JP3071023B2 - Pyridinostatin, a novel enzyme inhibitor, and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel enzyme inhibitor pyridinostatin and a method for producing the same.

More specifically, the present invention relates to a novel substance pyridinostatin having an enzyme inhibitory activity against pyroglutamyl peptidase, and to a method for producing pyridinostatin by culturing a microorganism belonging to the genus Streptomyces.

[0003]

BACKGROUND OF THE INVENTION As a known compound having similar physicochemical properties to the enzyme inhibitor pyridinostatin according to the present invention, fenuvenulin or 2-methylphenuvenuron [EC Taylor & F. Sowi
nski, "J. Org. Chem." 40, 2321-2332.
Page (1975)] and pyrimidotriazine compounds. The pyridinostatin according to the present invention is clearly different from these compounds in chemical structure, physicochemical properties and biological properties, and was confirmed to be a novel substance. .

Pyroglutamyl peptidase of the present invention, in which pyridinostatin exhibits enzyme inhibitory activity, is a peptide hormone having various physiological functions such as neurotensin, luteinizing hormone-releasing hormone, and thyroid-stimulating hormone-releasing hormone in vivo in animals. It is an enzyme that is involved in the degradation and metabolism of (bioactive peptides) and is involved in the regulation of the function of peptide hormones or in the development of new biological activities.

[0005] Numerous enzyme inhibitors have hitherto been reported.
Some of them have been put to practical use as pharmaceutical agents. However, there are few reports of specific enzyme inhibitors for pyroglutamyl peptidase. Therefore, an enzyme inhibitor having high specificity for pyroglutamyl peptidase is desired.

An object of the present invention is to provide a physiologically active substance having an enzyme inhibitory activity with high specificity for pyroglutamyl peptidase and a method for producing the same.

[0007]

Means for Solving the Problems The present inventors have been studying for the above purpose, and have separated a large number of microorganisms from soil and searched for a substance produced in the culture.
It has been found that certain microorganisms belonging to actinomycetes with the strain number of A-2289 strain produce a physiologically active substance having an inhibitory effect on pyroglutamyl peptidase in a culture. Upon further detailed investigation, they succeeded in isolating the bioactive substance from the culture and confirming its chemical structure, and found that the bioactive substance was a novel antibiotic, Named.

The present invention has been completed based on the above findings.

Therefore, according to the first aspect of the present invention, the following formula (I) Provided is an enzyme inhibitor pyridinostatin, which is a compound represented by the formula:

The pyridinostatin of the present invention has an enzyme inhibitory activity having high specificity for pyroglutamyl peptidase, and thus is useful for treating diseases related to the degradation or metabolism of peptide hormone in vivo in mammals. Is expected. And, pyridinostatin has some antibacterial activity against certain bacteria. Furthermore, pyridinostatin is expected to be useful as an immunomodulator and as a medicament for treating pituitary hormone secretion deficiency.

The enzyme inhibitor pyridinostatin according to the present invention has the following physicochemical and biological properties.

1) Physicochemical properties of pyridinostatin (1) Appearance: colorless needle-like crystal (2) Melting point: 188-190 ° C (3) Molecular formula: C ₁₁ H ₁₅ N ₅ O ₄ (4) Elemental analysis Value: C ₁₁ H ₁₅ N ₅ O ₄ Calculated: C 46.92%, H 5.33%, N 24.89
% Found: C, 46.54%, H, 5.60%, N, 24.64
% (5) Mass spectrum: FAB-MS (pos.) M / z 28
2 (M + H) ⁺ .

(6) Specific rotation: [D] _D ²³ −5.6 °
(C 1.0, methanol) (7) Ultraviolet absorption spectrum: shown in FIG. 1 of the accompanying drawings; λmax, nm (ε) (in methanol): 280 (4600) (8) Infrared absorption spectrum: FIG. (9) ¹ H-NMR spectrum (400 MHz, CDCl ₃ ): shown in FIG. 3 of the accompanying drawings; (10) ¹³ C-NMR spectrum (100 MHz, CDCl ₃ ): shown in FIG. 4 of the accompanying drawings; δ (ppm): 202.7 (s), 166.2 (s), 151.5 (s), 149.8 (s),
138.6 (s), 54.9 (s), 49.6 (t), 37.0 (q), 30.8 (q), 30.4
(q), 28.8 (q).

(11) Solubility: soluble in water, methanol, dimethyl sulfoxide and chloroform, but not soluble in n-hexane.

[0015] (12) Thin layer chromatography (silica gel; manufactured by Merck) expand Solvent Rf n-butanol - acetic acid - water (4: 1: 2) 0.72 chloroform - methanol - water (65: 25: 2 ) 0.95 (13) Color reaction: Positive for 10% sulfuric acid, iodine and molybdic acid reagents.

2) Enzyme Inhibitory Activity of Pyridinostatin The measurement of pyroglutamyl peptidase inhibitory activity is described in Anal.
Biochem., 53, 321 (1973).

That is, 5 mM L-pyroglutamic acid-β
-Naphthylamide 0.02 ml, buffer 0.2 M potassium phosphate buffer (pH 8.0)-0.1 M EDTA-0.01 M DTT = 5:
1: 1] To a mixed solution containing 0.1 ml and 0.6 ml of an aqueous solution containing a sample, 0.02 ml of a pyroglutamyl peptidase (manufactured by Shigma) solution was added, and the mixture was reacted at 37 ° C. for 60 minutes to give 16 mM NaIO ₄ . Add 01 ml and add 37
The reaction was performed at 15 ° C. for 15 minutes.

After the reaction, 0.1 ml of 0.5 M sodium citrate buffer of 2 mg / ml Fast Garnet GBC (manufactured by Shigma) was added, and the absorbance (a) at 525 nm was measured. At the same time, the absorbance (b) of the control when the same reaction was carried out without the sample was measured. At this time, the absorbances (a ') and (b') of each of the blind tests were measured.

The inhibition rate of pyroglutamyl peptidase was (1
− (Aa ′) / (bb ′)) × 100. 5
The concentration of the sample showing 0% inhibition was defined as the IC ₅₀ value.

The enzyme inhibitory activity of pyridinostatin on pyroglutamyl peptidase has an IC ₅₀ value of 1.80 μg.
/ Ml.

3) Antibacterial activity of pyridinostatin The pyridinostatin of the present invention has a concentration of 100 μg / ml.
Bacteria Shigella Zonnay (Shigella sonnei) JS11
764 and Proteus Bulgaris(Proteus vulgari
s) OX19 was found to exhibit antibacterial activity,
As far as testing is concerned, other bacteria and fungi
No antibacterial activity was observed.

4) Toxicity of pyridinostatin Pyridinostatin according to the present invention is 100
As a result of observation for 2 weeks by intravenous administration of mg / kg, no toxicity was observed.

According to a second aspect of the present invention, there is provided the above-mentioned formula (I), wherein a pyridinostatin-producing bacterium belonging to the genus Streptomyces is cultured, and pyridinostatin is collected from the culture. An enzyme inhibitory active substance represented
A method for producing pyridinostatin is provided.

An example of a pyridinostatin-producing bacterium used in the method of the present invention is a novel actinomycete isolated from a soil sample and has a strain number of SA-2289.

Next, the bacteriological properties of the strain SA-2289 will be described.

The SA-2289 strain which can be used in the method of the present invention is an actinomycete having a hyphae width of about 1 micron, and the spore chain on the aerial hyphae of the strain is Retinaculiaperti under an optical microscope. The aerial mycelium is sometimes ISP-medium no. 2 and others show ring branching. However, rod-shaped spores are observed under an electron microscope, and the spore surface is smooth. L, L-diaminopimelic acid is detected as a component of the cell wall, and no sporangia or other characteristic structures are observed under an electron microscope. Based on the above properties, this strain is considered to be an actinomycete belonging to the genus Streptomyces.

The following Tables 1, 2 and 3 show the culture properties, physiological properties, and the presence or absence of the ability to assimilate sugars of this strain.

For the description of the colors in the various media, the Color Harmony Manual of Container Corporation of America was used. All property tests were determined after 2 weeks culture at 27 ° C.

[0029] Although the above properties indicate that the present SA-2289 strain belongs to the genus Streptomyces, it can be classified into the genus Streptoverticillium in view of sometimes showing a ring-shaped branch in a specific medium. Conceivable. However, the expression of the ring branch may or may not be seen in plantations, etc., and at present, the genus Streptoverticillium is not included as an independent genus because it is included in the genus Streptomyces Due to the theory, the SA-2289 strain will be treated as belonging to the genus Streptomyces.

Accordingly, the pyridinostatin-producing bacteria which can be used in the method of the present invention include the present SA-2289 strain, and an actinomycete belonging to the genus Streptomyces having the same or similar pyridinostatin-producing ability as the present strain, or Includes all actinomycetes of the genus Streptoverticillium.

The above SA-2289 strain was applied for deposit with the Research Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology,
On the 0th, the micro-organization Kenbyo No. 12705 (FERM P-12)
705).

The SA-2289 strain is easily changed in its properties as seen in other actinomycetes, for example, SA-22.
All 89 strains or mutant strains (naturally occurring or inducible), transfectants or transgenics derived therefrom that produce the novel enzyme inhibitor pyridinostatin are used in the present invention. it can.

In the method of the present invention, the above bacteria are cultured in a medium containing nutrients that can be used by ordinary microorganisms. As the nutrient source, known nutrients conventionally used for culturing actinomycetes can be used. For example, as a carbon source, glucose, galactose, dextrin, starch, molasses,
Vegetable oils and the like can be used. As a nitrogen source, soybean flour, wheat germ, corn steep liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea, and the like can be used.

The method, and if necessary, it is effective to add sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other inorganic salts capable of producing ions. In addition, organic and inorganic substances that promote the growth of bacteria and promote the production of pyridinostatin can be appropriately added.

As a method for culturing pyridinostatin-producing bacteria, a culture method under aerobic conditions, particularly a submerged culture method is most suitable. A suitable temperature for culturing is 15 to 37 ° C, but in most cases, culturing is performed at around 26 to 30 ° C. The production of pyridinostatin varies depending on the culture medium and the culture conditions, but the maximum amount of the pyridinostatin usually reaches 2 to 7 days in both shake culture and tank culture. When the accumulated amount of pyridinostatin during the culture reaches the maximum, the culture is stopped, and the target substance pyridinostatin is isolated and purified from the culture solution.

In the method of the present invention, pyridinostatin is collected from the resulting culture of pyridinostatin-producing bacteria by a conventional separation means utilizing its properties, for example, filtration, adsorption or distribution column chromatography, gel separation, Purification can be performed by a filtration method, a dialysis method, a precipitation method, or the like, alone or in an appropriate combination. For example, pyridinostatin
It is adsorbed on an adsorbent such as DIAION HP-20 (manufactured by Mitsubishi Kasei) or Sepabeads SP206 (manufactured by Mitsubishi Kasei) and eluted with a mixture of an organic solvent and water. For example, 50
Pyridinostatin is eluted from the adsorbent with 50% methanol, 50% acetone or the like. In order to further purify pyridinostatin, chromatography or countercurrent distribution using Sephadex LH20 (manufactured by Pharmacia) or the like may be performed.

The tracing of the pyridinostatin during the culturing and purification steps was based on the determination of the anti-pyroglutamyl peptidase activity described above and / or by the simple determination of pyridinostatin on silica gel thin layer chromatography. one spot was performed based on the detection of _{[BuOH-AcOH-H 2 O (} 4:: 1 2) to the a developing solvent Rf value = 0.72].

Examples of the present invention will be described below, but these are merely examples and do not limit the present invention. Of course, many alternatives or modifications not illustrated herein may be used.

Example 1 As a seed medium and a production medium, soluble starch 1.0%, K _{2
HPO 4 0.2%, MgSO 4 ·} 7H 2 O 0.1
%, (NH ₄ ) ₂ SO ₄ 0.2%, Yeast extract 0.1%, FeSO ₄ .7H ₂ O 0.0001
%, MnCl ₂ .4H ₂ O 0.0001%, ZnSO
₄ · 7H ₂ O 0.0001%, calcium carbonate 0.2
% Dissolved in artificial seawater (Jamarin S, manufactured by Jamarin Laboratory). The pH before sterilization was 7.
Adjusted to 2 and used.

The above medium was placed in a 500 ml Erlenmeyer flask.
Each 10 ml was dispensed and sterilized at 120 ° C. for 20 minutes, and then Streptomyces sp. SA-2289 strain (FER)
MP-12705) was inoculated with 1 to 2 loops of a slant culture, and cultured at 27 ° C. at 180 rpm for 2 days. 2 ml of the seed culture was transferred to a 500 ml Erlenmeyer flask in which 110 ml of the medium was dispensed and sterilized, and cultured under the same conditions for 4 days. After completion of the culture, diatomaceous earth was added to the culture solution, followed by filtration to obtain a culture filtrate.

14 L of the culture filtrate obtained as described above is applied to a 1.2 L column of the adsorbent DIAION HP-20 (manufactured by Mitsubishi Kasei Co., Ltd.), washed with water, and 0 to 50% aqueous acetone (2.4 ×). (2.4 L). The eluate containing the active fraction was concentrated under reduced pressure to remove acetone, and then the adsorbent Sepabeads SP206 (manufactured by Mitsubishi Kasei) 50
The mixture was applied to a 0 ml column, washed with water, and eluted with a concentration gradient method of 0 to 90% aqueous methanol (1.5 × 1.5 L). The eluate containing the active fraction was lyophilized to give an active crude powder 45.
8 mg were obtained.

This powder was centrifuged by Centrifigal Partition Chromatograph-L. L. N (Centrifugal Pa
Purified by centrifugal liquid distribution chromatography using a solvent system of butanol-acetic acid-water (750: 50: 750) using rtition Chromatograph-LLN (Model NMF, manufactured by Miki Engineering Co., Ltd.). The upper layer of the solvent system was used as a stationary phase liquid, and the lower layer was used as a mobile phase liquid at 20 ° C. and 1,000 rpm.
The resulting active fraction was concentrated and freeze-dried to obtain 96 mg of an active crude powder containing pyridinostatin. This active powder was dissolved in a small amount of methanol, and Sephadex LH2 filled with methanol was used.
0 (Pharmacia Fine Chemicals) 700ml
, And chromatography was performed using methanol as a developing solution, and the active fraction was concentrated to give 47.5 of active powder.
mg was obtained.

Further, 40.2 mg of colorless needle crystals of pyridinostatin were isolated by crystallizing this active powder in methanol.

[0044]

The novel substance pyridinostatin of the present invention is
Decomposes and metabolizes peptide hormones (bioactive peptides) that have various physiological functions in vivo as substrates, and has enzyme inhibitory activity against pyroglutamyl peptidase, which is involved in the regulation of peptide hormone functions or the development of new active activities . From this, pyridinostatin is expected to be useful as a drug having a new physiological action. Pyridinostatin is also expected to be used as an immunomodulator and as a medicament effective for treating various pituitary hormone secretion disorders.

[0045]

[Brief description of the drawings]

[0046]

FIG. 1 shows an ultraviolet absorption spectrum of pyridinostatin in methanol.

[0047]

FIG. 2 shows an infrared absorption spectrum of pyridinostatin in a potassium bromide tablet.

[0048]

FIG. 3. 40 of pyridinostatin in deuterochloroform.
¹ shows a ¹ H-NMR spectrum at 0 MHz.

[0049]

FIG. 4. 10-pyridinostatin in deuterochloroform.
1 shows a ¹³ C-NMR spectrum at 0 MHz.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI A61P 5/00 A61P 5/00 31/04 31/04 37/00 37/00 (C12P 17/18 C12R 1: 465) (72 ) Inventor Chiaki Imada 3-32-15 Bunzo, Urawa City, Saitama Prefecture (72) Inventor Masahiro 3-25-10 Hiyoshi Honcho, Kohoku-ku, Yokohama City, Kanagawa Prefecture (58) Fields investigated (Int. Cl. ⁷ , DB name) C07D 487/04 147 C12N 9/99 C12P 17/18 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. The following formula (I) An enzyme inhibitor pyridinostatin, which is a compound represented by the formula: 2. The following formula, wherein a pyridinostatin-producing bacterium belonging to the genus Streptomyces is cultured, and pyridinostatin is collected from the culture. A method for producing an enzyme inhibitor pyridinostatin represented by the formula: