WO2021095741A1

WO2021095741A1 - Novel sugar derivative useful in freezing of cells

Info

Publication number: WO2021095741A1
Application number: PCT/JP2020/041967
Authority: WO
Inventors: 博之井嶋; 文靖小野; 梢吉田; 雄大蝶野; 奈菜白木川
Original assignee: 国立大学法人九州大学; 日産化学株式会社
Priority date: 2019-11-11
Filing date: 2020-11-10
Publication date: 2021-05-20
Also published as: JPWO2021095741A1

Abstract

Provided is a cryoprotectant capable of inhibiting cytotoxicity, even in the presence of a cryoprotectant such as DMSO.　Specifically provided is a compound represented by formula I-1 or I-2, or a salt or a solvate thereof. [In the formulas, n is 0 or 1; R₁ and R₂ are both H, or R₁ and R₂ together form =O; R₃and R₄ are both H, or R₃ and R₄ together form a bond; R₅ is a saturated or unsaturated aliphatic hydrocarbon group having 1-30 carbons; R₆ is H or -CH₂OH, with the caveat that if R₆ is -CH₂OH, n is 0; R₇ is H or a C_1-6alkyl, or is selected from the group consisting of the structures defined in the description; R₈ is H or -NR₉R₁₀; R₉ and R₁₀ are each independently H, a C_1-6alkyl or -COR₁₁; and R₁₁ is -OH or a C_1-6alkyl.]

Description

A novel sugar derivative useful for cell freezing

The present invention relates to a novel sugar derivative useful for freezing cells. More specifically, the present invention relates to a cell cryoprotectant capable of suppressing cytotoxicity even in the presence of a cell cryoprotectant such as DMSO.

Cells, tissues, organs, etc. are used in a wide range of fields such as elucidation of biological phenomena and medical fields. Therefore, the demand for a stable supply of cells and the like is increasing. As a method for stably supplying cells and the like, a technique for freezing cells has been attracting attention.

When freezing cells, it is common to use dimethyl sulfoxide (DMSO) as a cell cryoprotectant for the purpose of preventing cell damage due to ice crystal formation and the like. DMSO is thought to enable cell freezing as a result of suppressing intracellular ice crystal formation by passing through the cell membrane and substituting for intracellular water molecules. However, it is known that when DMSO is used, cytotoxicity such as a decrease in the number of living cells occurs when the cells are frozen and thawed. Therefore, development of a cell cryoprotectant (for example, trehalose, etc.) that replaces DMSO is underway (Patent Document 1).

International Publication No. 2012/067240

When trehalose or the like is used as a cell cryoprotectant, it is common to add a high dose of about 10% by mass or more to the cell freezing solution, so that problems such as cytotoxicity have not yet been solved. As described above, even when trehalose or the like is used, a satisfactory cell freeze-protecting agent or cell freezing method has not yet been obtained. On the other hand, if the mechanism of cytotoxicity by DMSO can be estimated, it is expected to lead to the development of new and useful cell cryoprotectants or cell freezing methods.

The present inventors hypothesized that DMSO disturbs the structure of the phospholipid bilayer as it passes through the cell membrane, that is, the cell membrane becomes fragile. Therefore, it was conceived that if it is possible to suppress such fragility of the cell membrane by a cell cryoprotectant such as DMSO, it will be possible to suppress cytotoxicity such as a decrease in the number of living cells. After repeated diligent studies based on this idea, the present inventor unexpectedly used a sugar derivative in which an aliphatic hydrocarbon group was introduced into sugar to obtain a cell cryoprotectant such as DMSO. We have found that it is possible to suppress cytotoxicity even in the presence of the present invention, and have completed the present invention.

Therefore, the present invention provides the following abstracts.
[1] Formula I-1 or I-2:

[During the ceremony,
n is 0 or 1;
Both R ₁ and R ₂ are H, or R ₁ and R ₂ together form = O;
Both R ₃ and R ₄ are H, or R ₃ and R ₄ together form a bond;
R ₅ is an aliphatic saturated or unsaturated hydrocarbon group of 1 to 30 carbon atoms;
R ₆ is H or -CH ₂ OH, where n is 0 if _{R 6} is -CH _{2 OH;}
R ₇ is H or C _1-6 alkyl, or:

(During the ceremony,
m is 0 or 1;
R _A is H or _-NR C _{R D;}
R _B is H or _-CH 2 OH, provided that when _{R B} is _-CH 2 OH, m is 0;
R _C and _{R D} are, independently, _H, be a _{C 1-6} alkyl or -COR _E;
R _E is -OH or _{C 1-6} alkyl)
Selected from the group consisting of;
R ₈ is H or -NR ₉ R ₁₀ ;
R ₉ and R ₁₀ are independently H, C _1-6 alkyl or -COR ₁₁ ;
R ₁₁ is -OH or C _1-6 alkyl]
The compound represented by, or a salt or solvate thereof.
[2] The compound according to [1], or a salt or solvate thereof, wherein _{R 3} and R _{4 form a bond together.}
[3] The compound according to [1] or [2], or a salt or solvate thereof, wherein n is 1.
[4] Below:

The compound according to any one of [1] to [3], or a salt or solvate thereof.
[5] Below:

The compound according to any one of [1] to [4], or a salt or solvate thereof.
[6] Below:

The compound according to any one of [1] to [5], or a salt or solvate thereof, selected from the group consisting of.
[7] A composition for cell freezing, which comprises the compound according to any one of [1] to [6], or a salt or solvate thereof.
[8] The composition for cell freezing according to [7], further comprising a cell membrane-permeable cell freeze-protecting agent.
[9] The cell freezing composition according to [7] or [8], wherein the cell is a primary cultured cell.
[10] ^{A solution for freezing cells, which comprises 1 × 10 -7} to 1 × 10 ⁻² mass% of the compound according to any one of [1] to [6], or a salt or solvate thereof.
[11] A composition for stabilizing a cell membrane, which comprises the compound according to any one of [1] to [6], or a salt or solvate thereof.
[12] A method for freezing cells, which comprises contacting the cells with the compound according to any one of [1] to [6], or a salt or solvate thereof.
[13] A method for stabilizing a cell membrane, which comprises contacting a cell with the compound according to any one of [1] to [6], or a salt or solvate thereof.

According to the present invention, it is possible to provide a novel sugar derivative useful for freezing cells. More specifically, it is possible to provide a cell cryoprotectant capable of suppressing cytotoxicity even in the presence of a cell cryoprotectant such as DMSO.

In Test Example 1, the result of evaluating the number of viable cells when the rat primary cultured hepatocytes were frozen and thawed together with the compound of Example 1 is shown. In Test Example 1, the result of evaluating the number of viable cells when the rat primary cultured hepatocytes were frozen and thawed together with the compound of Example 2 is shown. In Test Example 1, the result of evaluating the number of viable cells when the rat primary cultured hepatocytes were frozen and thawed together with the compound of Example 3 is shown. In Test Example 1, the result of evaluating the number of viable cells when the rat primary cultured hepatocytes were frozen and thawed together with the compound of Example 4 is shown. In Test Example 2, the results of evaluating the viable cell activity when rat primary cultured hepatocytes were frozen and thawed together with the compound of Example 2 and / or the compound of Comparative Example 1 and cultured for 1 to 5 days are shown. In Test Example 2, the results of evaluating the EROD activity when rat primary cultured hepatocytes were frozen and thawed together with the compound of Example 2 and / or the compound of Comparative Example 1 and cultured for 3 to 5 days are shown. In Test Example 2, the albumin-producing ability when rat primary cultured hepatocytes were frozen and thawed together with the compound of Example 2 and / or the compound of Comparative Example 1 and cultured for 1 to 3 days or 3 to 5 days was evaluated. The result is shown. In Test Example 2, the result of calculating the albumin production rate per living cell is shown. In Test Example 2, the results of freeze-thawing rat primary cultured hepatocytes together with the compound of Example 2 and / or the compound of Comparative Example 1 and evaluating the ammonia metabolic capacity when cultured for 1 day are shown. In Test Example 3, the results of evaluating the cell membrane fluidity when rat primary cultured hepatocytes were cultured together with the compound of Example 2 are shown. In Test Example 4, the result of evaluating the viable cell when the rat primary cultured hepatocyte was cultured together with the compound of Example 2 is shown. In Test Example 5, the results of evaluating the viable cell activity when the rat primary cultured hepatocytes were vortexed together with the compound of Example 1 to apply shear stress and then cultured for 1 day are shown. In Test Example 6, the results of evaluating the storage elastic modulus G ′ and the loss elastic modulus G ″ when the rat primary cultured hepatocytes were suspended in the medium (DHDM) containing the compound of Example 2 are shown. In Test Example 6, the storage elastic modulus G'and the loss elastic modulus G'when the rat primary cultured hepatocytes were suspended in the medium (DHDM) containing the compound of Example 2 were plotted on the complex surface. Is shown. In Test Example 7, the result of evaluating the action position of the compound of Example 2 in the cell is shown. The presumed mechanism (dehydration and concentration effect) of suppression of cytotoxicity by the compound represented by the formula I-1 or I-2, or a salt or solvate thereof is shown. The presumed mechanism of suppression of cytotoxicity (effect of suppressing ice crystal formation) by the compound represented by the formula I-1 or I-2, or a salt or solvate thereof is shown.

The present invention describes the formula I-1 or I-2:

(During the ceremony,
m is 0 or 1;
R _A is H or _-NR C _{R D;}
R _B is H or _-CH 2 OH, provided that when _{R B} is _-CH 2 OH, m is 0;
R _C and _{R D} are, independently, _H, be a _{C 1-6} alkyl or -COR _E;
R _E is -OH or _{C 1-6} alkyl)
Selected from the group consisting of;
R ₈ is H or -NR ₉ R ₁₀ ;
R ₉ and R ₁₀ are independently H, C _1-6 alkyl or -COR ₁₁ ;
R ₁₁ is -OH or C _1-6 alkyl]
Provided are a compound represented by, or a salt or solvate thereof.

The present invention is also a compound represented by the formula I-1 or I-2 for producing a cell cryoprotectant or a cell cryoprotective composition, or a cell membrane stabilizer or a cell membrane stabilizing composition, or a compound thereof. The use of salts or solvates is provided.

The present invention also provides a method for freezing cells, which comprises contacting the cells with a compound represented by the formula I-1 or I-2, or a salt or solvate thereof.

The present invention also provides a method for stabilizing a cell membrane, which comprises contacting a cell with a compound represented by the formula I-1 or I-2, or a salt or solvate thereof.

As used herein, the term "saturated or unsaturated aliphatic hydrocarbon group having 1 to 30 carbon atoms" refers to the carbonization of a saturated or unsaturated linear or branched chain containing 1 to 30 carbon atoms. It means a hydrogen group. The saturated or unsaturated aliphatic hydrocarbon group may be a cyclic type having a cyclic structure (including a spiro ring, a condensed ring, and a bridge) or an acyclic type having no cyclic structure. Saturated or unsaturated aliphatic hydrocarbon groups having 1 to 30 carbon atoms are not limited to these, but for example, methyl, ethyl, propyl, isopropyl, n-butyl, i-butyl, t-. Butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecil, icosyl, henicosyl, docosyl. , Tricosyl, Tetracosyl, Pentacosyl, Hexacosyl, Heptacosyl, Octacosyl, Nonacosyl, Triacontyl, CH _3- (CH ₂ ) ₅ CH = CH (CH ₂ ) _6- , CH _3- (CH ₂ ) ₅ CH = CH (CH ₂ ) _{_{_{_{7 -, CH 3 - (CH}}}} 2) 7 CH = CH (CH 2) 6 -, CH 3 - (CH 2) 7 CH = CH (CH 2) 7 -, CH 3 - (CH 2) 7 CH = CH _{_{_{_{(CH 2) 8 -, CH}}}} 3 - (CH 2) 5 CH = CH (CH 2) 8 -, CH 3 - (CH 2) 5 CH = CH (CH 2) 9 -, CH 3 - (CH 2) _{_{_{_{3 (CH 2 CH = CH)}}}} 2 (CH 2) 6 -, CH 3 - (CH 2) 3 (CH 2 CH = CH) 2 (CH 2) 7 -, CH 3 - (CH 2 CH = CH) 3 _{_{_{_{(CH 2) 6 -, CH}}}} 3 - (CH 2 CH = CH) 3 (CH 2) 7 -, CH 3 - (CH 2) 3 (CH 2 CH = CH) 3 (CH 2) 3 -, CH 3 _{_{_{_{- (CH 2) 3 (CH}}}} 2 CH = CH) 3 (CH 2) 4 -, CH 3 - (CH 2) 3 (CH = CH) 3 (CH 2) 6 -, CH 3 - (CH 2) 3 _{_{_{_{(CH = CH) 3 (CH}}}} 2) 7 -, CH 3 - (CH 2) 6 (CH 2 CH = CH) 2 (CH 2) 5 -, CH 3 - (CH 2) 6 (CH 2 CH = CH ) ₂ (CH ₂ ) _6- , CH _3- (CH ₂ ) ₆ (CH ₂ CH = CH) ₃ (CH ₂ ) _2- , CH _3- (CH ₂ ) ₆ (CH ₂ CH = CH) ₃ (CH) ₂ ) _3- , CH _3- (CH ₂ ) ₃ (CH ₂ CH = CH) ₄ (CH ₂ ) _2- , CH _3- (CH ₂ ) ₃ (CH ₂ CH = CH) ₄ (CH ₂ ) _3- , CH _3- (CH ₂ ) ₇ CH ₂ CH = CH (CH ₂₎ _{_{_{_{) 12 -, CH 3 - (}}}} CH 2) 7 CH 2 CH = CH (CH 2) 13 -, CH 3 -CH 2 (CH = CHCH 2) 6 -, CH 3 -CH 2 (CH = CHCH 2) 6 Acyclic aliphatic hydrocarbon groups such as CH _2- , CH _3- CH ₂ (CH = CHCH ₂ ) ₅ CH _2- , CH _3- CH ₂ (CH = CHCH ₂ ) ₅ (CH ₂ ) _{2-, etc.;} For example, cyclic aliphatic hydrocarbon groups such as cyclopentyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooxyl can be mentioned. The saturated or unsaturated aliphatic hydrocarbon group having 1 to 30 carbon atoms is preferably an aliphatic hydrocarbon group having 2 to 26 saturated or unsaturated carbon atoms, and more preferably a saturated or unsaturated aliphatic hydrocarbon group. It is an aliphatic hydrocarbon group having 3 to 22 carbon atoms, more preferably a saturated or unsaturated aliphatic hydrocarbon group having 3 to 20 carbon atoms. More specifically, 1 to 30 saturated or unsaturated aliphatic hydrocarbon groups are methyl, ethyl, propyl, isopropyl, n-butyl, undecyl, dodecyl, CH _3- (CH ₂ ) ₇ CH = CH ( _{_{_{_{CH 2) 7 -, CH 3}}}} - (CH 2) 7 CH = CH (CH 2) 8 - is preferably, propyl, _{_{_{undecyl, CH 3 - (CH 2)}}} 7 CH = CH (CH 2) 7 - Is more preferable.

As used herein, "C _1-6 alkyl" refers to a saturated linear or branched hydrocarbon group containing 1 to 6 carbon atoms (but not limited to, for example, for example. Means methyl, ethyl, propyl, isopropyl, n-butyl, i-butyl, t-butyl, pentyl, hexyl, etc.). Preferred C _1-6 alkyl is C _1-4 alkyl (including, for example, methyl, ethyl, propyl, butyl, isopropyl, etc.), more preferably methyl, ethyl.

As used herein, the term "salt" is, but is not limited to, an inorganic acid (but not limited to, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitrate). , Carbonate, phosphoric acid, etc.) or organic acids (but not limited to, for example, formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvate, oxalic acid, malic acid, maleic acid, etc. Maronic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranic acid, benzoic acid, silicate acid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p -Acid addition salts with toluenesulfonic acid, salicylic acid, etc.; organic bases (but not limited to, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, Salts with trimetamin, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, etc. Can be mentioned.

As used herein, "solvate" means an aggregate, complex, or the like of one or more solvent molecules and a compound represented by the formula I-1 or I-2. Examples of the solvent include, but are not limited to, water, methanol, ethanol, isopropanol, DMSO, acetic acid, ethyl acetate and the like.

As used herein, a wavy line that intersects a bond in a chemical structure.

Indicates the bond point of the atom to which the wavy bond is connected in the chemical structure to the rest of the molecule.

The compound represented by the formula I-1 or I-2 described in the present specification, or a salt or solvate thereof, may have one or more chiral atoms (for example, an asymmetric carbon atom). Therefore, the compounds represented by the formulas I-1 or I-2 described herein, or salts or solvates thereof, may be present as diastereomers, enantiomers, or mixtures thereof. .. If the stereochemistry of any particular chiral atom is not specified in the chemical structures shown herein, then all stereoisomers are intended. In one embodiment of the invention, stereochemistry is specified by a dark wedge that represents a particular configuration.

The compounds represented by the formulas I-1 or I-2 described herein, or salts or solvates thereof, or some chemical structures in these molecules are sugars (eg, 5 monosaccharides, 6). Since it has a structure similar to that of monosaccharides), it may be represented by, for example, a Haworth projection type, a chair conformation (chair form), or a boat conformation (boat foam). The compounds represented by the formulas I-1 or I-2 described herein, or salts or solvates thereof, or some of the chemical structures in these molecules are Haworth projections, chair conformations and / Or when represented by a boat conformation, stereochemistry is specified for such a representation.

In one embodiment of the invention, a compound represented by the formula I-1 or I-2 described herein, or a salt or solvate thereof, wherein both _{R 1} and R _{2 are H.} provide.

In one embodiment of the invention, a compound of formula I-1 or I-2 described herein, or a salt or solvate thereof _{, wherein R 3} and R _{4 together form a bond.} provide.

In one embodiment of the invention:

Provided are a compound represented by the formula I-1 described in the present specification, or a salt or solvate thereof.

In one embodiment of the invention, there is provided a compound of formula I-1 described herein, or a salt or solvate thereof _{, wherein R 8 is H.}

In one embodiment of the invention:

In one embodiment of the invention:

In one embodiment of the invention:

In one embodiment of the invention, R ₇ is a C _1-6 alkyl, preferably methyl, a compound of formula I-1 or I-2 described herein, or a salt or solvate thereof. Provide things.

In one embodiment of the present invention, R ₇ is selected from the group consisting of:

Provided are compounds represented by the formulas I-1 or I-2 described herein, or salts or solvates thereof, selected from the group consisting of.

Provided are a compound represented by the formula I-1 or I-2 described in the present specification, or a salt or solvate thereof.

In one embodiment of the present invention, _{R 5} is methyl, ethyl, propyl, isopropyl, n- butyl, undecyl, _{_{_{dodecyl, CH 3 - (CH 2)}}} 7 CH = CH (CH 2) 7 - , or _CH 3 - ( CH ₂ ) ₇ CH = CH (CH ₂ ) ₈ -Provides a compound represented by the formula I-1 or I-2 described herein, or a salt or solvate thereof.

In one embodiment of the present invention, _{R 5} is propyl, undecyl or _{_{_{CH 3 - (CH 2) 7}}} CH = CH (CH 2) 7 - in which formula I-1 or as described herein I- A compound represented by 2 or a salt or solvate thereof is provided.

In one embodiment of the invention:

Provided are compounds represented by the formula I-1 described herein, or salts or solvates thereof, selected from the group consisting of.

In one embodiment of the invention:

The compounds represented by the formulas I-1 or I-2 described herein, or salts or solvates thereof, are not limited thereto, but are, for example, by the methods described in the following general scheme. , Or a method similar to this.

General reaction scheme

[In these formulas, R 1 _~ R ₈ and n are as defined herein]

The compound represented by the formula 0-1 or 0-2 (commercially available, or can be synthesized, for example, by a known method or a method disclosed herein, or a method similar thereto). And the _{aldehyde represented by the formula R 5-} CHO in a suitable solvent (for example, but not limited to, N, N-dimethylformamide (DMF), tetrahydrofuran (THF), ethanol, etc.). Acids (but not limited to, for example, p-toluenesulfonic acid, copper (II) tetrafluoroborate, naphthion, etc.) and optionally water traps (which can remove water in the reaction system). As long as it is, the reaction is carried out at room temperature to the reflux temperature of the solvent in the presence of, for example, triethyl orthoformate, trimethyl orthoformate, molecular sieves, magnesium sulfate, etc.). Then, a suitable base (for example, but not limited to these, triethylamine, sodium hydrogencarbonate, etc.) is added at a suitable temperature, or the reaction solution is passed through basic silica gel or the like. Then, if necessary, by concentrating, purifying, or the like by a method known to those skilled in the art, the compound represented by the formula I-1 or I-2 described in the present specification, or a salt or solvate thereof. Can be obtained.

As the _{aldehyde represented by the formula R 5-} CHO, a commercially available aldehyde may be used. Alternatively, the _{aldehyde represented by the formula R 5-} CHO is not limited to these, but for example, the _{alcohol represented by the formula R 5-} CH ₂ OH can be used as a suitable solvent (not limited to these). However, in (eg, dichloromethane, etc.), suitable oxidizing agents (for example, but not limited to, 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO), etc.) and, if necessary, In the presence of a suitable reoxidant (for example, but not limited to, iodobenzene diacetate, N-chlorosuccinimide, etc.), the reaction is carried out at 0 ° C. to the reflux temperature of the solvent, if necessary. The solvent may be concentrated or purified by a method known to those skilled in the art, or may be obtained by other known methods.

When synthesizing a compound having a chiral atom, a racemate, a diastereomer or an enantiomer may be used as a starting material or an intermediate. The racemic mixture, the mixture of diastereomers and the like may be separated by a method known to those skilled in the art such as chromatography or crystallization.

In one embodiment of the present invention, it is a process for producing a compound represented by the formula I-1 or I-2 described in the present specification, or a salt or solvate thereof, wherein the formula 0-1 or a solvate thereof is produced. 0-2:

The compound represented by the formula is expressed in the presence of an acid.

In comprises reacting a compound represented process (in these _{_{formulas, R 1, R 2, R}} 5 ~ R 8 and n are is as defined herein) it provides.

As used herein, the term "cell" means that any cell derived from an animal, plant, etc. is included, and may be a normal cell or an abnormal cell, and is a primary culture. It may be a cell or a cell line. Further, the "cell" used in the present specification also includes a cell whose trait has been changed by genetic modification or the like, or a cell which has been subjected to other treatments.

As used herein, the term "abnormal cell" means, for example, a cell that has a lesion due to a disease, disorder, etc.; a cell that has trauma, etc .; a cell that has other abnormalities. When the "cell" used in the present specification is derived from an animal, the disease, disorder, etc. are not limited to these, and for example, cancer; autoimmune disease; inflammation such as inflammatory bowel disease, etc. Sexual diseases; allergic diseases such as atopic dermatitis and allergic rhinitis; metabolic diseases such as diabetes and obesity; neurodegenerative diseases such as Alzheimer's disease. The cancer is not limited to these, for example, breast cancer; biliary tract cancer; bladder cancer; brain tumor including glioma, myeloma, etc .; cervical cancer; chorionic villi cancer; colon cancer; endometrial Cancer; Esophageal cancer; Gastric cancer; Hematological neoplasms including acute lymphocytic, myeloid leukemia; T-cell acute lymphoblastic leukemia; Hairy cell leukemia; Chronic myeloid leukemia; Multiple myeloma; AIDS Related leukemia; adult T-cell leukemia; intraepithelial neoplasia including Bowen's disease, Paget's disease, etc .; liver cancer; lung cancer; lymphoma including Hodgkin's disease, lymphocytic lymphoma, etc .; neuroblastoma; oral cavity including squamous epithelial cancer, etc. Cancer; ovarian cancer; pancreatic cancer; prostate cancer; rectal cancer; smooth myoma, horizontal print myoma, liposarcoma, fibrosarcoma, osteosarcoma, etc. Skin cancer including flat epithelial cancer; testicular cancer including embryo tumor, stromal tumor, embryo cell tumor, etc .; thyroid cancer including thyroid cancer, medullary cancer, etc .; kidney cancer including adenocarcinoma, Wilms tumor, etc. Be done.

As used in the present specification, the "primary cultured cell" means a cell obtained by first seeding and culturing an organ, tissue, cell or the like collected from a living body. The primary cultured cells may be one type of cells or a mixture of two or more types of cells. Primary cultured cells can be passaged a finite number of times, and repeated passages may gradually lose their ability to divide.

When the "cell" used in the present specification is derived from an animal, the animal is not limited to these, and for example, ungulates such as mice, rats, hamsters and guinea pigs; and rabbits such as rabbits. Eyes; ungulates such as pigs, cows, goats, horses and sheep; cats such as dogs and cats; birds such as chickens, quail and turkeys; primates such as humans, monkeys, red-tailed monkeys, marmosets, orangutans and chimpanzees; Etc., but are preferably primates, and more preferably humans.

The cells derived from animals are not limited to these, for example, epidermal cells (keratinocytes, etc.), pigment cells (melanosites, etc.), basal cells, spinous cells, granule cells, keratinocytes, fibroblasts, etc. , Lymphocytes, B cells, T cells, cytotoxic T cells, natural killer T cells, regulatory T cells, helper T cells, myeloid cells, granulocytes, basic granulocytes, acidophilic granulocytes, neutrophils Granulocytes, hyperlobed neutrophils, monospheres, macrophages, reticular red cells, platelets, obese cells, macronuclear cells, dendritic cells, thyroid cells, thyroid epithelial cells, parafollicular cells, epithelial body cells, epithelial body main cells , Acidophilic cells, adrenal cells, chromium-affinitive cells, pineapple cells, nerve cells, glial cells, glial cells, glial blast cells, stellate cells, dilute glial cells, small glial cells, stellate cells, Betchell cells , Hydrus cells, gonad stimulating hormone-producing cells, adrenal cortex stimulating hormone-producing cells, thyroid stimulating hormone-producing cells, growth hormone-producing cells, prolactin-producing cells, pancreatic β cells, lung cells, type I lung cells, type II lung cells, Clara cells, cup cells, alveolar macrophages, myocardial cells, pericutaneous cells, vascular endothelial cells, gastric cells, gastric main cells, wall cells, cup cells, panate cells, G cells, D cells, I cells, K cells, S Cells, intestinal endocrine cells, intestinal chromium-affinitive cells, intestinal chromium-affinitive cell-like cells, APUD cells, hepatocytes, cupper cells, hepatic stellate cells, sinus endothelial cells, bone cells, osteoblasts, osteoclasts, ivory Blast cells, cement blast cells, enamel blast cells, cartilage cells, chondrocyte blast cells, skin cells, hair cells, keratinocytes, melanocytes, mother spot cells, muscle cells, myoblasts, myotube cells, fat cells, fibroblasts , Tendant cells, pedicle cells, parafilamentous cells, intragranular mesangial cells, extraglobulal mesangial cells, renal cells, compact spot cells, sperm, Sertri cells, Leidich cells, egg matrix cells, small hepatocytes, oval Examples include cells, mesenchymal stem cells, nerve stem cells, hematopoietic stem cells, cancer stem cells, pluripotent stem cells; cells derived from insects and the like. Examples of plant-derived cells include, but are not limited to, parenchyma cells, collenchyma cells, collenchyma cells, xylem cells, phloem cells, and epidermal cells.

As used herein, "pluripotent stem cell" means a cell having the ability to differentiate into all the tissues and cells constituting the living body. The pluripotent stem cells are not limited to these, for example, embryonic stem cells (ES cells); embryonic stem cells (ntES cells) derived from cloned embryos obtained by nuclear transplantation; sperm stem cells (GS cells). Pluripotent germ stem cells (mGS cells); Embryonic stem cells (EG cells); Artificial pluripotent stem cells (iPS cells); Pluripotent cells derived from cultured fibroblasts or bone marrow stem cells (Muse cells); Human ES Fusion cells of cells and somatic cells; pluripotent stem cells induced and selected by stress or cell stimulation; pluripotent stem cells established by culturing early embryos created by nuclear transplantation of somatic cell nuclei (Nature, 385,810 (1997); Science, 280, 1256 (1998); Nature Biotechnology, 17,456 (1999); Nature, 394,369 (1998); Nature Genetics, 22, 127 (1999); Proc. Natl. Acad. Sci. USA, 96, 14984 (1999); Nature Genetics, 24, 109 (2000); and the like.

In one embodiment of the invention, the cell is an animal-derived cell.

In one embodiment of the invention, the cell is an animal-derived primary cultured cell.

The compounds represented by the formulas I-1 or I-2 described herein, or salts or solvates thereof, may be useful for cell freezing. Therefore, in one embodiment of the present invention, there is provided a composition for cell freezing, which comprises the compound represented by the formula I-1 or I-2 described in the present specification, or a salt or solvate thereof. Further, in one embodiment of the present invention, a cell cryoprotectant containing a compound represented by the formula I-1 or I-2, or a salt or solvate thereof is provided.

The compound represented by the formula I-1 or I-2 described in the present specification, or a salt or solvate thereof, stabilizes the cell membrane (particularly, destabilizes the cell membrane such as a cell membrane-permeable cell cryoprotectant). It can stabilize the cell membrane, which can be destabilized by the substance that makes it. Therefore, in another embodiment of the present invention, there is provided a composition for stabilizing a cell membrane, which comprises the compound represented by the formula I-1 or I-2 described in the present specification, or a salt or solvate thereof. .. Further, in one embodiment of the present invention, there is provided a cell membrane stabilizer containing a compound represented by the formula I-1 or I-2, or a salt or solvate thereof.

The compositions described in the present specification (for example, cell freezing compositions, cell membrane stabilizing compositions) include the compounds represented by the formula I-1 or I-2 described in the present specification, or the compounds thereof. In addition to salts or solvates, basal medium can be added.

The basal medium is not particularly limited as long as the object of the present invention can be achieved, but for example, DHDM, DMEM, EMEM, IMDM (Iscover's Modified Dulvecco's Medium), GMEM (Glasgow's MEM), and the like. RPMI-1640, α-MEM, Ham's medium F-10, Ham's medium F-12, Ham's medium F12K, Medium 199, ATCC-CRCM30, DM-160, DM-201, BME, Glasgow, McCoy 's5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's MB752 / 1, CMRL-1066, Williams' media Examples thereof include 3 medium, E8 medium (Nature Methods, 2011, 8, 424-429), ReproFF, ReproFF2 and the like, and these may be used alone or in combination of two or more.

In addition, the compositions described in the present specification (for example, cell freezing compositions, cell membrane stabilizing compositions) include compounds represented by the formula I-1 or I-2 described in the present specification. Or, in addition to its salts or solvates, as required, for example, serum, growth factors, iron sources, polyamines, trace metals, sugars, organic acids, amino acids and derivatives thereof, reducing agents, vitamins, steroids, antibiotics, etc. , Buffers, inorganic salts, pH regulators, proteins (including enzymes, etc.), various activators / inhibitors, compounds represented by the formula I-1 or I-2 described in the present specification, or salts thereof. Alternatively, an additive such as a cell cryoprotectant other than the solvate can be added. The amount of the additive added is not particularly limited as long as the object of the present invention can be achieved, and can be appropriately selected by those skilled in the art.

The serum is not particularly limited as long as the object of the present invention can be achieved, and examples thereof include sera derived from mammals such as fetal bovine serum, calf serum, horse serum, and human serum, and these are singular. In addition, two or more types may be used in combination. In addition, when cells are used for medical purposes, since heterologous components may become an infection source or heterologous collagen of blood-borne pathogens, serum is used in the cell freezing composition described in the present specification. It may be preferable not to add. When serum is not added to the cell freezing composition described herein, for example, Knockout Serum Replacement (KSR) (Invitrogen), Chemically-defined Lipid predicted (Gibco), Glutamax (Gibco). Additives that substitute for serum such as may be added.

Growth factors include, but are not limited to, glucagon, insulin, insulin-like growth factor (IGF), epithelial growth factor (EGF), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF). ), Vascular endothelial cell growth factor (VEGF), granulocyte colony stimulator (G-CSF), granulocyte macrophage colony stimulator (GM-CSF), erythropoetin (EPO), thrombopoetin (TPO), hepatocellular growth factor (HGF) ), Thrombotic growth factor (PDGF), transforming growth factor beta (TGF-β), basic fibroblast growth factor (bFGF), etc., which can be used alone or in combination of two or more. You may use it.

The iron source is not limited to these, and examples thereof include transferrin, ferritin, iron (II) sulfate, and the like, which may be used alone or in combination of two or more.

Examples of polyamines include, but are not limited to, spermine, spermidine, norspermine, norspermidine, homospermidine, homospermidine, cadaverine, putrescine, agmatine, ornithine, and the like, and these are used alone. Also, two or more types may be used in combination.

The trace metal is not limited to these, and examples thereof include magnesium, zinc, cobalt, tin, molybdenum, nickel, selenium, sodium selenite, and the like. The above may be used in combination.

Examples of saccharides include, but are not limited to, glucose, galactose, fructose, sucrose and the like, and these may be used alone or in combination of two or more.

Examples of the organic acid include, but are not limited to, pyruvic acid, lactic acid, linoleic acid, linoleic acid and the like, and these may be used alone or in combination of two or more. ..

Amino acids and derivatives thereof are not limited to these, for example, glycine, L-alanine, L-serine, L-valine, L-leucine, L-isoleucine, L-arginine, L-lysine, L. -Asparagine, L-glutamine, L-aspartic acid, L-glutamic acid, L-methionine, L-cysteine, L-proline, L-threonine, L-histidine, L-tryptophane, L-phenylalanine, L-tyrosine, L- Examples thereof include carnitine, L-ornithine, glutathione (including reduced form), and these may be used alone or in combination of two or more.

The reducing agent is not limited to these, and examples thereof include 2-mercaptoethanol and dithiothreitol, which may be used alone or in combination of two or more.

The vitamins are not limited to these, but for example, vitamin A and its derivatives (including vitamin A acetate ester, etc.), vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B12, vitamins. Examples thereof include C, vitamin D, vitamin E and derivatives thereof (including DL-α-tocopherol acetate and the like), vitamin K, biotin, folic acid and the like, and these may be used alone or in combination of two or more. Good.

Examples of steroids include, but are not limited to, β-estradiol, progesterone, corticosterone, etc., and these may be used alone or in combination of two or more.

Examples of antibiotics include, but are not limited to, penicillin, streptomycin, gentamicin, kanamycin, etc., which may be used alone or in combination of two or more.

Examples of the buffering agent include, but are not limited to, sodium hydrogen carbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, HEPES, and the like, and even if they are used alone, two or more kinds thereof can be mentioned. May be used in combination.

The inorganic salt is not limited to these, for example, sodium chloride, calcium chloride, potassium chloride, copper sulfate, iron (II) nitrate, iron sulfate, magnesium chloride, magnesium sulfate, sodium hydrogen carbonate, phosphoric acid. Examples thereof include disodium hydrogen hydrogen and sodium dihydrogen phosphate, and these may be used alone or in combination of two or more.

The pH adjuster is not limited to these, and examples thereof include hydrochloric acid, nitric acid, phosphoric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate and the like. These may be used alone or in combination of two or more.

Examples of proteins (including enzymes) include, but are not limited to, human albumin, bovine serum albumin, superoxide dismutase, catalase, etc., and even if they are used alone, two or more kinds thereof May be used in combination.

The various activators / inhibitors are not limited to these, but are, for example, Wnt signal activators such as Wnt-3a, Wnt-5a, lithium chloride, and complement molecule C1q; Y-27632, Examples thereof include Rho-kinase (ROCK) inhibitors such as K-115 (ripasudil hydrochloride hydrate) and HA1077 (fasudil hydrochloride), which may be used alone or in combination of two or more.

The cell cryoprotectant other than the compound represented by the formula I-1 or I-2 described in the present specification or a salt or solvate thereof is not limited to these, and is, for example, glucose and fluctose. , Galactose, sucrose, maltose, trehalose, raffinose, cellulose, starch, xylitol, sorbitol, erythritol, mannitol, glycerol, polyvinyl alcohol, polyvinylpyrrolidone, ficol, polyethylene glycol, albumin, etc. , Ethylene glycol, propylene glycol, 1,3-propanediol, butylene glycol, isoprene glycol, dipropylene glycol, glycerin and other cell membrane-permeable cell cryoprotectants. May be used in combination.

In one embodiment of the present invention, there is provided the composition for cell freezing described herein, further comprising a cell freeze protectant.

In one embodiment of the present invention, there is provided a cell membrane stabilizing composition described herein, further comprising a cell membrane permeable cell cryoprotectant, preferably DMSO.

The pH of the compositions described herein (eg, cell freezing compositions, cell membrane stabilizing compositions) is not particularly limited as long as the object of the present invention can be achieved, but for example, about 4 It can be from about 10, preferably from about 5 to about 9, more preferably from about 6 to about 8, and even more preferably from about 6.5 to about 7.5.

The compositions described in the present specification (for example, cell freezing composition, cell membrane stabilizing composition) may be sterilized for the purpose of preventing contamination and the like. The method of sterilization is not limited to these, and examples thereof include ultraviolet irradiation, irradiation, heating, and filtration.

The cell freezing composition described in the present specification may be used as a cell freezing solution.

The compositions described herein (eg, cell freezing compositions, cell membrane stabilizing compositions) are, for example, dry solids (eg, solids, powders, etc.), even in the form of solutions. It may be in the form of.
When the cell freezing composition is in the form of a solution, it may be used as it is as a cell freezing solution, or a solution obtained by diluting with a solvent and adding the above-mentioned additives as necessary is used as a cell freezing solution. May be used as. Examples of the solvent used for dilution include water, a buffer solution, physiological saline, DMSO, the above-mentioned basal medium and the like, and these may be used alone or in combination of two or more.
When the cell freezing composition was in the form of a dry solid, it was dissolved in a solvent such as water, a buffer solution, a physiological saline solution, or the above-mentioned basal medium, and the above-mentioned additives were added as necessary. Can be used as a cell freezing solution.

Formula I-1 or the formula I-1 described in the present specification in the composition described in the present specification (for example, a cell freezing composition, a cell membrane stabilizing composition), or in a cell freezing solution obtained from the composition. The content of the compound represented by I-2, or a salt or solvate thereof, is, for example, about 1 × ^10-10 to about 1% by mass as a final concentration with respect to the total amount of the composition or the total amount of the solution. It is preferably about 1 × 10 ^-9 to about 1 × 10 ^-1 % by mass, more preferably about 1 × 10 ^-8 to about 1 × 10 ^-2 % by mass, and even more preferably about 1 × 10 ^-7 to about 1 ×. ^{It can be 10-2} % by mass.

In one embodiment of the present invention, 1 × 10 ^-7 to 1 × 10 ⁻² mass% of the compound represented by the formula I-1 or I-2 described in the present specification, or a salt or solvate thereof. A solution for freezing cells is provided.

As a method for freezing cells using the compound represented by the formula I-1 or I-2 described in the present specification, or a salt or solvate thereof, for example, described in the present specification in a solvent. For cell freezing, which is filled with a compound represented by the formula I-1 or I-2, or a salt or solvate thereof, in contact with cells (hereinafter, also referred to as "cell mixture for freezing"). The container may be placed in an environment suitable for slow freezing, vitrification freezing, and the like. The method by slow freezing is not limited to these, and examples thereof include a method using a program freezer and the like, a method using a bicell and the like, and the like. When using a program freezer or the like, for example, a cell freezing container filled with a freezing cell mixture is allowed to stand in a deep freezer or the like, the temperature is lowered to about -50 ° C, and then the freezing cell mixture is filled. The cell freezing container may be stored in liquid nitrogen. When using a bicelle or the like, for example, the bicelle or the like containing a cell freezing container filled with a cell mixture for freezing is allowed to stand in a deep freezer or the like at about -80 ° C for about half a day to about one day or more, and then. If necessary, it may be stored in liquid nitrogen.

The cell freezing container is not particularly limited as long as it can freeze the freezing cell mixture, but for example, a tube, a vial, a flask, a tissue culture flask, a dish, a petri dish, and a tissue. Examples thereof include culture dishes, multi-dish, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, petri dishes, trays, culture bags, roller bottles and the like.

The amount of the cell mixture for freezing to be filled in the cell freezing container can be appropriately changed in consideration of the capacity of the cell freezing container to be used, for example, about 0.001 to about 1000 mL, preferably about 0.001 to about 1000 mL. It may be from about 0.01 to about 100 mL, more preferably from about 0.1 to about 10 mL, even more preferably from about 0.2 to about 5 mL, and even more preferably from about 0.5 to about 3 mL. The number of cells contained in the freezing cell mixture is, for example, about 1 to about 1 x 10 ¹⁰ cells / mL, preferably about 1 x 10 ² to about 3 x 10 ⁹ cells / mL, more preferably about. 1 × 10 ³ to about 1 × 10 ⁹ cells / mL, more preferably about 1 × 10 ⁴ to about 3 × 10 ⁸ cells / mL, even more preferably about 1 × 10 ⁵ to about 1 × 10 ⁸ cells / mL. It may be / mL.

Examples of the method of thawing the frozen cell mixture for freezing include a method of rapidly thawing in a constant temperature water tank heated to about 37 ° C. to about 42 ° C. A medium or the like cooled to about 0 ° C. to about 10 ° C. is added to the thawed cell mixture for freezing, centrifugation is performed, and the supernatant is discarded. The compound represented by 2 or a salt or solvate thereof may be removed. Then, the cells may be cultured by suspending the cells in an appropriate medium and seeding them on a plate or the like. Alternatively, a medium having a gradual concentration corresponding to the osmotic resistance of the frozen cells is added, and the compound represented by the formula I-1 or I-2 described in the present specification, or a salt or solvate thereof, or the like is added. After removal, the cells may be cultured by suspending them in an appropriate medium and seeding them on a plate or the like.

The culture temperature of the cells after thawing is not particularly limited as long as the cells can be cultured, but can be, for example, about 30 to about 40 ° C., preferably about 37 ° C. The method can be cultured in an atmosphere of _{CO 2-} _{containing air, and the CO 2} concentration can be, for example, about 1 to about 10%, preferably about 2 to about 5%.

The number of cells to be cultured is not particularly limited, but for example, in a medium in a solution state, about 1 to about 1 × 10 ¹⁰ cells / mL, preferably about 10 to about 3 × 10 ⁹ cells / mL. , More preferably from about 1 × 10 ² to about 1 × 10 ⁹ cells / mL, even more preferably from about 5 × 10 ² to about 3 × 10 ⁸ cells / mL, even more preferably from about 1 × 10 ³ to. It may be about 1 × 10 ⁸ cells / mL. Alternatively, in solution medium, from about 1 × 10 ¹⁰ cells / cm ² , preferably from about 10 to about 3 × 10 ⁹ cells / cm ² , more preferably from about 1 × 10 ² to about 1 ×. 10 ⁹ cells / cm ² , more preferably about 5 × 10 ² to about 3 × 10 ⁸ cells / cm ² , even more preferably about 1 × 10 ³ to about 1 × 10 ⁸ cells / cm ^2. May be good.

The cells after thawing can be appropriately replaced with a medium by a method known or well known to those skilled in the art. The medium can be changed, for example, every 1 to about 7 days, preferably about 1 to about 4 days, and more preferably every 1 day.

Further, the cells after thawing can be appropriately subcultured by a method known or well known to those skilled in the art. Subculture can be performed, for example, every 1 to about 7 days, preferably every 2 to about 5 days, more preferably every 3 to about 4 days.

The thawed cells may be used as they are or after being passaged several times and then subjected to treatments known to those skilled in the art as necessary, and may be used, for example, in vitro, or the cells themselves may be used as a subject (for example,). , Mice, humans, etc.).

Hereinafter, the present invention will be described in more detail with reference to Examples, but these Examples do not limit the scope of the present invention at all.

Examples, reagents used as synthetic raw materials such as fluorescent labels, and sources are shown below:
1-dodecanal, 2,2,6,6-tetramethylpiperidin-1-oxyl and butylaldehyde (primary) were obtained from Wako Pure Chemical Industries, Ltd., and D- (+)-trehalose dihydrate, methyl. α-D-Glucopyranoside, p-toluenesulfonic acid monohydrate, triethyl orthoformate and iodobenzene diacetate were obtained from Tokyo Chemical Industry Co., Ltd., and were obtained from Tokyo Chemical Industry Co., Ltd. Was obtained from Sigma Aldrich Japan Co., Ltd.

N, N-dimethylformamide (DMF) (for ultra-dehydration and organic synthesis) and dichloromethane (for ultra-dehydration and organic synthesis) used as reaction solvents were obtained from Wako Pure Chemical Industries, Ltd.
Triethylamine (special grade), sodium sulfate (special grade), sodium hydrogen carbonate (special grade), sodium thiosulfate (special grade), diethyl ether (special grade), sodium chloride (special grade) and toluene (special grade) used in the reaction post-treatment and purification It was obtained from Wako Pure Chemical Industries, Ltd., and hexane (special grade), ethyl acetate (special grade), methanol (special grade) and chloroform (special grade) were obtained from Kanto Chemical Industries, Ltd.
Pure water was used as the water.
Deuterated chloroform (containing 0.05% TMS (tetramethylsilane)) and deuterated DMSO (containing 0.05% TMS (tetramethylsilane)) used for NMR measurement were obtained from Wako Pure Chemical Industries, Ltd.

In addition, the equipment and conditions used for various measurements and analyzes are shown below.
(1) 1H-NMR spectrum / equipment: JNM-ECS 400, manufactured by JEOL Ltd. (2) Mass spectrometer: JMS-700, manufactured by JEOL Ltd. (3) Elemental analyzer / equipment: Yanako CHN coder MT-5 type, MT-6 type, manufactured by Yanako Analytical Industry Co., Ltd.

[Example 1]

1-dodecanal (0.74 g, 4.0 mmоl), p-toluenesulfonic acid monohydrate in a DMF (20 mL) suspension solution of D- (+)-trehalose dihydrate (1.9 g, 5.0 mmol). Japanese product (190 mg, 1.0 mmol) and triethyl orthoformate (666 μL, 4.0 mmol) were added at room temperature. The flask containing the reaction solution was connected to a rotary evaporator, the bath temperature was set to 70 ° C., and the inside of the system was rotated for 5 hours while reducing the pressure to 220 hPa. After 5 hours, the mixture was allowed to cool to room temperature, and triethylamine (280 μL, 2.0 mmol) was added. The reaction solution was stirred at room temperature for 5 minutes and then concentrated under reduced pressure. After adding methanol (20 mL) and chloroform (20 mL) to the residue, the insoluble material was removed by filtration, and the filtrate was concentrated. Column chromatography of residue (aminopropyl silane modified silica gel, chloroform: methanol = 100: 0 to 70:30 (v / v), and silica gel, chloroform: methanol = 100: 0 to 70:30 (v / v)) The compound of Example 1 was obtained as a white solid.
Yield 51% (1.04 g), HRMS (FAB +): m / z calc'd for C24H45O11 (M + H) +: 509.2962; found: 509.2962. , 1H NMR (400MHz, DMSO-d6): δ5.05 (1H, d, J = 5.0Hz), 4.93-4.85 (3H, m), 4.83 (1H, d, J = 3) .7Hz), 4.76 (2H, d, J = 5.0Hz), 4.51 (1H, t, J = 5.0Hz), 4.34 (1H, t, J = 6.0Hz), 3 .92 (1H, dd, J = 4.6, 9.6Hz), 3.83 (1H, dt, J = 4.6, 9.6Hz), 3.71-3.60 (2H, m), 3.60-3.28 (5H, m), 3.28-3.20 (1H, m), 3.19-3.05 (2H, m), 1.58-1.44 (2H, m) ), 1.42-1.15 (18H, m), 0.86 (3H, J = 6.9Hz)

[Example 2]

Oleyl alcohol (6.3 g, 20 mmol) and 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) (0.31 g, 0.2 mmol) in a dichloromethane (20 mL) solution. , Iodobenzene diacetate (7.1 g, 22 mmol) was added at room temperature, and the mixture was stirred for 1 hour. After 1 hour, dichloromethane (100 mL) was added to the reaction solution, sodium thiosulfate (100 mL) was added, the organic layer was separated, and the mixture was extracted twice with dichloromethane (100 mL). The organic layers were combined and washed with saturated sodium hydrogen carbonate (100 mL) and saturated brine (100 mL). After washing, the organic layer was separated, sodium sulfate was added and dried, and then sodium sulfate was removed by filtration to concentrate the filtrate. The residue was purified by column chromatography (silica gel, hexane: ethyl acetate = 100: 0 to 95: 5 (v / v)) to give oleylaldehyde as a colorless liquid (yield 93% (5.0 g)). ..
D- (+)-trehalose dihydrate (1.9 g, 5.0 mmol) in DMF (20 mL) suspension solution with oleylaldehyde (1.07 g, 4.0 mmоl), p-toluenesulfonic acid monohydration The product (190 mg, 1.0 mmol) and triethyl orthoformate (666 μL, 4.0 mmol) were added at room temperature. The flask containing the reaction solution was connected to a rotary evaporator, the bath temperature was set to 70 ° C., and the inside of the system was rotated for 5 hours while reducing the pressure to 220 hPa. After 5 hours, the mixture was allowed to cool to room temperature, and triethylamine (280 μL, 2.0 mmol) was added. The reaction solution was stirred at room temperature for 5 minutes and then concentrated under reduced pressure. Chloroform / methanol (2 / 1, v / v) (50 mL) was added to the residue, the insoluble material was removed by filtration, and the filtrate was concentrated. Column chromatography of residue (aminopropyl silane modified silica gel, chloroform: methanol = 100: 0 to 70:30 (v / v), and silica gel, chloroform: methanol = 100: 0 to 70:30 (v / v)) The compound of Example 2 was obtained as a white solid.
Yield 51% (1.21 g), HRMS (FAB +): m / z calc'd for C30H55O11 (M + H) +: 591.3744; found: 591.3742. , 1H NMR (400MHz, DMSO-d6): δ5.40-5.27 (2H, m), 5.05 (1H, d, J = 5.0Hz), 4.93-4.85 (3H, m) ), 4.83 (1H, d, J = 3.2Hz) 4.76 (2H, d, J = 5.0Hz), 4.50 (1H, t, J = 5.0Hz), 4.34 ( 1H, t, J = 6.0Hz), 3.92 (1H, dd, J = 5.0, 10.1Hz), 3.83 (1H, dd, J = 5.0, 10.1Hz), 3 .71-3.60 (2H, m), 3.60-3.42 (3H, m), 3.42-3.28 (3H, m), 3.28-3.20 (1H, m) , 3.18-3.05 (2H, m), 2.04-1.89 (3H, m), 1.58-1.43 (2H, m), 1.40-1.15 (22H, 22H, m), 0.86 (3H, t, J = 6.9Hz)

[Example 3]

Butyraldehyde (0.29 g, 4.0 mmоl) and p-toluenesulfonic acid monohydrate in a DMF (20 mL) suspension solution of D- (+)-trehalose dihydrate (1.9 g, 5.0 mmol). A product (190 mg, 1.0 mmol) and triethyl orthoformate (666 μL, 4.0 mmol) were added at room temperature, and the mixture was heated and stirred at 70 ° C. for 5 hours. After 5 hours, the mixture was allowed to cool to room temperature, and triethylamine (280 μL, 2.0 mmol) was added. The reaction solution was stirred at room temperature for 5 minutes and then concentrated under reduced pressure. The residue was purified by column chromatography (aminopropylsilane modified silica gel, chloroform: methanol = 50:50 (v / v), and silica gel, chloroform: methanol = 100: 0 to 70:30 (v / v)). The obtained white solid was washed with diethyl ether (50 mL) and purified by column chromatography (silica gel, chloroform: methanol = 100: 0 to 70:30 (v / v)) to obtain the compound of Example 3 as a white solid. I got it in.
Yield 13% (0.21 g), HRMS (FAB +): m / z calc'd for C16H29O11 (M + H) +: 397.1710; found: 397.1710. , 1H NMR (400MHz, DMSO-d6): δ5.03 (1H, d, J = 5.0Hz), 4.92-4.80 (4H, m), 4.75 (2H, d, J = 5) .0Hz), 4.53 (1H, t, J = 5.0Hz), 4.33 (1H, t, J = 6.0Hz), 3.93 (1H, dd, J = 5.0, 9. 6Hz), 3.83 (1H, dt, J = 5.0, 9.6Hz), 3.72-3.61 (2H, m), 3.60-3.28 (5H, m), 3. 28-3.20 (1H, m), 3.19-3.06 (2H, m), 1.56-1.46 (2H, m), 1.42-1.28 (2H, m), 0.87 (3H, t, J = 7.3Hz)

[Example 4]

In a suspension solution of methyl α-D-glucopyranoside (7.8 g, 40 mmol) in DMF (50 mL), p-toluenesulfonic acid monohydrate (190 mg, 1 mmol), triethyl orthoformate (6.7 mL, 40 mmol), 1 -Dodecanal (7.4 g, 40 mmol) was added at room temperature. The flask containing the reaction solution was connected to a rotary evaporator, the bath temperature was set to 50 ° C., and the inside of the system was rotated for 6 hours while reducing the pressure to 50 hPa. After 6 hours, the mixture was allowed to cool to room temperature, saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was concentrated under reduced pressure. Toluene (200 mL) and water (200 mL) were added to the residue and shaken vigorously in a separating funnel. The organic layer was separated, dried over sodium sulfate, sodium sulfate was removed by filtration, and the solvent was distilled off under reduced pressure. Hexane (200 mL) was added to the residue and the mixture was stirred. The obtained suspension was filtered to obtain a white solid. Hexane (100 mL) was added to the obtained white solid, and the mixture was stirred while cooling in an ice bath. The obtained suspension was filtered, washed with chilled hexane, and the obtained powder was dried to obtain the compound of Example 4.
Yield 63% (9.1 g), Anal. calc'd fоr C19H36O6: C, 63.31, H, 10.07; found: C, 63.26, H, 10.06. , 1H NMR (400MHz, CDCl3): δ4.76 (1H, d, J = 3.7Hz), 4.54 (1H, t, J = 5.0Hz), 4.12 (1H, dd, J = 4) .6,5.0,10.1,10.5Hz), 3.85 (1H, dt, J = 2.3,9.2Hz), 3.69-3.46 (3H, m), 3. 43 (3H, s), 3.26 (1H, t, J = 9.2Hz), 2.74 (1H, d, J = 2.3Hz), 2.29 (1H, d, J = 9.6Hz) ), 1.73-1.56 (2H, m), 1.47-1.16 (18H, m), 0.88 (3H, t, J = 6.9Hz)

[Comparative Example 1]

The one manufactured by Tokyo Chemical Industry Co., Ltd. (D- (+)-trehalose dihydrate) was used.

Test example

Common items [reagents, etc.]
・ BuySell (manufactured by Japan Freezer Co., Ltd.)
・ Freezing vial (manufactured by Thermo Fisher SCIENTIFIC)
・ DHDM (Murakami S, Ijima H, Ono T, Kawakami K., “Development of co-culture system oh hepatocytes with bone marrow cells for expression and maintenance of hepatic functions.,” Int J Artif Organs, 27, 2, 118- Prepared with the following composition with reference to 126, 2004)

[Preparation of primary rat cultured hepatocytes]
SD rats (male) 6-8 weeks old were anesthetized and cannulated into the portal vein. After blood removal with preperfusion fluid (see Table 2), extracellular matrix components were digested with collagenase solution (see Table 2), the liver was separated from surrounding tissues, and the liver was suspended in DMEM. The liver was cut with a scalpel and sieved with a 150 μm / 45 μm mesh to remove undigested liver tissue and surrounding tissues. The obtained cells were suspended in DMEM, and non-parenchymal cells and hepatic parenchymal cells were separated by repeating centrifugation resuspension three times. The number of cells in the cell suspension was measured by the trypan blue dye exclusion method. The trypan blue solution was prepared by dissolving trypan blue in PBS at a concentration of 3.0 g / L.

Test Example 1: a compound of frozen viable cell number of evaluation after thawing ^{0-10 -2} wt% of Example 1 or 2, in DHDM containing a 10 wt% DMSO, and 1 × ¹⁰ 6 cells / mL Rat primary cultured hepatocytes were suspended so that 1 mL of cell suspension (1 × 10 ⁶ cells / vial) was added to the freezing vial. Freezing vials were placed in the bicelle and the cells were frozen in a deep freezer at -80 ° C. The freezing vial was then placed in liquid nitrogen. The freezing vial was placed in a warm bath at 37 ° C., thawed 1/2 of the suspension, rapidly diluted with DMEM (5 mL) cooled to about 4 ° C., and centrifuged (500 rpm, 90 seconds). The supernatant was removed and the cells were suspended in DHDM (0.5 mL). Then, the number of living cells was counted by the trypan blue pigment exclusion method. The results are shown in FIGS. 1A-1D.

Results The number of viable cells after freezing and thawing increased by using the compound of Examples 1, 2, 3 or 4 and DMSO as compared with the case where only DMSO was used (0% by mass). Further, in the compounds of Examples 1 to 4, the number of viable cells increased even ^{at least about 1 × 10 -6} % by mass, and the number of viable cells was the largest at ^{1 × 10 -6% by mass.} Therefore, the compounds represented by the formula I-1 or I-2 described in the present specification, or salts or solvates thereof, are frozen even in the presence of a cell membrane-permeable cell cryoprotectant such as DMSO. It is considered that cytotoxicity such as a decrease in the number of viable cells after thawing (particularly, the number of viable cells such as primary cultured cells) can be suppressed.
When the compounds of Examples 1 to 4 ^{were used in an amount of about 1 × 10 -7} to about 1 × 10 ⁻² mass%, the number of living cells was on the low concentration side (for example, about 1 × 10 ^-7 to about 1). It ^{may have a first peak on the x10-5} % by mass) side and a second peak on the high concentration side (eg, about ^1x10-4 to about ^1x10-2 % by weight).

Test Example 2: Culture evaluation after freeze-thaw 1 × 10 ^-6 % by mass of Example 2 compound, 1 × 10 ^-4 % by mass of Comparative Example 1 compound, or both, and 10% by mass of DMSO. Rat primary cultured hepatocytes were suspended in DHDM containing 1 × 10 ⁶ cells / mL, and 1 mL (1 × 10 ⁶ cells / vial) of cell suspension was added to a freezing vial. For comparison, a solution (unfrozen cells) in which rat primary cultured hepatocytes were similarly suspended in DHDM and not subjected to the freeze-thaw operation described later was also prepared. Freezing vials were placed in the bicelle and the cells were frozen in a deep freezer at -80 ° C. The freezing vial was then placed in liquid nitrogen. The freezing vial was placed in a warm bath at 37 ° C., 1/2 of the suspension was thawed, diluted rapidly with DMEM (5 mL) and centrifuged (500 rpm, 90 seconds). The supernatant was removed and the cells were suspended in DHDM (2.45 mL) supplemented with 10% FBS (recovery: about 20%, from 1 × 10 ⁶ cells / vial, 2.5 × 10 ⁴ cells /). cm ² and so as to estimate: seeded at 12.5 × ¹⁰ 4 cells- freeze number of viable cells / cm ^2). This cell suspension was mixed with a collagen film (Cell matrix Type IC manufactured by Nitta Gelatin Co., Ltd. with a pH 3.0 HCl aqueous solution at a ratio of 1: 9, 40 μL / well was placed on a well plate, and air-dried for 12 hours. After that, seeding was performed at 70 μL / well on DMEM 60 μL / well (washed twice) (2.5 × 10 ⁴ cells / cm ² ), and monolayer culture on collagen film (culture medium: 10% FBS). DHDM, 37 ° C./5% CO ₂ incubator) was started. After 4 hours, the medium was changed. After that, the medium is changed every other day, and the viable cell activity (1, 3, 5 days after seeding), drug metabolism (3, 5 days after seeding), and albumin production ability (after seeding) are changed by the method described later. 1 to 3 days and 3 to 5 days after sowing) and ammonia metabolism (1 day after sowing) were evaluated. The results are shown in FIGS. 2A-2E.

[Evaluation of living cell activity]
The culture medium of rat primary cultured hepatocytes was replaced with DHDM supplemented with 10% Cell Counting Kit-8 (Dougen Kagaku Kenkyusho) (80 μL / well-96 well plate), and _{incubated at 37 ° C. and 5% CO 2} for 2 hours. did. Then, 8 μl / well of 0.1 M HCl aqueous solution was added to stop the reaction, and the absorbance (450 nm) of 70 μl / well of the medium was measured to evaluate the viable cell activity.
[Evaluation of drug metabolism (EROD)]
The culture medium was replaced with 2 μM 3MC-added medium (80 μL / well-96 well plate) and _{incubated at 37 ° C. under 5% CO 2} for 24 hours. Then, it was replaced with DHDM supplemented with 10 μM ER (7-Ethoxyresorufin) (80 μL / well-96 well plate), and _{incubated at 37 ° C. and 5% CO 2} for 1 hour. The fluorescence intensity (excitation wavelength: 530 nm, fluorescence wavelength: 580 nm) of 75 μL / well of the medium was measured, and the drug-metabolizing activity was evaluated.

[Evaluation of albumin production ability]
The albumin production rate of hepatocytes was determined as one index of the protein synthesis ability of the liver.
Culture media 1 to 3 days after sowing and 3 to 5 days after sowing were collected and analyzed using the ELISA method (protein detector ELISA kit HRP / ABTS system, Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA). did. Rat albumin standards and antibodies purchased from ICN Pharmaceuticals (Aurora, OH, USA) were used.

[Evaluation of ammonia metabolism]
The culture medium was replaced with 1 mM NH ₄ Cl-added medium (70 μL / well-96 well plate) and _{incubated at 37 ° C. under 5% CO 2} for 4 hours. Then, the medium was collected, and the ammonia concentration in the medium was measured using Ammonia Test Wako (Fujifilm Wako Pure Chemical Industries, Ltd.) to evaluate the ammonia metabolic capacity of the cells after freezing and thawing.

Results The viable cell activity on

days

1, 3 and 5 after seeding was higher when DMSO and the compound of Example 2 were used as compared with the case where only DMSO was used (Fig. 2A). Therefore, it is considered that the compound represented by the formula I-1 or I-2 described in the present specification or a salt or solvate thereof has a small effect on the viable cell activity as compared with the case where DMSO alone is used. Be done. In addition, the compound of Example 2 had higher viable cell activity on the 5th day after seeding as compared with the compound of Comparative Example 1 (FIG. 2A). Furthermore, the viable cell activity was similar between the case where the compound of Example 2 and the compound of Comparative Example 1 were used in combination and the case where the compound of Example 2 was used alone (FIG. 2A).
When DMSO and the compound of Example 2 were used, they showed EROD activity comparable to or higher than that of unfrozen cells (Fig. 2B). Therefore, it is considered that the compound represented by the formula I-1 or I-2 described in the present specification or a salt or solvate thereof does not have a negative effect on drug metabolism activity and the like.
Compared with the case of using DMSO alone, when the compound of DMSO and Example 2 was used, the albumin-producing ability was higher 1 to 3 days after sowing and 3 to 5 days after sowing (Fig. 2C). .. Therefore, the compound represented by the formula I-1 or I-2 described in the present specification or a salt or solvate thereof has a small effect on the protein synthesis ability of the cell as compared with the case where DMSO alone is used. it is conceivable that. In addition, the compound of Example 2 had higher albumin-producing ability 1 to 3 days after sowing and 3 to 5 days after sowing as compared with the compound of Comparative Example 1 (FIG. 2C). Furthermore, when the compound of Example 2 and the compound of Comparative Example 1 were used in combination, the albumin-producing ability was equal to or higher than that of unfrozen cells (FIG. 2C). In addition, the albumin production rate and the albumin production rate per living cell (albumin production rate divided by the living cell activity) showed the same tendency (FIGS. 2C and 2D).
Compared with the case of using DMSO alone, when DMSO and the compound of Example 2 were used, the ammonia metabolism ability was higher on the first day after sowing (Fig. 2E). Therefore, the compound represented by the formula I-1 or I-2 described in the present specification or a salt or solvate thereof has a small effect on the ammonia metabolism ability of the cell as compared with the case where DMSO alone is used. it is conceivable that.

Test Example 3: Evaluation of cell membrane fluidity Rat primary so as to be ^{1 × 10 6} ^{cells / mL in DHDM containing 10-7} % by weight (16 nM) of the compound of Example 2 and 10% by weight of DMSO. The cultured hepatocytes were suspended and cryopreserved in the same manner as in Test Example 1. The cell membrane fluidity of the cell suspension thawed by the same method as that described in Test Example 1 was evaluated using the MarkerGene (registered trademark) membrane fluidity assay (Cosmo Bio Co., Ltd.). The results are shown in FIG.

Results When the primary cultured hepatocytes were frozen by adding the compound of Example 2, the fluidity of the cell membrane was increased as compared with the case where DMSO alone was used. Cooling cells reduces membrane fluidity and / or domain formation, resulting in increased leakage of intracellular solutes and / or irreversible structural changes that can impair cell function (H Sieme et al). , Sperm Membrane Behavior during Cooling and Cryopreservation, Reprod Domest Anim, Suppl 3: 20-6, 2015). On the other hand, under conditions where the fluidity of the membrane increases at low temperatures, such a phenomenon can be suppressed and suitable for cryopreservation (Phillip H Purdy et al, Membrane modification strategies for cryopreservation, Methods Mol Biol., 1257). : 337-42, 2015). Therefore, the compound represented by the formula I-1 or I-2 described in the present specification or a salt or solvate thereof can increase the fluidity of the cell membrane, and thus may be useful for cryopreservation of cells. Conceivable.

Test Example 4: Evaluation of cytotoxicity for non-freeze-thawed cells Collagen coat (Cell matrix Type IC manufactured by Nitta Gelatin Co., Ltd. and pH 3.0 HCl aqueous solution are mixed 1: 9 and then on a well plate. Was added to the cells at 40 μL / well, air-dried for 1 hour, and then washed twice with DMEM at 60 μL / well), and rat primary cultured hepatocytes were added to the wells at a cell density of ^{2.5 × 10 4} cells / cm ^2. The seeds were sown and monolayer culture was started. After 4 hours, the medium was changed. On the first day after sowing, ^{the compound was replaced with DHDM or DHDM containing 0 to 10-3} % by mass of the compound of Example 2 and 10% by mass of DMSO, and _{incubated at 37 ° C. and 5% CO 2} for 3 hours. did. Then, it was replaced with DHDM (80 μL / well-96 well plate) supplemented with 10% Cell Counting Kit-8 (Institute of Dominant Chemistry), and _{incubated at 37 ° C. under 5% CO 2} for 2 hours. Then, 8 μl / well of 0.1 M HCl aqueous solution was added to stop the reaction, and the absorbance (450 nm) of 70 μl / well of the medium was measured to evaluate the living cells. The results are shown in FIG.

Results Rat primary cultured hepatocytes are viable cells when they contain at least 10% by weight DMSO and ^0-10-4 % by weight of the compound of Example 2 as compared to when they contain only 10% by weight DMSO. No decrease was observed. Therefore, when the compound represented by the formula I-1 or I-2 described in the present specification or a salt or solvate thereof is contained at ^{a concentration of at least 10-4} % by mass, cells that have not been frozen and thawed. It is considered that the cytotoxicity is low.

Test Example 5: Evaluation of Resistance to Shear Stress ^{1 × 10 6} cells / mL in DHDM containing 0 to 1 × 10 ^-6 mass% of the compound of Example 1 and 10 mass% DMSO. Rat primary cultured hepatocytes were suspended in. For comparison, a solution in which rat primary cultured hepatocytes were similarly suspended in DHDM was also prepared. The resulting cell suspension was allowed to stand on ice for 10 minutes. The medium of the cell suspension was replaced with DHDM and shear stress was applied by vortexing for 30-60 seconds. Then, a collagen coat (Cell matrix Type IC manufactured by Nitta Gelatin Co., Ltd.) was mixed with a pH 3.0 HCl aqueous solution at a ratio of 1: 9 so as to be ^{2.5 × 10 4 cells / mL, and then placed on a well plate.} Cells were seeded on 40 μL / well, air-dried for 1 hour, and washed twice with DMEM 60 μL / well), and monolayer culture on a collagen coat (culture medium: DHDM, 37 ° C./5% CO ₂ incubator). ) Was started, and the viable cell activity on the first day after seeding was evaluated by measuring the absorbance. The results are shown in FIG.

Results Compared with the case of using DMSO alone (0% by mass), the use of the compound of Example 1 together with DMSO resulted in higher viable cell activity in cells after shear stress was applied. Then, by using the compound of Example 1, the viable cell activity was comparable to that of the medium alone (DHDM). Therefore, it is considered that the compound represented by the formula I-1 or I-2 described in the present specification or a salt or solvate thereof has resistance to stimuli such as shear stress.

Test Example 6: Evaluation of dynamic viscoelasticity ^{3 × 10 7} cells of primary cultured rat liver in DHDM (5 mL) containing 1 × 10 ^-6 mass% of the compound of Example 2 and 10 mass% DMSO. The cells were suspended. For comparison, a solution in which rat primary cultured hepatocytes were similarly suspended in DHDM and a solution in which rat primary cultured hepatocytes were similarly suspended in DHDM containing 10% by mass of DMSO were also prepared. The resulting cell suspension was allowed to stand on ice for 15 minutes. The cell suspension was centrifuged (300 rpm, 90 seconds) and the cells were suspended in DHDM (1 mL). Further centrifugation (500 rpm, 90 seconds) was performed to remove the supernatant, 200 μL of cell pellet was placed on a measuring dish, and dynamic viscoelasticity was measured by a rheometer (manufactured by Antonio Par, MCR 302) (strain dispersion measurement, angular frequency). 1Hz, strain: logarithmic elevation 1-100%, CP-25). Usually, G'can indicate the elastic modulus based on Hooke's law, and G'' can indicate the elastic modulus based on Newton's law. Generally, G'is understood as a solid element and G'is a liquid element, and when G'and G'are expressed on the complex plane, the viscoelasticity is integrated according to the inclination and the distance from the origin. It is possible to evaluate in a targeted manner. The results are shown in FIG. 6A (relationship diagram between storage elastic modulus and loss elastic modulus) and FIG. 6B (comprehensive evaluation of viscoelasticity).

Results It is considered that cells in which DMSO alone is added to DHDM tend to have a liquid component, and cells containing only DHDM tend to have a solid component. The cells co-added with the compound of Example 2 and DMSO showed viscoelasticity similar to that of DHDM-only cells. Therefore, the compound represented by the formula I-1 or I-2 described in the present specification, or a salt or solvate thereof, is a cell even in the presence of a cell membrane-permeable cell cryoprotectant such as DMSO. It is considered that it may have the original viscoelasticity.

Test Example 7: Evaluation of action position on cells The action position on cells was evaluated using a fluorescently labeled Example compound.

<Synthesis of oleyltrehalose-fluorescein (Oleyl-Treh-FL) (fluorescent label of the compound of Example 2)>
Fluorescein isothiocyanate and Isomer I (195 mg, 0.5 mmоl) were added to a solution of the compound of Example 2 (295 mg, 0.5 mmol) in DMF (2 mL), and the mixture was stirred at room temperature for 7 days. After 7 days, the DMF was removed under reduced pressure and the residue was column chromatographed (silica gel, hexane: ethyl acetate = 40: 60-20 / 80 (v / v), chloroform: methanol = 90: 10-80: 20 (). Purification with v / v)) gave the desired product, oleyl-fluorescein, as a reddish brown solid. : Yield 8% (40 mg), HRMS (FAB +): m / z calc'd for C51H66NO16S (M + H) ⁺ : 980.4102; found: 980.4102. , Anal. calc'd fоr C51H65NO16S (+ 1 / 2H ₂ O): C, 61.93, H, 6.73, N, 1.42; found: C, 61.97, H, 6.72, N, 1.44 ..

<Synthesis of trehalose-fluorescein (Treh-FL) (comparative target, fluorescent label of trehalose)>
To a solution of D- (+)-trehalose dihydrate (95 mg, 0.25 mmol) in DMF (2 mL), add fluorosane isothiocyanate and Isomer I (78 mg, 0.2 mmоl) and heat at 70 ° C. for 5 hours. did. After 5 hours, DMF is removed under reduced pressure and the residue is purified by column chromatography (silica gel, ethyl acetate: methanol = 90/10 to 85/15 (v / v)) to give the desired trehalose-fluorescein. Obtained as a reddish-brown solid .: Yield 10% (30 mg), HRMS (FAB +): m / z silica gel for C33H34NO16S (M + H) ⁺ : 732.598; found: 732.159., Anal.calc'd fоr C33H33NO16S: C, 54.17, H, 4.55, N, 1.91; found: C, 54.16, H, 4.85, N, 1.83.

<Synthesis of oleyl-fluorescein (Oleyl-FL) (comparison target, fluorescent label of oleyl group)>
Fluorescein isothiocyanate and Isomer I (195 mg, 0.5 mmоl) were added to a solution of oleyl alcohol (158 mg, 0.5 mmol) in DMF (2 mL), and the mixture was stirred at room temperature for 7 days. After 7 days, the DMF was removed under reduced pressure, the residue was purified by column chromatography (silica gel, hexane: ethyl acetate = 80: 20 (v / v)), and the target oleyl-fluorescein was a light brown solid. Obtained as. : Yield 43% (140 mg), HRMS (FAB +): m / z calc'd for C39H48NO6S (M + H) ⁺ : 658.3202; found: 658.3203. , Anal. calc'd fоr C39H47NO6S (+ 1 / 3H ₂ O): C, 70.56, H, 7.24, N, 2.11; found: C, 70.46, H, 7.24, N, 2.11. .. , ^{1 1} H NMR (400 MHz, CDCl ₃ ): δ11.50 (1H, br), 10.13 (2H, br), 8.70-8.75 (2H, br), 7.24 (1H, d, J = 8.2Hz), 6.67 (2H, d, J = 1.8Hz), 6.62-6.49 (4H, m), 5.43-5.19 (2H, m), 4. 51 (2H, br), 2.06-1.85 (3H, m), 1.82-1.68 (2H, m), 1.50-1.10 (22H, m), 0.83 ( 3H, J = 6.4Hz).

<Evaluation method>
^{1 × 10 6} cells / in PBS containing 0.001% by weight (16 μM) of oleyltrehalose-fluorescein (Oleyl-Treh-FL, fluorescent label of the compound of Example 2) and 10% by weight of DMSO. Rat primary hepatocytes were suspended to make mL. For comparison, solutions in which rat primary hepatocytes were suspended in 16 μM fluorescein (FL), 16 μM trehalose-fluorescein (Treh-FL) or 16 μM oleyl-fluorescein (Oleyl-FL) were also prepared. The obtained cell suspension was allowed to stand on ice for 1 hour and washed once with DHDM. The cells were suspended in DHDM again and the cells were observed using a fluorescence microscope. The results are shown in FIG.

Results Fluorescein, trehalose-fluorescein (Treh-FL), and oleyl-fluorescein (Oleyl-FL) did not show fluorescence in cells. Therefore, it is considered that fluorescein, trehalose alone, or oleyl group alone does not localize around the cell membrane. On the other hand, fluorescently labeled oleyltrehalose (Oleyl-Treh-FL, a fluorescently labeled compound of the compound of Example 2) showed fluorescence in the cell contour. Therefore, it is considered that the compound represented by the formula I-1 or I-2 described in the present specification or a salt or solvate thereof is localized around the cell membrane.

The compounds of formula I-1 or I-2, or a salt or solvate thereof described herein, the hydrophobic group (preferably in the molecule, as defined by R ₅ in formula I It has an aliphatic hydrocarbon group) and a portion similar to sugar. Based on such chemical structural characteristics and the results of Test Examples 1 to 6, the hydrophobic group in the molecule and / or the portion similar to the sugar in the molecule (preferably, the hydrophobicity in the molecule). It is considered that the group can suppress the destabilization of the cell membrane (that is, stabilize the cell membrane) which may be caused by the cell membrane-permeable cell cryoprotectant. Then, from the result of Test Example 7, in the stabilization of the cell membrane, the compound represented by the formula I-1 or I-2 described in the present specification, or a salt or solvate thereof is localized around the cell membrane. It is thought that this may be due to the fact that it acts directly on the cell membrane.
Furthermore, based on the results of Test Example 1 and the like, when cells are frozen and thawed together with the compound represented by the formula I-1 or I-2 described in the present specification, or a salt or solvate thereof, the number of viable cells is determined. The first peak is on the low concentration side (for example, about 1 × 10 ^-7 to about 1 × 10 ^-5 % by mass), and the high concentration side (for example, about 1 × 10 ^-4 to about 1 × 10 ⁻² mass%). Can have a second peak. It is considered that the first peak on the low concentration side may be due to the stabilization of the cell membrane as described above. The second peak on the high concentration side is the dehydration concentration effect of the compound represented by the formula I-1 or I-2 described in the present specification or a salt or solvate thereof, and the formula I described in the present specification. The effect of suppressing ice crystal formation by the compound represented by -1 or I-2 or a salt or solvate thereof, and / or the compound represented by the formula I-1 or I-2 described in the present specification or a salt thereof or a salt thereof. It is believed that this may be due to the cytoprotective effect of moieties similar to sugars (eg glucose, trehalose, etc.) in the solvate (FIGS. 8A, 8B).

Claims

Formula I-1 or I-2:

[During the ceremony,
n is 0 or 1;
Both R 1 and R 2 are H, or R 1 and R 2 together form = O;
Both R 3 and R 4 are H, or R 3 and R 4 together form a bond;
R 5 is an aliphatic saturated or unsaturated hydrocarbon group of 1 to 30 carbon atoms;
R 6 is H or -CH 2 OH, where n is 0 if R 6 is -CH 2 OH;
R 7 is H or C 1-6 alkyl, or:

(During the ceremony,
m is 0 or 1;
R A is H or -NR C R D;
R B is H or -CH 2 OH, provided that when R B is -CH 2 OH, m is 0;
R C and R D are, independently, H, be a C 1-6 alkyl or -COR E;
R E is -OH or C 1-6 alkyl)
Selected from the group consisting of;
R 8 is H or -NR 9 R 10 ;
R 9 and R 10 are independently H, C 1-6 alkyl or -COR 11 ;
R 11 is -OH or C 1-6 alkyl]
The compound represented by, or a salt or solvate thereof.
The compound according to claim 1, or a salt or solvate thereof, wherein R 3 and R 4 form a bond together.
The compound according to claim 1 or 2, or a salt or solvate thereof, wherein n is 1.
Less than:

The compound according to any one of claims 1 to 3, or a salt or solvate thereof.
Less than:

The compound according to any one of claims 1 to 4, or a salt or solvate thereof.
Less than:

The compound according to any one of claims 1 to 5, or a salt or solvate thereof, selected from the group consisting of.
A composition for cell freezing, which comprises the compound according to any one of claims 1 to 6, or a salt or solvate thereof.
The composition for cell freezing according to claim 7, further comprising a cell membrane-permeable cell freeze-protecting agent.
The cell freezing composition according to claim 7 or 8, wherein the cell is a primary cultured cell.
A solution for freezing cells containing the compound according to any one of claims 1 to 6 in an amount of 1 × 10 -7 to 1 × 10 −2% by mass, or a salt or solvate thereof.
A composition for stabilizing a cell membrane containing the compound according to any one of claims 1 to 6, or a salt or solvate thereof.
A method for freezing cells, which comprises contacting the cells with the compound according to any one of claims 1 to 6, or a salt or solvate thereof.
A method for stabilizing a cell membrane, which comprises contacting a cell with the compound according to any one of claims 1 to 6, or a salt or solvate thereof.