JP2002068973A - Stem cell differentiation-inducing accelerator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To he a stem cell differentiation-including accelerator comprising a cyclohexenone long-chain alcohol derivative accelerating the differentiation induction of stem cells to cells exerting a specific biofunction, and so capable of giving medicines useful as prophylactic and therapeutic agents for diseases caused by the regression/decrease of various tissues or cells or by cell death, e.g. neuropathy, osteopathy, circulatory diseases and myopathy. SOLUTION: This stem cell differentiation-inducing accelerator comprises, as the active ingredient, a cyclohexenone long-chain alcohol derivative represented by formula (1) (wherein R1, R2 and R3 arc each H or methyl; and X is a 10-28C straight-chain or branched alkylene or alkenylene).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention relates to an agent for promoting stem cell differentiation which promotes the induction of stem cell differentiation.

[0002]

2. Description of the Related Art Alzheimer's disease and Pick's disease (Pick), mainly in the cerebral cortex, Parkinson's disease and Huntington's disease, mainly in the basal ganglia, spinocerebellar degeneration with the cerebellum, and spinal cord. In neurological diseases such as amyotrophic lateral sclerosis and bone diseases such as osteoporosis and fractures, tissues and organs are affected by the degeneration or decrease of cells such as nerve cells and osteoblasts or the death of cells. It is thought to lose function and develop.

On the other hand, stem cells typified by bone marrow stem cells, neural stem cells, epidermal stem cells, etc. are undifferentiated cells. Has greatly contributed to the maintenance of These stem cells can also be obtained from ES cells,
Cells have the ability to differentiate into any organ or tissue. Therefore, in recent years, attempts have been made to improve and treat the pathological condition by transplanting ES cells or stem cells into a tissue that has lost its function and specifically differentiating into cells expressing individual biological functions. There is a demand for the development of substances that promote the induction of differentiation of ES cells and stem cells.

On the other hand, in neurons and osteoblasts,
In the process of differentiating from each stem cell, differentiation promoting factors such as bone morphogenetic factor (BMP), brain-derived neurotrophic factor (BDNF), and fibroblast growth factor (bFGF) are known. Although it is conceivable to use it for the prevention and treatment of the disease, since such factors are peptides having a large molecular weight, they are easily degraded in vivo and cannot pass through the blood-brain barrier. Problem.

[0005] Therefore, there is a demand for the development of a synthetic compound that promotes differentiation induction from a stem cell to a mature cell and is small in molecular weight and easy to handle.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention to promote the induction of differentiation of stem cells, and to provide a low-dose useful for preventing and treating diseases caused by cell regression / decrease or cell death such as bone disease and neurological disease. To provide a molecular substance.

[0007]

Means for Solving the Problems In view of such circumstances, the present inventors have conducted various searches for low-molecular compounds that promote differentiation of stem cells into cells that express specific biological functions, and as a result, the following formula (1) The present inventors have found that a long-chain alcohol having a cyclohexenone skeleton represented by the formula (1) has an excellent stem cell differentiation induction promoting action, and completed the present invention.

That is, the present invention provides the following general formula (1)

[0009]

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Wherein R ¹ , R ² and R ³ each represent a hydrogen atom or a methyl group, and X represents a linear or branched alkylene or alkenylene group having 10 to 28 carbon atoms.
The present invention provides a stem cell differentiation induction promoter comprising a cyclohexenone long-chain alcohol derivative represented by the following formula:

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The agent for promoting stem cell differentiation of the present invention refers to a substance which promotes the induction of differentiation of stem cells into cells expressing a specific biological function. Here, the term “stem cells” refers to undifferentiated cells having the ability to differentiate into cells expressing individual biological functions, and undifferentiated cells obtained from ES cells, as well as directions for differentiating into specific organs and tissues. This includes undifferentiated progenitor cells that have been attached but whose morphology cannot be identified. In addition, cells that express a biological function in a specific tissue are not particularly limited, but include, for example, bone cells, nerve cells, blood vessels, muscles, and the like, and are preferably nerve cells.

In the cyclohexenone long-chain alcohol derivative represented by the general formula (1), X is a linear or branched alkylene or alkenylene group having 10 to 28 carbon atoms. In the case of the above, an alkyl group having 1 to 10 carbon atoms can be used as the side chain.
As the side chain alkyl group, for example, methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group,
Isopentyl group, neopentyl group, tert-pentyl group,
Examples include a hexyl group, an isohexyl group, a heptyl group, an octyl group, a nonyl group, and a decyl group. Of these, a methyl group is particularly preferred. The substitution of the side chain with a linear alkylene group or alkenylene group (meaning an alkene structure having at least one carbon-carbon double bond) is preferably at the 3- and / or 7-position. Of these Xs, the number of carbon atoms is 1
A linear alkylene group having 0 to 28 is more preferable, and a linear alkylene group having 10 to 18 carbon atoms is particularly preferable. R ¹ , R ² and R ³ each represent a hydrogen atom or a methyl group, but it is more preferred that at least one of them is a methyl group.

The compound represented by the above general formula (1) may be in the form of a pharmaceutically acceptable salt, or a solvent or hydrate thereof. The compound (1) may have various isomers, and these isomers are also included in the present invention.

The cyclohexenone long-chain alcohol derivative represented by the general formula (1) can be produced, for example, by the following production method A or production method B:
It can be manufactured according to

[0015]

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Wherein R ^1a , R ^2a and R ^3a represent a hydrogen atom or a methyl group, at least one of which represents a methyl group, Ph represents a phenyl group, and X, R ¹ , R ² and R ³ is the same as above)

That is, phenylsulfinate is reacted with cyclohexenone (2) or methyl-substituted 2-cyclohexen-1-one (3) in the presence of an acid to give compound (4), which is then reacted with ethylene glycol. To give a ketal compound (5), followed by reaction with ω-halogenoalkanol or ω-halogenoalkenol to give a compound (6), which is treated with an acid to remove the protecting group to give a compound (1). Can be

The methyl-substituted 2 used here as a raw material
-Cyclohexen-1-one (3) is obtained by reacting a methyl-substituted cyclohexanone with a trialkylsilyl halide in the presence of butyllithium, and then oxidizing in the presence of a palladium-based catalyst.

First, the reaction of cyclohexenone (2) or methyl-substituted 2-cyclohexen-1-one (3) with a phenylsulfinate, for example, sodium phenylsulfinate, is carried out by the presence of an acid such as hydrochloric acid, sulfuric acid or phosphoric acid. Below, 0
It is preferably performed at a temperature of 100 ° C. for 5 to 40 hours.

The ω-halogenoalkanol to be reacted with the ketal compound (5) is preferably ω-bromoalkanol. The reaction between the ketal compound (5) and the ω-halogenoalkanol is carried out in the presence of a metal compound such as butyllithium.
It is preferable to carry out under low temperature conditions.

The removal of the phenylsulfonyl group and the ketal protecting group from the obtained compound (6) is preferably carried out by reacting an acid such as paratoluenesulfonic acid.

[0022]

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Wherein X ¹ represents an alkylene or alkenylene group having 9 to 27 carbon atoms, Ac represents an acyl group,
R ¹ , R ² , R ³ and Ph are the same as described above.

That is, the compound (7) [for example, Synthe
sis, 1996, Nov.] with ω-bromoalcohol (8) to give compound (9), and then the phenylsulfonyl group is eliminated to give compound (10), which is protected with this hydroxy group. The compound (11) is oxidized to a compound (12), and then the hydroxy protecting group is eliminated to obtain a compound (1a).

The reaction between the compound (7) and the ω-bromoalcohol (8) is preferably carried out in the presence of a metal compound such as butyllithium at a low temperature.

The removal of the phenylsulfonyl group from the compound (9) is carried out, for example, by reacting a phosphate or the like in the presence of sodium amalgam.

The hydroxy-protecting group of compound (10) is preferably an acetyl group or the like. The protection reaction is carried out, for example, by reacting compound (10) with acetic anhydride.

The oxidation reaction of the compound (11) is carried out by reacting an alkyl hydroperoxide such as t-butyl hydroperoxide in the presence of a metal compound such as ruthenium trichloride.

The elimination reaction of the protecting group of the compound (12) is preferably carried out by hydrolysis in the presence of a base such as potassium carbonate.

The cyclohexenone long-chain alcohol derivative (1) of the present invention thus obtained specifically induces neural stem cells to differentiate into nerve cells as shown in the test examples described below. It is useful as a preventive and therapeutic agent for diseases caused by regression / decrease or cell death of various tissues and cells.

Since the cyclohexenone long-chain alcohol derivative (1) of the present invention has a low molecular weight, it can be administered either orally or parenterally (intramuscular, subcutaneous, intravenous, suppository, etc.).

When preparing an oral preparation, excipients and, if necessary, a binder, a disintegrant, a lubricant, a coloring agent, a flavoring agent, etc. are added, and then tablets, clothing, Tablets, granules, capsules, solutions, syrups, elixirs, oily or aqueous suspensions, and the like. Excipients include, for example, lactose, corn starch, sucrose, glucose, sorbit, crystalline cellulos and the like. As the binder, for example, polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose,
Gum arabic, tragacanth, gelatin, shellac, hydroxypropylcellulose, hydroxypropylstarch, polyvinylpyrrolidone and the like can be mentioned.

Examples of the disintegrant include starch, agar, gelatin-free, crystalline cellulose, calcium carbonate, sodium hydrogen carbonate, calcium citrate, dextran, pectin and the like. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, hydrogenated vegetable oil and the like. As the coloring agent, those permitted to be added to pharmaceuticals can be used. As the flavoring agent, cocoa powder, peppermint brain, aromatic acid, peppermint oil, dragon brain, cinnamon powder and the like can be used. In these tablets, the granules may be sugar-coated, gelatin-coated and optionally coated as needed.

When preparing an injection, a pH adjuster, a buffer, a stabilizing agent, a preservative and the like are added, if necessary, to give a subcutaneous, intramuscular or intravenous injection according to a conventional method. Injections
After storing the solution in a container, it may be prepared as a solid preparation by freeze-drying or the like to prepare a preparation for business use. Also, one dose may be stored in a container, or multiple doses may be stored in the same container.

The dose of the compound of the present invention as a pharmaceutical is usually 0.01 to 1000 mg per day for an adult human,
Preferably, a daily dose in the range of 0.1 to 100 mg is administered once a day or divided into 2 to 4 times.

[0036]

The present invention will be described in more detail with reference to the following examples.

Production Example 1 (1) 7 ml of N, N-diisopropylamine in 20 ml of TH
At −78 ° C., 35.4 ml of a 1.4 M n-butyllithium solution is added dropwise to the F solution. The solution is stirred at 0 ° C. for 30 minutes. 10 ml of 4-methylcyclohexane-1-one 4 ml
The above-mentioned diisopropylaminolithium (LDA) solution is added dropwise to the THF solution at -78 ° C. After stirring at -78 ° C for 1 hour, 6.5 ml of trimethylsilyl chloride is added dropwise. After stirring at room temperature for 1 hour, the solution is poured into aqueous sodium hydrogen carbonate and extracted with ether. The organic layer is washed with saturated saline and dried over magnesium sulfate. After evaporating the solvent under reduced pressure, the residue is distilled under reduced pressure to obtain 5.83 g of 4-methyl-1- (trimethylsilyloxy) -1-cyclohexene.
(Yield: 96%)

4-Methyl-1- (trimethylsilyloxy) -1-cyclohexene Molecular weight: 184 (C ₁₀ H ₂₀ OSi) TLC: (hexane-ethyl acetate: 8-2) Rf = 0.8 ¹ H-NMR (200 MHz, CDCl 2) _{3) δ: 0.17 (s,} 9H, Si- (CH 3) 3),
0.94 (d, J = 6.2Hz, 3H, H-7), 1.2-1.43 (m, 1H, H-4),
1.57-1.76 (m, 3H, H-3, 6), 1.88-2.14 (m, 3H, H-5),
4.8-4.83 (m, 1H, H-2). ¹³ C-NMR (50 MHz, CDCl ₃ ) δ: 0.3 (Si- (CH ₃ ) ₃ ), 21.2 (C-
7), 28.3 (C-4), 29.6 (C-5), 31.3 (C-6), 32.3 (C-3),
103.5 (C-2), 150.1 (C-1). IR (NaCl): 3052, 3021, 2954, 2926, 1670, 1457, 137
1, 1252, 1190, 1046,892, 844.

(2) A catalytic amount of palladium acetate was added to a solution of 3.53 g of 4-methyl-1- (trimethylsilyloxy) -1-cyclohexene in 70 ml of dimethylsulfoxide (DMSO), and oxygen was introduced for 6 hours, followed by stirring. Water is added at 0 ° C., and the mixture is filtered and extracted with ether. The solvent is distilled off from the organic layer under reduced pressure, and the residue is dissolved in hexane-water and extracted with hexane. The hexane layer is washed with saturated saline and dried over magnesium sulfate. The solvent is distilled off under reduced pressure to obtain an oil of 4-methyl-2-cyclohexen-1-one. (Yield 72%)

[0040] 4-Methyl-2-cyclohexen-1-one Molecular _{weight: 110 (C 7 H 10 O} ) TLC :( hexane - ethyl ^{acetate: 8-2) Rf = 0.35 1 H} -NMR (200MHz, CDCl 3) δ ： 1.15 (d, J = 7.1Hz, 3H, H-7),
1.56-1.76 (m, 1H, H-5a), 2.1 (dqa, Jgem = 13.3Hz, ³ J =
4.9Hz, 1H, H-5e), 2.26-2.48 (m, 2H, H-6), 2.49-2.62
(m, 1H, H-4), 5.94 (dd, ³ J = 10.1Hz, ⁴ J = 2.5Hz, 1H, H-
2), 6.79 (ddd, ³ J = 10.1Hz, ³ J = 2.7Hz, ⁴ J = 1.5Hz, 1H, H-
3). ¹³ C-NMR (50 MHz, CDCl ₃ ) δ: 20.1 (C-7), 29.6 (C-5), 3
0.9 (C-4), 36.8 (C-6), 128.4 (C-2), 156.2 (C-3), 1
99.7 (C-1). IR (NaCl): 3025, 2958, 2932, 1683, 1617, 1458, 139
1, 1375, 1251, 1094,1053, 1016, 828, 750.

(3) 3.0 g of sodium benzenesulfinate is added to a solution of 1.52 g of 4-methyl-2-cyclohexen-1-one and 9 ml of water. 1N in this solution
18 ml of hydrochloric acid are added dropwise. After stirring at room temperature for 24 hours, the precipitated crystals are filtered and washed with water, isopropanol and cold ether. Recrystallized from isopropanol to give 4-methyl-3- (phenylsulfonyl) -cyclohexane-1 as white crystals.
-Get on. (Yield 72%)

4-methyl-3- (phenylsulfonyl)
-Cyclohexane-1-one Molecular weight: 252 (C ₁₃ H ₁₆ O ₃ S) Melting point: 71-74 ° C. TLC: (hexane-ethyl acetate: 6-4) Rf = 0.2 ¹ H-NMR (200 MHz, CDCl ₃ ), -trans δ: 1.32 (d, J = 6.9Hz, 3H, H-7), 1.5-1.7 (m,
1H, H-5), 2.15-2.3 (m, 1H, H-5), 2.35-2.5 (m, 3H,
H-4,6), 2.55-2.68 (m, 2H, H-2), 3.17 (ddd, J = 8Hz,
J = 6.6Hz, J = 6.4Hz, 1H, H-3), 7.52-7.72 (m, 3H, Ha
r.-3 ', 4'), 7.83-7.9 (m, 2H, Har.-2 '), -cisδ: 1.44 (d, J = 7.1Hz, 3H, H-7), 1.75-1.9 (m , 1
H, H-5), 1.95-2.1 (m, 1H, H-5), 2.23-2.5 (m, 3H, H
-4, 6), 2.73-2.9 (m, 2H, H-2), 3.34 (dt, J = 12.9Hz,
J = 4Hz, 1H, H-3), 7.52-7.72 (m, 3H, H ar.-3 ', 4'),
. 7.83-7.9 (m, 2H, H ar.-2 ') 13 C-NMR (50MHz, CDCl 3), -transδ: 20.3 (C-7), 28.5 (C-4), 30.4 (C-5) , 37.9
(C-6 or -2), 38.6 (C-2 or -6), 66.3 (C-3), 128.6
(C ar.-2 'or -3'), 129.1 (C ar.-3 'or -2'), 133.9
(C ar.-4 '), 137.2 (C ar.-1'), 206.6 (C-1) .-cisδ: 13 (C-7), 27.2 (C-4), 31.1 (C-5), 35.9 (C-
6 or -2), 36.9 (C-2 or -6), 64.6 (C-3), 128.3 (C a
r.-2 'or -3'), 129.1 (C ar.-3 'or -2'), 133.9 (C a
r.-4 '), 138 (C ar.-1'), 206.6 (C-1) .MS (EI): 111.1 (M-SO ₂ Ph, 88), 110.1 (27), 83, 15 (3
2), 77.1 (29), 69.1 (36), 55.2 (100)

(4) A solution prepared by dissolving 2.45 g of 4-methyl-3- (phenylsulfonyl) -cyclohexane-1-one in 40 ml of benzene was added with 0.7 of 1,2-ethanediol.
ml and 0.2 g of para-toluenesulfonic anhydride are added. Heat the reaction to reflux for 4 hours. After the reaction, 2 M aqueous sodium hydrogen carbonate solution is added, and the mixture is extracted three times with ethyl acetate. The organic layer is washed with saturated saline and dried over magnesium sulfate.
After evaporating the solvent under reduced pressure, the residue is recrystallized from ether to obtain white crystals of 1,1- (ethylenedioxy) -4-methyl-3- (phenylsulfonyl) -cyclohexane. (Yield 97%)

1,1-ethylenedioxy-4-methyl-
3-phenylsulfonyl-cyclohexane Molecular weight: 296 (C ₁₅ H ₂₀ O ₄ S) Melting point: 105-106 ° C. TLC: (hexane-ethyl acetate: 6-4) Rf = 0.3 ¹ H-NMR (200 MHz, CDCl ₃ ), -transδ: 1.23 (d, J = 6.1Hz, 3H, H-7), 1.37-1.77 (m,
6H, H-2a, 4, 5, 6), 1.84 (ddd, J _gem = 12.9Hz, ³ J = 3.7
Hz, ⁴ J = 2.7Hz, 1H, H-2e), 3.02 (ddd, ³ J = 13Hz, ³ J = 10.3
Hz, ³ J = 3.7Hz, 1H, H-3), 3.71-3.91 (m, 4H, O-CH ₂ -CH ₂
-O), 7.48-7.67 (m, 3H, Har.-3 ', 4'), 7.8-7.88 (m,
2H, H ar.-2 ') -cisδ: 1.18 (d, J = 6.9Hz, 3H, H-7), 1.37-1.77 (m,
4H, H-5, 6), 1.84 (ddd, J _gem = 13Hz, ³ J = 3.7Hz, ⁴ J = 2.
7Hz, 1H, H-2e), 2.02 (t, J = 13Hz, 1H, H-2a), 2.30-2.
45 (m, 1H, H-4), 3.29 (dt, ³ J = 13Hz, ³ J = 3.7Hz, 1H, H-
3), 3.71-3.91 (m, 4H, O-CH ₂ -CH ₂ -O), 7.48-7.67 (m, 3
H, H ar.-3 ', 4'), 7.8-7.88 (m, 2H, H ar.-2 '). ¹³ C-NMR (50 MHz, CDCl ₃ ), -transδ: 20.4 (C-7), 31.9 (C-4), 32.6 (C-5), 34.1
(C-6), 35.8 (C-2), 64.4 (CH ₂ -O), 66.8 (C-3), 107.9
(C-1), 128.6 (C ar.-3 'or -2'), 129 (C ar.-2 'or
-3 '), 133.5 (C ar.-4'), 138 (C ar.-1 ') .- cisδ: 12.4 (C-7), 26.7 (C-4), 29.2 (C-5, 6) , 32
(C-2), 64.1 (C-3), 64.4 (CH ₂ -O), 108.2 (C-1), 128.
3 (C ar.-2 ', 3'), 133.5 (C ar.-4 '), 138.5 (C ar.-
1 '). IR (KBr): 3060, 2968, 2938, 1583, 1448, 1301, 1267,
1158, 1144, 1082, 1023, 939, 916, 838, 749, 718,
689. Elemental analysis (%): Calculated: C; 60.79, H; 6.8. Analytical value: C; 60.5, H; 6.9.

(5) 1,1- (ethylenedioxy) -4
To a solution of 560 mg of -methyl-3- (phenylsulfonyl) -cyclohexane and 4 mg of triphenylmethane in 5 ml of THF was added dropwise a solution of 1.8 ml of n-butyllithium at -78 ° C under a stream of argon. After stirring for 10 minutes, react at room temperature for 1 hour. 1 ml of HMPT was added, the mixture was cooled again to -78 ° C, and 205 mg of 14-bromo-1-tetradecanol was added.
Add ml THF solution dropwise. After reacting at −20 ° C. for 2 hours, the reaction solution is poured into a saturated ammonium chloride solution. The solution is extracted with ether, and the organic layer is washed with water and saturated saline and dried over magnesium sulfate. After evaporating the solvent under reduced pressure, hexane
-Purified by silica gel column chromatography with ethyl acetate to afford 1,1- (ethylenedioxy)-as a colorless oil.
3- (14-hydroxytetradecyl) -4-methyl-
3- (phenylsulfonyl) -cyclohexane is obtained.
(Yield: 98%)

1,1- (ethylenedioxy) -3- (1
4-hydroxy-tetradecyl) -4-methyl-3- (phenylsulfonyl) - cyclohexane Molecular _{weight: 508 (C 29 H 48 O} 5 S) TLC: (hexane-AcOEt: 60-40) Rf = 0.22 1 H-NMR ( 200 MHz), δ: 1.13 (d, J = 6Hz, 3H, H-21); 1.
28 (s large, 20H, H-9a H-18); 1.43-1.6 (m, 9H, H-
4, 5, 7, 8, 19); 1.67 (m, 1H, H-2); 1.89 (dd, J _gem
= 12.5Hz, J = 3Hz, 1H, H-6e); 2.14 (t large, J = 12.5Hz,
1H, H-6a); 2.43 (dd, J _gem = 13.8 Hz, ⁴ J = 2.5Hz, 1H,
H-2); 3.63 (t, J = 6.5 Hz, 2H, H-20); 3.83-3.97 (m,
4H, O-CH ₂ -CH ₂ -O), 7.49-7.68 (m, 3H, H ar.-3 ', 4');
7.80-7.88 (m, 2H, Har.-2 '). ¹³ C-NMR (50 MHz), δ: 16.1 (C-21); 24.4 (C-18); 2
5.6 (C-5 or -7); 25.8 (C-7 or -5); 29.5 (C-9 to C-1
7); 30.3 (C-8); 32.7 (C-19); 34.9 (C-6); 35.5 (C-
4); 36.2 (C-2); 62.8 (C-20); 63.9 and 65.1 (O-CH _2-
CH ₂ -O); 71.2 (C-3); 108.4 (C-1); 128.7 (C ar.-3 ');
130.1 (C ar.-2 '); 133.3 (C ar.-4'); 136.8 (C ar.
-1 ') IR (NaCl): 3510 (m large, OH); 3063 (f, CH); 292
6, 2853 (f, CH); 1585 (f, CC); 1447 (m); 1286, 1
140 (F, SO ₂ ); 1096, 1083 (m, O-CH ₂ ); 723, 693 (m) MS (Cl-NH ₃ ): 526.4 (MNH ₄ , 16); 369.4 (MH ₂ -SO ₂ Ph , 2
8); 370.4 (MH-SO ₂ Ph, 25); 367.3 (M-SO ₂ Ph, 100); 31
1.3 (7); 307.3 (8); 305.3 (9); 175 (17); 159.9 (1
1); 98.9 (35); 94 (6); 78 (11). Elemental analysis (%): Calculated C: 67.98, H: 9.37 Analytical value C: 67.4, H: 9.1

(6) 1,1- (ethylenedioxy) -3
-(14-hydroxytetradecyl) -4-methyl-3
To a solution of 235 mg of-(phenylsulfonyl) -cyclohexane in 20 ml of chloroform and 4 ml of acetone is added 20 mg of paratoluenesulfonic acid. Mix for 24 hours 50
React at ° C. 10 ml of saturated aqueous sodium hydrogen carbonate solution is added, and the mixture is extracted with dichloromethane. The organic layer is washed with saturated saline and dried over magnesium sulfate. After evaporating the solvent under reduced pressure, the residue is purified by silica gel column chromatography with hexane-ethyl acetate to obtain 3- (14-hydroxytetradecyl) -4-methyl-2-cyclohexen-1-one as a colorless oil. (Yield: 75%)

3- (14-hydroxytetradecyl)-
4-methyl-2-cyclohexen-1-one Molecular _{weight: 322 (C 21 H 38 O} 2) TLC: (hexane = AcOEt: 6-4) Rf = 0.3 MS (EI): 322.2 (M +, 37); 304.1 _{(MH 2 O, 12);} 292.1
(21); 164.9 (C ₁₁ H ₁₇ O, 9); 151 (C ₁₀ H ₁₅ O, 4); 138.1
(12); 137 (C ₉ H ₁₃ O, 43); 96 (30); 94.9 (24); 81 (2
4); 78.9 (13); 69 (15); 67 (25); 55 (37). Elemental analysis (%): Calculated C: 78.20, H: 11.88 Analytical value C: 78.6, H: 11.9

Production Example 2 In the same manner as in Production Example 1, 3- (15-hydroxypentadecyl) 4-methyl-2-cyclohexen-1-one (compound 2) was synthesized.

Production Example 3 3- (12-acetoxypentadecyl) -2,4,4-
132 mg of trimethyl-2-cyclohexen-1-one
(0.36 mmol, 1 equivalent) in a methanol solution (8 ml) containing 3 drops of water and 74 mg of K ₂ CO ₃ (0.54 mmol)
1, 1.5 equivalents). After stirring at room temperature for 2 hours and 30 minutes, the pH was adjusted to 7 with 5% HCl, extracted with ether, dried over magnesium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography and eluted with hexane-ethyl acetate (8-2 to 7-3) to give 3- (12-hydroxydodecyl) -2,4,4- as a colorless oil.
94 mg (yield 81%) of trimethyl-2-cyclohexen-1-one (compound 3) was obtained.

3- (12-hydroxydodecyl) -2,
4,4-trimethyl-2-cyclohexen-1-one TLC: (hexane-AcOEr: 7-3) Rf = 0.2 GC: 40-280 ° C (20 ° C / min) 12min, 99%; ¹ H-NMR (200 MHz), δ: 1.13 (s, 6H, H-19, 20); 1.26
(s, br, 16H, H-9 toH-16); 1.35-169 (m, 4H, H-8, 1
7); 1.73 (s, 3H, H-21); 1.77 (t, J = 7.5 Hz, 2H, H-5);
2.11-2.19 (m, 2H, H-7); 2.43 (t, J = 6.8 Hz, 2H, H-
. 6); 3.61 (t, J = 6.8 Hz, 2H, H-18) 13 C-NMR (50 MHz), δ: 11.4 (C-21); 25.7 (C-16); 2
6.8 (C-19, 20); 28.8 (C-8); 29.5 (C-9 to C-15); 30.
45 (C-7); 32.7 (C-17); 34.2 (C-5); 36.2 (C-4); 37.
3 (C-6); 62.9 (C-18); 130.4 (C-2); 165.4 (C-3); 19
9 (C-1) IRν: 3440 (broad OH); 2925, 2852 (w, CH); 1666
(w, C = 0); 1605 (s, C = C); 1467 (s, CH)

Production Example 4 The following compound was obtained in the same manner as in Production Example 3. The numerical value in parentheses is the TL in hexane: ethyl acetate = 7: 3.
The Rf value of C is shown. (1) 3- (15-hydroxypentadecyl) -2,
4,4-trimethyl-2-cyclohexen-1-one (compound 4) (Rf = 0.29) (2) 3- (18-hydroxypentadecyl) 2,4
4-trimethyl-2-cyclohexen-1-one (compound 5) (Rf = 0.25)

Test Example The generation of neural stem cells from ES cells was performed by Weiss and Reynolds.
(1996). That is, the striatum was excised from the mouse fetus, the cells were dispersed in a culture solution containing EGF (20 ng / ml), and cultured in a 5% CO ₂ and 37 ° C. environment for 5 days. After that, Dissociation Medium (S
Centrifugation at 400 rpm for 5 minutes in Neuroscience (Neuros), a mass of neural stem cells.
pheres). The Neurospheres are dispersed in a culture solution and cultured under the same conditions.
I got A sterilized cover glass placed in a 24-well plate was treated with a polyornithine solution (30 μg / ml) overnight, rinsed three times with a phosphate buffer, and each cover glass was treated with Ne.
Planted urospheres to have 20 to 50 urospheres. Compounds 1 to 5 obtained in Production Examples 1 to 4 adjusted to a concentration of 10 −6 M with ethanol were applied and cultured for a period (usually 24 hours) during which Neurospheres differentiated sufficiently. Well differentiated Neuro
The ospheres were fixed with 4% para-formaldehyde, washed with a phosphate buffer, added with 0.5% Triton-X100 for 5 minutes, and washed again with a phosphate buffer. Mouse monoclonal antibody anti-MAP2 (2a +
2b) (Sigma), mouse monoclonal antibody anti-04 (Boeringher) for labeling oligodendrocytes and rabbit polyclonal antibody anti-GFAP (DAKO) for astrocyte labeling were added at room temperature. Cultured overnight or at 4 ° C overnight. After adding an anti-mouse IgM antibody and a fluorescent indicator and culturing at room temperature for 1 hour, washing with a phosphate buffer, a cover glass was placed on a confocal microscope to observe the differentiation of Neurospheres. As a result, it was shown that Compounds 1 to 5 have an effect of inducing differentiation of neurospheres, which are clusters of neural stem cells, into neural cells.

[0054]

[Table 1]

[0055]

Industrial Applicability The cyclohexenone long-chain alcohol derivative of the present invention promotes the induction of differentiation of stem cells into cells expressing a specific biological function, so that a medicine containing the same can be used for regression of various tissues and cells. Diseases caused by reduction or cell death, for example, Alzheimer's disease, Pick's disease (Pick), Parkinson's disease, Huntington's disease, spinal cerebellar degeneration, neuropathy such as amyotrophic lateral sclerosis, bone disease such as osteoporosis, fracture, and narrowing It is useful as a preventive and therapeutic drug for cardiovascular diseases such as heart disease, retinopathy, and arterial occlusion, muscular dystrophy, and muscular diseases such as congenital myopathy.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Shoji Yamada 1-26-11, Midori, Sumida-ku, Tokyo Meiji Dairy Products Pharmaceutical Division (72) Inventor Yukie Suma 1-26-11, Midori, Sumida-ku, Tokyo Meiji Dairies Corporation Pharmaceutical Division (72) Inventor Hirohito Suzuki 1-26-11 Midori, Sumida-ku, Tokyo Meiji Dairies Corporation Pharmaceutical Division F-term (reference) 4C206 AA01 AA02 CB23 MA01 MA04 NA14 ZA01 ZA96 ZC41

Claims (2)

[Claims] [Claim 1] The following general formula (1) [Wherein, R ¹ , R ² and R ³ each represent a hydrogen atom or a methyl group, and X represents a linear or branched alkylene or alkenylene group having 10 to 28 carbon atoms]. An agent for inducing stem cell differentiation comprising a long-chain alcohol derivative as an active ingredient. 2. The stem cell differentiation-inducing agent according to claim 1, which differentiates stem cells to induce them into nerve cells.