JPH08231420A - Protein dephosphorylase inhibitor - Google Patents

Abstract

PURPOSE: To obtain a novel protein dephosphorylase inhibitor which contains a specific physiologically active substance as an active ingredient and is useful as a treating agent for diseases relating to protein dephosphorylase or as a reagent. CONSTITUTION: This inhibitor contains, as an active ingredient, a physiologically active substance selected from NK374186A, NK374186B, NK374186B3, NK374186C3, NK374186A3, NK374186D3, NK374186E3, NK374186H, NK374186I, NK374186P, NK374186PP or a salt thereof. Among these physiologically active substances, NK374186A, B, B3, C3 and the like are obtained by culturing a microorganism belonging to the genus Penicillium capable of producing these substances, for example, NK374186 strain (FERM 12285 and FERM 3870) and the product is collected. The concentration of the substance is 0.1-70wt.% in the preparation. The daily dose is suitably 0.01-800mg/adult.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a protein phosphatase inhibitor. Since protein dephosphorylation is a basic regulatory mechanism of biological functions, its inhibitors are useful as therapeutic agents and reagents for diseases based on abnormal dephosphorylation.

[0002]

2. Description of the Related Art Phosphorylation and dephosphorylation of proteins in living organisms are known to be one of the basic regulatory mechanisms of biological functions. Many drugs that inhibit protein phosphorylation have been reported, and are known to exhibit anticancer effects and the like. However, inhibitors of protein phosphatase, which also play an essential role in cell proliferation and differentiation, are, for example, okadaic acid [Proc.
Natl. Acad. Sci. , USA, 85,176
8-1771 (1988)], and there are few reports.

[0003]

The above-described okadaic acid has drawbacks such as strong cytotoxicity and is lacking in practicality. Inhibitors against protein phosphatase are useful as pharmaceuticals for diseases involving protein phosphatase. It is also useful, for example, as a reagent for elucidating the mechanisms of cell growth and differentiation and for investigating the action of phosphorylase. The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a novel protein phosphatase inhibitor. Another object of the present invention is to
An object of the present invention is to provide a reagent for inhibiting a protein phosphatase.

[0004]

That is, the gist of the present invention is to provide NK37418 represented by the chemical formula of claim 1.
6A, NK374186B, NK374186B3, N
K374186C3, NK374186A3, NK37
4186D3, NK374186E3, NK37418
6H, NK374186I, NK374186P and N
A protein phosphatase inhibitor comprising at least one physiologically active substance selected from K374186PP or a pharmacologically acceptable salt thereof as an active ingredient. Yet another aspect of the present invention resides in a protein phosphatase inhibiting reagent comprising the above-mentioned physiologically active substance or a pharmacologically acceptable salt thereof as an active ingredient.

Hereinafter, the present invention will be described in detail. The physiologically active substance of the present invention comprises N represented by the chemical formula according to claim 1.
K374186A, NK374186B, NK3741
86B3, NK374186C3, NK374186A
3, NK374186D3, NK374186E3, N
K374186H, NK374186I, NK3741
86P and NK374186PP (hereinafter NK3741)
It may be called 86. At least one selected from
Kinds of physiologically active substances or pharmacologically acceptable salts thereof.

The NK374186s in the present invention are represented by the following chemical formula.

[0007]

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[0008]

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[0009]

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[0010]

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[0011]

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[0012]

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[0013]

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[0014]

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[0015]

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[0016]

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[0017]

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Biologically active substances NK374186A, NK3
74186B, NK374186B3 and NK3741
86C3 can be produced by the method described in JP-A-5-194571.

That is, it belongs to the genus Penicillium and has the above-mentioned physiologically active substances NK374186A and NK374186.
B, the NK374186 strain (Microtechnical Laboratories No. 12285 and Microtechnical Research Laboratories No. 3870) having the ability to produce NK374186B3 and NK374186C3 is cultured, and a physiologically active substance is produced and accumulated in the culture. It can be obtained by sampling.

Further, a physiologically active substance 374186A3, N
K374186D3 and NK374186E3 can also be obtained by culturing the NK374186 strain, producing and accumulating these physiologically active substances in the culture, and collecting these physiologically active substances.

On the other hand, a physiologically active substance NK374186H,
NK374186I, NK374186P and NK37
4186PP can be produced by a chemical conversion method using NK374186B as a raw material described in JP-A-5-194571. Bioactive substance NK37418
Class 6 may be a pharmacologically acceptable salt, such as sodium, potassium, calcium or ammonium salts.

Next, the protein phosphatase inhibitory activity of the physiologically active substances NK374186s will be described. VHR protein phosphatase and protein tyrosine phosphatase CD4 as human cell-derived protein phosphatases
5 was used to examine the protein phosphatase inhibitory activity of the physiologically active substances NK374186s. Any NK374
186 also showed inhibitory activity.

Next, the toxicity of the physiologically active substances NK374186 will be described. 100 mg / kg of physiologically active substances NK374186 were added to CDF-mice (8 weeks old, male).
Was administered intraperitoneally once a day and continuously for 10 days. NK374186s are 3.5% DMSO + 6.5
% Tween 80 + 90% physiological saline and used in vials. Observed for 10 days, any NK
No deaths were found in 374186.

Since the physiologically active substances NK374186 inhibit the protein phosphatase which plays an essential role in cell growth and differentiation, the NK374186s may be further used as an inhibitor for diseases involving protein dephosphorylation. It is useful as a medicine against It is also useful as a reagent for elucidating the mechanism of cell growth and differentiation, and further for examining the action of phosphorylase.

Conventional methods can be applied to the preparation and administration of the physiologically active substances NK374186 as pharmaceuticals. That is, administration methods such as injection, oral administration, and rectal administration are possible. The preparation may take the form of injections, powders, tablets, suppositories and the like. As long as NK374186 is not adversely affected, various auxiliaries used for medicine, that is, carriers and other auxiliaries, such as stabilizers, preservatives, soothing agents, and emulsifiers, may be used in the formulation. Used as needed.

In the formulation, the physiologically active substance NK374
The content of 186 can be varied widely depending on the form of preparation and the like. Generally, NK374186 is contained in an amount of 0.01 to 100% by weight, preferably 0.1 to 70% by weight, and the rest is usually a pharmaceutical. And other auxiliaries used for The dose of NK374186 varies depending on symptoms and the like, but is 0.01 to 8 per adult per day.
It is about 00 mg. 1 if continuous dosing is required
It is preferable to reduce daily usage.

On the other hand, when the physiologically active substance NK374186 is used as a reagent, it is dissolved in, for example, an organic solvent such as dimethyl sulfoxide or a surfactant.
It is diluted in various buffers such as Tris buffer and used for an enzyme reaction or the like.

[0028]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist.

Example 1 (Human cell-derived protein phosphatase VHR inhibition test) The inhibitory activity of NK374186s was measured according to the following method. That is, the human cell-derived protein phosphatase was prepared from Escherichia coli into which the VHR protein phosphatase gene was introduced.

A series of dilution ratios of NK374186 were added to this protein phosphatase, and paranitrophenyl phosphate (Sigma) was used as a substrate.
For 30 minutes. After the reaction, 1N sodium hydroxide was added, and the absorbance of the generated paranitrophenol was measured by E.
It was measured with an IA reader (Model 2550: manufactured by Bio-Rad), and this was used as an index for determining the presence or absence of NK374186 activity.

The 50% inhibitory concentration was calculated and is shown in Table 1. From this, it was found that NK374186s exhibited an inhibitory activity on human cell-derived protein phosphatase VHR.

Example 2 (Inhibition test of protein tyrosine phosphatase CD45) The protein tyrosine phosphatase CD45 prepared from the cell membrane of the cultured cell Ball-1 was used and the above-mentioned Example 1 was carried out.
The inhibitory activity of NK374186s was measured in the same manner as described above. Then, the 50% inhibitory concentration was calculated and shown in Table 1.
From this, NK374186P and NK374186P
P was found to show an inhibitory activity on the protein tyrosine phosphatase CD45 in particular.

[0033]

[Table 1]

Next, NK374186 used in the present invention.
The production method of the class will be described. Reference Example 1 (NK374
186A3, NK374186D3 and NK37418
Production method of 6E3) <Fermentation> Seed culture medium 1 having the composition shown in Table 2 below
00 ml was dispensed into a 500 ml Erlenmeyer flask and autoclaved at 120 ° C. for 20 minutes. This was followed by NK374186 strain (Microtechnical Lab.
No. 5) was inoculated and cultured at 27 ° C. under the condition of 200 revolutions / minute for 2 days, which was used as a primary seed.

[0035]

[Table 2]

In the secondary seed culture, 20 liters of a medium having the same composition as the primary is charged into a 30-liter jar fermenter, sterilized, and 200 ml of the primary seed obtained by the above method is aseptically sterilized. And cultivated for 3 days at 25 ° C. while aerating at 20 liters of air per minute and stirring at 250 rpm to obtain a secondary seed.

In the main culture, 150 liters of a production medium having a composition in which the glycerin concentration of the seed culture medium was changed to 1.0% was placed in a 200-liter tank, sterilized, and then the secondary seed mother obtained by the above method was sterilized. Aseptically transplant 2 liters,
150 liters of air per minute at 25 ° C.
The culture was performed for 4 days while stirring at 0 rotation. Filtration was performed using a filter press from the culture solution obtained from the four 200-liter tanks to separate the filtrate from the cells.

<Purification>
After adding 3 liters and performing stirring extraction for 3 hours, the bacterial cells and the extract were separated by suction filtration. An equal amount of water was added to the obtained extract. This solution was passed through a 20 liter HP-20 column, washed with 50% methanol, and then washed with 60% acetone 20%.
Elution was performed with 1 liter to obtain fraction 1, and then eluted with 30 liter of 80% acetone to obtain fraction 2.

The fraction 1 was concentrated to dryness, dissolved in 0.2 liter of water, passed through a 2 liter HP-20ss column, and eluted with acetone having a concentration gradient of 50% to 70%. In this order, fractions 1A and 1B were obtained. Fraction 1A
And Fraction 1B were respectively chloroform: methanol = 1:
NK374186A3 from fractions 1A to 800 mg, and NK374186A3 from fractions 1B to NK.
130 mg of 374186D3 was obtained.

On the other hand, fraction 2 was concentrated to dryness, and then applied to a 5 liter silica gel column, and washed with 5 liters of chloroform. Then, chloroform: methanol = 5
The fraction was developed at 0: 1 to obtain fraction 2A. A silica gel column developing Fraction 2A with hexane: acetone = 3: 1, L
Apply NK374186E3 to the H-20 column sequentially.
00 mg was obtained. The physicochemical properties of each component were obtained as follows.

NK374186A3: MW = 735 (FAB-Negative Spect)
FIG. 1 shows an ultraviolet absorption spectrum, FIG. 2 shows a hydrogen nuclear magnetic resonance spectrum measured in deuterated dimethyl sulfoxide, and FIG. 3 shows a carbon nuclear magnetic resonance spectrum measured in deuterated dimethyl sulfoxide.

NK374186D3: MW = 675 (FAB-Negative Spect)
FIG. 4 shows an ultraviolet absorption spectrum, FIG. 5 shows a hydrogen nuclear magnetic resonance spectrum measured in deuterated dimethyl sulfoxide, and FIG. 6 shows a carbon nuclear magnetic resonance spectrum measured in deuterated dimethyl sulfoxide.

NK374186E3: MW = 595 (FAB-Negative Spect)
FIG. 7 shows an ultraviolet absorption spectrum, FIG. 8 shows a hydrogen nuclear magnetic resonance spectrum measured in deuterated dimethyl sulfoxide, and FIG. 9 shows a carbon nuclear magnetic resonance spectrum measured in deuterated dimethyl sulfoxide.

Reference Example 2 (Production of NK374186H) NK374186B (42.6 mg, 0.065 mmol)
l) was dissolved in pyridine (6.5 mL) and acetic anhydride (3
(0.7 μL, 0.325 mmol) and stirred at room temperature for 24 hours. The reaction solution is concentrated under reduced pressure, and the obtained residue is subjected to silica gel column chromatography (silica gel 5).
g, eluted with 2% methanol-chloroform) to obtain NK374186H (36.0 mg, 79%). Table 3 shows ¹ H-NMR and FA of NK374186H.
The measurement result of B-MS is shown.

[0045]

[Table 3]

Reference Example 3 (Production of starting material compound (b1) for production of NK374186I)

[0047]

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NK374186B (52.7 mg, 0.
080 mmol) and 4-dimethylaminopyridine (1
0.1 mg, 0.083 mmol) in methylene chloride (0.7 mL) and triethylamine (15.0 μL, 0.108 mmol) at −40 ° C. under an argon atmosphere.
l) and methanesulfonyl chloride (7.4 μL, 0.00
96 mmol) was added. After stirring for 1.5 hours under ice cooling, triethylamine (15.0 µ
L, 0.108 mmol) and methanesulfonyl chloride (7.4 μL, 0.096 mmol) were added, and the mixture was stirred at 5 ° C. overnight.

To the reaction solution was added a saturated aqueous solution of sodium hydrogen carbonate, and the mixture was extracted with methylene chloride. The obtained organic layer was washed with saturated saline and dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (silica gel 8 g, eluted with 0 to 2% methanol-chloroform) to obtain the above compound (b1) (45.6 mg, 78%). That FA
The measurement result of B-MS is m / z: 734 [(M +
H) ⁺ ].

Reference Example 4 (Production of NK374186I) The compound (b1) (45.0 mg, 0.061 mm)
ol) in tetrahydrofuran (0.7 mL)
At room temperature, 1,8-diazabicyclo [5.4.0] undecene (10.1 μL, 0.067 mmol) was added and stirred. After 1.5 hours, further 1,8-diazabicyclo [5.4.0] undecene (5.1 μL, 0.034 m
mol) and stirred for 4 hours.

Ethyl acetate was added to the reaction mixture, and the mixture was washed successively with a 1N aqueous hydrochloric acid solution, water and saturated brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the obtained residue was purified by silica gel column chromatography (silica gel 3 g, eluted with 25-35% ethyl acetate-hexane) to give NK374186I (21.4 m
g, 61%). ¹ H of NK374186I in Table 4
-Shows the measurement results of NMR and FAB-MS.

[0052]

[Table 4]

Reference Example 5 (Production of starting compound (c1) of NK374186P)

[0054]

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NK374186B (61.2 mg, 0.
093 mmol) and 1H-tetrazole (32.6 m
g, 0.465 mmol) in methylene chloride (2 mL) and dissolve in N, N-dimethyl-1,5-
Dihydro-2,4,3-benzodioxaphosphepin-
3-Amine (24.1 μL, 0.112 mmol) was added, and the mixture was stirred at room temperature.

After 30 minutes, N, N-dimethyl-
1,5-dihydro-2,4,3-benzodioxaphosphepin-3-amine (12.0 μL, 0.056 mmol
l) was added and stirred for 20 minutes. After adding water (50 μL) and stirring for 10 minutes, the mixture was cooled to −40 ° C., 3-chloroperbenzoic acid (140 mg, 0.811 mmol) was added, and the mixture was returned to room temperature and further stirred for 10 minutes. A 10% aqueous sodium sulfite solution (10 mL) was added to the reaction solution, and the mixture was stirred and extracted with ethyl acetate.

The obtained organic layer was washed with a saturated aqueous solution of sodium hydrogen carbonate and further with a saturated saline solution, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography (silica gel 15 g, 1
% Methanol-chloroform).
Compound (c1) (39.4 mg, 51%) was obtained. The FAB-MS measurement result was: m / z: 838 [(M +
H) ⁺ ], 860 [(M + Na) ⁺ ].

Reference Example 6 (Production of NK374186P) The compound (c1) (29.1 mg, 0.035 mm)
ol) in 80% aqueous ethanol (5 mL),
% Palladium on carbon (water-containing 50%, 10 mg) was added, and the mixture was stirred for 40 minutes while passing hydrogen gas. The reaction solution was filtered through celite to remove palladium carbon. Concentrated aqueous ammonia (0.1 mL) was added to the obtained filtrate, and the mixture was concentrated under reduced pressure. After dissolving in water (1 mL) and freeze-drying, NK3
74186P was converted to an ammonium salt (25.9 mg, 96
%). Table 5 shows ¹ H-N of NK374186P.
The measurement results of MR and FAB-MS are shown.

[0059]

[Table 5]

Reference Example 7 (Production of starting compound (c3) of NK374186PP)

[0061]

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NK374186B (65.5 mg, 0.
100 mmol) and 1H-tetrazole (50.0 m
g, 0.714 mmol) in methylene chloride (2 mL) and dissolve in N, N-dimethyl-1,5-
Dihydro-2,4,3-benzodioxaphosphepin-
3-Amine (0.104 mL, 0.482 mmol) was added, and the mixture was stirred at room temperature for 15 minutes. Water (0.05 mL)
And stirred for 15 minutes, then cooled to -40 ° C,
Chloroperbenzoic acid (148mg, 0.858mmol)
Was added, and the mixture was returned to room temperature and stirred for another 15 minutes.

A 10% aqueous solution of sodium sulfite (10 mL) was added to the reaction solution, and the mixture was stirred and extracted with ethyl acetate. The obtained organic layer was washed with a saturated aqueous solution of sodium hydrogencarbonate and then with saturated saline, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (silica gel 15 g, eluted with 2% methanol-chloroform) to obtain the previous compound (c3) (93.8 mg, 93%). That F
The measurement result of AB-MS is m / z: 1020 [(M +
H) ⁺ ], 1042 [(M + Na) ⁺ ]

Reference Example 8 (Production of NK374186PP) The compound (c3) (80.0 mg, 0.079 mm
ol) in 75% aqueous ethanol (20 mL).
0% palladium carbon (water 50%, 40 mg) was added,
The mixture was stirred for 1 hour while passing hydrogen gas. The reaction solution was filtered through celite to remove palladium carbon. Concentrated aqueous ammonia (0.6 mL) was added to the obtained filtrate, and the mixture was concentrated under reduced pressure. After dissolving in water (1 mL) and freeze-drying, NK3
74186PP with ammonium salt (65.1 mg, 93
%). Table 6 of NK374186PP ¹ H-
The measurement results of NMR and FAB-MS are shown.

[0065]

[Table 6]

[0066]

According to the present invention described above, NK37
The 4186s or pharmacologically acceptable salts thereof are useful as protein phosphatase inhibitors, reagents for protein phosphatase inhibition, and the like.

[Brief description of the drawings]

FIG. 1 is an ultraviolet absorption spectrum of NK374186A3.

FIG. 2 is a hydrogen nuclear magnetic resonance spectrum measured in deuterated dimethyl sulfoxide of NK374186A3.

FIG. 3 is a carbon nuclear magnetic resonance spectrum measured in deuterated dimethyl sulfoxide of NK374186A3.

FIG. 4 is an ultraviolet absorption spectrum of NK374186D3.

FIG. 5 is a hydrogen nuclear magnetic resonance spectrum measured in heavy dimethyl sulfoxide of NK374186D3.

FIG. 6 is a carbon nuclear magnetic resonance spectrum measured in deuterated dimethyl sulfoxide of NK374186D3.

FIG. 7 is an ultraviolet absorption spectrum of NK374186E3.

FIG. 8 is a hydrogen nuclear magnetic resonance spectrum measured in heavy dimethyl sulfoxide of NK374186E3.

FIG. 9 is a carbon nuclear magnetic resonance spectrum measured in deuterated dimethyl sulfoxide of NK374186E3.

Claims (2)

[Claims] 1. NK37418 represented by the following chemical formula:
6A, NK374186B, NK374186B3, N
K374186C3, NK374186A3, NK37
4186D3, NK374186E3, NK37418
6H, NK374186I, NK374186P and N
A protein phosphatase inhibitor comprising, as an active ingredient, at least one physiologically active substance selected from K374186PP or a pharmacologically acceptable salt thereof. Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image 2. A reagent for inhibiting a protein phosphatase, comprising as an active ingredient at least one of the physiologically active substance according to claim 1 or a pharmacologically acceptable salt thereof.