JPH07242545A - Protein phosphatase inhibitor and antitumor agent - Google Patents

Abstract

(57) [Summary] [Structure] Formula (I) A protein dephosphorylating enzyme inhibitor and an antitumor agent containing the tetronic acid derivative substance represented by [Effect] A protein dephosphorylating enzyme inhibitor and an antitumor agent can be provided.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a protein phosphatase inhibitor and an antitumor agent containing a tetronic acid derivative substance as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Protein phosphorylation and dephosphorylation are one of the basic regulatory mechanisms of biological functions. Many drugs that inhibit protein phosphorylase have been reported as antitumor agents, but there are few reports about inhibitors for protein dephosphorylating enzymes that also play an essential role in cell growth.

In the field of antitumor agents, inhibitors of protein phosphatase have a new mechanism of action (Proc. Natl. Aca
d. Sci. 86: 9946-9950; (1989), Proc. Natl. Acad. Sc
i. 89: 12170-12174; (1992). (See Reference), and further development of a protein phosphatase inhibitor having excellent specificity and effectiveness is desired.

[0004]

An object of the present invention is to provide a protein phosphatase inhibitor and an antitumor agent.

[0005]

Means for Solving the Problems As a result of searching for a substance that inhibits the dephosphorylation activity of a protein dephosphorylating enzyme, the present inventors found that a specific tetronic acid derivative substance is a dephosphorylating enzyme of a protein dephosphorylating enzyme. The inventors have found that the acid activity is inhibited and completed the present invention. That is, the present invention has the formula (I)

[0006]

[Chemical 3]

A protein dephosphorylating enzyme inhibitor or an antitumor agent containing the tetronic acid derivative substance represented by as an active ingredient. Hereinafter, the present invention will be described in detail. The active ingredient of the protein phosphatase inhibitor and the antitumor agent of the present invention has the formula (I)

[0008]

[Chemical 4]

It is a tetronic acid derivative substance represented by the structural formula: This tetronic acid derivative substance is, for example, a microorganism that belongs to the genus Streptomyces and has the ability to produce a tetronic acid derivative substance having protein dephosphorylating enzyme inhibitory activity, is cultured in a medium, and the tetronic acid derivative substance is cultured from the culture. It can be manufactured by collecting. Examples of such microorganisms include Streptomyces sp. 88-682. The mycological properties of this strain are as follows. (I) Morphology Morphology was observed with a microscope and an electron microscope for growth on sucrose-nitrate agar and starch-inorganic salt agar. The 88-682 strain showed a form belonging to the genus Streptomyces. Its morphological characteristics are as follows. 1. Basal hyphae: Well grown on agar and branched. 2. Aerial mycelium: Well settled, spore stalks are simply branched, mature sporophytes form straight chains with 10 or more spores in each spore chain. According to the observation with an electron microscope, the spore surface is smooth, and the spore itself is cylindrical, and its size is 0.85.
˜1.19 × 0.58 to 0.66 μm. No formation of sporangia, chlamydospores, sclerotium, etc. is observed. (II) Properties on various media Prepare various media according to the Japan Patent Office Industrial Examination Standards and inoculate them.
The results observed after the day are described below. (1) Sucrose / nitrate agar medium Growth: Medium.

Aerial mycelium: formed well and exhibited a yellowish white color. Good spore settlement. Backside color: yellowish white. Soluble dye: Does not form. (2) Glucose / asparagine agar medium Growth: Moderate.

Aerial mycelium: Does not form. Backside color: light green to blackish brown. Soluble pigment: pale yellow. (3) Glycerin / asparagine agar medium Growth: Moderate.

Aerial mycelium: Moderate formation with white to light gray color. No spore settlement is observed. Backside color: blackish brown. Soluble pigment: brown. (4) Starch / inorganic salt agar medium Growth: Good.

Aerial mycelium: formed well, and had a yellowish white color. Moderate spore settlement. Backside color: light tan to blackish brown. Soluble pigment: pale yellow. (5) Tyrosine agar medium Growth: Good.

Aerial mycelium: Good formation, white to light gray color. No spore settlement is observed. Backside color: blackish brown. Soluble pigment: blackish brown. (6) Nutrient agar medium Growth: Good.

Aerial mycelium: Does not form. Backside color: light brown. Soluble dye: Does not form. (7) Yeast / malt agar medium Growth: Good.

Aerial mycelium: formed well and exhibited a yellowish white color. No spore settlement is observed. Backside color: blackish brown. Soluble pigment: brown. (8) Oatmeal agar medium Growth: Good.

Aerial mycelium: Good formation and yellowish white color. No spore settlement is observed. Backside color: pale brown to pale blackish brown. Soluble pigment: pale yellow. (9) Peptone-Yeast-Iron Agar Medium Growth: Good.

Aerial mycelium: Does not form. Backside color: blackish brown. Soluble pigment: blackish brown. (III) Physiological properties (1) Growth temperature range: 11 ° C to 37 ° C. (2) Optimal growth temperature: 28 ° C to 32 ° C. (3) Starch hydrolysis: positive. (4) Skim milk powder: Positive coagulability.

Peptone negative. (5) Generation of melanin-like pigment: Tyrosine agar medium positive. Peptone, yeast, iron agar medium positive. (6) Liquefaction of gelatin: Negative. (IV) Utilization of various carbon sources The results of culturing strain 88-682 by adding various sugars to Pridham-Gothriep agar medium (manufactured by Difco) are as follows.

L-arabinose: + D-xylose: + D-glucose: ++ D-fructose: + sucrose:-inositol: + L-rhamnose:-raffinose: ++ D-mannitol:-Note) + +: grow well +: Grows −: does not grow (V) Diaminopimelic acid in all cells As a result of analysis of diaminopimelic acid in all cells, L, L-diaminopimelic acid was detected.

The mycological properties of strain 88-682 are as described above, but the features of this strain are summarized as follows. 1. It grows well on aerial mycelia, and the color tone changes from yellowish white to light gray. Spore stalk is simple branch, more than 10 spores are linked and spore linkage is straight. The spores are cylindrical and the surface is smooth. It has a form belonging to the genus Streptomyces. 2. The reverse side of the colony of bacteria mainly exhibits a single brown to a blackish brown color. 3. Form a melanin-like pigment. 4. Utilizes various sugars except sucrose, rhamnose and mannitol. 5. It has L, L-diaminopimelic acid.

The above various characteristics are described in "International Journal of Systematic Bacteriolog".
y) ”, Vol. 18, pp. 69-189, 279-392, (1968
), Vol. 19, pp. 391-512 (1969) and Vol. 22,
As a result of comparison with many known strains reported on pages 265 to 394 (1972) and "Bergey's Manual of Systematic Bacteriology", Volume 4 (1989), Streptomyces nashbilensis (Stre
ptomycesnashvillensis) was found to be very similar, but there were some differences in the color of the aerial hyphae and the use of carbon source from 88-682 strain, and a definitive basis for the same species was obtained. Since it was not present, this strain was named Streptomyces sp. 88-682 strain. This strain was collected and isolated from soil in Kuroishi City, Aomori Prefecture, and has been deposited as FERM P-14189 at the Institute of Biotechnology, Institute of Industrial Science and Technology.

The Streptomyces sp. Strain 88-682, like other actinomycetes of the genus Streptomyces, is likely to change its properties, and is an artificial mutagenizing means using, for example, ultraviolet rays, X-rays, radiation, artificial mutagens and the like. It is possible to easily mutate, and even if such a mutation is used, all those capable of producing a tetronic acid derivative substance having an activity of inhibiting the dephosphorylation activity of protein dephosphorylating enzyme should be used. You can

As a medium used for culturing the above-mentioned microorganism, a medium containing nitrogen sources, inorganic salts, vitamins and other nutrient sources may be used. Here, as the nitrogen source, for example, ammonium sulfate, ammonium chloride,
It may be an inorganic nitrogen source such as ammonium nitrate or ammonium phosphate, or an organic nitrogen source such as urea, yeast extract, peptone, glutamic acid or sodium glutamate. The pH of the medium is 6 to 10, preferably 6.5 to 7.
5. The culture temperature is usually 10 to 35 ° C, preferably 20 to 27 ° C.

The culture can be appropriately determined depending on the growth condition of the microorganisms and the like, but it is usually carried out for 3 to 14 days under aerobic conditions. The isolation / purification of the tetronic acid derivative substance from the culture is a method commonly used for isolating / purifying microbial metabolites from the culture, for example, extraction with an organic solvent, adsorption and elution with an adsorbent, and chromatography. Etc. can be implemented in an appropriate combination.

The administration method when the tetronic acid derivative substance obtained by the above microorganisms or the like is used as a protein dephosphorylating enzyme inhibitor or an antitumor agent based on the inhibitory action of its protein dephosphorylating enzyme activity is as follows: is there. When used as an injection, the active ingredient contains a pH adjuster, a buffer,
Stabilizers, excipients, etc. may be added. Also, lyophilization can be carried out by a conventional method, so that a lyophilized injection can be used. Furthermore, a pH adjusting agent, a buffering agent, a stabilizing agent, an isotonic agent, and a narcotics sugar are added to this active ingredient, and subcutaneous, intramuscular, or intravenous injection in the form of a solution or suspension is carried out by a conventional method. It can also be used as an agent.

On the other hand, when used as a solid preparation, usual excipients, stabilizers, and if necessary, binders, disintegrating agents, lubricants, coloring agents, flavoring agents, flavoring agents, etc. are added to the present active ingredient. However, tablets, granules, powders, capsules and the like can be prepared by a conventional method. Although the dose of the substance of the present active ingredient varies depending on the administration method and symptoms, it is usually preferably 0.1 to 1000 mg / kg as a single dose for an adult, and is administered three times a day or three or more times a day depending on the symptoms.

[0028]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples. (Production Example 1) A medium (pH: 2% glucose, 1% soluble starch, 0.1% meat extract, 0.4% yeast, 0.2% sodium chloride, 0.005% dibasic potassium phosphate and 2.5% soybean flour)
7.0) was inoculated with the actinomycete strain 88-682 and shake-cultured at 28 ° C for 96 hours. 350 ml of this culture broth was inoculated into 36 liters of a medium having the same composition, and agitated at 300 revolutions per minute while performing aeration at 18 liters per minute at 28 ° C. for 96 hours.

The above culture solution was separated into bacterial cells and supernatant by a high speed centrifuge (15000 rpm), and the bacterial cells were extracted with 12 L of 70% acetone. After concentration under reduced pressure, pH of the resulting aqueous solution
It was adjusted to 3.0 and extraction was repeated twice with 5 L of ethyl acetate. After extraction, all ethyl acetate was combined and concentrated under reduced pressure,
43.7 g of brown syrup was obtained. Dissolve 43.7 g of the obtained syrup in 30 ml of chloroform, infiltrate a silica gel column (diameter: 6 cm, length: 90 cm) prepared with chloroform, first flow 2 L of chloroform, and then change the mixing ratio of chloroform-methanol solution sequentially. (99: 1, 19: 1,
(9: 1, 4: 1, 1: 1) 1 L each, and finally 2 L of methanol.

The activity was eluted in the fraction of chloroform-methanol solution (4: 1). This was concentrated under reduced pressure to obtain 12.3 g of white powder. Further, this white powder was dissolved in a chloroform-methanol solution (1: 1),
It was applied to a column (diameter 4.5 cm, length 120 cm) of Sephadex LH-20 (manufactured by Pharmacia) prepared with the same solution and eluted with a chloroform-methanol solution (1: 1). Fractions of 10 ml each were collected, and fraction No. 39-48, which was the main active fraction, was collected, and 1.1 g of white powder was obtained from this fraction by concentration under reduced pressure.

Finally, an ODS column (diameter 2 cm, length 25 cm;
High performance liquid chromatography was used for preparative separation. The solvent used was tetrahydrofuran-water (2: 3), and the flow rate was 6.
The Rt. At 0 ml / min was 14.1 min. As a result, about 170 mg of a purified standard product was obtained as a white powder. The physicochemical properties of the obtained purified sample were examined and the results were as follows. (a) Molecular formula: C ₂₁ H ₃₆ O ₅ (b) Molecular weight: 368 (c) Melting point: 225 to 227 ° C (d) Appearance: White powder (e) Property of substance: acidic substance

[0032]

(G) Solubility: Easily soluble in hot methanol. Soluble in ethyl acetate, chloroform and methanol. Insoluble in water. (h) Color reaction: Positive in the color reaction by I ₂ . Negative to ferric chloride. (i) UV absorption spectrum: λmax (MeOH) (ε); 230 nm (2570), 265 nm (2755)
(Figure 1) (j) Infrared absorption spectrum: 3400, 2920, 1710, 1640,
1460 cm ^-1 (Fig. 2) (k) Nuclear magnetic resonance spectrum: ¹ H-NMR result measured in deuterated methanol ... as shown in Fig. 3.

Results of ¹³ C-NMR measured in deuterated methanol ... As shown in FIG. As a result of determining the structure of this substance based on the above physicochemical properties, the substance has the formula (I)

[0035]

[Chemical 5]

It was a tetronic acid derivative substance represented by the structural formula: (Test Example 1) The activity of the tetronic acid derivative substance obtained in Production Example 1 was measured according to the following method. First, pGEX-VHR
A solubilized extract was adsorbed on glutathione-agarose gel from Escherichia coli into which the gene was introduced, and eluted with 5 mM glutathione to prepare a protein dephosphorylating enzyme. 3.125, 6.25, 12.5 and 25.0 μ in this protein dephosphorylating enzyme
Add the tetronic acid derivative substance to a concentration of g / ml,
Paranitrophenyl phosphate (manufactured by Sigma) was used as a substrate and reacted at 37 ° C for 30 minutes. After the reaction, 1N sodium hydroxide was added, and the absorbance of the produced para-nitrophenol was measured with an EIA reader (Model 2550: manufactured by Bio-Rad). Based on this measurement result, the inhibition rate of the protein dephosphorylating enzyme activity at each concentration of the tetronic acid derivative substance was calculated. The results are shown in Table 1.

[0037]

[Table 1]

As a result, Streptomyces sp.
It was revealed that the tetronic acid derivative substance produced from the 8-682 strain inhibits the activity of protein dephosphorylating enzyme.

[0039]

INDUSTRIAL APPLICABILITY According to the present invention, a protein phosphatase inhibitor and an antitumor agent can be provided.

[Brief description of drawings]

FIG. 1 is a chart of an ultraviolet absorption spectrum (methanol) of a tetronic acid derivative substance.

FIG. 2 is a chart of an infrared absorption spectrum (Neat) of a tetronic acid derivative substance.

FIG. 3 ¹ H-NMR (methano) of tetronic acid derivative substances
ld ₄ ) is a chart showing the results.

FIG. 4 ¹³ C-NMR (methano) of tetronic acid derivative substance
ld ₄ ) is a chart showing the results.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1: 645) (72) Inventor Takuya Hamaguchi 3-24-1 Takada, Toshima-ku, Tokyo Taisho product Yaku Co., Ltd.

Claims (2)

[Claims] 1. Formula (I): A protein dephosphorylating enzyme inhibitor containing, as an active ingredient, a tetronic acid derivative substance represented by: 2. Formula (I): An antitumor agent containing as an active ingredient a tetronic acid derivative substance represented by: