JPH1192364A - Therapeutic agent for diabetes - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a therapeutic agent having a low side effect, low toxicity and high safety by using dephostatin as an active component. SOLUTION: The objective therapeutic agent is produced by including 0.01-100 wt.% of dephostatin of the formula as an active component in the agent. The daily administration rate for adult is usually 0.01-1,000 mg/kg in one or divided doses. It can be used in the form of peroral agent, injection, suppository, poultice, etc., and peroral agent is preferable from the viewpoint of the physical load on the patient. It has remarkable suppressing effect on the release of free fatty acid from cultured fat cell by epinephrine stimulation without accompanying cell damage.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic agent for diabetes.

[0002]

2. Description of the Related Art Diabetes is a disease with a large number of people affected.
The number is increasing every year. In addition, serious complications such as retinopathy, renal failure, and myocardial infarction may progress as chronic complications, and it is necessary to diagnose and treat the disease at an early stage.

[0003] Examples of the therapeutic agent for diabetes include insulin, synthetic hypoglycemic agents and the like.

[0004] However, the insulin is inactivated by proteolytic enzymes such as pepsin secreted in the stomach and the like, and thus has a drawback that the insulin cannot be administered orally. Further, there is a disadvantage that excessive administration of the insulin and the synthetic hypoglycemic agent causes harmful symptoms to the human body such as hypoglycemia.

[0005] Therapeutic agents for non-insulin-dependent diabetes mellitus (NIDDM), which account for 95% or more of diabetic patients in Japan, include synthetic drugs such as sulfonylurea drugs, biguanides, and α-glucosidase inhibitors. However, the synthetic drugs have a drawback that they are not satisfactory in terms of therapeutic effects, safety, and the like.

[0006] As a new therapeutic agent for the non-insulin-dependent diabetes mellitus, an insulin action ameliorating agent characterized by activating the insulin receptor on the cell surface and enhancing the effect of insulin has recently been developed. Although a thiazolidine derivative is recognized as an active ingredient, development of a safer substance having stronger activity as a therapeutic agent for diabetes is desired.

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above prior art, and has as its object to provide an effective and safe therapeutic agent for diabetes.

[0008]

Means for Solving the Problems The present inventors expect that a substance that inhibits tyrosine phosphatase directly activates the tyrosine kinase of the insulin receptor, and is considered to be useful as a therapeutic agent for diabetes. , Earnestly studied. As a result, defostatin conventionally known as an immunomodulator having an inhibitory activity on tyrosine phosphatase (substrate antagonistic inhibition), an antitumor agent and the like (Japanese Patent Laid-Open No. 6-25628)
No. 1) has found a completely new fact that it exhibits an excellent diabetes therapeutic effect on diabetes, particularly non-insulin-dependent diabetes. The present invention has been completed based on this fact.

That is, the present invention relates to the following [1] Formula (I):

[0010]

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A therapeutic agent for diabetes comprising, as an active ingredient, defostatin represented by the following formula or a pharmaceutically acceptable salt thereof, and [2] a formula (II):

[0012]

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[0013] The present invention relates to a therapeutic agent for diabetes, comprising, as an active ingredient, an isomer of 3,4-dihydroxydefostatin represented by the following formula or a pharmaceutically acceptable salt thereof.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION

 Formula (I) of the present invention:

[0015]

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The defostatin represented by the formula (I) is a known compound, and is disclosed in JP-A-6-256281 and J. Antib.
Streptomyces sp. MJ742 belonging to the genus Streptomyces described in iot., 1995, 46, 9 , 1342-1346.
It can be obtained by culturing the NF5 strain (FERM P-13223). Also, defostatin having a tyrosine phosphatase inhibitory activity equivalent to that of natural defostatin recovered from the culture can be produced by a synthetic method (J. Chem. Soc. Commun. 1994, pp. 437).

Further, the formula (II) of the present invention:

[0018]

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The 3,4-dihydroxydefostatin isomer represented by the formula is a known compound having better stability in a culture medium than defostatin, and is disclosed in J. Antibiot., 199.
5, 48, 12 , 1460-1466.

Pharmaceutically acceptable salts of said defostatin or said 3,4-dihydroxydefostatin isomer are described, for example, in J. Antibiot., 1995, 48, 12 , 1460-146.
It can be produced by the method described in 6.

Examples of the pharmacologically acceptable salt of the above-mentioned defostatin or the above 3,4-dihydroxydefostatin isomer include alkali metal salts such as lithium salt, sodium salt and potassium salt. These pharmacologically acceptable salts can be used alone or in combination of two or more. Among these, from the viewpoint of biocompatibility,
Alkali metal salts of phenolic hydroxyl groups are preferred, with sodium and potassium salts being particularly preferred.

The defostatin or the 3,4-dihydroxydefostatin isomer was used as described below in the experimental examples using 3T3-L1 cells derived from mouse fetuses in cultured fats without cell damage, as shown in the experimental results. It shows a remarkable inhibitory effect on the release of free fatty acids by epinephrine stimulation from cells. For this reason, it is useful as a therapeutic agent for diabetes in mammals (humans, horses, dogs, cats, etc.). Further, the defostatin or the 3,4-dihydroxydefostatin isomer is more than a conventional drug.
Fewer side effects, low toxicity, a high safety compounds, for example, LD ₅₀ (50% lethal dose) of Deformation statin
Is 100 mg / kg or more (ICR mouse) by blood administration, and LD ₅₀ (lethal dose of 50%) of 3,4-dihydroxydefostatin isomer is 100 m / kg by blood administration.
g / kg or more (ICR mice).

The above-mentioned defostatin, 3,4-dihydroxydefostatin isomer or a pharmaceutically acceptable salt thereof can be used alone or in combination of two or more.

The dosage of the above-mentioned defostatin, 3,4-dihydroxydefostatin isomer or a pharmaceutically acceptable salt thereof may vary depending on the condition, body weight, age and the like of the patient. 0.01-1000
The dose is preferably about mg / kg, which can be administered once or in several divided doses.

The diabetes to which the therapeutic agent for diabetes of the present invention is applied is particularly effective in non-insulin-dependent diabetes, and the therapeutic effect is remarkable.

The dosage form of the therapeutic agent for diabetes of the present invention is not particularly limited as long as it is generally used as a therapeutic agent. Such dosage forms include, for example, oral preparations, injections, suppositories, cataplasms and the like, but oral preparations are desirable from the viewpoint of physical burden on patients.

The antidiabetic agent of the present invention may further include, in addition to defostatin, 3,4-dihydroxydefostatin isomer or a pharmaceutically acceptable salt thereof,
It may contain any organic or inorganic solid or liquid ingredient suitable for oral, enteral or other parenteral administration.

The optional ingredients include, for example, crystalline cellulose, gelatin, lactose, sucrose, starch, corn starch, dextrin, mannitol, magnesium stearate, talc, vegetable and animal fats, oils, gums, poly Alkylene glycol, gum arabic,
Excipients such as pectin, binders, disintegrants, lubricants, flavoring agents, extenders, coloring agents, stabilizers, isotonic agents, solubilizers, dispersants, dissolution aids, emulsifiers, humectants, Antioxidants and the like.

The content of the above-mentioned defostatin, 3,4-dihydroxydefostatin isomer or a pharmaceutically acceptable salt thereof in the preparation varies depending on the dosage form, base components and the like, so that it is generally limited. However, it is usually preferred that the amount is 0.01 to 100% by weight in the preparation.

The antidiabetic agent of the present invention can be produced by a generally employed method for preparing a drug.

As described above, the therapeutic agent for diabetes of the present invention has the effect of improving the insulin sensitivity of a patient, and exhibits a remarkable therapeutic effect on non-insulin-dependent diabetes mellitus, particularly among diabetes.

[0032]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Production Example 1 [3,4-dihydroxydefos
Production of Tatin Isomers] According to the method described in J. Antibiot. 1995, 48, 12 , pp. 1460-1466, 3,4-dihydroxydefostatin isomers were produced.

First, 5.02 g (33 mmol) of 3,4-dimethoxyaniline (manufactured by Aldrich) was dissolved in 100 mL of dichloromethane, and 2.9 mL (36 mmol) of pyridine and trifluoroacetic acid (Wako Pure Chemical Industries, Ltd.) were added to the dichloromethane solution. 5.0 mL (36 mmo)
l) was added under 0 ° C conditions, and the mixture was stirred for 2 hours.

100 mL of dichloromethane was added to the reaction solution, and the organic layer was washed with 100 mL of 1N hydrochloric acid, dried over sodium sulfate, and dried using a vacuum evaporator.

A column (4 cm) equipped with silica gel (trade name: Wakogel C-200, manufactured by Wako Pure Chemical Industries, Ltd.)
φ × 20 cm, developing solution: hexane / ethyl acetate = 1/1
(Volume ratio: v / v)) and a pale yellow powder (1)
4.53 g (17.8 mmol) of 3,4-dimethoxy-N-trifluoroacetylaniline was obtained (yield 53.
5%).

(1) 4.53 g of 3,4-dimethoxy-N-trifluoroacetylaniline was dissolved in 10 mL of dimethylformamide, and a mixed solution of 0.7 g of 60% sodium hydride (manufactured by Aldrich) and 5 mL of dimethylformamide. Then, the mixture was stirred at 0 ° C for 1 hour. 2.0 mL of methyl iodide was added at -23 ° C, and the mixture was further stirred at room temperature of about 20 ° C for 1 hour.

The whole reaction solution was cooled to 4 ° C. with 1N hydrochloric acid 10
The mixture was added dropwise to 0 mL, and extracted twice with 100 mL of ethyl acetate. The N-methylated product thus produced was subjected to a column (3 cmφ × 30 cm) equipped with silica gel (trade name: Wakogel C-200, manufactured by Wako Pure Chemical Industries, Ltd., developing solution: hexane / ethyl acetate = 3/1 to 1 /). 1 (volume ratio: v / v)) and (2) 2.96 g of 3,4-dimethoxy-N-methyl-N-trifluoroacetylaniline (11.0 mmol)
1) was obtained. The yield was 61.8%.

Aluminum chloride (Wako Pure Chemical Industries, Ltd.)
2.53 g (19.0 mmol) and 19.0 mL of ethanethiol were dissolved in 30 mL of dichloromethane, and the solution was dissolved at 0 ° C.
Then, (2) 3,4-dimethoxy-N-methyl-N-trifluoroacetylaniline (1.0 g) was added, and the mixture was stirred for 5 hours, and (2) 3,4-dimethoxy-N-methyl-N-trifluoroacetyl The methoxy group of aniline was converted to a hydroxyl group. The solution was dried using a vacuum evaporator, and a column (3 cmφ × 30 cm, equipped with silica gel (manufactured by Wako Pure Chemical Industries, Ltd., trade name: Wakogel C-200)) was used.
Developing solution: hexane / ethyl acetate = 5: 1 (volume ratio: v /
Purification in v)) yielded (3) 3,4-dihydroxy-N-methyl-N-trifluoroacetylaniline.

Before carrying out the nitrosation of the amino group, the TBSO-formation, which can be removed under mild conditions,
(3) The hydroxyl group of 3,4-dihydroxy-N-methyl-N-trifluoroacetylaniline was protected.

(3) 3,4-dihydroxy-N-methyl-N-trifluoroacetylaniline 507 mg
(2.15 mmol) was dissolved in 15 mL of dichloromethane and 2,6-lutidine (manufactured by Wako Pure Chemical Industries, Ltd.)
90 mL (16.3 mmol) tert-butyldimethylsilyl sulfate trifluoromethane (manufactured by Aldrich)
After 1.78 mL (7.74 mmol) was added at 0 ° C., the mixture was stirred at room temperature for 12 hours.

50 mL of cold water at 4 ° C. was added to the reaction solution, and the mixture was extracted twice with 100 mL of ethyl acetate. Column (3 cmφ × 30 cm) equipped with silica gel (trade name: Wakogel C-200, manufactured by Wako Pure Chemical Industries, Ltd., developing solution: hexane / ethyl acetate = 20/1 to 10/1 (volume ratio: v /
v)), and (4) 3,4-bis [(tert-butyldimethylsilyl) oxy] -N-methyl-N-trifluoroacetylaniline 390 mg (1.0 mmol)
1) was obtained.

(4) The protection of the amino group of 3,4-bis [(tert-butyldimethylsilyl) oxy] -N-methyl-N-trifluoroacetylaniline by the trifluoroacetyl group was removed with potassium carbonate. Product (4) 3,4-bis [(tert-butyldimethylsilyl) oxy] -N-methyl-N-trifluoroacetylaniline in 10 mL of aqueous potassium carbonate-methanol
(7% potassium carbonate: methanol = 2: 5 (volume ratio: v
/ V)) and stirred at room temperature for 3 hours. The reaction solution was extracted with dichloromethane, and a column (3 cmφ × 30 cm, hexane / ethyl acetate =) equipped with silica gel (trade name: Wakogel C-200, manufactured by Wako Pure Chemical Industries, Ltd.)
15/1 to 10/1 (volume ratio: v / v)),
(5) 196 mg of 3,4-bis [(tert-butyldimethylsilyl) oxy] -N-methylaniline (0.6 mg
5 mmol) (yield 65.0%).

Next, using sodium nitrate, (5)
The amino group of 3,4-bis [(tert-butyldimethylsilyl) oxy] -N-methylaniline was nitrosated. (5) 3,4-bis [(tert-butyldimethylsilyl) oxy] -N-methylaniline 196
mg was dissolved in 15 mL of tetrahydrofuran, and 3 mL of 1N hydrochloric acid and 45 mg of equimolar sodium nitrate were added at 0 ° C. After the reaction solution was stirred at 0 ° C. for 3 hours, it was extracted with 100 mL of dichloromethane, and a column (3 cmφ × 30 cm, equipped with silica gel (trade name: Wakogel C-200, manufactured by Wako Pure Chemical Industries, Ltd.); developing solution: hexane / (6) 3,4-bis [(tert-butyldimethylsilyl) oxy] -N-methyl-N-nitrosoaniline (ethyl acetate = 15/1 to 10: 1 (volume ratio: v / v)) 182 mg (0.5 mmol) were obtained (yield 85.0).
%).

The obtained (6) 3,4-bis [(tert
-Butyldimethylsilyl) oxy] -N-methyl-N-
Nitrosoaniline was dissolved in 6 mL of tetrahydrofuran, 1.5 mL of sodium fluoride-hydrofluoric acid buffer (pH 4.98) was added at 0 ° C, and the mixture was stirred at room temperature for 24 hours. The reaction solution was extracted three times with chloroform, and the organic layer was dried over sodium sulfate and then dried. Silica gel (Wako Pure Chemical Industries, Ltd., trade name: Wakogel C-200)
(Developing solution: hexane / ethyl acetate = 3)
/ 1 to 1/1 (volume ratio: v / v)).
92.1 mg of dihydroxydefostatin (0.5 mm
ol) was obtained (yield 99.0%).

Defostatin is available from J. Chem. Soc., Ch.
em.Commun. 1994 Synthesized by the method described in pp.437 was used.

EXPERIMENTAL EXAMPLES 1-3 As a system for testing the effect of defostatin and 3,4-dihydroxydefostatin on diabetes,
A test system was used to examine the suppression of free fatty acid release from cultured adipocytes treated with epinephrine (BBRC _, 995
_, 14 _, No. 3, pp. 1095-1099). This system is highly sensitive as a system for determining the effects of diabetes at in vitro levels,
Known as a simple predictive test method for hypoglycemia without using radioactive glucose (Biol. Pharm. Bull. 1995, 18, p.
p.719-722).

The cells were 3T3-L1 cells (HSRRB JC) obtained from Human Science Research Resource Bank.
Using RB9014), culture was continued in a Dulbecco's modified MEM medium supplemented with 10% bovine serum at 37 ° C. in a 5% CO ₂ environment.

2 × 10 ⁶ / mL of 3T3-L1 cells were added to 20 mg of bovine serum albumin (manufactured by Wako Pure Chemical Industries, Ltd.) and KRB buffer (120 mM NaCl, 1.27).
mM CaCl ₂ , 1.2 mM MgSO ₄ , 4.75 m
M KCl, 1.2 mM KH ₂ PO ₄ , 24 mM Ma
Drug dissolved in HCO ₃ (pH 7.4) (Experimental example 1: Defostatin 20 μg / mL (10 ⁻⁴ M), Experimental example 2:
3,4-dihydroxydefostatin isomer 2 μg / m
L, Experimental Example 3: 20 μg / mL isomer of 3,4-dihydroxydefostatin) at 37 ° C. for 30 minutes.

Epinephrine (10 ⁻⁵ M) was added to the above culture solution, the mixture was further cultured at 37 ° C. for 3 hours, and 0.5 mL of ice-cold water was added to stop the reaction. 1200r using a centrifuge
centrifugation at pm. for 10 minutes, and using an FFA kit Wako (manufactured by Wako Pure Chemical Industries, Ltd., trade name: NEFA-C Test Wako), titer the absorbance at 550 nm as an index of the concentration of free fatty acids in the extracellular fluid. Tech Multi Scan Monitor (Flow Laboratory Co., Ltd., trade name:
(Multiscan Plus MKII). In addition, vanadium oxide (manufactured by Wako Pure Chemical Industries, Ltd.), which is known as a tyrosine phosphatase inhibitor (an analog of phosphorus), is replaced with 5 × 10 ^{− in place of} defostatin and 3,4-dihydroxydefostatin isomers. those used in ⁴ M as a positive control, the absorbance was measured 550nm extracellular fluid. Table 1 shows the results.

[0051]

[Table 1]

From the results of Experimental Examples 1 to 3, the addition of defostatin and 3,4-dihydroxydefostatin isomers showed that compared to epinephrine alone (no drug),
Release of free fatty acids from T3-L1 cells was significantly suppressed.

Also, from the results of Experimental Examples 1 to 3 and the positive control, in Experimental Examples 1 to 3, an effect equivalent to that of vanadium oxide sulfate was expressed at a concentration of about 1/5 to 1/10. The effect of defostatin and the 3,4-dihydroxydefostatin isomer was much stronger than vanadium oxide sulfate.

When the 3T3-L1 cells in Experimental Examples 1 to 3 were observed under a microscope, no cell damage occurred, and the suppression of fatty acid release seen in Experimental Examples 1 to 3 was not due to cytotoxicity. Confirmed that there is no.

From the above, it was strongly suggested that defostatin and 3,4-dihydroxydefostatin isomers are effective for improving hyperglycemia due to diabetes.

[0056]

Industrial Applicability According to the present invention, there is provided a therapeutic agent for diabetes, especially for non-insulin dependent diabetes. The defostatin of the present invention and its pharmacologically acceptable salt and the 3,4-dihydroxydefostatin isomer and its pharmacologically acceptable salt can be used safely and effectively for diabetes, especially for non-insulin-dependent diabetes. It has a therapeutic effect and can improve hyperglycemia due to diabetes.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Takashi Yamashita Beppu 899-1-101 Beppu-cho, Kakogawa-shi, Hyogo

Claims (2)

[Claims] 1. A compound of formula (I): Or a pharmacologically acceptable salt thereof as an active ingredient. 2. A compound of formula (II): A therapeutic agent for diabetes, comprising, as an active ingredient, a 3,4-dihydroxydefostatin isomer represented by the formula: or a pharmaceutically acceptable salt thereof.