WO2019117262A1 - Composition for promoting cell adhesion and method for culturing cells using same - Google Patents
Composition for promoting cell adhesion and method for culturing cells using same Download PDFInfo
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- WO2019117262A1 WO2019117262A1 PCT/JP2018/045960 JP2018045960W WO2019117262A1 WO 2019117262 A1 WO2019117262 A1 WO 2019117262A1 JP 2018045960 W JP2018045960 W JP 2018045960W WO 2019117262 A1 WO2019117262 A1 WO 2019117262A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C251/00—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton
- C07C251/72—Hydrazones
- C07C251/86—Hydrazones having doubly-bound carbon atoms of hydrazone groups bound to carbon atoms of six-membered aromatic rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
- C07C281/06—Compounds containing any of the groups, e.g. semicarbazides
- C07C281/08—Compounds containing any of the groups, e.g. semicarbazides the other nitrogen atom being further doubly-bound to a carbon atom, e.g. semicarbazones
- C07C281/14—Compounds containing any of the groups, e.g. semicarbazides the other nitrogen atom being further doubly-bound to a carbon atom, e.g. semicarbazones the carbon atom being further bound to a carbon atom of a six-membered aromatic ring
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
Definitions
- 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. .
- 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.
- 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.
- problems such as low cell adhesion ratio or long time to cell adhesion may occur.
- Non-patent Document 1 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.
- Patent Documents 1 and 2 methods for promoting cell adhesion using chemically synthesized low molecular weight compounds and polymers have been reported.
- 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.
- 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.
- composition for promoting cell adhesion comprising a compound represented by the following general formula (I), or a salt thereof:
- 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.
- 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.
- the composition according to [9], wherein the normal cell line is HUVEC, C3H10T1 / 2 or HEK293.
- the composition according to [9], wherein the cancer cell line is HeLa cells, A431, HCT116, or MCF7.
- the composition of [9], wherein the stem cells are human mesenchymal stem cells (hMSCs) or human induced pluripotent stem cells (hiPSCs).
- hMSCs human mesenchymal stem cells
- hiPSCs human induced pluripotent stem cells
- the medium according to [13], wherein the cells are selected from the group consisting of normal cell lines, cancer cell lines and stem cells.
- the medium according to [14], wherein the normal cell line is HUVEC, C3H10T1 / 2 or HEK293.
- the cancer cell line is HeLa cells, A431, HCT116, or MCF7.
- the stem cells are human mesenchymal stem cells (hMSCs) or human induced pluripotent stem cells (hiPSCs).
- 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.
- the cells are selected from the group consisting of normal cell lines, cancer cell lines and stem cells.
- the normal cell line is HUVEC, C3H10T1 / 2 or HEK293.
- the cancer cell line is HeLa cells, A431, HCT116, or MCF7.
- the stem cells are human mesenchymal stem cells (hMSCs) or human induced pluripotent stem cells (hiPSCs).
- 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.
- 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.
- halogen atom is a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.
- halogeno group is fluoro, chloro, bromo or iodo.
- 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.
- 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.
- alkyl 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,
- carbamoyl group (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
- 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.
- an alkoxycarbonylamino group eg, a carbonylamino group substituted with an alkoxy group (as defined in the above (12)
- an alkylsulfonyl group eg, a sulfonyl group substituted with an alkyl group (as defined as the “alkyl group” in the above (26)
- 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
- they may be the same or different.
- the compounds of formula (I) may be in the form of salts.
- 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.
- 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.
- a usual post treatment such as extraction after extraction with an organic solvent to obtain the desired compound of the present invention.
- it can be separated and purified by any purification method such as recrystallization, column chromatograph, thin layer chromatograph, liquid chromatograph or the like.
- reaction mixture 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.
- it can be separated and purified by any purification method such as recrystallization, column chromatograph, thin layer chromatograph, liquid chromatograph or the like.
- composition for promoting cell adhesion 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):
- 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.
- R 1 is an alkyl group having 1 to 10 carbon atoms which may have a substituent (more preferably a substituent
- 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
- R 3 is a hydrogen atom.
- 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
- 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
- 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.
- R 1 is an aryl group which may have a substituent (more preferably, an aryl group having no substituent, particularly preferably a phenyl group)
- 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)
- R 3 is a hydrogen atom.
- 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
- 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.
- 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
- 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 ⁇ M or more, preferably 0.01 ⁇ M or more, more preferably 0.1 ⁇ M or more, more preferably as a concentration at which the desired effect of the present invention can be exerted. May be 1 ⁇ M or more, particularly preferably 10 ⁇ M or more.
- the upper limit value of the concentration may be usually 100 ⁇ M or less, preferably 50 ⁇ M or less, particularly preferably 20 ⁇ M or less, but is not limited thereto.
- 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.
- 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.
- 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.
- C3H10T1 / 2 mouse embryonic fibroblasts
- HUVEC human umbilical vein endothelial cells
- HEK293 human fetal kidney cells
- MDBK bovine kidney-derived cells
- MDCK MDCK
- Vero Vero
- NIH 3 T 3 mamouse fetal fibroblasts
- HepaRG hepatocytes, registered trademark
- human primary culture hepatocytes and the like can be mentioned.
- 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, S
- 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.
- ES cells embryonic stem cells
- iPSCs induced pluripotent stem cells
- neural stem cells hematopoietic stem cells
- mesenchymal stem cells mesenchymal stem cells
- hepatic stem cells pancreatic stem cells
- pancreatic stem cells pancreatic stem cells
- muscle stem cells reproductive Examples include stem
- the precursor cells are cells in the process of differentiating from the stem cells to specific somatic cells or germ cells.
- stem cells human mesenchymal stem cells (hMSCs) or human induced pluripotent stem cells (hiPSCs) are particularly preferable.
- composition of the present invention can be replaced with the term “agent of the present invention” or "cell adhesion promoter of the present invention”.
- 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.
- the culture medium 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 ⁇ M or more, preferably 0.01 ⁇ M or more More preferably, it may be 0.1 ⁇ M or more, more preferably 1 ⁇ M or more, and particularly preferably 10 ⁇ M or more. Also, the upper limit value of the concentration may be usually 100 ⁇ M or less, preferably 50 ⁇ M or less, particularly preferably 20 ⁇ M 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.
- 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), ⁇ MEM (alpha Modified Eagle's Minimum Essential Medium; ⁇ 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
- 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”.
- 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.
- cell culture conditions for example, temperature, carbon dioxide concentration, culture period, etc.
- 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”.
- the concentration (%) of CO 2 in the CO 2 incubator is shown by volume% of CO 2 in the atmosphere.
- PBS phosphate buffered saline (manufactured by Sigma Aldrich Japan)
- FBS fetal calf serum (manufactured by Biological Industries).
- (w / v) represents weight per volume.
- 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.
- 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.
- 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.
- 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.
- 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 ⁇ M:
- 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).
- 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.
- the medium is washed three times with 500 ⁇ L 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 ⁇ L 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.
- 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%
- trypsin-1 mmol / L EDTA ethylenediaminetetraacetic acid
- trypan blue manufactured by Wako Pure Chemical Industries, Ltd.
- 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 ⁇ M.
- 8000 cells / 100 ⁇ L / well were seeded on a 96-well adhesive plate (Corning # 3585).
- a cell suspension containing DMSO was seeded (final concentration of DMSO 0.1%).
- the luminescence intensity 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.
- the compound-free RLU value ATP measurement, luminescence intensity
- those showing values of 100% or less are "-"
- those showing values of 103% or more are " ⁇ ”
- 120% or more are shown in Table 2
- those having a value of “o” are shown in Table 2 as “o”.
- the blanks indicate that the test has not been conducted.
- 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.
- HUVEC Human Umbilical Vein Endothelial Cells
- trypsin-1 mmol / L EDTA ethylenediaminetetraacetic acid
- the above cells were adjusted to a final concentration of 10 ⁇ M 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 ⁇ L / well were seeded on a 96-well adhesion plate (Corning # 3585).
- 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.
- 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 ⁇ M, 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 ⁇ L / 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.
- 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).
- trypsin-1 mmol / L EDTA ethylenediaminetetraacetic acid
- 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 ⁇ M
- 8000 cells / 100 ⁇ L / well were seeded on a 96-well culture plate (Corning # 351172).
- a cell suspension containing DMSO was seeded (final concentration of DMSO 0.1%).
- 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 ⁇ L / well of medium was added.
- 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 253G1 strain
- mTeSR (registered trademark) 1 medium manufactured by STEMCELL TECHNOLOGIES
- the resulting hiPSCs were suspended in the same medium to 1.0 ⁇ 10 4 cells / mL.
- a solution of k-1: B-1 or k-1: J-1 dissolved in DMSO is added to a final concentration of 10 ⁇ M, and a 12-well flat bottom plate (Corning # 351143) is added to 1
- the cells were seeded at 0 mL / well.
- 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 ⁇ L 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.
- CellTiter-Glo registered trademark
- Luminescent Cell Viability Assay Promega
- 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.
- hiPSC Human induced pluripotent stem cells
- 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.
- a solution of k-1: B-1 or k-1: J-1 dissolved in DMSO is added to a final concentration of 10 ⁇ M, and a 12-well flat bottom plate (Corning # 351143) is added to 1
- the cells were seeded at 0 mL / well.
- 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 ⁇ L 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.
- CellTiter-Glo registered trademark
- Luminescent Cell Viability Assay Promega
- 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
- StemFit medium manufactured by Ajinomoto Co.
- the resulting hiPSCs were suspended in the same medium to 2.0 ⁇ 10 4 cells / mL.
- a solution of k-1: B-1 or k-1: J-1 dissolved in DMSO is added to a final concentration of 10 ⁇ M, and a 6-well flat bottom plate (Corning # 351146) is added to 1 .5 ml / well was seeded.
- 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.
- PBS PBS
- 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 ⁇ L 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.
- FBS-containing D-MEM High Glucose
- PE Mouse anti-Human TRA-1-60 Antigen and Alexa Fluor (registered trademark) 647 Mouse anti-SSEA-4 both manufactured by BD Bioscience
- 5 ⁇ L of each was added and allowed to stand at room temperature for 30 minutes.
- 5 ⁇ L 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.
- adhesion of adherent cells to a culture vessel can be promoted, whereby the culture of adherent cells can be made efficient.
- the present invention is useful in the field of research and medicine using adherent cells.
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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
本発明は、接着性細胞の細胞接着を促進するための組成物等に関し、詳細には、細胞接着を促進するための組成物、および当該組成物を用いることを特徴とする細胞の培養方法に関する。
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. .
近年、動物体内の様々な細胞を生体外にて増殖、分化或いは維持させるための技術が発展してきている。本技術は、細胞の機能及び構造を詳細に研究するため、医薬品等の薬効及び毒性を評価するため、また、再生医療を実施するために不可欠なものとなっている。動物由来の細胞は、その性状から浮遊性細胞と接着性細胞に大きく二分される。浮遊性細胞は、生育・増殖に足場を必要としない細胞であり、接着性細胞は、生育・増殖に足場を必要とする細胞であるが、生体を構成する大部分の細胞は後者の接着性細胞である。ここで、接着性細胞は培養する際に容器に接着させることがまず必要となるが、細胞の接着の割合が低い、或いは細胞の接着までに時間がかかるなどの問題が生じることがある。このような問題が生じた場合、細胞を用いた研究を迅速かつ効率的に実施できない。そのため、細胞の培養容器への接着を促進する手法が求められており、例えば、細胞外マトリックスを利用して細胞接着を促進する方法が報告されている(非特許文献1)。また、化学合成された低分子化合物やポリマーを用いて、細胞の接着を促進する方法が報告されている(特許文献1、2)。
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).
本発明は、接着細胞の培養容器への接着を促進させ得る培地添加用組成物の提供を目的とする。
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.
本発明者らは、上記課題に対して鋭意検討した結果、特定の化合物を培地に添加することにより、接着性細胞の培養容器への接着を極めて良好に促進し得ることを見出し、かかる知見に基づいてさらに研究を進めることによって本発明を完成するに至った。すなわち、本発明は以下の通りである。
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]下記一般式(I)で示される化合物、またはその塩を含む、細胞接着を促進するための組成物:
[1] A composition for promoting cell adhesion, comprising a compound represented by the following general formula (I), or a salt thereof:
{式中、Xは、単結合、-CH2COO-、-CONH-、または-NHCO-であり、R1は、置換基を有していてもよい炭素数1~10のアルキル基、置換基を有していてもよいアリール基、または-Y-NH-Z-Arであり(式中、Y、およびZは、単結合、または置換基を有していてもよい炭素数1~6のアルキレン基であり、Arは、置換基を有していてもよいアリール基である)、R2は、置換基を有していてもよい炭素数1~6のアルキル基であり、R3は、水素原子またはヒドロキシル基である。}。
[2]Xが、-NHCO-である、[1]記載の組成物。
[3]R2が、置換基を有していてもよいメチル基またはエチル基であり、R3が水素原子である、[1]または[2]に記載の組成物。
[4]R1が、-Y-NH-Z-Arである、[1]~[3]のいずれかに記載の組成物。
[5]Arが、ハロゲン原子、水酸基もしくはメチル基で置換されていてもよいフェニル基である、[4]記載の組成物。
[6]Yが、メチレン基、エチリデン基またはプロピリデン基である、[4]または[5]に記載の組成物。
[7]化合物が、以下からなる群より選択される化合物またはその塩である、[1]~[6]のいずれかに記載の組成物。 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.
[2]Xが、-NHCO-である、[1]記載の組成物。
[3]R2が、置換基を有していてもよいメチル基またはエチル基であり、R3が水素原子である、[1]または[2]に記載の組成物。
[4]R1が、-Y-NH-Z-Arである、[1]~[3]のいずれかに記載の組成物。
[5]Arが、ハロゲン原子、水酸基もしくはメチル基で置換されていてもよいフェニル基である、[4]記載の組成物。
[6]Yが、メチレン基、エチリデン基またはプロピリデン基である、[4]または[5]に記載の組成物。
[7]化合物が、以下からなる群より選択される化合物またはその塩である、[1]~[6]のいずれかに記載の組成物。 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.
および、
and,
[8]化合物が、以下で示される化合物またはその塩である、[1]~[5]のいずれかに記載の組成物。
[8] The composition according to any one of [1] to [5], wherein the compound is a compound shown below or a salt thereof.
および、
and,
[9]細胞が、正常細胞株、がん細胞株および幹細胞からなる群から選択される、[1]~[8]のいずれかに記載の組成物。
[10]正常細胞株が、HUVEC、C3H10T1/2またはHEK293である、[9]記載の組成物。
[11]がん細胞株が、HeLa細胞、A431、HCT116、またはMCF7である、[9]記載の組成物。
[12]幹細胞が、ヒト間葉系幹細胞(hMSC)またはヒト人工多能性幹細胞(hiPSC)である、[9]記載の組成物。
[13][1]~[8]のいずれかに記載の組成物を含む、細胞を培養するための培地。
[14]細胞が、正常細胞株、がん細胞株および幹細胞からなる群から選択される、[13]記載の培地。
[15]正常細胞株が、HUVEC、C3H10T1/2またはHEK293である、[14]記載の培地。
[16]がん細胞株が、HeLa細胞、A431、HCT116、またはMCF7である、[14]記載の培地。
[17]幹細胞が、ヒト間葉系幹細胞(hMSC)またはヒト人工多能性幹細胞(hiPSC)である、[14]記載の培地。
[18][1]~[8]のいずれかに記載の組成物を培地に添加することを含む、細胞の培養容器への接着を促進するための方法。
[19]細胞が、正常細胞株、がん細胞株および幹細胞からなる群から選択される、[18]記載の方法。
[20]正常細胞株が、HUVEC、C3H10T1/2またはHEK293である、[19]記載の方法。
[21]がん細胞株が、HeLa細胞、A431、HCT116、またはMCF7である、[19]記載の方法。
[22]幹細胞が、ヒト間葉系幹細胞(hMSC)またはヒト人工多能性幹細胞(hiPSC)である、[19]記載の方法。 [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).
[10]正常細胞株が、HUVEC、C3H10T1/2またはHEK293である、[9]記載の組成物。
[11]がん細胞株が、HeLa細胞、A431、HCT116、またはMCF7である、[9]記載の組成物。
[12]幹細胞が、ヒト間葉系幹細胞(hMSC)またはヒト人工多能性幹細胞(hiPSC)である、[9]記載の組成物。
[13][1]~[8]のいずれかに記載の組成物を含む、細胞を培養するための培地。
[14]細胞が、正常細胞株、がん細胞株および幹細胞からなる群から選択される、[13]記載の培地。
[15]正常細胞株が、HUVEC、C3H10T1/2またはHEK293である、[14]記載の培地。
[16]がん細胞株が、HeLa細胞、A431、HCT116、またはMCF7である、[14]記載の培地。
[17]幹細胞が、ヒト間葉系幹細胞(hMSC)またはヒト人工多能性幹細胞(hiPSC)である、[14]記載の培地。
[18][1]~[8]のいずれかに記載の組成物を培地に添加することを含む、細胞の培養容器への接着を促進するための方法。
[19]細胞が、正常細胞株、がん細胞株および幹細胞からなる群から選択される、[18]記載の方法。
[20]正常細胞株が、HUVEC、C3H10T1/2またはHEK293である、[19]記載の方法。
[21]がん細胞株が、HeLa細胞、A431、HCT116、またはMCF7である、[19]記載の方法。
[22]幹細胞が、ヒト間葉系幹細胞(hMSC)またはヒト人工多能性幹細胞(hiPSC)である、[19]記載の方法。 [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).
本発明によれば、細胞増殖において基板などへの接着が必須である接着性細胞の培養容器への接着を促進することができる。これにより、当該細胞を効率的に接着させることが可能となり、当該細胞を用いた研究を従来よりも迅速かつ効率的に実施することが可能となる。なお、本発明を用いて培養される接着性細胞が幹細胞(例、人工多能性幹細胞)の場合、本発明は該幹細胞の培養容器への接着を促進させつつも、当該培養の間、該幹細胞の未分化状態を好適に保つことができる。
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.
本明細書において使用する用語を以下に定義する。
The terms used in the present specification are defined below.
「ハロゲン原子」とは、フッ素原子、塩素原子、臭素原子、ヨウ素原子である。「ハロゲノ基」とは、フルオロ、クロロ、ブロモ、ヨードである。
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.
「アルキル基」とは、直鎖または分岐状のアルキル基を意味し、具体的には、メチル、エチル、n-プロピル、イソプロピル、n-ブチル、イソブチル、sec-ブチル、tert-ブチル、n-ペンチル、イソペンチル、tert-ペンチル、ネオペンチル、2-ペンチル、3-ペンチル、n-ヘキシル、2-ヘキシル、n-ヘプチル、n-オクチル、n-ノニル、n-デシル等の基が挙げられる。
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.
「アリール基」とは、少なくとも1つの環が芳香族であり、各環が5~8の環原子を有する単環式、二環式、三環式および四環式炭素環式基が挙げられ、具体的には、フェニル、インデニル、ナフチル、フルオレニル等が挙げられる。特に、アリール基は、C6-10の芳香族のフェニル、インデニル、ナフチルであり得る。
"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.
「アルキレン基」とは、直鎖または分岐状のアルキレン基を意味し、具体的には、メチレン、エチレン、n-プロピレン、イソプロピレン、プロピリデン、n-ブチレン、sec-ブチレン、tert-ブチレン、n-ペンチレン、sec-ペンチレン、tert-ペンチレン、n-ヘキシレン、sec-ヘキシレン、tert-ヘキシレン等の基が挙げられる。
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.
「アルキル基」、「アリール基」及び「アルキレン基」は、置換基を有していてもよく、そのような置換基としては、例えば以下が挙げられる。
(1)ハロゲノ基、
(2)ヒドロキシ基、
(3)シアノ基、
(4)ニトロ基、
(5)カルボキシル基、
(6)アルケニル基(C2-10アルケニル基;例、ビニル、アリル、プロペニル、ブテニル、ペンテニル、へキセニル、ヘプテニル、ブタジエニル、ヘキサトリエニル、およびその各異性体)、
(7)アルキニル基(C2-10アルキニル基;例、エチニル、プロピニル、ブチニル、ペンチニル、ヘキシニル、および、その各異性体)、
(8)ハロゲノアルキル基(例、モノフルオロメチル、ジフルオロメチル、トリフルオロメチル、モノフルオロエチル、ジフルオロエチル、トリフルオロエチル、クロロメチル、クロロエチル、ジクロロエチル、およびその各異性体)、
(9)環状アルキル基(環中にヘテロ原子を含んでもよい)(例、シクロプロピル、シクロブチル、シクロペンチル、シクロヘキシル、テトラヒドロフラニル、テトラヒドロピラニル、アジリジニル、アゼチジニル、ピロリジニル、ピペリジニル、モルホリニル)、
(10)アリール基(例、フェニル、ナフチル)、
(11)ヘテロアリール基(例、ピリジル、ピリダジニル、ピリミジニル、ピラジニル、フリル、チオフェニル、ピロリル、ピラゾリル、イミダゾリル、トリアゾリル(例、1,2,3-トリアゾリル、1,2,4-トリアゾリル)、テトラゾリル、オキサゾリル、イソオキサゾリル、チアゾリル、イソチアゾリル、オキサジアゾリル(例、1,2,3-オキサジアゾリル、1,2,4-オキサジアゾリル、1,3,4-オキサジアゾリル)、チアジアゾリル(例、1,2,3-チアジアゾリル、1,2,4-チアジアゾリル、1,3,4-チアジアゾリル)、ベンゾフリル、ベンゾチオフェニル、インドリル、イソインドリル、ベンゾオキサゾリル、ベンゾチアゾリル、ベンズイミダゾリル、インダゾリル、ベンズイソオキサゾリル、ベンズイソチアゾリル、ベンゾオキサジアゾリル、ベンゾチアジアゾリル、プリニル、キノリニル、イソキノリニル、シンノリニル、フタラジニル、キナゾリニル、キノキサリニル、プテリジニル、イミダゾオキサゾリル、イミダゾチアゾリル、イミダゾイミダゾリル)、
(12)アルコキシ基(例、メトキシ、エトキシ、n-プロポキシ、イソプロポキシ、n-ブトキシ、イソブトキシ、sec-ブトキシ、tert-ブトキシ、n-ペンチルオキシ、イソペンチルオキシ、tert-ペンチルオキシ、ネオペンチルオキシ、2-ペンチルオキシ、3-ペンチルオキシ、n-ヘキシルオキシ、2-ヘキシルオキシ)、
(13)アルキルチオ基(例、メチルチオ、エチルチオ、n-プロピルチオ、イソプロピルチオ、n-ブチルチオ、イソブチルチオ、sec-ブチルチオ、tert-ブチルチオ、n-ペンチルチオ、イソペンチルチオ、tert-ペンチルチオ、ネオペンチルチオ、2-ペンチルチオ、3-ペンチルチオ、n-ヘキシルチオ、2-ヘキシルチオ)、
(14)アリール基(上記(10)と同義)で置換された、アルコキシ基(上記(12)と同義)、
(15)アリール基(上記(10)と同義)で置換された、アルキルチオ基(上記(13)と同義)、
(16)ヘテロアリール基(上記(11)と同義)で置換された、アルコキシ基(上記(12)と同義)、
(17)ヘテロアリール基(上記(11)と同義)で置換された、アルキルチオ基(上記(13)と同義)、
(18)環状アルキル(環中にヘテロ原子を含んでもよい)オキシ基(例、シクロプロピルオキシ、シクロブチルオキシ、シクロペンチルオキシ、シクロヘキシルオキシ、テトラヒドロフラニルオキシ、テトラヒドロピラニルオキシ、アジリジニルオキシ、アゼチジニルオキシ、ピロリジニルオキシ、ピペリジニルオキシ、モルホリニルオキシ)、
(19)アリールオキシ基(例、アリール基(上記(10)と同義)が酸素原子に結合した基)、
(20)ヘテロアリールオキシ基(例、ヘテロアリール基(上記(11)と同義)が酸素原子に結合した基)、
(21)ハロゲノアルコキシ基(例、ハロゲノアルキル基(上記(8)と同義)が酸素原子に結合した基)、
(22)ハロゲノアルキルチオ基(例、ハロゲノアルキル基(上記(8)と同義)が硫黄原子に結合した基)、
(23)ヒドロキシ基で置換された、アルコキシ基(上記(12)と同義)、
(24)アルコキシ基(上記(12)と同義)で置換された、アルコキシ基(上記(12)と同義)、
(25)アミノ基、
(26)アルキル基でモノまたはジ置換されたアミノ基、
ここで、「アルキル基」とは、C1-6アルキル基が挙げられ、具体的には、メチル、エチル、n-プロピル、イソプロピル、n-ブチル、イソブチル、sec-ブチル、tert-ブチル、n-ペンチル、イソペンチル、tert-ペンチル、ネオペンチル、2-ペンチル、3-ペンチル、n-ヘキシル、2-ヘキシル等が挙げられる。
(27)カルバモイル基、
(28)アルキル基(上記(26)における「アルキル基」と同義)でモノまたはジ置換されたカルバモイル基(例、メチルカルバモイル、エチルカルバモイル、ジメチルカルバモイル、ジエチルカルバモイル、エチルメチルカルバモイル)
(29)スルファモイル基、
(30)アルキル基(上記(26)における「アルキル基」と同義)でモノまたはジ置換されたスルファモイル基(例、メチルスルファモイル、エチルスルファモイル、ジメチルスルファモイル、ジエチルスルファモイル、エチルメチルスルファモイル)、
(31)アルカノイル基(例、水素原子若しくはアルキル基(上記(26)における「アルキル基」と同義)が炭素原子に結合したカルボニル基)、
(32)アロイル基(例、アリール基(上記(10)と同義)が炭素原子に結合したカルボニル基)、
(33)アルキルスルホニルアミノ基(例、アルキル基(上記(26)における「アルキル基」と同義)で置換されたスルホニルアミノ基)
(34)アリールスルホニルアミノ基(例、アリール基(上記(10)と同義)で置換されたスルホニルアミノ基)、
(35)へテロアリールスルホニルアミノ基(例、ヘテロアリール基(上記(11)と同義)で置換されたスルホニルアミノ基)、
(36)アシルアミノ基(例、アシル基で置換されたアミノ基)、
ここで、「アシル基」とは、C1-6アルキル基、またはC6-10アリール基を有するアシル基である。ここで、「C1-6アルキル基」とは、上記「アルキル基」のうち、炭素数が1~6のものであり、「C6-10アリール基」とは、上記「アリール基」のうち、炭素数が6~10のものである。アシル基としては、具体的には、アセチル基、プロピオニル基、ブチロイル基、イソブチロイル基、バレロイル基、イソバレロイル基、ピバロイル基、ヘキサノイル基、アクリロイル基、メタクリロイル基、クロトノイル基、イソクロトノイル基、ベンゾイル基、ナフトイル基等が挙げられる、
(37)アルコキシカルボニルアミノ基(例、アルコキシ基(上記(12)と同義)で置換されたカルボニルアミノ基)、
(38)アルキルスルホニル基(例、アルキル基(上記(26)における「アルキル基」と同義)で置換されたスルホニル基)、
(39)アルキルスルフィニル基(例、アルキル基(上記(26)における「アルキル基」と同義)で置換されたスルフィニル基)、
(40)アルコキシカルボニル基(例、メトキシカルボニル基、エトキシカルボニル基)、等が挙げられる。
置換基が2以上存在する場合は、それらは同一でも異なっていてもよい。 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.
(1)ハロゲノ基、
(2)ヒドロキシ基、
(3)シアノ基、
(4)ニトロ基、
(5)カルボキシル基、
(6)アルケニル基(C2-10アルケニル基;例、ビニル、アリル、プロペニル、ブテニル、ペンテニル、へキセニル、ヘプテニル、ブタジエニル、ヘキサトリエニル、およびその各異性体)、
(7)アルキニル基(C2-10アルキニル基;例、エチニル、プロピニル、ブチニル、ペンチニル、ヘキシニル、および、その各異性体)、
(8)ハロゲノアルキル基(例、モノフルオロメチル、ジフルオロメチル、トリフルオロメチル、モノフルオロエチル、ジフルオロエチル、トリフルオロエチル、クロロメチル、クロロエチル、ジクロロエチル、およびその各異性体)、
(9)環状アルキル基(環中にヘテロ原子を含んでもよい)(例、シクロプロピル、シクロブチル、シクロペンチル、シクロヘキシル、テトラヒドロフラニル、テトラヒドロピラニル、アジリジニル、アゼチジニル、ピロリジニル、ピペリジニル、モルホリニル)、
(10)アリール基(例、フェニル、ナフチル)、
(11)ヘテロアリール基(例、ピリジル、ピリダジニル、ピリミジニル、ピラジニル、フリル、チオフェニル、ピロリル、ピラゾリル、イミダゾリル、トリアゾリル(例、1,2,3-トリアゾリル、1,2,4-トリアゾリル)、テトラゾリル、オキサゾリル、イソオキサゾリル、チアゾリル、イソチアゾリル、オキサジアゾリル(例、1,2,3-オキサジアゾリル、1,2,4-オキサジアゾリル、1,3,4-オキサジアゾリル)、チアジアゾリル(例、1,2,3-チアジアゾリル、1,2,4-チアジアゾリル、1,3,4-チアジアゾリル)、ベンゾフリル、ベンゾチオフェニル、インドリル、イソインドリル、ベンゾオキサゾリル、ベンゾチアゾリル、ベンズイミダゾリル、インダゾリル、ベンズイソオキサゾリル、ベンズイソチアゾリル、ベンゾオキサジアゾリル、ベンゾチアジアゾリル、プリニル、キノリニル、イソキノリニル、シンノリニル、フタラジニル、キナゾリニル、キノキサリニル、プテリジニル、イミダゾオキサゾリル、イミダゾチアゾリル、イミダゾイミダゾリル)、
(12)アルコキシ基(例、メトキシ、エトキシ、n-プロポキシ、イソプロポキシ、n-ブトキシ、イソブトキシ、sec-ブトキシ、tert-ブトキシ、n-ペンチルオキシ、イソペンチルオキシ、tert-ペンチルオキシ、ネオペンチルオキシ、2-ペンチルオキシ、3-ペンチルオキシ、n-ヘキシルオキシ、2-ヘキシルオキシ)、
(13)アルキルチオ基(例、メチルチオ、エチルチオ、n-プロピルチオ、イソプロピルチオ、n-ブチルチオ、イソブチルチオ、sec-ブチルチオ、tert-ブチルチオ、n-ペンチルチオ、イソペンチルチオ、tert-ペンチルチオ、ネオペンチルチオ、2-ペンチルチオ、3-ペンチルチオ、n-ヘキシルチオ、2-ヘキシルチオ)、
(14)アリール基(上記(10)と同義)で置換された、アルコキシ基(上記(12)と同義)、
(15)アリール基(上記(10)と同義)で置換された、アルキルチオ基(上記(13)と同義)、
(16)ヘテロアリール基(上記(11)と同義)で置換された、アルコキシ基(上記(12)と同義)、
(17)ヘテロアリール基(上記(11)と同義)で置換された、アルキルチオ基(上記(13)と同義)、
(18)環状アルキル(環中にヘテロ原子を含んでもよい)オキシ基(例、シクロプロピルオキシ、シクロブチルオキシ、シクロペンチルオキシ、シクロヘキシルオキシ、テトラヒドロフラニルオキシ、テトラヒドロピラニルオキシ、アジリジニルオキシ、アゼチジニルオキシ、ピロリジニルオキシ、ピペリジニルオキシ、モルホリニルオキシ)、
(19)アリールオキシ基(例、アリール基(上記(10)と同義)が酸素原子に結合した基)、
(20)ヘテロアリールオキシ基(例、ヘテロアリール基(上記(11)と同義)が酸素原子に結合した基)、
(21)ハロゲノアルコキシ基(例、ハロゲノアルキル基(上記(8)と同義)が酸素原子に結合した基)、
(22)ハロゲノアルキルチオ基(例、ハロゲノアルキル基(上記(8)と同義)が硫黄原子に結合した基)、
(23)ヒドロキシ基で置換された、アルコキシ基(上記(12)と同義)、
(24)アルコキシ基(上記(12)と同義)で置換された、アルコキシ基(上記(12)と同義)、
(25)アミノ基、
(26)アルキル基でモノまたはジ置換されたアミノ基、
ここで、「アルキル基」とは、C1-6アルキル基が挙げられ、具体的には、メチル、エチル、n-プロピル、イソプロピル、n-ブチル、イソブチル、sec-ブチル、tert-ブチル、n-ペンチル、イソペンチル、tert-ペンチル、ネオペンチル、2-ペンチル、3-ペンチル、n-ヘキシル、2-ヘキシル等が挙げられる。
(27)カルバモイル基、
(28)アルキル基(上記(26)における「アルキル基」と同義)でモノまたはジ置換されたカルバモイル基(例、メチルカルバモイル、エチルカルバモイル、ジメチルカルバモイル、ジエチルカルバモイル、エチルメチルカルバモイル)
(29)スルファモイル基、
(30)アルキル基(上記(26)における「アルキル基」と同義)でモノまたはジ置換されたスルファモイル基(例、メチルスルファモイル、エチルスルファモイル、ジメチルスルファモイル、ジエチルスルファモイル、エチルメチルスルファモイル)、
(31)アルカノイル基(例、水素原子若しくはアルキル基(上記(26)における「アルキル基」と同義)が炭素原子に結合したカルボニル基)、
(32)アロイル基(例、アリール基(上記(10)と同義)が炭素原子に結合したカルボニル基)、
(33)アルキルスルホニルアミノ基(例、アルキル基(上記(26)における「アルキル基」と同義)で置換されたスルホニルアミノ基)
(34)アリールスルホニルアミノ基(例、アリール基(上記(10)と同義)で置換されたスルホニルアミノ基)、
(35)へテロアリールスルホニルアミノ基(例、ヘテロアリール基(上記(11)と同義)で置換されたスルホニルアミノ基)、
(36)アシルアミノ基(例、アシル基で置換されたアミノ基)、
ここで、「アシル基」とは、C1-6アルキル基、またはC6-10アリール基を有するアシル基である。ここで、「C1-6アルキル基」とは、上記「アルキル基」のうち、炭素数が1~6のものであり、「C6-10アリール基」とは、上記「アリール基」のうち、炭素数が6~10のものである。アシル基としては、具体的には、アセチル基、プロピオニル基、ブチロイル基、イソブチロイル基、バレロイル基、イソバレロイル基、ピバロイル基、ヘキサノイル基、アクリロイル基、メタクリロイル基、クロトノイル基、イソクロトノイル基、ベンゾイル基、ナフトイル基等が挙げられる、
(37)アルコキシカルボニルアミノ基(例、アルコキシ基(上記(12)と同義)で置換されたカルボニルアミノ基)、
(38)アルキルスルホニル基(例、アルキル基(上記(26)における「アルキル基」と同義)で置換されたスルホニル基)、
(39)アルキルスルフィニル基(例、アルキル基(上記(26)における「アルキル基」と同義)で置換されたスルフィニル基)、
(40)アルコキシカルボニル基(例、メトキシカルボニル基、エトキシカルボニル基)、等が挙げられる。
置換基が2以上存在する場合は、それらは同一でも異なっていてもよい。 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.
式(I)に示される化合物は、塩の形態であってもよい。前記式(I)で表される化合物の塩としては、例えば、塩酸及び臭化水素酸等の無機酸との塩ならびに酢酸、プロピオン酸、酒石酸、フマル酸、マレイン酸、リンゴ酸、シュウ酸、コハク酸、クエン酸及び安息香酸等の有機酸との塩が挙げられる。
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.
[ケトンおよびヒドラジドの組合せからなる化合物の合成]
一般的には、下記式で表されるように、ケトンとヒドラジド、それぞれを1当量ずつ用い、トルエン、1,4-ジオキサン、N、N-ジメチルホルムアミド、ジメチルスルホキシド等の溶媒中、100℃以上の温度範囲で、1時間から3日間反応を行なうのが好ましい。 [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.
一般的には、下記式で表されるように、ケトンとヒドラジド、それぞれを1当量ずつ用い、トルエン、1,4-ジオキサン、N、N-ジメチルホルムアミド、ジメチルスルホキシド等の溶媒中、100℃以上の温度範囲で、1時間から3日間反応を行なうのが好ましい。 [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.
[反応式1]
[Reaction Formula 1]
反応終了後の反応混合物は、蒸留水を加えて析出させる、又は析出しない場合は、有機溶媒抽出後濃縮といった通常の後処理を行ない、目的の本発明化合物を得ることができる。また、精製の必要が生じたときには、再結晶、カラムクロマトグラフ、薄層クロマトグラフ、液体クロマトグラフ分取等の任意の精製方法によって分離、精製することができる。
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.
[k-1誘導体の合成]
一般的に、2’,4’-ジヒドロキシ-3’-メチルプロピオフェノン(k-1)と、所望の一級アミン、あるいはその塩を用いて、それぞれを1当量ずつ用い、トルエン、1,4-ジオキサン、N、N-ジメチルホルムアミド、ジメチルスルホキシド等の溶媒中、100℃以上の温度範囲で、1時間から24時間反応を行なうのが好ましい。アミンの塩としては、塩酸塩、p-トルエンスルホン酸塩、トリフルオロ酢酸塩等を用いることが出来る。 [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.
一般的に、2’,4’-ジヒドロキシ-3’-メチルプロピオフェノン(k-1)と、所望の一級アミン、あるいはその塩を用いて、それぞれを1当量ずつ用い、トルエン、1,4-ジオキサン、N、N-ジメチルホルムアミド、ジメチルスルホキシド等の溶媒中、100℃以上の温度範囲で、1時間から24時間反応を行なうのが好ましい。アミンの塩としては、塩酸塩、p-トルエンスルホン酸塩、トリフルオロ酢酸塩等を用いることが出来る。 [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.
また、上記の手法を使用しない場合には、2’,4’-ジヒドロキシ-3’-メチルプロピオフェノン(k-1)を、アンモニアのメタノール溶液に溶解させ、アンモニアガスを注入しながら撹拌することで、イミン(k-1’)を合成し、その後、対応するイソシアネートと反応させることでも、合成できる。
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.
反応終了後の反応混合物は、蒸留水を加えた後に、溶媒抽出後濃縮といった通常の後処理を行ない、目的の本発明化合物を得ることができる。また、精製の必要が生じたときには、再結晶、カラムクロマトグラフ、薄層クロマトグラフ、液体クロマトグラフ分取等の任意の精製方法によって分離、精製することができる。
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.細胞接着を促進するための組成物
本発明は、以下の化合物を含む、細胞接着を促進するための組成物(以下、「本発明の組成物」と称することがある)を提供する。本発明の組成物は、細胞培養用の培地に添加することにより、細胞(特に接着細胞)の培養容器への細胞接着を促進することができる。 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.
本発明は、以下の化合物を含む、細胞接着を促進するための組成物(以下、「本発明の組成物」と称することがある)を提供する。本発明の組成物は、細胞培養用の培地に添加することにより、細胞(特に接着細胞)の培養容器への細胞接着を促進することができる。 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.
本発明の組成物に含まれる化合物は、一般式(I):
The compounds included in the composition of the present invention have the general formula (I):
{式中、Xは、単結合、-CH2COO-、-CONH-、または-NHCO-であり、R1は、置換基を有していてもよい炭素数1~10のアルキル基、置換基を有していてもよいアリール基、または-Y-NH-Z-Arであり(式中、Y、およびZは、単結合、または置換基を有していてもよい炭素数1~6のアルキレン基であり、Arは、置換基を有していてもよいアリール基である)、R2は、置換基を有していてもよい炭素数1~6のアルキル基であり、R3は、水素原子またはヒドロキシル基である。}で表される化合物、またはその塩である(以下、式(I)で示される化合物またはその塩を総称して、単に「本発明の組成物に含まれる化合物」または「本発明の組成物中の上記化合物」等と称する場合がある)。
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
一態様において、前記一般式(I)中のXが単結合である場合は、R1は、置換基を有していてもよい炭素数1~10のアルキル基(より好ましくは、置換基を有していない炭素数1~10のアルキル基、特に好ましくは、オクチル基)であり、R2は、置換基を有していてもよい炭素数1~6のアルキル基(より好ましくは、置換基を有していない炭素数1~6のアルキル基、特に好ましくは、エチル基)であり、R3は、水素原子である。
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.
また、Xが-CH2COO-である場合は、R1は、置換基を有していてもよい炭素数1~10のアルキル基(好ましくは、置換基を有していてもよい炭素数1~6のアルキル基、特に好ましくは、エチル基)であり、R2は、置換基を有していてもよい炭素数1~6のアルキル基(より好ましくは、置換基を有していない炭素数1~6のアルキル基、特に好ましくは、エチル基)であり、R3は、水素原子であるか、あるいは、R1は、置換基を有していてもよい炭素数1~10のアルキル基(より好ましくは、置換基を有している炭素数1~6のアルキル基、特に好ましくは、ベンジル基)であり、R2は、置換基を有していてもよい炭素数1~6のアルキル基(より好ましくは、置換基を有していない炭素数1~6のアルキル基、特に好ましくは、エチル基)であり、R3は、水素原子である。
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.
また、Xが-CONH-である場合は、R1は、置換基を有していてもよいアリール基(より好ましくは、置換基を有していないアリール基、特に好ましくは、フェニル基)であり、R2は、置換基を有していてもよい炭素数1~6のアルキル基(より好ましくは置換基を有していない炭素数1~6のアルキル基、特に好ましくは、エチル基)であり、R3は、水素原子である。
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.
また、Xが-NHCO-である場合は、R1は、-Y-NH-Z-Arであり、ここで、Yが、単結合である場合は、Zは、単結合または置換基を有していてもよいアルキレン基(より好ましくは、単結合または置換基を有していないアルキレン基、特に好ましくは、単結合またはメチレン基)であり、Arは置換基(より好ましくは、ハロゲン原子、メチル基、水酸基、またはメトキシ基)を有していてもよいアリール基(より好ましくは、水酸基を有するアリール基または置換基を有していないアリール基、特に好ましくはフェニル基または水酸基を有するフェニル基)であり、R2は、置換基を有していてもよい炭素数1~6のアルキル基(より好ましくは、置換基を有していない炭素数1~6のアルキル基、特に好ましくは、エチル基)であり、R3は、水素原子である。
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.
また、Xが-NHCO-、かつ、Yが、置換基を有していてもよい炭素数1~6のアルキレン基(より好ましくは、置換基を有していない炭素数1~6のアルキレン基、特に好ましくはメチレン基、エチリデン基またはプロピリデン基)である場合は、Zは、単結合であり、Arは、置換基を有していてもよいアリール基(より好ましくは、置換基を有しているアリール基、特に好ましくは、ハロゲノ基、メチル基、水酸基もしくはエトキシ基を有するフェニル基、またはナフチル基)であり、R2は、置換基を有していてもよい炭素数1~6のアルキル基(より好ましくは、置換基を有していない炭素数1~6のアルキル基、特に好ましくは、メチル基、エチル基、またはイソプロピル基)であり、R3は、水素原子またはヒドロキシル基である。
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.
本発明の組成物中の上記化合物の配合量は、本発明の組成物を培地に添加した際に、当該培地が、本発明の所望の効果を発揮できる濃度となるものであれば特に限定されない。なお、本発明の所望の効果を発揮できる濃度としては、上記化合物の培地中の濃度の下限値は、通常0.001μM以上、好ましくは0.01μM以上、より好ましくは0.1μM以上、さらに好ましくは1μM以上、特に好ましくは10μM以上であり得る。また、その濃度の上限値は、通常100μM以下、好ましくは50μM以下、特に好ましくは20μM以下であり得るが、これらに限定されない。
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.
本発明の組成物は、提供時あるいは保存時に任意の形状であり得る。当該組成物は、錠剤、丸剤、カプセル剤、顆粒剤のような製剤化された固体、適切な溶媒並びに溶解剤で溶解した溶液あるいは懸濁液のような液体、又は基板や担体に結合させた状態であり得る。製剤化される際の添加物としては、p-ヒドロキシ安息香酸エステル類等の防腐剤;乳糖、ブドウ糖、ショ糖、マンニット等の賦形剤;ステアリン酸マグネシウム、タルク等の滑沢剤;ポリビニルアルコール、ヒドロキシプロピルセルロース、ゼラチン等の結合剤;脂肪酸エステル等の界面活性剤;グリセリン等の可塑剤等が挙げられる。これらの添加物は上記のものに限定されることはなく、当業者が利用可能であれば自由に選択することができる。
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.
本発明の組成物の使用が好適である細胞種としては、接着性細胞の細胞であり得る。そのような細胞の例としては、生体を構成する体細胞、正常細胞株、がん細胞株、前駆細胞、幹細胞、生体から分離され人為的に遺伝子改変が成された細胞、生体から分離され人為的に核が交換された細胞等の細胞が挙げられるが、これらに限定されない。なお、これらの細胞の由来も特に限定されないが、ラット、マウス、ウサギ、モルモット、リス、ハムスター、ハタネズミ、カモノハシ、イルカ、クジラ、イヌ、ネコ、ヤギ、ウシ、ウマ、ヒツジ、ブタ、ゾウ、コモンマーモセット、リスザル、アカゲザル、チンパンジー、ヒト等の哺乳動物由来の細胞が好ましい。また、細胞が由来する組織または臓器も、本発明の所望の効果を得られる限り特に限定されないが、組織の例としては、皮膚、腎臓、脾臓、副腎、肝臓、肺、卵巣、膵臓、子宮、胃、結腸、小腸、大腸、脾臓、膀胱、前立腺、精巣、胸腺、筋肉、結合組織、骨、軟骨、血管組織、血液、心臓、眼、脳または神経組織等の組織が挙げられる。また、臓器の例としては、肝臓、肺、腎臓、心臓、膵臓、胃、脾臓、小腸、大腸、生殖器等の臓器が挙げられる。
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.
本発明において、正常細胞株の例としては、C3H10T1/2(マウス胚線維芽細胞)、HUVEC(ヒト臍帯静脈内皮細胞)、HEK293(ヒト胎児腎細胞)、MDBK(ウシ腎臓由来細胞)、MDCK(イヌ腎臓尿細管上皮細胞)、Vero、NIH3T3(マウス胎児線維芽細胞)、HepaRG(肝細胞、登録商標)、ヒト初代培養肝細胞等が挙げられる。これらの中では、特にC3H10T1/2、HUVECまたはHEK293が好ましい。
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.
また、がん細胞株の例としては、以下に限定されるものではないが、ヒト乳がん細胞株としてHBC-4、BSY-1、BSY-2、MCF-7、MCF-7/ADR RES、HS578T、MDA-MB-231、MDA-MB-435、MDA-N、BT-549、T47D、ヒト子宮頸がん細胞株としてHeLa、ヒト肺がん細胞株としてA549、EKVX、HOP-62、HOP-92、NCI-H23、NCI-H226、NCI-H322M、NCI-H460、NCI-H522、DMS273、DMS114、ヒト大腸がん細胞株としてCaco-2、COLO-205、HCC-2998、HCT-15、HCT-116、HT-29、KM-12、SW-620、WiDr、ヒト前立腺がん細胞株としてDU-145、PC-3、LNCaP、ヒト中枢神経系がん細胞株としてU251、SF-295、SF-539、SF-268、SNB-75、SNB-78、SNB-19、ヒト卵巣がん細胞株としてOVCAR-3、OVCAR-4、OVCAR-5、OVCAR-8、SKOV3、IGROV-1、ヒト腎がん細胞株としてRXF-631L、ACHN、UO-31、SN-12C、A498、CAKI-1、RXF-393L、786-0、TK-10、ヒト胃がん細胞株としてMKN45、MKN28、St-4、MKN-1、MKN-7、MKN-74、皮膚がん細胞株としてA431、LOX-IMVI、LOX、MALME-3M、SK-MEL-2、SK-MEL-5、SK-MEL-28、UACC-62、UACC-257、M14、白血病細胞株としてCCRF-CRM、K562、MOLT-4、HL-60TB、RPMI8226、SR、UT7/TPO、Jurkat等が挙げられる。これらの中では、HeLa、A431、HCT116およびMCF7が特に好ましい。
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.
また、本明細書において「幹細胞」とは、自分自身を複製する能力と他の複数系統の細胞に分化する能力を兼ね備えた細胞を意味し、その例としては、以下に限定されるものではないが、胚性幹細胞(ES細胞)、胚性腫瘍細胞、胚性生殖幹細胞、人工多能性幹細胞(iPSC)、神経幹細胞、造血幹細胞、間葉系幹細胞、肝幹細胞、膵幹細胞、筋幹細胞、生殖幹細胞、腸幹細胞、癌幹細胞、毛包幹細胞などが挙げられる。多能性幹細胞としては、前記幹細胞のうち、ES細胞、胚性生殖幹細胞、人工多能性幹細胞が挙げられる。前駆細胞とは、前記幹細胞から特定の体細胞や生殖細胞に分化する途中の段階にある細胞である。本発明において、幹細胞としては、ヒト間葉系幹細胞(hMSC)またはヒト人工多能性幹細胞(hiPSC)等が特に好ましい。
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.
本明細書において「本発明の組成物」との語は、「本発明の剤」または「本発明の細胞接着促進剤」との語に置き換えることができる。
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.培地
本発明は、本発明の組成物を含む細胞培養用の培地(以下、「本発明の培地」と称することがある)を提供する。本発明の培地を用いることにより、細胞(特に接着細胞)の培養容器への細胞接着を促進することができる。 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.
本発明は、本発明の組成物を含む細胞培養用の培地(以下、「本発明の培地」と称することがある)を提供する。本発明の培地を用いることにより、細胞(特に接着細胞)の培養容器への細胞接着を促進することができる。 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.
本発明の培地に有効成分として含まれる上記化合物の濃度は、本発明の所望の効果を得られる限り特に限定されないが、その濃度の下限値は、通常0.001μM以上、好ましくは0.01μM以上、より好ましくは0.1μM以上、さらに好ましくは1μM以上、特に好ましくは10μM以上であり得る。また、その濃度の上限値は、通常100μM以下、好ましくは50μM以下、特に好ましくは20μM以下であり得る。
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.
本発明の培地は、本発明の組成物が配合されている以外は、公知の培地の組成と同様とすることができる。
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.
一態様において、本発明の培地は、市販される培地に、本発明の組成物を添加することにより調製することができる。本発明の組成物を添加することにより本発明の培地とし得る市販の培地としては、所望の効果を得られる限り特に限定されないが、例えば、ダルベッコ改変イーグル培地(Dulbecco’s Modified Eagle’s Medium;DMEM)、ハムF12培地(Ham’s Nutrient MixtureF12)、DMEM/F12培地、マッコイ5A培地(McCoy’s 5A medium)、イーグルMEM(Eagle’s Minimum Essential Medium;EMEM)、αMEM(alpha Modified Eagle’s Minimum Essential Medium;αMEM)、MEM(Minimum Essential Medium)、RPMI1640培地、イスコフ改変ダルベッコ培地(Iscove’s Modified Dulbecco’s Medium;IMDM)、MCDB131培地、ウィリアム培地E、IPL41培地、Fischer’s培地、StemPro34(インビトロジェン社製)、X-VIVO 10(ケンブレックス社製)、X-VIVO 15(ケンブレックス社製)、HPGM(ケンブレックス社製)、StemSpan H3000(ステムセルテクノロジー社製)、StemSpanSFEM(ステムセルテクノロジー社製)、StemlineII(シグマアルドリッチ社製)、QBSF-60(クオリティバイオロジカル社製)、StemProhESCSFM(インビトロジェン社製)、Essential8(登録商標)培地(ギブコ社製)、mTeSR1或いは2培地(ステムセルテクノロジー社製)、TeSR-E8培地(ステムセルテクノロジー社製)、リプロFF或いはリプロFF2(リプロセル社製)、Primate ES Cell Medium(リプロセル社製)、PSGro hESC/iPSC培地(システムバイオサイエンス社製)、NutriStem(登録商標)培地(バイオロジカルインダストリーズ社製)、StemFit(登録商標)培地(味の素社製)、CSTI-7培地(細胞科学研究所社製)、MesenPRO RS培地(ギブコ社製)、MF-Medium(登録商標)間葉系幹細胞増殖培地(東洋紡株式会社製)、間葉系幹細胞用培地(プロモセル社製)、Sf-900II(インビトロジェン社製)、Opti-Pro(インビトロジェン社製)等の培地が挙げられる。
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.
また、上記の培地に、ナトリウム、カリウム、カルシウム、マグネシウム、リン、塩素、各種アミノ酸、各種ビタミン、抗生物質、血清、脂肪酸、糖、細胞増殖因子、分化誘導因子、細胞接着因子、抗体、酵素、サイトカイン、ホルモン、レクチン、細胞外マトリックス、生理活性物質等を目的に応じて添加してもよい。
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.
本発明の培地における細胞の培養は、細胞培養に一般的に用いられるシャーレ、フラスコ、プラスチックバック、テフロン(登録商標)バック、ディッシュ、ペトリデッシュ、組織培養用ディッシュ、マルチディッシュ、マイクロプレート、マイクロウエルプレート、マルチプレート、マルチウエルプレート、チャンバースライド、チューブ、トレイ、培養バック、ローラーボトル等の培養容器を用いて実施することができる。一態様において、培養容器として、培養容器の表面が、細胞との接着性を向上させる目的で人工的に処理(例えば、細胞外マトリクス等によるコーティング処理)されているものを用いてもよい。このような容器の例としては、マトリゲル(登録商標)、Geltrex(登録商標)、コラーゲン、ゼラチン、ポリ-L-リジン、ポリ-D-リジン、ラミニン、フィブロネクチン、ビトロネクチン、テネイシン、セレクチン、ヒアルロン酸、フィブリン等をコーティングした容器が挙げられるが、これに限定されない。
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.
本発明の培地により細胞接着を促進させることができる細胞は、「1.細胞接着を促進するための組成物」において説明した細胞と同様である。
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.細胞接着を促進するための方法
本発明は、本発明の組成物を培地に添加することを含む、細胞接着を促進するための方法(以下、「本発明の方法」と称することがある)を提供する。本発明の方法は、細胞(特に接着細胞)の培養容器への細胞接着を促進することができる。 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.
本発明は、本発明の組成物を培地に添加することを含む、細胞接着を促進するための方法(以下、「本発明の方法」と称することがある)を提供する。本発明の方法は、細胞(特に接着細胞)の培養容器への細胞接着を促進することができる。 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.
本発明の方法において用いられる培地は、所望の効果を得られる限り特に限定されない。また、本発明の方法における細胞培養条件(例えば、温度、二酸化炭素濃度、培養期間等)は自体公知の方法を用いればよく、あるいは、目的に応じて、適宜改変してもよい。例えば、細胞を培養する際の温度は、動物細胞であれば通常25℃~39℃、好ましくは33℃~39℃(例、37℃)である。二酸化炭素濃度は、通常、培養の雰囲気中、4体積%~10体積%であり、4体積%~6体積%が好ましい。培養期間は、通常1乃至35日間であるが、培養の目的に合わせて適宜設定すればよい。
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.
本発明の方法において用いられる培地は、本発明の培地を用いればよい。
The medium used in the method of the present invention may be the medium of the present invention.
本発明の方法における、上述した有効成分となる化合物の濃度および好適な細胞種等は、「1.細胞接着を促進するための組成物」において説明したものと同様である。
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”.
以下に、本発明の細胞接着促進用組成物の有用性について、以下の実施例において具体的に説明するが、本発明はこれらのみに限定されるものではない。なお、CO2インキュベーターにおけるCO2の濃度(%)は、雰囲気中のCO2の体積%で示した。また、PBSはリン酸緩衝生理食塩水(シグマアルドリッチジャパン社製)を意味し、FBSは牛胎児血清(Biological Industries社製)を意味する。また、(w/v)は、1体積あたりの重量を表わす。
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.
[参考例1]化合物の合成
本明細書において2’,4’-ジヒドロキシ-3’-メチルプロピオフェノン(「k-1」とも称することがある)および2’,4’-ジヒドロキシ-3’-メチルアセトフェノン(「k-2」とも称することがある)の2種のケトンについては自体公知の方法により合成が可能である(Sum TH et al., Tetrahedron. 2015 Jul 1;71(26-27): 4557-4564.)。また、2-(フェニルアミノ)アセトヒドラジド(「H-1」とも称することがある)および4-フェニルセミカルバジド(「H-10」とも称することがある)の2種のヒドラジンも、自体公知の方法により合成が可能である(Samal RP et al., Chem Biol Drug Des. 2013 Jun;81(6):715-29.)。 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. 2015Jul 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.).
本明細書において2’,4’-ジヒドロキシ-3’-メチルプロピオフェノン(「k-1」とも称することがある)および2’,4’-ジヒドロキシ-3’-メチルアセトフェノン(「k-2」とも称することがある)の2種のケトンについては自体公知の方法により合成が可能である(Sum TH et al., Tetrahedron. 2015 Jul 1;71(26-27): 4557-4564.)。また、2-(フェニルアミノ)アセトヒドラジド(「H-1」とも称することがある)および4-フェニルセミカルバジド(「H-10」とも称することがある)の2種のヒドラジンも、自体公知の方法により合成が可能である(Samal RP et al., Chem Biol Drug Des. 2013 Jun;81(6):715-29.)。 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
[k-1:H-1]
2’,4’-ジヒドロキシ-3’-メチルプロピオフェノン(k-1)(100mg、0.555mmol)、2-(フェニルアミノ)アセトヒドラジド(H-1)(110mg、0.666mmol)をDMSO(1.1mL)に溶解させ、100℃で14時間撹拌した。放冷後、蒸留水(11mL)を加え、再度100℃で撹拌後、熱時ろ過してk-1:H-1(70.1mg、0.214mmol、収率39%)を薄黄色固体として得た。 [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.
2’,4’-ジヒドロキシ-3’-メチルプロピオフェノン(k-1)(100mg、0.555mmol)、2-(フェニルアミノ)アセトヒドラジド(H-1)(110mg、0.666mmol)をDMSO(1.1mL)に溶解させ、100℃で14時間撹拌した。放冷後、蒸留水(11mL)を加え、再度100℃で撹拌後、熱時ろ過してk-1:H-1(70.1mg、0.214mmol、収率39%)を薄黄色固体として得た。 [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]
N-ブロモ酪酸メチル(8.0g、44mmol)、アニリン(8.0mL、88mmol)をトルエン(10mL)に溶解させ、5時間加熱還流した。放冷後、反応溶液を水(30mL)、2M塩酸(25mL)、水(30mL)、飽和炭酸水素ナトリウム水溶液(30mL)、ブライン(30mL)で順次洗浄して、無水硫酸ナトリウムで乾燥した。ろ過、減圧下濃縮して得られた残渣を中圧シリカゲルカラムクロマトグラフィー(シリカゲル100g、酢酸エチル/ヘキサン=1/99~10/90)で精製し、中間化合物(化合物番号105)(5.11g、26.4mmol、収率60%)を黄色液体として得た。 [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.
N-ブロモ酪酸メチル(8.0g、44mmol)、アニリン(8.0mL、88mmol)をトルエン(10mL)に溶解させ、5時間加熱還流した。放冷後、反応溶液を水(30mL)、2M塩酸(25mL)、水(30mL)、飽和炭酸水素ナトリウム水溶液(30mL)、ブライン(30mL)で順次洗浄して、無水硫酸ナトリウムで乾燥した。ろ過、減圧下濃縮して得られた残渣を中圧シリカゲルカラムクロマトグラフィー(シリカゲル100g、酢酸エチル/ヘキサン=1/99~10/90)で精製し、中間化合物(化合物番号105)(5.11g、26.4mmol、収率60%)を黄色液体として得た。 [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.
上記のようにして得られた中間化合物(化合物番号105)(5.11g、26.4mmol)をメタノール(26mL)に溶解させ、ヒドラジン・1水和物(12.8mL、264mmol)を加えて室温で4.5時間撹拌した。水(150mL)を加えて、塩化メチレン(30mLx5)で抽出した。有機層を飽和食塩水(100mL)で洗浄し、無水硫酸マグネシウムで乾燥、ろ過、減圧下濃縮して得られた固体をIPEで懸濁洗浄し、H-7(4.60g、23.8mmol、収率90%)を無色固体として得た。
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’-ジヒドロキシ-3’-メチルプロピオフェノン(k-1)(50mg、0.28mmol)、2-(フェニルアミノ)ブタンヒドラジド(H-7)(54mg、0.28mmol)をDMSO(0.55mL)に溶解させ100℃で17時間撹拌した。放冷後、蒸留水(6mL)を加えてデカンテーションし、残渣を塩化メチレン(0.5mL)に溶解させた。ヘキサン(0.5mL)を加えて析出した固体をろ取してk-1:H-7(56.8mg、0.160mmol、収率57%)を無色固体として得た。
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’-ジヒドロキシ-3’-メチルプロピオフェノン(k-1)(100mg,0.55mmol)、4-フェニルセミカルバジド(H-10)(109mg,0.721mmol)をDMSO(1.1mL)に溶解させ100℃で3.5時間撹拌した。放冷後、水(20mL)、酢酸エチル(20mL)を加えて分液し、有機層を飽和食塩水(20mL)で洗浄した。無水硫酸ナトリウムで乾燥、ろ過、減圧下濃縮し、得られた残渣を中圧シリカゲルカラムクロマトグラフィー(シリカゲル10g、酢酸エチル/ヘキサン=10/90~50/50)で精製した。得られた固体をIPEで懸濁洗浄してk-1:H-10(19.1mg、0.0610mmol、収率11%)を無色固体として得た。 [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.
2’,4’-ジヒドロキシ-3’-メチルプロピオフェノン(k-1)(100mg,0.55mmol)、4-フェニルセミカルバジド(H-10)(109mg,0.721mmol)をDMSO(1.1mL)に溶解させ100℃で3.5時間撹拌した。放冷後、水(20mL)、酢酸エチル(20mL)を加えて分液し、有機層を飽和食塩水(20mL)で洗浄した。無水硫酸ナトリウムで乾燥、ろ過、減圧下濃縮し、得られた残渣を中圧シリカゲルカラムクロマトグラフィー(シリカゲル10g、酢酸エチル/ヘキサン=10/90~50/50)で精製した。得られた固体をIPEで懸濁洗浄してk-1:H-10(19.1mg、0.0610mmol、収率11%)を無色固体として得た。 [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’-ジヒドロキシ-3’-メチルアセトフェノン(k-2)(80mg、0.48mmol)、2-(フェニルアミノ)ブタンヒドラジド(H-7)(121mg、0.626mmol)をDMSO(0.55mL)に溶解させ100℃で17時間撹拌した。放冷後、蒸留水(20mL)、酢酸エチル(20mL)を加えて分液し、有機層を飽和食塩水(20mL)で洗浄した。無水硫酸ナトリウムで乾燥、ろ過、減圧下濃縮して得られた残渣を中圧シリカゲルカラムクロマトグラフィー(シリカゲル10g、酢酸エチル/ヘキサン=5/95~50/50)で精製してk-2:H-7(109mg、0.319mmol、収率66%)を薄黄色固体として得た。 [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.
2’,4’-ジヒドロキシ-3’-メチルアセトフェノン(k-2)(80mg、0.48mmol)、2-(フェニルアミノ)ブタンヒドラジド(H-7)(121mg、0.626mmol)をDMSO(0.55mL)に溶解させ100℃で17時間撹拌した。放冷後、蒸留水(20mL)、酢酸エチル(20mL)を加えて分液し、有機層を飽和食塩水(20mL)で洗浄した。無水硫酸ナトリウムで乾燥、ろ過、減圧下濃縮して得られた残渣を中圧シリカゲルカラムクロマトグラフィー(シリカゲル10g、酢酸エチル/ヘキサン=5/95~50/50)で精製してk-2:H-7(109mg、0.319mmol、収率66%)を薄黄色固体として得た。 [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’-ジヒドロキシ-3’-メチルアセトフェノン(k-2)(80mg、0.48mmol)、2-(フェニルアミノ)アセトヒドラジド(H-1)(95.4mg、0.578mmol)をDMSO(1.0mL)に溶解させ100℃で15時間撹拌した。放冷後、蒸留水(10mL)を加えて析出した固体をろ取、塩化メチレンで懸濁洗浄してk-2:H-1(80.4mg、0.257mmol、収率53%)を黄色固体として得た。 [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.
2’,4’-ジヒドロキシ-3’-メチルアセトフェノン(k-2)(80mg、0.48mmol)、2-(フェニルアミノ)アセトヒドラジド(H-1)(95.4mg、0.578mmol)をDMSO(1.0mL)に溶解させ100℃で15時間撹拌した。放冷後、蒸留水(10mL)を加えて析出した固体をろ取、塩化メチレンで懸濁洗浄してk-2:H-1(80.4mg、0.257mmol、収率53%)を黄色固体として得た。 [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]
ヒドラジン一水和物(0.26mL、5.3mmol)を塩化メチレン(5.3mL)に溶解させ、氷冷下ベンジルイソシアネート(101)(0.324 mL、2.63 mmol)をゆっくり加えた。室温で3時間撹拌し、析出した固体をIPEで洗浄後、減圧下乾燥し、102(352 mg、2.13 mmol、収率82%)を無色固体として得た。 [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.
ヒドラジン一水和物(0.26mL、5.3mmol)を塩化メチレン(5.3mL)に溶解させ、氷冷下ベンジルイソシアネート(101)(0.324 mL、2.63 mmol)をゆっくり加えた。室温で3時間撹拌し、析出した固体をIPEで洗浄後、減圧下乾燥し、102(352 mg、2.13 mmol、収率82%)を無色固体として得た。 [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.
上記のようにして得られた102(155mg、0.938mmol)、2’,4’-ジヒドロキシ-3’-メチルプロピオフェノン(k-1)(130mg、0.72mmol)をDMSO(1.4mL)に溶解させ100℃で3.5時間撹拌した。放冷後、蒸留水(10mL)を加え、析出した固体をろ取、中圧シリカゲルカラムクロマトグラフィー(シリカゲル10g、酢酸エチル/ヘキサン=10/90~55/45)にて精製した。得られた固体を塩化メチレンで懸濁洗浄後、減圧下乾燥し、k-1:I-1(55mg、0.17mmol、収率24%)を無色固体として得た。
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]
2-ブロモプロピオン酸メチル(109)(500mg、3.0mmol)をDMSO(6mL)に溶解させ、アニリン(0.36mL、3.9mmol)、炭酸カリウム(0.54g、3.9mmol)を加えて室温で21時間撹拌した。酢酸エチル(30mL)、水(50mL)を加えて分液し、有機層を飽和食塩水(30mL)で洗浄、無水硫酸ナトリウムで乾燥した。ろ過、減圧下濃縮して得られた残渣を中圧シリカゲルクロマトグラフィー(シリカゲル30g、酢酸エチル/ヘキサン=2/98~15/85)で精製し、110(343mg、1.91mmol、収率64%)を薄黄色液体として得た。 [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.
2-ブロモプロピオン酸メチル(109)(500mg、3.0mmol)をDMSO(6mL)に溶解させ、アニリン(0.36mL、3.9mmol)、炭酸カリウム(0.54g、3.9mmol)を加えて室温で21時間撹拌した。酢酸エチル(30mL)、水(50mL)を加えて分液し、有機層を飽和食塩水(30mL)で洗浄、無水硫酸ナトリウムで乾燥した。ろ過、減圧下濃縮して得られた残渣を中圧シリカゲルクロマトグラフィー(シリカゲル30g、酢酸エチル/ヘキサン=2/98~15/85)で精製し、110(343mg、1.91mmol、収率64%)を薄黄色液体として得た。 [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.
上記のようにして得られた110(340mg、1.9mmol)をメタノール(3.8mL)に溶解させ、ヒドラジン一水和物(0.18mL、3.8mmol)を加えて60℃で24時間撹拌した。ヒドラジン一水和物(0.36mL、7.4mmol)を追加して60℃で更に17時間撹拌した。反応溶液を減圧下濃縮して得られた残渣を中圧シリカゲルクロマトグラフィー(アミンシリカゲル10g、酢酸エチル/塩化メチレン=0/100~20/80)で精製し、111(321mg、1.79mmol、収率94%)を無色固体として得た。
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.
上記のようにして得られた111(168mg、0.937mmol)、2’,4’-ジヒドロキシ-3’-メチルプロピオフェノン(k-1)(130mg、0.72mmol)をDMSO(1.4mL)に溶解させ100℃で19時間撹拌した。放冷後、蒸留水(15mL)を加え、析出した固体をろ取、乾燥して得られた黄色固体を塩化メチレンで懸濁洗浄後、減圧下乾燥し、k-1:B-1(173mg、0.507mmol、収率70%)を薄黄色固体として得た。
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]
3-ベンジルオキシアニリン(129)(2.44g、12.2mmol)をDMF(24mL)に溶解させ、酢酸ナトリウム(1.10g、13.5mmol)、ブロモ酢酸エチル(128)(1.49mL、13.5mmol)を加えて室温で4時間撹拌した。水(300mL)、酢酸エチル(150mL)を加えて分液し、有機層を飽和食塩水(100mL)で洗浄した。無水硫酸ナトリウムで乾燥、ろ過、減圧下濃縮して得られた残渣を中圧シリカゲルカラムクロマトグラフィー(シリカゲル50g、酢酸エチル/ヘキサン=2/98~10/90)にて精製し、130(2.82g、9.88mmol、収率81%)を薄黄色固体として得た。 [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.
3-ベンジルオキシアニリン(129)(2.44g、12.2mmol)をDMF(24mL)に溶解させ、酢酸ナトリウム(1.10g、13.5mmol)、ブロモ酢酸エチル(128)(1.49mL、13.5mmol)を加えて室温で4時間撹拌した。水(300mL)、酢酸エチル(150mL)を加えて分液し、有機層を飽和食塩水(100mL)で洗浄した。無水硫酸ナトリウムで乾燥、ろ過、減圧下濃縮して得られた残渣を中圧シリカゲルカラムクロマトグラフィー(シリカゲル50g、酢酸エチル/ヘキサン=2/98~10/90)にて精製し、130(2.82g、9.88mmol、収率81%)を薄黄色固体として得た。 [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.
上記のようにして得られた130(1.0g、3.5mmol)をメタノール(18mL)に懸濁させ、10%Pd/C(0.1g)を加えて水素雰囲気下室温で5時間撹拌した。反応溶液をセライトろ過し、ろ液を減圧下濃縮して得られた残渣を中圧シリカゲルカラムクロマトグラフィー(シリカゲル30g、酢酸エチル/ヘキサン=3/97~35/65)にて精製し、131(295mg、1.51mmol、収率43%)を薄黄色液体として得た。
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.
上記のようにして得られた131(0.29g、1.5mmol)をエタノール(3.5mL)に溶解させ、ヒドラジン一水和物(0.32mL、6.5mmol)を加えて60℃で18時間撹拌した。反応溶液を減圧下濃縮し、得られた残渣を中圧シリカゲルクロマトグラフィー(アミンシリカゲル10g、メタノール/塩化メチレン=1/99~10/90)で精製した。得られた固体をIPEで懸濁洗浄後、減圧下乾燥し、132(239mg、1.32mmol、収率88%)を薄黄色固体として得た。
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.
上記のようにして得られた132(130mg、0.72mmol)、2’,4’-ジヒドロキシ-3’-メチルプロピオフェノン(k-1)(100mg、0.55mmol)をDMSO(1.1mL)に溶解させ100℃で21時間撹拌した。放冷後、反応溶液をそのまま中圧シリカゲルカラムクロマトグラフィー(シリカゲル10g、酢酸エチル/塩化メチレン=5/95~50/50)にて精製した。得られた固体を水、およびIPEで懸濁洗浄後、減圧下乾燥し、k-1:D-1(95.9mg、0.279mmol、収率51%)を無色固体として得た。
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]
氷冷したTHF(10mL)に水素化アルミニウム(1.0g、26mmol)、3-シアノフェノール(148)(0.63g、5.3mmol)を順次加え、室温で1.5時間、60℃で3.5時間撹拌した。放冷後、水素化アルミニウム(1.0g、26mmol)、THF(10mL)を追加して60℃で更に16時間撹拌した。反応溶液を氷冷し、水(1.5mL)、15%水酸化ナトリウム水溶液 (1.5mL)、水(4.5mL)を順次加えて室温で3時間撹拌した。懸濁溶液をセライトろ過し、ろ液を減圧下濃縮して得られた残渣を中圧シリカゲルカラムクロマトグラフィー(アミンシリカゲル10g、メタノール/塩化メチレン=0/100~8/92)にて精製した。得られた固体をIPEで懸濁洗浄後、減圧下乾燥し、149(477mg、3.87mmol、収率73%)を無色固体として得た。 [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.
氷冷したTHF(10mL)に水素化アルミニウム(1.0g、26mmol)、3-シアノフェノール(148)(0.63g、5.3mmol)を順次加え、室温で1.5時間、60℃で3.5時間撹拌した。放冷後、水素化アルミニウム(1.0g、26mmol)、THF(10mL)を追加して60℃で更に16時間撹拌した。反応溶液を氷冷し、水(1.5mL)、15%水酸化ナトリウム水溶液 (1.5mL)、水(4.5mL)を順次加えて室温で3時間撹拌した。懸濁溶液をセライトろ過し、ろ液を減圧下濃縮して得られた残渣を中圧シリカゲルカラムクロマトグラフィー(アミンシリカゲル10g、メタノール/塩化メチレン=0/100~8/92)にて精製した。得られた固体をIPEで懸濁洗浄後、減圧下乾燥し、149(477mg、3.87mmol、収率73%)を無色固体として得た。 [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.
上記のようにして得られた149(200mg、1.6mmol)を塩化メチレン(2mL)、水(2mL)に溶解させ、炭酸水素ナトリウム(0.27g、3.2mmol)を加えて氷冷下クロロギ酸フェニル(136)(0.22mL、1.7mmol)をゆっくり滴下した。室温で20時間撹拌し、酢酸エチル(20mL)、水(20mL)を加えて分液した。有機層を飽和食塩水(20mL)で洗浄し、無水硫酸マグネシウムで乾燥、ろ過、減圧下濃縮して得られた残渣を中圧シリカゲルカラムクロマトグラフィー(シリカゲル10g、酢酸エチル/ヘキサン=5/95~35/65)にて精製し150(369mg、1.52mmol、収率95%)を無色液体として得た。
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.
上記のようにして得られた150(365mg、1.50mmol)をアセトニトリル(3.8mL)に懸濁させ、ヒドラジン一水和物(0.18mL、3.8mmol)を加えて室温で2.5時間、ヒドラジン一水和物(0.18mL、3.8mmol)を追加して55℃で更に20時間撹拌した。反応溶液を減圧下濃縮し、得られた固体をIPE/塩化メチレン(3/1)で懸濁洗浄後、減圧下乾燥し、151(233mg、1.29mmol、収率86%)を無色固体として得た。
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.
上記のようにして得られた151(130mg、0.72mmol)、2’,4’-ジヒドロキシ-3’-メチルプロピオフェノン(k-1)(100mg、0.55mmol)をDMSO(1.1mL)に溶解させ100℃で15時間撹拌した。放冷後、反応溶液をそのまま中圧シリカゲルカラムクロマトグラフィー(シリカゲル10g、酢酸エチル/塩化メチレン=10/90~55/45)にて精製した。得られた精製物に水を加えて析出した固体をろ取後、減圧下乾燥し、k-1:J-1(102mg、0.297mmol、収率54%)を無色固体として得た。
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.
[実施例1]間葉系幹細胞に対する作用
ヒト骨髄由来間葉系幹細胞(hMSC、PromoCell社製)を、間葉系幹細胞増殖培地2(PromoCell社製)を用いて7乃至10日間前培養した。得られたhMSCを同培地に2.0×105 cells/mLとなるように懸濁した。引き続き、終濃度10μMとなるように、DMSOに溶解したk-1:H-1、k-1:H-7、k-1:H-10、k-1:I-1、k-1:B-1、またはk-1:J-1溶液を添加し、24ウェル平底プレート(コーニング社製、#35114)に0.5mL/ウェルずつ播種した。対照としては、DMSOを終濃度0.1%となるよう添加した。本プレートを37℃、5% CO2インキュベーターにて20分間培養した。その後、培地を除いた後500μLのPBSで3回洗浄してPBSを除去後、0.25 w/v %トリプシン-1mmol/L EDTA(エチレンジアミン四酢酸)溶液(和光純薬工業社製)をPBSで5倍希釈した溶液を400μL添加して37℃にて2~5分間インキュベートして細胞を剥離させた。更に同容量の培地を添加して別の容器へと移し、400gで4分間遠心して上清を除去した。その後、200μLのPBSで細胞を再懸濁した後、200μLのATP試薬(CellTiter-Glo(登録商標) Luminescent Cell Viability Assay, Promega社製)を添加し懸濁させ、15分間室温で静置した後、100μLの懸濁液を96ウェル平底プレートに分注し、FlexStation3(Molecular Devices社製)にて発光強度(RLU値)を測定した。ここで培地のみの発光値を差し引くことで生細胞の数を測定した。化合物無添加(対照)のRLU値(ATP測定、発光強度)を100%としたときの、それぞれの化合物添加時の相対値を表1に示す。 [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%.
ヒト骨髄由来間葉系幹細胞(hMSC、PromoCell社製)を、間葉系幹細胞増殖培地2(PromoCell社製)を用いて7乃至10日間前培養した。得られたhMSCを同培地に2.0×105 cells/mLとなるように懸濁した。引き続き、終濃度10μMとなるように、DMSOに溶解したk-1:H-1、k-1:H-7、k-1:H-10、k-1:I-1、k-1:B-1、またはk-1:J-1溶液を添加し、24ウェル平底プレート(コーニング社製、#35114)に0.5mL/ウェルずつ播種した。対照としては、DMSOを終濃度0.1%となるよう添加した。本プレートを37℃、5% CO2インキュベーターにて20分間培養した。その後、培地を除いた後500μLのPBSで3回洗浄してPBSを除去後、0.25 w/v %トリプシン-1mmol/L EDTA(エチレンジアミン四酢酸)溶液(和光純薬工業社製)をPBSで5倍希釈した溶液を400μL添加して37℃にて2~5分間インキュベートして細胞を剥離させた。更に同容量の培地を添加して別の容器へと移し、400gで4分間遠心して上清を除去した。その後、200μLのPBSで細胞を再懸濁した後、200μLのATP試薬(CellTiter-Glo(登録商標) Luminescent Cell Viability Assay, Promega社製)を添加し懸濁させ、15分間室温で静置した後、100μLの懸濁液を96ウェル平底プレートに分注し、FlexStation3(Molecular Devices社製)にて発光強度(RLU値)を測定した。ここで培地のみの発光値を差し引くことで生細胞の数を測定した。化合物無添加(対照)のRLU値(ATP測定、発光強度)を100%としたときの、それぞれの化合物添加時の相対値を表1に示す。 [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%.
表1に示される通り、化合物k-1:H-1、k-1:H-7、k-1:H-10、k-1:I-1、k-1:B-1及びk-1:J-1は、細胞接着促進作用を有することが明らかになった。
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.
[実施例2]各種細胞株に対する作用
各種ヒト由来細胞を、次に示す各々の培地を用いて培養した。ヒト子宮頸がん由来細胞株HeLa(American Type Culture Collection(ATCC)社製、10%ウシ胎児血清(FBS、Corning社製)含有Dulbecco’s Modified Eagle’s Medium;DMEM(和光純薬工業社製))、ヒト上皮様細胞がん由来細胞株A431(ATCC社製、10% FBS及び1%MEM非必須アミノ酸溶液(MEM Non-Essential Amino Acids solution;NEAA、和光純薬工業社製)含有Eagle's Minimum Essential Medium;EMEM(和光純薬工業社製))、ヒト結腸腺がん由来細胞株HCT116(DSファーマバイオメディカル社製、10%FBS含有McCoy’s 5A Medium(シグマアルドリッチ社製))、ヒト乳腺がん由来細胞株MCF7(ATCC社製、10% FBS及び1%MEM非必須アミノ酸溶液(NEAA、和光純薬工業社製)含有EMEM(和光純薬工業社製))、ヒト胎児腎細胞由来細胞株HEK293(ATCC社製、10%FBS含有DMEM(和光純薬工業社製))。対数増殖期にある細胞をPBSにて洗浄後、0.25w/v %トリプシン-1mmol/L EDTA(エチレンジアミン四酢酸)溶液(和光純薬工業社製)を添加して37℃にて1~5分間インキュベートして剥離し、各々の培地を添加、遠心して上清を除去した。その後、同培地で再懸濁した後、一部をトリパンブルー(和光純薬工業社製)で懸濁してTC-20(BIO-RAD社製)にて生細胞数をカウントした。 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).
各種ヒト由来細胞を、次に示す各々の培地を用いて培養した。ヒト子宮頸がん由来細胞株HeLa(American Type Culture Collection(ATCC)社製、10%ウシ胎児血清(FBS、Corning社製)含有Dulbecco’s Modified Eagle’s Medium;DMEM(和光純薬工業社製))、ヒト上皮様細胞がん由来細胞株A431(ATCC社製、10% FBS及び1%MEM非必須アミノ酸溶液(MEM Non-Essential Amino Acids solution;NEAA、和光純薬工業社製)含有Eagle's Minimum Essential Medium;EMEM(和光純薬工業社製))、ヒト結腸腺がん由来細胞株HCT116(DSファーマバイオメディカル社製、10%FBS含有McCoy’s 5A Medium(シグマアルドリッチ社製))、ヒト乳腺がん由来細胞株MCF7(ATCC社製、10% FBS及び1%MEM非必須アミノ酸溶液(NEAA、和光純薬工業社製)含有EMEM(和光純薬工業社製))、ヒト胎児腎細胞由来細胞株HEK293(ATCC社製、10%FBS含有DMEM(和光純薬工業社製))。対数増殖期にある細胞をPBSにて洗浄後、0.25w/v %トリプシン-1mmol/L EDTA(エチレンジアミン四酢酸)溶液(和光純薬工業社製)を添加して37℃にて1~5分間インキュベートして剥離し、各々の培地を添加、遠心して上清を除去した。その後、同培地で再懸濁した後、一部をトリパンブルー(和光純薬工業社製)で懸濁してTC-20(BIO-RAD社製)にて生細胞数をカウントした。 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).
DMSOに溶解したk-1:H-1、k-1:H-7、k-1:H-10、k-2:H-7、k-2:H-1、k-1:I-1、k-1:B-1、k-1:D-1、又はk-1:J-1溶液を、終濃度10μMとなるように前述の各種細胞が懸濁された各培地に添加し、8000cells/100 μL/ウェルずつ96穴接着プレート(コーニング社製、#3585)に播種した。対照としてはDMSOを添加した細胞懸濁液を播種した(DMSO終濃度0.1%)。本プレートを37℃、5%CO2インキュベーターにて30分から2時間静置培養後、培養液をアスピレートし、各々の培地を用いて2回洗浄を行うことにより培養プレートに接着していない細胞を取り除いた後、各々の培地を100μL/ウェル添加した。続いて、培養液に対してATP試薬100μL(CellTiter-Glo(登録商標)Luminescent Cell Viability Assay, Promega社製)を添加・懸濁し、10分間室温で静置した。さらに、EnSpire(PerkinElmer社製)にて発光強度(RLU値)を測定し、培地のみの発光値を差し引くことでプレートに接着する細胞数を測定した。化合物無添加のRLU値(ATP測定、発光強度)を100%とした際に、100%以下の値を示すものを「-」、103%以上の値を示すものを「△」、120%以上の値を示すものを「○」、200%以上の値を示すものを「◎」として表2に示す。なお、空欄は試験未実施を示す。
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.
表2に示される通り、本発明組成物は、各種細胞株に対して細胞接着促進効果を有することが示された。
As shown in Table 2, the composition of the present invention was shown to have a cell adhesion promoting effect on various cell lines.
[実施例3]本発明組成物の正常細胞への作用
ヒト臍帯静脈内皮細胞(Human Umbilical Vein Endothelial Cells:HUVEC)は、Endothelial Cell Growth Medium (Ready-to-use)(PromoCell社製)の培地にて培養を行った。対数増殖期にある上記細胞をPBSにて洗浄後、0.25w/v %トリプシン-1mmol/L EDTA(エチレンジアミン四酢酸)溶液(和光純薬工業社製)を添加して37℃にて3分間インキュベートして剥離し、培地を添加、遠心して上清を除去した。その後、同培地で再懸濁した後、一部をトリパンブルー(和光純薬工業社製)で懸濁してTC-20(BIO-RAD社製)にて生細胞数をカウントした。 [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).
ヒト臍帯静脈内皮細胞(Human Umbilical Vein Endothelial Cells:HUVEC)は、Endothelial Cell Growth Medium (Ready-to-use)(PromoCell社製)の培地にて培養を行った。対数増殖期にある上記細胞をPBSにて洗浄後、0.25w/v %トリプシン-1mmol/L EDTA(エチレンジアミン四酢酸)溶液(和光純薬工業社製)を添加して37℃にて3分間インキュベートして剥離し、培地を添加、遠心して上清を除去した。その後、同培地で再懸濁した後、一部をトリパンブルー(和光純薬工業社製)で懸濁してTC-20(BIO-RAD社製)にて生細胞数をカウントした。 [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).
DMSOに溶解したk-1:I-1、k-1:B-1、k-1:D-1、又はk-1:J-1溶液を、終濃度10μMとなるように前述の細胞が懸濁された各培地に添加し、8000cells/100 μL/ウェルずつ96穴接着プレート(コーニング社製、#3585)に播種した。対照としてはDMSOを添加した細胞懸濁液を播種した(DMSO終濃度0.1%)。本プレートを37℃、5%CO2インキュベーターにて30分静置培養後、培養液をアスピレートし、各々の培地を用いて2回洗浄を行うことにより培養プレートに接着していない細胞を取り除いた後、培地を100μL/ウェル添加した。続いて、培養液に対してATP試薬100μL(CellTiter-Glo(登録商標)Luminescent Cell Viability Assay, Promega社製)を添加・懸濁し、10分間室温で静置した。さらに、EnSpire(PerkinElmer社製)にて発光強度(RLU値)を測定し、培地のみの発光値を差し引くことでプレートに接着する細胞数を測定した。化合物無添加のRLU値(ATP測定、発光強度)を100%としたときの、それぞれの化合物添加時の相対値を表3に示す。
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%.
表3に示される通り、k-1:I-1、k-1:B-1、k-1:D-1、又はk-1:J-1を培地へ添加することにより、正常細胞であるHUVECの細胞接着が促進することが明らかとなった。
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.
[実施例4]HEK293細胞株に対する濃度依存的作用
DMSOに溶解させたk-1:J-1溶液を、終濃度0.1、1、10μMとなるように培地へ添加し、更にHEK293細胞を各培地に8000cells/100 μL/ウェルの細胞濃度で播種できるよう添加後、96穴接着プレート(コーニング社製、#3585)に播種した。対照としてはDMSOを添加した細胞懸濁液を播種した(DMSO終濃度0.1%)。37℃、5%CO2インキュベーターにて1時間静置培養後、培養液をアスピレートし、培地を用いて2回洗浄を行い培養プレートに接着していない細胞を取り除いた後、培地を100μL/ウェル添加した。続いて、培養液に対してATP試薬100μL(CellTiter-Glo(登録商標)Luminescent Cell Viability Assay, Promega社製)を添加・懸濁し、10分間室温で静置した後、EnSpire(PerkinElmer社製)にて発光強度(RLU値)を測定し、培地のみの発光値を差し引くことでプレートに接着する細胞数を測定した。化合物無添加のRLU値(ATP測定、発光強度)を1とした際の化合物添加時の相対的RLU値を図1に示す。 [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.
DMSOに溶解させたk-1:J-1溶液を、終濃度0.1、1、10μMとなるように培地へ添加し、更にHEK293細胞を各培地に8000cells/100 μL/ウェルの細胞濃度で播種できるよう添加後、96穴接着プレート(コーニング社製、#3585)に播種した。対照としてはDMSOを添加した細胞懸濁液を播種した(DMSO終濃度0.1%)。37℃、5%CO2インキュベーターにて1時間静置培養後、培養液をアスピレートし、培地を用いて2回洗浄を行い培養プレートに接着していない細胞を取り除いた後、培地を100μL/ウェル添加した。続いて、培養液に対してATP試薬100μL(CellTiter-Glo(登録商標)Luminescent Cell Viability Assay, Promega社製)を添加・懸濁し、10分間室温で静置した後、EnSpire(PerkinElmer社製)にて発光強度(RLU値)を測定し、培地のみの発光値を差し引くことでプレートに接着する細胞数を測定した。化合物無添加のRLU値(ATP測定、発光強度)を1とした際の化合物添加時の相対的RLU値を図1に示す。 [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.
図1に示される通り、k-1:J-1は、濃度依存的にHEK293の細胞接着を促進した。
As shown in FIG. 1, k-1: J-1 promoted cell adhesion of HEK 293 in a concentration-dependent manner.
[実施例5]本発明組成物の線維芽細胞株C3H10T1/2への作用
マウス胚線維芽細胞C3H10T1/2(DSファーマバイオメディカル社製)は、10(v/v)%FBS(Corning社製)とL-グルタミン-ペニシリン-ストレプトマイシン安定化溶液(Sigma-Aldrich社製)を含むBME培地(サーモフィッシャーサイエンティフィック社製)を用いて培養を行った。対数増殖期にある上記細胞をPBSにて洗浄後、0.25w/v %トリプシン-1mmol/L EDTA(エチレンジアミン四酢酸)溶液(和光純薬工業社製)を添加して37℃にて3分間インキュベートして剥離し、培地を添加、遠心して上清を除去した。その後、同培地で再懸濁した後、一部をトリパンブルー(和光純薬工業社製)で懸濁してTC-20(BIO-RAD社製)にて生細胞数をカウントした。 [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).
マウス胚線維芽細胞C3H10T1/2(DSファーマバイオメディカル社製)は、10(v/v)%FBS(Corning社製)とL-グルタミン-ペニシリン-ストレプトマイシン安定化溶液(Sigma-Aldrich社製)を含むBME培地(サーモフィッシャーサイエンティフィック社製)を用いて培養を行った。対数増殖期にある上記細胞をPBSにて洗浄後、0.25w/v %トリプシン-1mmol/L EDTA(エチレンジアミン四酢酸)溶液(和光純薬工業社製)を添加して37℃にて3分間インキュベートして剥離し、培地を添加、遠心して上清を除去した。その後、同培地で再懸濁した後、一部をトリパンブルー(和光純薬工業社製)で懸濁してTC-20(BIO-RAD社製)にて生細胞数をカウントした。 [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).
DMSOに溶解したk-1:I-1、k-1:B-1、又はk-1:J-1溶液を、終濃度10μMとなるように前述の細胞が懸濁された各培地に添加し、8000cells/100 μL/ウェルずつ96穴培養プレート(コーニング社製、#351172)に播種した。対照としてはDMSOを添加した細胞懸濁液を播種した(DMSO終濃度0.1%)。本プレートを37℃、5%CO2インキュベーターにて30分静置培養後、培養液をアスピレートし、上記の培地を用いて2回洗浄を行うことにより培養プレートに接着していない細胞を取り除いた後、培地を100μL/ウェル添加した。続いて、培養液に対してATP試薬100μL(CellTiter-Glo(登録商標)Luminescent Cell Viability Assay, Promega社製)を添加・懸濁し、10分間室温で静置した。さらに、EnSpire(PerkinElmer社製)にて発光強度(RLU値)を測定し、培地のみの発光値を差し引くことでプレートに接着する細胞数を測定した。化合物無添加のRLU値(ATP測定、発光強度)を100%としたときの、それぞれの化合物添加時の相対値を表4に示す。
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%.
表4に示される通り、k-1:I-1、k-1:B-1、又はk-1:J-1を培地へ添加することにより、正常細胞であるC3H10T1/2の細胞接着が促進することが明らかとなった。
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.
[実施例6]本発明組成物のヒト人工多能性幹細胞への作用
ヒト人工多能性幹細胞(hiPSC、iPSアカデミアジャパン社製)253G1株を、mTeSR(登録商標)1培地(STEMCELL TECHNOLOGIES社製)を用いて7乃至14日間培養した。得られたhiPSCを同培地に1.0×104cells/mLとなるように懸濁した。引き続き、終濃度が10μMとなるように、DMSOに溶解したk-1:B-1、またはk-1:J-1溶液を添加し、12ウェル平底プレート(コーニング社製、#351143)に1.0mL/ウェルずつ播種した。対照としては、DMSOを終濃度が0.1%となるよう添加した。本プレートを37℃、5% CO2インキュベーターにて24時間培養した。培養後、培地を除き500μLの培地を新たに添加し、続いて同量のATP試薬(CellTiter-Glo(登録商標) Luminescent Cell Viability Assay, Promega社製)を添加・懸濁し、10分間室温で静置した後、96ウェル平底プレートに200μL/ウェルで分注し、EnSpire(PerkinElmer社製)にて発光強度(RLU値)を測定し、培地のみの発光値を差し引くことでプレートに接着する細胞数を測定した。化合物無添加のRLU値(ATP測定、発光強度)を100%としたときの、それぞれの化合物添加時の相対値を表5に示す。 [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%.
ヒト人工多能性幹細胞(hiPSC、iPSアカデミアジャパン社製)253G1株を、mTeSR(登録商標)1培地(STEMCELL TECHNOLOGIES社製)を用いて7乃至14日間培養した。得られたhiPSCを同培地に1.0×104cells/mLとなるように懸濁した。引き続き、終濃度が10μMとなるように、DMSOに溶解したk-1:B-1、またはk-1:J-1溶液を添加し、12ウェル平底プレート(コーニング社製、#351143)に1.0mL/ウェルずつ播種した。対照としては、DMSOを終濃度が0.1%となるよう添加した。本プレートを37℃、5% CO2インキュベーターにて24時間培養した。培養後、培地を除き500μLの培地を新たに添加し、続いて同量のATP試薬(CellTiter-Glo(登録商標) Luminescent Cell Viability Assay, Promega社製)を添加・懸濁し、10分間室温で静置した後、96ウェル平底プレートに200μL/ウェルで分注し、EnSpire(PerkinElmer社製)にて発光強度(RLU値)を測定し、培地のみの発光値を差し引くことでプレートに接着する細胞数を測定した。化合物無添加のRLU値(ATP測定、発光強度)を100%としたときの、それぞれの化合物添加時の相対値を表5に示す。 [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%.
表5に示される通り、k-1:B-1、またはk-1:J-1溶液を培地へ添加することにより、ヒト人工多能性幹細胞である253G1細胞株の細胞接着が促進されることが明らかとなった。
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.
[実施例7]本発明組成物のヒト人工多能性幹細胞への作用
ヒト人工多能性幹細胞(hiPSC、iPSアカデミアジャパン社製)201B7株を、StemFit培地(味の素社製)を用いて7乃至14日間培養した。得られたhiPSCを同培地に1.0×104cells/mLとなるように懸濁した。引き続き、終濃度が10μMとなるように、DMSOに溶解したk-1:B-1、またはk-1:J-1溶液を添加し、12ウェル平底プレート(コーニング社製、#351143)に1.0mL/ウェルずつ播種した。対照としては、DMSOを終濃度が0.1%となるよう添加した。本プレートを37℃、5% CO2インキュベーターにて24時間培養した。培養後、培地を除き500μLの培地を新たに添加し、続いて同量のATP試薬(CellTiter-Glo(登録商標) Luminescent Cell Viability Assay, Promega社製)を添加・懸濁し、10分間室温で静置した後、96ウェル平底プレートに200μL/ウェルで分注し、EnSpire(PerkinElmer社製)にて発光強度(RLU値)を測定し、培地のみの発光値を差し引くことでプレートに接着する細胞数を測定した。化合物無添加のRLU値(ATP測定、発光強度)を100%としたときの、それぞれの化合物添加時の相対値を表6に示す。 [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%.
ヒト人工多能性幹細胞(hiPSC、iPSアカデミアジャパン社製)201B7株を、StemFit培地(味の素社製)を用いて7乃至14日間培養した。得られたhiPSCを同培地に1.0×104cells/mLとなるように懸濁した。引き続き、終濃度が10μMとなるように、DMSOに溶解したk-1:B-1、またはk-1:J-1溶液を添加し、12ウェル平底プレート(コーニング社製、#351143)に1.0mL/ウェルずつ播種した。対照としては、DMSOを終濃度が0.1%となるよう添加した。本プレートを37℃、5% CO2インキュベーターにて24時間培養した。培養後、培地を除き500μLの培地を新たに添加し、続いて同量のATP試薬(CellTiter-Glo(登録商標) Luminescent Cell Viability Assay, Promega社製)を添加・懸濁し、10分間室温で静置した後、96ウェル平底プレートに200μL/ウェルで分注し、EnSpire(PerkinElmer社製)にて発光強度(RLU値)を測定し、培地のみの発光値を差し引くことでプレートに接着する細胞数を測定した。化合物無添加のRLU値(ATP測定、発光強度)を100%としたときの、それぞれの化合物添加時の相対値を表6に示す。 [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%.
表6に示される通り、k-1:B-1、またはk-1:J-1溶液を培地へ添加することにより、ヒト人工多能性幹細胞である201B7細胞株の細胞接着が促進されることが明らかとなった。
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.
[実施例8]本発明組成物使用時のヒト人工多能性幹細胞の未分化性の評価
ヒト人工多能性幹細胞(hiPSC、iPSアカデミアジャパン社製)253G1株を、StemFit培地(味の素社製)を用いて7乃至14日間培養した。得られたhiPSCを同培地に2.0×104cells/mLとなるように懸濁した。引き続き、終濃度が10μMとなるように、DMSOに溶解したk-1:B-1、またはk-1:J-1溶液を添加し、6ウェル平底プレート(コーニング社製、#351146)に1.5mL/ウェルずつ播種した。対照としては、DMSOを終濃度が0.1%となるよう添加した。本プレートを37℃、5% CO2インキュベーターにて120時間培養した。培養中は、24、72時間後に上記化合物を含まないStemFit培地を用いて培地交換を行った。培養後、培地を除き、1mL/ウェルのPBS(-)(富士フィルム和光純薬社製)を添加し洗浄した後、1mL/ウェルの0.5mmol/L-EDTA/PBS溶液(ナカライテスク社製)を添加し、37℃、5% CO2インキュベーターにて10分間静置した。10分後、剥離した細胞を15mL遠沈管に回収し、200g、3分間の条件で遠心分離(多本架恒温遠心機 EIX-136、トミー精工社製)を行った。その後、上清を除去し、200μLのTrypLE(登録商標) Select(1×)(ギブコ社製)を添加し、37℃の恒温槽中に2分間静置した。2分後、恒温槽中から15mL遠沈管を取り出し、5mLの10%FBS含有D-MEM(High Glucose)(富士フィルム和光純薬社製)を添加し、200g、3分間の条件で遠心分離を行った。遠心分離後上清を除去し、5mLの2%FBS含有PBS(-)(富士フィルム和光純薬社製)を添加し、200G、3分間の条件で遠心分離を行った。遠心分離後上清を除去し、100μLの2%FBS含有PBS(-)を添加し細胞を懸濁後、1.5mLエッペンドルフチューブに細胞懸濁液を分注した。分注後の細胞懸濁液に対し、細胞染色抗体である、PE Mouse anti-Human TRA-1-60 AntigenとAlexa Fluor(登録商標)647 Mouse anti-SSEA-4(共にBD Bioscience社製)を5μLずつ添加し、室温下に30分間静置した。また、Isotoype Controlとして、PE Mouse IgM, κ Isotype ControlとAlexa Fluor(登録商標) 647 Mouse IgG3, κ Isotoype Control(共にBD Bioscience社製)を5μLずつ添加し、室温下に30分間静置した。30分後、各1.5mLエッペンドルフチューブに1.0mLの2%FBS含有PBS(-)を添加し、2000g、3分間の条件で遠心分離(微量高速冷却遠心機 MX-307、トミー精工社製)後、上清を除去した。この操作を計2回行った後、300μLの2%FBS含有PBS(-)を添加し、測定用サンプルとした。測定は、BD FACSAria(登録商標) III セルソーター(BD Bioscience社製)を用いて行い、上記の抗体に対するダブルポジティブな割合を算出することで、未分化性維持の評価とした。DMSO、k-1:B-1、k-1:J-1を添加して培養したhiPSCにおける、TRA-1-60とSSEA-4のダブルポジティブの割合の結果を表7に示す。 [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.
ヒト人工多能性幹細胞(hiPSC、iPSアカデミアジャパン社製)253G1株を、StemFit培地(味の素社製)を用いて7乃至14日間培養した。得られたhiPSCを同培地に2.0×104cells/mLとなるように懸濁した。引き続き、終濃度が10μMとなるように、DMSOに溶解したk-1:B-1、またはk-1:J-1溶液を添加し、6ウェル平底プレート(コーニング社製、#351146)に1.5mL/ウェルずつ播種した。対照としては、DMSOを終濃度が0.1%となるよう添加した。本プレートを37℃、5% CO2インキュベーターにて120時間培養した。培養中は、24、72時間後に上記化合物を含まないStemFit培地を用いて培地交換を行った。培養後、培地を除き、1mL/ウェルのPBS(-)(富士フィルム和光純薬社製)を添加し洗浄した後、1mL/ウェルの0.5mmol/L-EDTA/PBS溶液(ナカライテスク社製)を添加し、37℃、5% CO2インキュベーターにて10分間静置した。10分後、剥離した細胞を15mL遠沈管に回収し、200g、3分間の条件で遠心分離(多本架恒温遠心機 EIX-136、トミー精工社製)を行った。その後、上清を除去し、200μLのTrypLE(登録商標) Select(1×)(ギブコ社製)を添加し、37℃の恒温槽中に2分間静置した。2分後、恒温槽中から15mL遠沈管を取り出し、5mLの10%FBS含有D-MEM(High Glucose)(富士フィルム和光純薬社製)を添加し、200g、3分間の条件で遠心分離を行った。遠心分離後上清を除去し、5mLの2%FBS含有PBS(-)(富士フィルム和光純薬社製)を添加し、200G、3分間の条件で遠心分離を行った。遠心分離後上清を除去し、100μLの2%FBS含有PBS(-)を添加し細胞を懸濁後、1.5mLエッペンドルフチューブに細胞懸濁液を分注した。分注後の細胞懸濁液に対し、細胞染色抗体である、PE Mouse anti-Human TRA-1-60 AntigenとAlexa Fluor(登録商標)647 Mouse anti-SSEA-4(共にBD Bioscience社製)を5μLずつ添加し、室温下に30分間静置した。また、Isotoype Controlとして、PE Mouse IgM, κ Isotype ControlとAlexa Fluor(登録商標) 647 Mouse IgG3, κ Isotoype Control(共にBD Bioscience社製)を5μLずつ添加し、室温下に30分間静置した。30分後、各1.5mLエッペンドルフチューブに1.0mLの2%FBS含有PBS(-)を添加し、2000g、3分間の条件で遠心分離(微量高速冷却遠心機 MX-307、トミー精工社製)後、上清を除去した。この操作を計2回行った後、300μLの2%FBS含有PBS(-)を添加し、測定用サンプルとした。測定は、BD FACSAria(登録商標) III セルソーター(BD Bioscience社製)を用いて行い、上記の抗体に対するダブルポジティブな割合を算出することで、未分化性維持の評価とした。DMSO、k-1:B-1、k-1:J-1を添加して培養したhiPSCにおける、TRA-1-60とSSEA-4のダブルポジティブの割合の結果を表7に示す。 [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.
表7に示される通り、k-1:B-1、またはk-1:J-1溶液を培地へ添加しても、ヒト人工多能性幹細胞の未分化性は維持されることが明らかとなった。即ち、本発明組成物を用いた幹細胞の培養は、その未分化性に対して影響を及ぼさないことが明らかとなった。
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.
本発明によれば、接着細胞の培養容器への接着を促進することができ、これにより接着細胞の培養を効率化することができる。従って、本発明は、接着細胞を用いる研究や医療の分野において有益である。
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.
本出願は、日本で出願された特願2017-239103(出願日:2017年12月13日)を基礎としており、その内容は本明細書に全て包含されるものである。
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)
- 下記一般式(I)で示される化合物、またはその塩を含む、細胞接着を促進するための組成物:
{式中、Xは、単結合、-CH2COO-、-CONH-、または-NHCO-であり、R1は、置換基を有していてもよい炭素数1~10のアルキル基、置換基を有していてもよいアリール基、または-Y-NH-Z-Arであり(式中、Y、およびZは、単結合、または置換基を有していてもよい炭素数1~6のアルキレン基であり、Arは、置換基を有していてもよいアリール基である)、R2は、置換基を有していてもよい炭素数1~6のアルキル基であり、R3は、水素原子またはヒドロキシル基である。}。 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. }. - Xが、-NHCO-である、請求項1記載の組成物。 The composition according to claim 1, wherein X is -NHCO-.
- R2が、置換基を有していてもよいメチル基またはエチル基であり、R3が水素原子である、請求項1または2に記載の組成物。 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.
- R1が、-Y-NH-Z-Arである、請求項1~3のいずれか一項に記載の組成物。 The composition according to any one of claims 1 to 3, wherein R 1 is -Y-NH-Z-Ar.
- Arが、ハロゲン原子、水酸基もしくはメチル基で置換されていてもよいフェニル基である、請求項4記載の組成物。 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.
- Yが、メチレン基、エチリデン基またはプロピリデン基である、請求項4または5に記載の組成物。 The composition according to claim 4 or 5, wherein Y is a methylene group, an ethylidene group or a propylidene group.
- 細胞が、正常細胞株、がん細胞株および幹細胞からなる群から選択される、請求項1~8のいずれか一項に記載の組成物。 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.
- 正常細胞株が、HUVEC、C3H10T1/2またはHEK293である、請求項9記載の組成物。 The composition according to claim 9, wherein the normal cell line is HUVEC, C3H10T1 / 2 or HEK293.
- がん細胞株が、HeLa細胞、A431、HCT116、またはMCF7である、請求項9記載の組成物。 The composition according to claim 9, wherein the cancer cell line is HeLa cells, A431, HCT116, or MCF7.
- 幹細胞が、ヒト間葉系幹細胞(hMSC)またはヒト人工多能性幹細胞(hiPSC)である、請求項9記載の組成物。 10. The composition of claim 9, wherein the stem cells are human mesenchymal stem cells (hMSCs) or human induced pluripotent stem cells (hiPSCs).
- 請求項1~8のいずれか一項に記載の組成物を含む、細胞を培養するための培地。 A culture medium for culturing cells, comprising the composition according to any one of claims 1 to 8.
- 細胞が、正常細胞株、がん細胞株および幹細胞からなる群から選択される、請求項13記載の培地。 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.
- 正常細胞株が、HUVEC、C3H10T1/2またはHEK293である、請求項14記載の培地。 The medium according to claim 14, wherein the normal cell line is HUVEC, C3H10T1 / 2 or HEK293.
- がん細胞株が、HeLa細胞、A431、HCT116、またはMCF7である、請求項14記載の培地。 The medium according to claim 14, wherein the cancer cell line is HeLa cells, A431, HCT116, or MCF7.
- 幹細胞が、ヒト間葉系幹細胞(hMSC)またはヒト人工多能性幹細胞(hiPSC)である、請求項14記載の培地。 The medium according to claim 14, wherein the stem cells are human mesenchymal stem cells (hMSCs) or human induced pluripotent stem cells (hiPSCs).
- 請求項1~8のいずれか一項に記載の組成物を培地に添加することを含む、細胞の培養容器への接着を促進するための方法。 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.
- 細胞が、正常細胞株、がん細胞株および幹細胞からなる群から選択される、請求項18記載の方法。 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.
- 正常細胞株が、HUVEC、C3H10T1/2またはHEK293である、請求項19記載の方法。 20. The method of claim 19, wherein the normal cell line is HUVEC, C3H10T1 / 2 or HEK293.
- がん細胞株が、HeLa細胞、A431、HCT116、またはMCF7である、請求項19記載の方法。 20. The method of claim 19, wherein the cancer cell line is HeLa cells, A431, HCT116, or MCF7.
- 幹細胞が、ヒト間葉系幹細胞(hMSC)またはヒト人工多能性幹細胞(hiPSC)である、請求項19記載の方法。 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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