JP2003095949A - Saccharide uptake promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new saccharide uptake promoter. SOLUTION: This new saccharide uptake promoter for cells is characterized by containing a histone deacetylase inhibiter.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a sugar uptake promoter.

[0002]

BACKGROUND OF THE INVENTION Diabetes mellitus is insulin-dependent diabetes mellitus (type 1 diabetes) that occurs as a result of relative or absolute lack of insulin action, and non-insulin-dependent diabetes mellitus that occurs as a failure of normal metabolic response of peripheral tissues to the action of insulin. (Type 2 diabetes).

At present, drugs such as insulin preparations, sulfonylurea preparations and biguanide preparations are selected and treated depending on the type of pathological condition, but further development of new preparations is expected from the viewpoint of side effects. .

[0004]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel sugar uptake promoter.

[0005]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors have found that the object can be achieved by inhibiting the deacetylation of histone, and the present invention has been completed. Came to do.

[0006] That is, the present invention is a cellular glucose uptake promoter characterized by containing a histone deacetylase inhibitor.

The present invention also provides the formula (I)

[0008]

[Chemical 3]

[Wherein R ¹ , R ² and R ³ are the same or different and each is a hydrogen atom, a halogen atom, a C ₁ -C ₆ alkyl group, a hydroxyl group, a nitro group, a sulfonyl group, NR ⁵ R ⁶ (in the formula, R ⁵
And R ⁶ are the same or different and each represents a hydrogen atom or a C ₁ -C ₆ alkyl group. ) Or S (CH ₂ ) pR ⁷ (in the formula, R ⁷ represents a hydrogen atom, a C _{1 to} C ₆ alkyl group, a hydroxyl group, a phenyl group or a C _{2 to} C ₇ alkoxycarbonyl group, and p is an integer of 0 to 3). R ⁴ represents a hydrogen atom, a nitro group or a sulfonyl group, and n represents an integer of 4 to 6. ] The sugar uptake | capture promoter characterized by containing the compound represented by these, or its pharmaceutically acceptable salt.

The present invention also provides the formula (I)

[0011]

[Chemical 4]

[Wherein R ¹ , R ² and R ³ are the same or different and each is a hydrogen atom, a halogen atom, a C ₁ -C ₆ alkyl group, a hydroxyl group, a nitro group, a sulfonyl group, NR ⁵ R ⁶ (in the formula, R ⁵
And R ⁶ are the same or different and each represents a hydrogen atom or a C ₁ -C ₆ alkyl group. ) Or S (CH ₂ ) pR ⁷ (in the formula, R ⁷ represents a hydrogen atom, a C _{1 to} C ₆ alkyl group, a hydroxyl group, a phenyl group or a C _{2 to} C ₇ alkoxycarbonyl group, and p is an integer of 0 to 3). R ⁴ represents a hydrogen atom, a nitro group or a sulfonyl group, and n represents an integer of 4 to 6. ] A compound represented by the following formula or a pharmaceutically acceptable salt thereof and containing a function of promoting sugar uptake.

The present invention also provides a method for promoting glucose uptake into cells, which is characterized by inhibiting histone deacetylase.

(Histone deacetylase inhibitor) The histone deacetylase inhibitor means a compound which acts on histone deacetylase and inhibits deacetylation of histone.

The inhibitory activity is determined by the method of Sue et al. (CANCER RESEARC
H 60, 3137-3142 (2000)).
The amount of acetylated histones in cells treated with the sample for a certain period of time (treatment group) was examined by polyacrylamide gel electrophoresis, and the difference from the amount of acetylated histones in cells treated with additive-free medium (non-treatment group) was examined. An inhibitor can be selected as an index.

Further, the inhibitory activity is determined by the method of Quon et al.
atl. Acad.Sci.Vol.95,3356-3361 (1998)) or Furumai's method (Proc.Natl.Acad.Sci.Vol.98 (1), 87-92 (200)
It can also be measured by 1)). Histone deacetylase is allowed to act on [ ³ H] acetylated histone, and an inhibitor can be selected by using the change in the amount of free acetic acid with or without the addition of a sample as an index.

The histone deacetylase inhibitor, which is the active ingredient of the present invention, is obtained by the method of Quon et al. (HL60 cells (1.0 × 10 ⁷
It is preferable that the IC ₅₀ is 5 μM or less when measured according to the method (using a crudely purified enzyme from cells).

As the histone deacetylase inhibitor,
Examples thereof include trichostatin A (TSA) s, scriptides, trapoxin As, trapoxin Bs, chlamidocins, sodium butyrate and the like. A compound represented by the formula (I) is preferable, and a compound of n = 5 in the formula (I) is more preferable. In the formula (I), the halogen atom means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

The C ₁ -C ₆ alkyl group means a linear, branched or cyclic alkyl group, for example, methyl group, ethyl group, propyl group, isopropyl group, cyclopropyl group, butyl group, isobutyl group. Group, sec-butyl group, cyclobutyl group, cyclopropylmethyl group, pentyl group, isopentyl group, cyclopentyl group, cyclobutylmethyl group, 1-ethylpropyl group and the like.

The C ₂ -C ₇ alkoxycarbonyl group means a linear or branched alkoxycarbonyl group, for example, a methoxycarbonyl group, an ethoxycarbonyl group, a propoxycarbonyl group, an isopropoxycarbonyl group,
Examples thereof include butoxycarbonyl group, isobutoxycarbonyl group, pentyloxycarbonyl group and hexyloxycarbonyl group.

The pharmaceutically acceptable salts include salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, ammonia, methylamine, dimethylamine, cyclopentylamine. And the like, salts with mineral acids such as sulfuric acid, hydrochloric acid and phosphoric acid, and salts with organic acids such as acetic acid, oxalic acid, lactic acid, fumaric acid and maleic acid.

The histone deacetylase inhibitor may be purchased from a reagent maker or the like, or may be synthetically produced.

For example, the compound having a novel structure among the compounds represented by the formula (I) can be produced by the following general production method.

[General manufacturing method]

[0025]

[Chemical formula 5] [Reaction formula 1]

(In the formula, R ¹ , R ² , R ³ , R ⁴ and n have the same meanings as described above.) A known compound of the formula (II) is replaced with an aminocarboxylic acid represented by the formula (III). By reacting 1 to 20 equivalents with an inert solvent (for example, N-methyl-α-pyrrolidinone (NMP), N, N-dimethylformamide (DMF), etc.) at 100 to 200 ° C. to give a compound of the formula (IV) Obtain the compound. A compound of formula (IV) is prepared by mixing 1 to 1.5 equivalents of ethyl chloroformate and 1 to 1.5 equivalents of N-methylmorpholine in an inert solvent (such as diethyl ether or tetrahydrofuran) at -15 to 0 ° C.
After reacting with the method described in J. Chem. Soc., Pe.
rkin Trans. 1 1975, 846-848) and a solution of 1 to 1.5 equivalents of hydroxyamine in methanol is added and reacted at room temperature to obtain a compound represented by the formula (I).

(Sugar uptake enhancer) The sugar uptake enhancer means a drug which increases the uptake of monosaccharides such as glucose into cells. The sugar uptake promoting activity can be measured according to the method of Yonemitsu et al. (Diabetes, 50,1093- (1974)) or the method of Moody et al. (Horm Metab Res. 6, 12-16 (1974)). For example, after exposing the cells to a sample for a certain period of time, the amount of uptake into the cells is measured with labeled glucose or the like (for example, ¹⁴ C-labeled 2-deoxyglucose). The exposure time of the sample is preferably 20 hours or more, more preferably 6 to 9 days.

The activity of promoting sugar uptake of the present invention is that by inhibiting histone deacetylation, the UNCOUPLING PROTEIN
It is speculated that the transcription of 2 (UCP2) is promoted, and further sugar uptake is promoted. The present inventors have confirmed that UCP2 transcription is dose-dependently increased by adding Compound 1 described below to cells.

The action of promoting sugar uptake may be that of the promoter of the present invention alone or that of enhancing the effect of hormones (insulin etc.) which act on sugar uptake.

The sugar uptake enhancer may be a reagent or a drug having a sugar uptake promoting action. A drug based on a sugar uptake promoting action means a drug that regulates a hyperglycemic state to a normal blood glucose state by promoting uptake of sugar into cells, and for example, a therapeutic / preventive agent for diabetes, diabetic retinopathy, Examples include prophylactic drugs for complications such as diabetic nephropathy and diabetic peripheral neuropathy.

The pharmaceutical composition of the present invention is orally prepared by adjusting it into a suitable pharmaceutical composition form (solid preparation such as tablets, pills, capsules, granules, dry syrups) using appropriately known carriers, diluents and the like. Can be used parenterally.

In order to produce solid agents, various additives such as excipients, disintegrants, binders, lubricants and coating bases are used, and stirring granulation method, fluidized bed granulation method and crushing granulation method are used. It can be manufactured by the method. Excipients include mannitol, xylitol, sorbitol, glucose, sucrose, lactose, crystalline cellulose and the like. As the disintegrant, low-substituted hydroxypropyl cellulose, carboxymethyl cellulose,
Examples thereof include carboxymethyl cellulose calcium and sodium carboxymethyl cellulose. Examples of the binder include methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, gelatin, gum arabic, ethyl cellulose and the like. As a lubricant, stearic acid,
Examples thereof include magnesium stearate and calcium stearate. In addition, an antioxidant, a coating agent, a coloring agent, a flavoring agent, a surfactant, a plasticizer and the like can be added if necessary.

Formulation Example Formulation (in 1 tablet) Compound 1 Lactose 40 mg Corn starch 49.75 mg Crystalline cellulose 17 mg Carmellose calcium 17 mg Hydroxypropyl cellulose 5.25 mg Magnesium stearate 1 mg Total compound, lactose, corn starch, crystalline cellulose, carmellose calcium are uniformly added. Mix, add 10% aqueous solution of hydroxypropylcellulose to this, dry after association, and then sift the granules with 30M sieve to make uniform granules, add magnesium stearate, and tablet into tablets .

Test Example 1 [Sugar uptake promotion experiment] L6 cells were cultured in medium 1. The cultured cells were suspended in Medium 2 and seeded on a 24-well plate at 1 × 10 ⁵ (cells / well) to induce differentiation. Change the culture medium every 2 days,
Four days after the induction of differentiation, a sample (final concentration: 1 μM) was added to the culture medium, and cells for 10 days after the induction of differentiation were used for the sugar uptake test. The culture supernatant was removed and washed once with HEPES buffer. 250 μl of HEPES buffer was poured into a 24-well plate and starved for 3 hours. When measuring the promoting effect during insulin stimulation, insulin was added at a final concentration of 100 nM and incubated at 37 ° C. for 5 hours at 5% CO ₂ . The sugar uptake amount was measured using labeled 2-deoxyglucose. 250 μl of 100 μM 2-deoxyglucose solution (2 μCi 2- [ ¹⁴ C] Deoxyglucose) was added, and the mixture was incubated at 37 ° C. for 5 hours at 5% CO ₂ . After washing 3 times with phosphate buffered saline, 300 μl of 1% SDS solution
The cell suspension suspended in 5 ml of scintillator was added and mixed, and the radiation dose was measured with a scintillation counter (manufactured by Beckman Coulter, Inc.). FIG. 1 shows the relative uptake amount when the sugar uptake amount of cells (control) to which the sample was not added was 100%. It was confirmed that Compound 1, which is an active ingredient of the accelerator of the present invention, has a higher sugar uptake promoting activity than Comparative Compounds 1, 2, and 3. The compositions of the mediums 1 and 2 and the HEPES buffer solution are as follows. Test Example 2 L6 cells were cultured using Medium 1. The medium was changed to medium 2 and seeded in a 96-well plate at 2 × 10 ⁴ (pieces / well) to induce differentiation. The culture medium was changed every two days during the induction of differentiation. 4 days after the induction of differentiation, the sample was added to the medium,
In the glucose uptake test, cells 10 days after induction of differentiation were used. The culture supernatant was removed and washed once with HEPES buffer. 50 μl of HEPES buffer was poured into a 96-well plate and starved for 3 hours. 100 μM 2-deoxyglucose solution (2 μC
50 μl of i2- [ ¹⁴ C] Deoxyglucose) was added, and the mixture was incubated at 37 ° C. in 5% CO ₂ for 1 hour. After washing three times with phosphate buffered saline, 100 μl of scintillator was added to the cell suspension suspended in 30 μl of 1% SDS solution and mixed well, and the radiation dose was measured with a scintillation counter (Perkin Elmer). FIG. 2 shows the relative uptake amount when the sugar uptake amount of cells (control) to which the sample was not added was 100%.

Compound 2 which is an active ingredient of the accelerator of the present invention
Was confirmed to have a higher activity of promoting sugar uptake as compared with Comparative Compounds 1 and 2. The compositions of the mediums 1 and 2 and the HEPES buffer solution are the same as in Test Example 1.

[Brief description of drawings]

FIG. 1 shows the results of an experiment of promoting L6 cell glucose uptake of Compound 1 in an insulin-stimulated state and an unstimulated state.

FIG. 2 shows the results of an experiment for promoting glucose uptake of L6 cells of compound 2 in a state without insulin stimulation.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Yoshiyoshi Takayama             3-24-1, Takada, Toshima-ku, Tokyo Taisho product             Yaku Co., Ltd. F term (reference) 4C086 AA01 AA02 BC27 MA01 MA04                       NA14 ZC35

Claims (4)

[Claims] 1. A sugar uptake enhancer comprising a histone deacetylase inhibitor. 2. A histone deacetylase inhibitor is represented by the formula (I): [Wherein R ¹ , R ² and R ³ are the same or different and each is a hydrogen atom,
Halogen atom, C ₁ -C ₆ alkyl group, hydroxyl group, nitro group, sulfonyl group, NR ⁵ R ⁶ (In the formula, R ⁵ and R ⁶ are the same or different and represent a hydrogen atom or a C ₁ -C ₆ alkyl group. ) Or S (CH ₂ ) pR ⁷ (wherein R ⁷ is a hydrogen atom, C
₁ -C ₆ alkyl group, a hydroxyl group, a phenyl group or a C ₂ -C ₇ alkoxycarbonyl group, p is an integer of 0 to 3. ), R ⁴ represents a hydrogen atom, a nitro group or a sulfonyl group, and n represents an integer of 4 to 6. ] The sugar uptake | capture promoter of Claim 1 which is a compound represented by these, or its pharmaceutically acceptable salt. 3. Formula (I): [Wherein R ¹ , R ² and R ³ are the same or different and each is a hydrogen atom,
Halogen atom, C ₁ -C ₆ alkyl group, hydroxyl group, nitro group, sulfonyl group, NR ⁵ R ⁶ (In the formula, R ⁵ and R ⁶ are the same or different and represent a hydrogen atom or a C ₁ -C ₆ alkyl group. ) Or S (CH ₂ ) pR ⁷ (wherein R ⁷ is a hydrogen atom, C
₁ -C ₆ alkyl group, a hydroxyl group, a phenyl group or a C ₂ -C ₇ alkoxycarbonyl group, p is an integer of 0 to 3. ), R ⁴ represents a hydrogen atom, a nitro group or a sulfonyl group, and n represents an integer of 4 to 6. ] The compound containing the compound or its pharmaceutically acceptable salt represented by these, and the pharmaceutical based on a sugar uptake promotion function. 4. A method for promoting glucose uptake into cells, which comprises inhibiting histone deacetylase.