COO-, -CONH-, or -NHCO-, R ₁ is an optionally substituted alkyl group having 1 to 10 carbon atoms, substituted An aryl group which may have a group or —Y—NH—Z—Ar, wherein Y and Z each have a single bond or a carbon number of 1 to 6 which may have a substituent Ar is an aryl group which may have a substituent), R ₂ is an alkyl group having 1 to 6 carbon atoms which may have a substituent, R ₃ Is a hydrogen atom or a hydroxyl group. Or a salt thereof (hereinafter collectively referred to as a compound represented by the formula (I) or a salt thereof, simply “compound contained in the composition of the present invention” or “a composition of the present invention May be referred to as "the above-mentioned compound in the

In one aspect, when X in the general formula (I) is a single bond, R ₁ is an alkyl group having 1 to 10 carbon atoms which may have a substituent (more preferably a substituent And R ₂ is an alkyl group having 1 to 6 carbon atoms which may have a substituent (more preferably substituted). R ₁ is an alkyl group having 1 to 6 carbon atoms, particularly preferably an ethyl group, and R ₃ is a hydrogen atom.

When X is -CH ₂ COO-, R ₁ is an alkyl group having 1 to 10 carbon atoms which may have a substituent (preferably, the number of carbons which may have a substituent) R ₂ is an alkyl group of 1 to 6, particularly preferably an ethyl group, and R ₂ is an alkyl group having 1 to 6 carbon atoms which may have a substituent (more preferably has no substituent) R ₁ is an alkyl group having 1 to 6 carbon atoms, particularly preferably an ethyl group, and R ₃ is a hydrogen atom, or R ₁ is a C 1 to 10 carbon atom which may have a substituent. R ₂ is an alkyl group (more preferably, an alkyl group having 1 to 6 carbon atoms having a substituent, particularly preferably a benzyl group), and R ₂ is an optionally substituted carbon atom having 1 to 6 carbon atoms 6 alkyl groups (more preferably, alkyl groups having 1 to 6 carbon atoms which have no substituent, Particularly preferred is an ethyl group), and R ₃ is a hydrogen atom.

When X is -CONH-, R ₁ is an aryl group which may have a substituent (more preferably, an aryl group having no substituent, particularly preferably a phenyl group) And R ₂ is an alkyl group having 1 to 6 carbon atoms which may have a substituent (more preferably an alkyl group having 1 to 6 carbon atoms which does not have a substituent, particularly preferably an ethyl group) And R ₃ is a hydrogen atom.

Also, when X is -NHCO-, R ₁ is -Y-NH-Z-Ar, where, when Y is a single bond, Z has a single bond or a substituent Optionally substituted alkylene group (more preferably a single bond or an alkylene group having no substituent, particularly preferably a single bond or a methylene group), Ar is a substituent (more preferably a halogen atom, An aryl group optionally having a methyl group, a hydroxyl group or a methoxy group (more preferably, an aryl group having a hydroxyl group or an aryl group having no substituent, particularly preferably a phenyl group or a phenyl group having a hydroxyl group And R ₂ is an alkyl group having 1 to 6 carbon atoms which may have a substituent (more preferably, an alkyl group having 1 to 6 carbon atoms which does not have a substituent, particularly preferably Echi A group), _{R 3} is hydrogen atom.

In addition, X is -NHCO- and Y is an alkylene group having 1 to 6 carbon atoms which may have a substituent (more preferably, an alkylene group having 1 to 6 carbon atoms which does not have a substituent) And particularly preferably a methylene group, an ethylidene group or a propylidene group), Z is a single bond, Ar is an aryl group which may have a substituent (more preferably, it has a substituent) Aryl group, particularly preferably a halogeno group, a methyl group, a phenyl group having a hydroxyl group or an ethoxy group, or a naphthyl group), and R ₂ is an optionally substituted C 1 to C 6 carbon atom alkyl group (more preferably an alkyl group having 1 to 6 carbon atoms having no substituent, particularly preferably, a methyl group, an ethyl group or an isopropyl group) is, R ₃ is a hydrogen atom or a hydroxyl It is.

The compounding amount of the above-mentioned compound in the composition of the present invention is not particularly limited as long as the medium has a concentration at which the desired effect of the present invention can be exhibited when the composition of the present invention is added to the medium. . The lower limit of the concentration of the above-mentioned compound in the medium is usually 0.001 μM or more, preferably 0.01 μM or more, more preferably 0.1 μM or more, more preferably as a concentration at which the desired effect of the present invention can be exerted. May be 1 μM or more, particularly preferably 10 μM or more. The upper limit value of the concentration may be usually 100 μM or less, preferably 50 μM or less, particularly preferably 20 μM or less, but is not limited thereto.

The compositions of the present invention may be in any form as provided or stored. The composition may be bound to a formulated solid such as a tablet, pill, capsule or granule, a suitable solvent and a liquid such as a solution or suspension dissolved with a solubilizer, or a substrate or carrier. Can be Additives at the time of formulation include preservatives such as p-hydroxybenzoic acid esters; excipients such as lactose, glucose, sucrose, and mannitol; lubricants such as magnesium stearate and talc; polyvinyl Binders such as alcohol, hydroxypropyl cellulose and gelatin; surfactants such as fatty acid esters; and plasticizers such as glycerin. These additives are not limited to those described above, and can be freely selected by those skilled in the art if available.

Cell types suitable for use in the composition of the present invention may be cells of adherent cells. Examples of such cells include somatic cells, normal cell lines, cancer cell lines, progenitor cells, stem cells, cells isolated from living bodies and artificially modified genetically, and artificial cells isolated from living bodies. And cells such as cells whose nuclei have been exchanged, but are not limited thereto. The origin of these cells is also not particularly limited, but rat, mouse, rabbit, guinea pig, squirrel, hamster, vole, platypus, dolphin, whale, dog, cat, goat, cow, horse, sheep, pig, elephant, common Preferred are cells derived from mammals such as marmosets, squirrel monkeys, rhesus monkeys, chimpanzees and humans. Also, the tissue or organ from which the cells are derived is not particularly limited as long as the desired effect of the present invention can be obtained, and examples of the tissue include skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, Tissues such as stomach, colon, small intestine, large intestine, spleen, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, blood vessel tissue, blood, heart, eye, brain or nerve tissue can be mentioned. Further, examples of the organ include organs such as liver, lung, kidney, heart, pancreas, stomach, spleen, small intestine, large intestine, genital organ and the like.

In the present invention, examples of normal cell lines include C3H10T1 / 2 (mouse embryonic fibroblasts), HUVEC (human umbilical vein endothelial cells), HEK293 (human fetal kidney cells), MDBK (bovine kidney-derived cells), MDCK (MDCK) Canine renal tubular epithelial cells), Vero, NIH 3 T 3 (mouse fetal fibroblasts), HepaRG (hepatocytes, registered trademark), human primary culture hepatocytes and the like can be mentioned. Among these, C3H10T1 / 2, HUVEC or HEK293 are particularly preferred.

In addition, examples of cancer cell lines include, but are not limited to, human breast cancer cell lines such as HBC-4, BSY-1, BSY-2, MCF-7, MCF-7 / ADR RES, HS578T. , MDA-MB-231, MDA-MB-435, MDA-N, BT-549, T47D, HeLa as a human cervical cancer cell line, A549 as a human lung cancer cell line, EKVX, HOP-62, HOP-92, NCI-H23, NCI-H226, NCI-H322M, NCI-H460, NCI-H522, DMS273, DMS114, human colon cancer cell lines Caco-2, COLO-205, HCC-2998, HCT-15, HCT-116 , HT-29, KM-12, SW-620, WiDr, DU-1 as a human prostate cancer cell line 5, PC-3, LNCaP, U251, SF-295, SF-539, SF-268, SNB-75, SNB-78, SNB-19, human ovarian cancer cell line as human central nervous system cancer cell line OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SKOV3, IGROV-1, human kidney cancer cell line RXF-631L, ACHN, UO-31, SN-12C, A498, CAKI-1, RXF -393L, 786-0, TK-10, MKN45, MKN28, St-4, MKN-1, MKN-7, MKN-74 as human gastric cancer cell lines, A431, LOX-IMVI, LOX, as skin cancer cell lines MALME-3M, SK-MEL-2, SK-MEL-5, SK-MEL-28, UACC-62, UACC-257 M14, CCRF-CRM, K562 as leukemia cell lines, MOLT-4, HL-60TB, RPMI8226, SR, UT7 / TPO, Jurkat, and the like. Among these, HeLa, A431, HCT116 and MCF7 are particularly preferred.

Furthermore, in the present specification, “stem cell” means a cell having both the ability to replicate itself and the ability to differentiate into cells of multiple other lineages, and examples thereof include, but are not limited to: But embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPSCs), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, reproductive Examples include stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells and the like. Pluripotent stem cells include ES cells, embryonic germ stem cells, and induced pluripotent stem cells among the stem cells. The precursor cells are cells in the process of differentiating from the stem cells to specific somatic cells or germ cells. In the present invention, as stem cells, human mesenchymal stem cells (hMSCs) or human induced pluripotent stem cells (hiPSCs) are particularly preferable.

In the present specification, the term "composition of the present invention" can be replaced with the term "agent of the present invention" or "cell adhesion promoter of the present invention".

2. Media The present invention provides a culture medium for cell culture (hereinafter sometimes referred to as “the culture medium of the present invention”) containing the composition of the present invention. By using the medium of the present invention, cell adhesion of cells (especially adherent cells) to a culture vessel can be promoted.

The concentration of the compound contained as an active ingredient in the culture medium of the present invention is not particularly limited as long as the desired effect of the present invention can be obtained, but the lower limit of the concentration is usually 0.001 μM or more, preferably 0.01 μM or more More preferably, it may be 0.1 μM or more, more preferably 1 μM or more, and particularly preferably 10 μM or more. Also, the upper limit value of the concentration may be usually 100 μM or less, preferably 50 μM or less, particularly preferably 20 μM or less.

The medium of the present invention can be the same as the composition of a known medium except that the composition of the present invention is blended.

In one aspect, the medium of the present invention can be prepared by adding the composition of the present invention to a commercially available medium. The commercially available medium which can be used as the medium of the present invention by adding the composition of the present invention is not particularly limited as long as the desired effect can be obtained, for example, Dulbecco's Modified Eagle's Medium; DMEM), Ham's F12 medium (Ham's Nutrient Mixture F12), DMEM / F12 medium, McCoy's 5A medium (McCoy's 5A medium), Eagle MEM (Eagle's Minimum Essential Medium; EMEM), α MEM (alpha Modified Eagle's Minimum Essential Medium; α MEM) , MEM (Minimum Essential Medium), RPMI 1640 medium, chair F) Modified Dulbecco's Medium (IMDM), MCDB 131 Medium, William Medium E, IPL 41 Medium, Fischer's Medium, StemPro 34 (Invitrogen), X-VIVO 10 (Kenbrex), X-VIVO 15 (Kenbrex) Made by Brecks, HPGM (made by Ken Brecks), StemSpan H3000 (made by Stemcell Technology), StemSpanSFEM (made by Stemcell Technology), Stemline II (made by Sigma Aldrich), QBSF-60 (made by Quality Biological), StemProhESCSFM (Manufactured by Invitrogen), Essential 8 (registered trademark) medium (manufactured by Gibco), mTeSR1 Or 2 medium (Stemcell Technology), TeSR-E8 medium (Stemcell Technology), ReproFF or ReproFF2 (Reprocell), Primate ES Cell Medium (Reprocell), PSGro hESC / iPSC medium (System Bio) Science), NutriStem (registered trademark) culture medium (manufactured by Biological Industries), StemFit (registered trademark) culture medium (manufactured by Ajinomoto), CSTI-7 culture medium (manufactured by Cell Science Research Institute), MesenPRO RS medium (Gibco) MF-Medium (registered trademark) mesenchymal stem cell growth medium (Toyobo Co., Ltd.), medium for mesenchymal stem cells (Promocell), Sf-900 II (Invitrogen), Opti-Pro (Invitrogen) Made) And the like.

In the above-mentioned medium, sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various vitamins, antibiotics, serum, fatty acids, sugars, cell growth factors, differentiation inducers, cell adhesion factors, antibodies, enzymes, Cytokines, hormones, lectins, extracellular matrices, physiologically active substances and the like may be added according to the purpose.

The culture of cells in the medium of the present invention may be carried out using a petri dish, flask, plastic bag, Teflon bag, dish, petri dish, dish for tissue culture, multidish, microplate, microwell, which is generally used for cell culture. It can be carried out using culture vessels such as plates, multiplates, multiwell plates, chamber slides, tubes, trays, culture bags, roller bottles and the like. In one embodiment, the culture vessel may be artificially treated (for example, coated with an extracellular matrix or the like) in order to improve the adhesion to cells. Examples of such containers are Matrigel (registered trademark), Geltrex (registered trademark), collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, vitronectin, tenascin, selectin, hyaluronic acid, Although the container which coated fibrin etc. is mentioned, it is not limited to this.

The cells capable of promoting cell adhesion by the medium of the present invention are the same as the cells described in “1. Composition for promoting cell adhesion”.

3. Method for promoting cell adhesion The present invention provides a method for promoting cell adhesion (hereinafter sometimes referred to as "the method of the invention"), which comprises adding the composition of the present invention to a culture medium. provide. The method of the present invention can promote cell adhesion of cells (especially adherent cells) to a culture vessel.

The culture medium used in the method of the present invention is not particularly limited as long as the desired effect can be obtained. In addition, cell culture conditions (for example, temperature, carbon dioxide concentration, culture period, etc.) in the method of the present invention may be a method known per se, or may be appropriately modified according to the purpose. For example, the temperature at which the cells are cultured is usually 25 ° C. to 39 ° C., preferably 33 ° C. to 39 ° C. (eg, 37 ° C.) for animal cells. The carbon dioxide concentration is usually 4% by volume to 10% by volume, preferably 4% by volume to 6% by volume, in the culture atmosphere. The culture period is usually 1 to 35 days, but may be appropriately set according to the purpose of culture.

The medium used in the method of the present invention may be the medium of the present invention.

The concentration of the compound serving as the active ingredient, the suitable cell type and the like in the method of the present invention are the same as those described in “1. Composition for promoting cell adhesion”.

Hereinafter, the utility of the composition for promoting cell adhesion of the present invention will be specifically described in the following examples, but the present invention is not limited to these. In addition, the concentration (%) of CO ₂ in the CO ₂ incubator is shown by volume% of CO _{2 in} the atmosphere. Further, PBS means phosphate buffered saline (manufactured by Sigma Aldrich Japan), and FBS means fetal calf serum (manufactured by Biological Industries). Also, (w / v) represents weight per volume.

Reference Example 1 Synthesis of Compounds 2 ', 4'-Dihydroxy-3'-methylpropiophenone (sometimes referred to as "k-1") and 2', 4'-dihydroxy-3 'in the present specification. Two ketones of 2-methylacetophenone (sometimes referred to as "k-2") can be synthesized by a method known per se (Sum TH et al., Tetrahedron. 2015 Jul 1; 71 (26-27) ): 4557-4564. Also, two hydrazines such as 2- (phenylamino) acetohydrazide (sometimes also referred to as "H-1") and 4-phenyl semicarbazide (sometimes referred to as "H-10") are also methods known per se. (Samal RP et al., Chem Biol Drug Des. 2013 Jun; 81 (6): 715-29.).

[k-1: H-1]
2 ', 4'-Dihydroxy-3'-methylpropiophenone (k-1) (100 mg, 0.555 mmol), 2- (phenylamino) acetohydrazide (H-1) (110 mg, 0.666 mmol) in DMSO It was dissolved in (1.1 mL) and stirred at 100 ° C. for 14 hours. After allowing to cool, distilled water (11 mL) is added, and after stirring again at 100 ° C., hot filtration is performed to obtain k-1: H-1 (70.1 mg, 0.214 mmol, yield 39%) as a light yellow solid Obtained.

[k-1: H-7]
Methyl N-bromobutyrate (8.0 g, 44 mmol), aniline (8.0 mL, 88 mmol) was dissolved in toluene (10 mL) and heated to reflux for 5 hours. After cooling, the reaction solution was washed successively with water (30 mL), 2 M hydrochloric acid (25 mL), water (30 mL), saturated aqueous sodium hydrogen carbonate solution (30 mL), brine (30 mL) and dried over anhydrous sodium sulfate. The residue obtained by filtration and concentration under reduced pressure is purified by medium pressure silica gel column chromatography (silica gel 100 g, ethyl acetate / hexane = 1/99 to 10/90), and the intermediate compound (Compound No. 105) (5.11 g) , 26.4 mmol, yield 60%) as a yellow liquid.

The intermediate compound (Compound No. 105) (5.11 g, 26.4 mmol) obtained as described above is dissolved in methanol (26 mL), hydrazine monohydrate (12.8 mL, 264 mmol) is added, and room temperature is obtained. The mixture was stirred for 4.5 hours. Water (150 mL) was added and extracted with methylene chloride (30 mL × 5). The organic layer is washed with saturated brine (100 mL), dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The solid obtained is suspended and washed with IPE, and H-7 (4.60 g, 23.8 mmol, Yield 90%) was obtained as a colorless solid.

2 ', 4'-Dihydroxy-3'-methylpropiophenone (k-1) (50 mg, 0.28 mmol), 2- (phenylamino) butanehydrazide (H-7) (54 mg, 0.28 mmol) in DMSO It was dissolved in (0.55 mL) and stirred at 100 ° C. for 17 hours. After cooling, distilled water (6 mL) was added and decanted, and the residue was dissolved in methylene chloride (0.5 mL). Hexane (0.5 mL) was added and the precipitated solid was collected by filtration to give k-1: H-7 (56.8 mg, 0.160 mmol, yield 57%) as a colorless solid.

[k-1: H-10]
2 ', 4'-Dihydroxy-3'-methylpropiophenone (k-1) (100 mg, 0.55 mmol), 4-phenyl semicarbazide (H-10) (109 mg, 0.721 mmol) in DMSO (1.1 mL) ) And stirred at 100 ° C. for 3.5 hours. After allowing to cool, water (20 mL) and ethyl acetate (20 mL) were added for liquid separation, and the organic layer was washed with saturated brine (20 mL). The extract was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the obtained residue was purified by medium pressure silica gel column chromatography (silica gel 10 g, ethyl acetate / hexane = 10/90 to 50/50). The obtained solid was suspended and washed with IPE to give k-1: H-10 (19.1 mg, 0.0610 mmol, 11% yield) as a colorless solid.

[k-2: H-7]
2 ', 4'-Dihydroxy-3'-methylacetophenone (k-2) (80 mg, 0.48 mmol), 2- (phenylamino) butanehydrazide (H-7) (121 mg, 0.626 mmol) in DMSO (0 The mixture was dissolved in .55 mL) and stirred at 100.degree. C. for 17 hours. After allowing to cool, distilled water (20 mL) and ethyl acetate (20 mL) were added for liquid separation, and the organic layer was washed with saturated brine (20 mL). The residue obtained by drying over anhydrous sodium sulfate, filtration and concentration under reduced pressure is purified by medium pressure silica gel column chromatography (silica gel 10 g, ethyl acetate / hexane = 5/95 to 50/50) to obtain k-2: H -7 (109 mg, 0.319 mmol, 66% yield) was obtained as a pale yellow solid.

[k-2: H-1]
2 ′, 4′-Dihydroxy-3′-methylacetophenone (k-2) (80 mg, 0.48 mmol), 2- (phenylamino) acetohydrazide (H-1) (95.4 mg, 0.578 mmol) in DMSO It was dissolved in (1.0 mL) and stirred at 100 ° C. for 15 hours. After allowing to cool, distilled water (10 mL) is added, and the precipitated solid is collected by filtration, suspended and washed with methylene chloride to give k-2: H-1 (80.4 mg, 0.257 mmol, yield 53%) as yellow Obtained as a solid.

[K-1: I-1]
Hydrazine monohydrate (0.26 mL, 5.3 mmol) was dissolved in methylene chloride (5.3 mL) and benzyl isocyanate (101) (0.324 mL, 2.63 mmol) was slowly added under ice-cooling. The mixture was stirred at room temperature for 3 hours, and the precipitated solid was washed with IPE and dried under reduced pressure to give 102 (352 mg, 2.13 mmol, yield 82%) as a colorless solid.

102 (155 mg, 0.938 mmol), 2 ′, 4′-dihydroxy-3′-methylpropiophenone (k-1) (130 mg, 0.72 mmol) obtained as described above, DMSO (1.4 mL) ) And stirred at 100 ° C. for 3.5 hours. After leaving to cool, distilled water (10 mL) was added, and the precipitated solid was collected by filtration and purified by medium pressure silica gel column chromatography (silica gel 10 g, ethyl acetate / hexane = 10/90 to 55/45). The obtained solid was suspended and washed with methylene chloride and then dried under reduced pressure to give k-1: I-1 (55 mg, 0.17 mmol, yield 24%) as a colorless solid.

[K-1: B-1]
Dissolve methyl 2-bromopropionate (109) (500 mg, 3.0 mmol) in DMSO (6 mL) and add aniline (0.36 mL, 3.9 mmol), potassium carbonate (0.54 g, 3.9 mmol) Stir at room temperature for 21 hours. Ethyl acetate (30 mL) and water (50 mL) were added for separation, and the organic layer was washed with saturated brine (30 mL) and dried over anhydrous sodium sulfate. The residue obtained by filtration and concentration under reduced pressure is purified by medium pressure silica gel chromatography (silica gel 30 g, ethyl acetate / hexane = 2/98 to 15/85), and 110 (343 mg, 1.91 mmol, yield 64% ) As a pale yellow liquid.

Dissolve 110 (340 mg, 1.9 mmol) obtained as above in methanol (3.8 mL), add hydrazine monohydrate (0.18 mL, 3.8 mmol) and stir at 60 ° C. for 24 hours did. Hydrazine monohydrate (0.36 mL, 7.4 mmol) was added and stirred at 60 ° C. for additional 17 hours. The residue obtained by concentrating the reaction solution under reduced pressure is purified by medium pressure silica gel chromatography (10 g of amine silica gel, ethyl acetate / methylene chloride = 0/100 to 20/80), and 111 (321 mg, 1.79 mmol, 94%) was obtained as a colorless solid.

111 (168 mg, 0.937 mmol), 2 ′, 4′-dihydroxy-3′-methylpropiophenone (k-1) obtained as described above (130 mg, 0.72 mmol) in DMSO (1.4 mL) ) And stirred at 100 ° C. for 19 hours. After allowing to cool, distilled water (15 mL) is added, and the precipitated solid is collected by filtration, dried, suspended and washed with methylene chloride, and dried under reduced pressure, k-1: B-1 (173 mg) 0.507 mmol, 70% yield) was obtained as a pale yellow solid.

[K-1: D-1]
Dissolve 3-benzyloxyaniline (129) (2.44 g, 12.2 mmol) in DMF (24 mL) and add sodium acetate (1.10 g, 13.5 mmol), ethyl bromoacetate (128) (1.49 mL, 13) .5 mmol) was added and stirred at room temperature for 4 hours. Water (300 mL) and ethyl acetate (150 mL) were added for liquid separation, and the organic layer was washed with saturated brine (100 mL). The residue obtained by drying over anhydrous sodium sulfate, filtration and concentration under reduced pressure was purified by medium pressure silica gel column chromatography (silica gel 50 g, ethyl acetate / hexane = 2/98 to 10/90) to obtain 130 (2. 82 g, 9.88 mmol, 81% yield) were obtained as a pale yellow solid.

130 (1.0 g, 3.5 mmol) obtained as described above was suspended in methanol (18 mL), 10% Pd / C (0.1 g) was added, and stirred at room temperature under a hydrogen atmosphere for 5 hours . The reaction solution is filtered through Celite, and the filtrate is concentrated under reduced pressure. The residue obtained is purified by medium pressure silica gel column chromatography (silica gel 30 g, ethyl acetate / hexane = 3/97 to 35/65), 131 ( 295 mg, 1.51 mmol, 43% yield) were obtained as a pale yellow liquid.

Dissolve 131 (0.29 g, 1.5 mmol) obtained as above in ethanol (3.5 mL), add hydrazine monohydrate (0.32 mL, 6.5 mmol) and Stir for hours. The reaction solution was concentrated under reduced pressure, and the obtained residue was purified by medium pressure silica gel chromatography (10 g of amine silica gel, methanol / methylene chloride = 1/99 to 10/90). The obtained solid was suspended and washed with IPE and then dried under reduced pressure to give 132 (239 mg, 1.32 mmol, yield 88%) as a pale yellow solid.

132 (130 mg, 0.72 mmol) obtained as described above, 2 ', 4'-dihydroxy-3'-methylpropiophenone (k-1) (100 mg, 0.55 mmol) in DMSO (1.1 mL) ) And stirred at 100 ° C. for 21 hours. After allowing to cool, the reaction solution was directly purified by medium pressure silica gel column chromatography (silica gel 10 g, ethyl acetate / methylene chloride = 5/95 to 50/50). The obtained solid was suspended and washed with water and IPE, and then dried under reduced pressure to give k-1: D-1 (95.9 mg, 0.279 mmol, 51% yield) as a colorless solid.

[K-1: J-1]
Aluminum hydride (1.0 g, 26 mmol) and 3-cyanophenol (148) (0.63 g, 5.3 mmol) were sequentially added to ice-cold THF (10 mL), and the mixture was stirred at room temperature for 1.5 hours at 60 ° C. Stir for .5 hours. After allowing to cool, aluminum hydride (1.0 g, 26 mmol) and THF (10 mL) were added, and the mixture was further stirred at 60 ° C. for 16 hours. The reaction solution was ice-cooled, water (1.5 mL), 15% aqueous sodium hydroxide solution (1.5 mL) and water (4.5 mL) were sequentially added, and the mixture was stirred at room temperature for 3 hours. The suspension was filtered through celite, and the filtrate was concentrated under reduced pressure. The residue thus obtained was purified by medium pressure silica gel column chromatography (10 g of amine silica gel, methanol / methylene chloride = 0/100 to 8/92). The obtained solid was suspended and washed with IPE and then dried under reduced pressure to give 149 (477 mg, 3.87 mmol, 73% yield) as a colorless solid.

Dissolve 149 (200 mg, 1.6 mmol) obtained as described above in methylene chloride (2 mL) and water (2 mL), add sodium hydrogen carbonate (0.27 g, 3.2 mmol), and cool under ice cooling. Acid phenyl (136) (0.22 mL, 1.7 mmol) was slowly added dropwise. It stirred at room temperature for 20 hours, ethyl acetate (20 mL) and water (20 mL) were added, and it liquid-separated. The organic layer is washed with saturated brine (20 mL), dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue obtained is medium pressure silica gel column chromatography (silica gel 10 g, ethyl acetate / hexane = 5 / 95- Purification at 35/65) gave 150 (369 mg, 1.52 mmol, yield 95%) as a colorless liquid.

150 (365 mg, 1.50 mmol) obtained as described above is suspended in acetonitrile (3.8 mL), hydrazine monohydrate (0.18 mL, 3.8 mmol) is added, and it is 2.5 at room temperature At that time, hydrazine monohydrate (0.18 mL, 3.8 mmol) was added and stirred at 55 ° C. for another 20 hours. The reaction solution is concentrated under reduced pressure, and the obtained solid is suspended and washed with IPE / methylene chloride (3/1) and then dried under reduced pressure to give 151 (233 mg, 1.29 mmol, yield 86%) as a colorless solid. Obtained.

151 (130 mg, 0.72 mmol), 2 ', 4'-dihydroxy-3'-methylpropiophenone (k-1) (100 mg, 0.55 mmol) obtained as described above, DMSO (1.1 mL) ) And stirred at 100 ° C. for 15 hours. After allowing to cool, the reaction solution was directly purified by medium pressure silica gel column chromatography (silica gel 10 g, ethyl acetate / methylene chloride = 10/90 to 55/45). Water was added to the obtained purified product, and the precipitated solid was collected by filtration and dried under reduced pressure to give k-1: J-1 (102 mg, 0.297 mmol, 54% yield) as a colorless solid.

[Example 1] Action on mesenchymal stem cells Human bone marrow-derived mesenchymal stem cells (hMSC, manufactured by PromoCell) were precultured for 7 to 10 days using mesenchymal stem cell growth medium 2 (manufactured by PromoCell). The resulting hMSCs were suspended in the same medium to 2.0 × 10 ⁵ cells / mL. Subsequently, k-1: H-1, k-1: H-7, k-1: H-10, k-1: I-1, k-1 dissolved in DMSO so as to give a final concentration of 10 μM: A B-1 or k-1: J-1 solution was added, and 0.5 mL / well was seeded in a 24-well flat bottom plate (Corning # 35114). As a control, DMSO was added to a final concentration of 0.1%. The plate was incubated at 37 ° C. in a 5% CO ₂ incubator for 20 minutes. After removing the medium, the medium is washed three times with 500 μL of PBS to remove PBS, and then 0.25 w / v% trypsin-1 mmol / L EDTA (ethylenediaminetetraacetic acid) solution (Wako Pure Chemical Industries, Ltd.) in PBS The cells were detached by adding 400 μL of a 5-fold diluted solution and incubating at 37 ° C. for 2 to 5 minutes. Further, the same volume of medium was added, transferred to another container, and centrifuged at 400 g for 4 minutes to remove the supernatant. Then, after resuspending cells with 200 μL of PBS, add 200 μL of ATP reagent (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega), suspend, and allow to stand at room temperature for 15 minutes. 100 μL of the suspension was aliquoted into a 96-well flat bottom plate, and the luminescence intensity (RLU value) was measured with FlexStation 3 (manufactured by Molecular Devices). Here, the number of living cells was measured by subtracting the luminescence value of the medium alone. The relative values at the time of addition of each compound are shown in Table 1 when the RLU value (ATP measurement, luminescence intensity) of no compound addition (control) is 100%.

As shown in Table 1, compounds k-1: H-1, k-1: H-7, k-1: H-10, k-1: I-1, k-1: B-1 and k- 1: J-1 was found to have cell adhesion promoting activity.

Example 2 Effects on Various Cell Lines Various human-derived cells were cultured using each of the following media. Human cervical cancer-derived cell line HeLa (manufactured by American Type Culture Collection (ATCC), 10% fetal bovine serum (FBS, manufactured by Corning)) Dulbecco's Modified Eagle's Medium; DMEM (Wako Pure Chemical Industries, Ltd.) ), Eagle 'containing Eagle's cell line A431 derived from human epithelial-like cell carcinoma (manufactured by ATCC, 10% FBS and 1% MEM non-essential amino acid solution (NEAA, manufactured by Wako Pure Chemical Industries, Ltd.)) s Minimum Essential Medium; EMEM (Wako Pure Chemical Industries, Ltd.), human colon adenocarcinoma cell line HCT116 (DS Pharma Biomedical Co., Ltd., McCoy's 5A Me with 10% FBS) ium (Sigma-Aldrich), human breast cancer cell line MCF7 (ATCC, 10% FBS and 1% MEM non-essential amino acid solution (NEAA, Wako Pure Chemical Industries)) containing EMEM (Wako Pure Chemical Industries) Company humane), human embryonic kidney cell-derived cell line HEK293 (ATCC company, DMEM containing 10% FBS (Wako Pure Chemical Industries)). After washing the cells in logarithmic growth phase with PBS, add 0.25 w / v% trypsin-1 mmol / L EDTA (ethylenediaminetetraacetic acid) solution (manufactured by Wako Pure Chemical Industries, Ltd.) to 1 to 5 at 37 ° C. Incubate for min, detach, add each medium, centrifuge and remove supernatant. Thereafter, after resuspension in the same medium, a part was suspended in trypan blue (manufactured by Wako Pure Chemical Industries, Ltd.), and the number of living cells was counted by TC-20 (manufactured by BIO-RAD).

K-1: H-1, k-1: H-7, k-1: H-10, k-2: H-7, k-2: H-1, k-1: I- dissolved in DMSO 1, k-1: B-1, k-1: D-1, or k-1: J-1 solution is added to each medium in which the various cells are suspended to a final concentration of 10 μM. , 8000 cells / 100 μL / well were seeded on a 96-well adhesive plate (Corning # 3585). As a control, a cell suspension containing DMSO was seeded (final concentration of DMSO 0.1%). After stationary culture of this plate in a 5% CO ₂ incubator at 37 ° C for 30 minutes to 2 hours, the culture solution is aspirated and cells not adhered to the culture plate are washed twice using each medium. After removal, 100 μL / well of each culture medium was added. Subsequently, 100 μL of an ATP reagent (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture solution and suspended, and allowed to stand at room temperature for 10 minutes. Furthermore, the luminescence intensity (RLU value) was measured with EnSpire (manufactured by PerkinElmer), and the number of cells adhering to the plate was measured by subtracting the luminescence value of the medium alone. When the compound-free RLU value (ATP measurement, luminescence intensity) is 100%, those showing values of 100% or less are "-", those showing values of 103% or more are "Δ", 120% or more Those having a value of “o” are shown in Table 2, and those having a value of 200% or more are shown in Table 2 as “o”. The blanks indicate that the test has not been conducted.

As shown in Table 2, the composition of the present invention was shown to have a cell adhesion promoting effect on various cell lines.

[Example 3] Effects of the composition of the present invention on normal cells Human umbilical vein endothelial cells (Human Umbilical Vein Endothelial Cells: HUVEC) are used as a medium of Endothelial Cell Growth Medium (Ready-to-use) (PromoCell). Culture was performed. The above cells in logarithmic growth phase are washed with PBS, added with 0.25 w / v% trypsin-1 mmol / L EDTA (ethylenediaminetetraacetic acid) solution (manufactured by Wako Pure Chemical Industries, Ltd.), and kept at 37 ° C. for 3 minutes. Incubate and detach, add media, centrifuge and remove supernatant. Thereafter, after resuspension in the same medium, a part was suspended in trypan blue (manufactured by Wako Pure Chemical Industries, Ltd.), and the number of living cells was counted by TC-20 (manufactured by BIO-RAD).

The above cells were adjusted to a final concentration of 10 μM with a solution of k-1: I-1, k-1: B-1, k-1: D-1 or k-1: J-1 dissolved in DMSO. The suspension was added to each culture medium, and 8000 cells / 100 μL / well were seeded on a 96-well adhesion plate (Corning # 3585). As a control, a cell suspension containing DMSO was seeded (final concentration of DMSO 0.1%). After incubating this plate for 30 minutes in a 5% CO ₂ incubator at 37 ° C., the culture solution was aspirated, and cells not adhered to the culture plate were removed by washing twice using each medium. Thereafter, 100 μL / well of medium was added. Subsequently, 100 μL of an ATP reagent (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture solution and suspended, and allowed to stand at room temperature for 10 minutes. Furthermore, the luminescence intensity (RLU value) was measured with EnSpire (manufactured by PerkinElmer), and the number of cells adhering to the plate was measured by subtracting the luminescence value of the medium alone. Table 3 shows relative values when each compound was added, where RLU value (ATP measurement, luminescence intensity) with no compound added is 100%.

As shown in Table 3, by adding k-1: I-1, k-1: B-1, k-1: D-1, or k-1: J-1 to the culture medium, It became clear that cell adhesion of certain HUVECs was promoted.

[Example 4] Concentration dependent action on HEK293 cell line k-1: J-1 solution in DMSO is added to the culture medium to a final concentration of 0.1, 1 or 10 μM, and HEK293 cells are further added. After adding so as to be able to inoculate each culture medium at a cell concentration of 8000 cells / 100 μL / well, the cells were seeded on a 96-well adhesion plate (Corning # 3585). As a control, a cell suspension containing DMSO was seeded (final concentration of DMSO 0.1%). After stationary culture at 37 ° C. in a 5% CO ₂ incubator for 1 hour, the culture solution is aspirated, washed twice with medium to remove cells not adhered to the culture plate, and then 100 μl / well of medium Added. Subsequently, 100 μL of ATP reagent (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) is added to the culture solution and suspended, allowed to stand at room temperature for 10 minutes, and then added to EnSpire (manufactured by PerkinElmer). The luminescence intensity (RLU value) was measured, and the number of cells adhering to the plate was determined by subtracting the luminescence value of the medium alone. The relative RLU value at the time of compound addition when the compound-free RLU value (ATP measurement, luminescence intensity) is 1 is shown in FIG.

As shown in FIG. 1, k-1: J-1 promoted cell adhesion of HEK 293 in a concentration-dependent manner.

[Example 5] Effect of the composition of the present invention on fibroblast cell line C3H10T1 / 2 Mouse embryonic fibroblast C3H10T1 / 2 (manufactured by DS Pharma Biomedical Co., Ltd.) is 10 (v / v)% FBS (manufactured by Corning) Culturing was carried out using BME medium (manufactured by Thermo Fisher Scientific Co.) containing L) -glutamine-penicillin-streptomycin stabilization solution (manufactured by Sigma-Aldrich). The above cells in logarithmic growth phase are washed with PBS, added with 0.25 w / v% trypsin-1 mmol / L EDTA (ethylenediaminetetraacetic acid) solution (manufactured by Wako Pure Chemical Industries, Ltd.), and kept at 37 ° C. for 3 minutes. Incubate and detach, add media, centrifuge and remove supernatant. Thereafter, after resuspension in the same medium, a part was suspended in trypan blue (manufactured by Wako Pure Chemical Industries, Ltd.), and the number of living cells was counted by TC-20 (manufactured by BIO-RAD).

Add k-1: I-1, k-1: B-1 or k-1: J-1 solution dissolved in DMSO to each medium in which the above-mentioned cells were suspended to a final concentration of 10 μM Then, 8000 cells / 100 μL / well were seeded on a 96-well culture plate (Corning # 351172). As a control, a cell suspension containing DMSO was seeded (final concentration of DMSO 0.1%). After 30 minutes of stationary culture of this plate in a 5% CO ₂ incubator at 37 ° C., the culture solution was aspirated and cells not adhered to the culture plate were removed by washing twice using the above-mentioned medium Thereafter, 100 μL / well of medium was added. Subsequently, 100 μL of an ATP reagent (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture solution and suspended, and allowed to stand at room temperature for 10 minutes. Furthermore, the luminescence intensity (RLU value) was measured with EnSpire (manufactured by PerkinElmer), and the number of cells adhering to the plate was measured by subtracting the luminescence value of the medium alone. Table 4 shows relative values when each compound was added, where RLU value (ATP measurement, luminescence intensity) with no compound added is 100%.

As shown in Table 4, addition of k-1: I-1, k-1: B-1 or k-1: J-1 to the culture medium results in cell adhesion of C3H10T1 / 2, which is a normal cell. It became clear to promote.

[Example 6] Effects of the composition of the present invention on human induced pluripotent stem cells Human induced pluripotent stem cells (hiPSC, iPS manufactured by Academia Japan) 253G1 strain, mTeSR (registered trademark) 1 medium (manufactured by STEMCELL TECHNOLOGIES) And cultured for 7 to 14 days. The resulting hiPSCs were suspended in the same medium to 1.0 × 10 ⁴ cells / mL. Subsequently, a solution of k-1: B-1 or k-1: J-1 dissolved in DMSO is added to a final concentration of 10 μM, and a 12-well flat bottom plate (Corning # 351143) is added to 1 The cells were seeded at 0 mL / well. As a control, DMSO was added to a final concentration of 0.1%. The plate was cultured at 37 ° C. in a 5% CO ₂ incubator for 24 hours. After culture, remove the medium, add 500 μL of medium afresh, add and suspend the same amount of ATP reagent (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, Promega), and allow to stand for 10 minutes at room temperature. After placing, aliquot 200 μl / well into a 96-well flat bottom plate, measure the luminescence intensity (RLU value) with EnSpire (manufactured by PerkinElmer), subtract the luminescence value of the medium alone, and attach the number of cells to the plate Was measured. Table 5 shows relative values when each compound was added, where RLU value (ATP measurement, luminescence intensity) with no compound added is 100%.

As shown in Table 5, addition of the k-1: B-1 or k-1: J-1 solution to the medium promotes cell adhesion of the 253G1 cell line, a human induced pluripotent stem cell. It became clear.

[Example 7] The effect of the composition of the present invention on human induced pluripotent stem cells Human induced pluripotent stem cells (hiPSC, iPS manufactured by Academia Japan Co., Ltd.) 201B7 strain, 7 to 7 using StemFit medium (manufactured by Ajinomoto Co.) It was cultured for 14 days. The resulting hiPSCs were suspended in the same medium to 1.0 × 10 ⁴ cells / mL. Subsequently, a solution of k-1: B-1 or k-1: J-1 dissolved in DMSO is added to a final concentration of 10 μM, and a 12-well flat bottom plate (Corning # 351143) is added to 1 The cells were seeded at 0 mL / well. As a control, DMSO was added to a final concentration of 0.1%. The plate was cultured at 37 ° C. in a 5% CO ₂ incubator for 24 hours. After culture, remove the medium, add 500 μL of medium afresh, add and suspend the same amount of ATP reagent (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, Promega), and allow to stand for 10 minutes at room temperature. After placing, aliquot 200 μl / well into a 96-well flat bottom plate, measure the luminescence intensity (RLU value) with EnSpire (manufactured by PerkinElmer), subtract the luminescence value of the medium alone, and attach the number of cells to the plate Was measured. Table 6 shows relative values when each compound is added, where RLU value (ATP measurement, luminescence intensity) with no compound added is 100%.

As shown in Table 6, addition of the k-1: B-1 or k-1: J-1 solution to the medium promotes cell adhesion of the human induced pluripotent stem cell 201B7 cell line It became clear.

[Example 8] Evaluation of undifferentiated human artificial pluripotent stem cells when using the composition of the present invention Human artificial pluripotent stem cells (hiPSC, manufactured by iPS Academia Japan) 253G1 strain, StemFit medium (manufactured by Ajinomoto Co.) And cultured for 7 to 14 days. The resulting hiPSCs were suspended in the same medium to 2.0 × 10 ⁴ cells / mL. Subsequently, a solution of k-1: B-1 or k-1: J-1 dissolved in DMSO is added to a final concentration of 10 μM, and a 6-well flat bottom plate (Corning # 351146) is added to 1 .5 ml / well was seeded. As a control, DMSO was added to a final concentration of 0.1%. The plate was cultured at 37 ° C. in a 5% CO ₂ incubator for 120 hours. During the culture, medium exchange was performed using StemFit medium not containing the above-mentioned compound after 24, 72 hours. After culture, the medium is removed and 1 mL / well of PBS (-) (Fujifilm Wako Pure Chemical Industries, Ltd.) is added and washed, then 1 mL / well of 0.5 mmol / L-EDTA / PBS solution (Nacalai Tesque, Inc.) ) Was added and allowed to stand for 10 minutes in a 37 ° C., 5% CO ₂ incubator. After 10 minutes, the detached cells were collected in a 15 mL centrifuge tube, and centrifuged at 200 g for 3 minutes (multiple tension thermostat EIX-136, manufactured by Tomy Seiko Co., Ltd.). The supernatant is then removed and 200 μL of TrypLE®   Select (1 ×) (Gibco) was added, and left in a thermostat at 37 ° C. for 2 minutes. After 2 minutes, remove the 15 mL centrifuge tube from the thermostat, add 5 mL of 10% FBS-containing D-MEM (High Glucose) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and centrifuge at 200 g for 3 minutes. went. After centrifugation, the supernatant was removed, and 5 mL of PBS (−) containing 2% FBS (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and centrifugation was performed under the conditions of 200 G for 3 minutes. After centrifugation, the supernatant was removed, and 100 μL of 2% FBS-containing PBS (−) was added to suspend the cells, and then the cell suspension was aliquoted into 1.5 mL eppendorf tubes. For cell suspension after dispensing, PE Mouse anti-Human TRA-1-60 Antigen and Alexa Fluor (registered trademark) 647 Mouse anti-SSEA-4 (both manufactured by BD Bioscience), which are cell staining antibodies, 5 μL of each was added and allowed to stand at room temperature for 30 minutes. In addition, 5 μL each of PE Mouse IgM, I Isotype Control and Alexa Fluor (registered trademark) 647 Mouse IgG 3, Is Isotomype Control (both manufactured by BD Bioscience) were added as Isotoype Control, and allowed to stand at room temperature for 30 minutes. After 30 minutes, 1.0 mL of PBS containing 2% FBS (-) was added to each 1.5 mL eppendorf tube, and centrifuged under the conditions of 2000 g for 3 minutes (micro high-speed cooling centrifuge MX-307, Tomy Seiko Co., Ltd.) After that, the supernatant was removed. After this operation was performed twice in total, 300 μL of PBS (−) containing 2% FBS was added to obtain a measurement sample. The measurement was performed using a BD FACSAria (registered trademark) III cell sorter (manufactured by BD Bioscience), and the double positive ratio to the above antibody was calculated to evaluate the maintenance of undifferentiated state. The results of the double positive ratio of TRA-1-60 and SSEA-4 in hiPSCs cultured by adding DMSO, k-1: B-1, k-1: J-1 are shown in Table 7.

As shown in Table 7, it is clear that the undifferentiated nature of human induced pluripotent stem cells is maintained even if the k-1: B-1 or k-1: J-1 solution is added to the culture medium. became. That is, it became clear that the culture of stem cells using the composition of the present invention had no effect on its undifferentiated nature.

According to the present invention, adhesion of adherent cells to a culture vessel can be promoted, whereby the culture of adherent cells can be made efficient. Thus, the present invention is useful in the field of research and medicine using adherent cells.

This application is based on patent application No. 2017-239103 filed in Japan (filing date: December 13, 2017), the contents of which are incorporated in full herein.

Claims (22)

1. A composition for promoting cell adhesion, comprising a compound represented by the following general formula (I), or a salt thereof:
   

   

   
   Wherein X is a single bond, -CH ₂ COO-, -CONH-, or -NHCO-, R ₁ is an optionally substituted alkyl group having 1 to 10 carbon atoms, substituted An aryl group which may have a group or —Y—NH—Z—Ar, wherein Y and Z each have a single bond or a carbon number of 1 to 6 which may have a substituent Ar is an aryl group which may have a substituent), R ₂ is an alkyl group having 1 to 6 carbon atoms which may have a substituent, R ₃ Is a hydrogen atom or a hydroxyl group. }.
2. The composition according to claim 1, wherein X is -NHCO-.
3. The composition according to claim 1 or 2, wherein R ₂ is a methyl group or an ethyl group which may have a substituent, and R ₃ is a hydrogen atom.
4. The composition according to any one of claims 1 to 3, wherein R ₁ is -Y-NH-Z-Ar.
5. The composition according to claim 4, wherein Ar is a phenyl group optionally substituted by a halogen atom, a hydroxyl group or a methyl group.
6. The composition according to claim 4 or 5, wherein Y is a methylene group, an ethylidene group or a propylidene group.
7. The composition according to any one of claims 1 to 6, wherein the compound is a compound selected from the group consisting of: or a salt thereof.
   

   

   
   

   

   
   
   

   

   
   
   
   

   

   
   
   

   

   
   
   and,
   

   
8. The composition according to any one of claims 1 to 5, wherein the compound is a compound shown below or a salt thereof.
   

   

   
   
   

   

   
   
   and,
   

   
9. The composition according to any one of claims 1 to 8, wherein the cells are selected from the group consisting of normal cell lines, cancer cell lines and stem cells.
10. The composition according to claim 9, wherein the normal cell line is HUVEC, C3H10T1 / 2 or HEK293.
11. The composition according to claim 9, wherein the cancer cell line is HeLa cells, A431, HCT116, or MCF7.
12. 10. The composition of claim 9, wherein the stem cells are human mesenchymal stem cells (hMSCs) or human induced pluripotent stem cells (hiPSCs).
13. A culture medium for culturing cells, comprising the composition according to any one of claims 1 to 8.
14. The medium according to claim 13, wherein the cells are selected from the group consisting of normal cell lines, cancer cell lines and stem cells.
15. The medium according to claim 14, wherein the normal cell line is HUVEC, C3H10T1 / 2 or HEK293.
16. The medium according to claim 14, wherein the cancer cell line is HeLa cells, A431, HCT116, or MCF7.
17. The medium according to claim 14, wherein the stem cells are human mesenchymal stem cells (hMSCs) or human induced pluripotent stem cells (hiPSCs).
18. A method for promoting the adhesion of cells to a culture vessel, comprising adding the composition according to any one of claims 1 to 8 to a culture medium.
19. 19. The method of claim 18, wherein the cells are selected from the group consisting of normal cell lines, cancer cell lines and stem cells.
20. 20. The method of claim 19, wherein the normal cell line is HUVEC, C3H10T1 / 2 or HEK293.
21. 20. The method of claim 19, wherein the cancer cell line is HeLa cells, A431, HCT116, or MCF7.
22. 20. The method of claim 19, wherein the stem cells are human mesenchymal stem cells (hMSCs) or human induced pluripotent stem cells (hiPSCs).

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