KR0161149B1 - Novel n-acetyl-ñô-d-glucosamidase inhibitor and process for the preparation thereof - Google Patents

Abstract

The present invention relates to a novel N-acetyl-β-D-glucosaminidate inhibitor and a method for preparing the same, wherein grammestatin, a bioactive substance isolated and purified from a culture solution of the MR970 strain of Streptomyces genus, is N-acetyl- Its strong inhibitory activity against β-D-glucosaminidate has the potential to be usefully used for the treatment of cancer as an immunomodulator.

Description

Novel N-acetyl-β-D-glycosaminidate inhibitors and methods for their preparation

1 is an infrared absorption spectrum of nagmestatin which is an N-acetyl-β-D-glycosaminidate inhibitor according to the present invention,

2 is a hydrogen ( ¹ H) -nuclear magnetic resonance spectrum of the nammestatin,

3 is the carbon ( ¹ C) -nuclear magnetic resonance spectrum of nagmestatin.

The present invention relates to an N-acetyl-β-D-glycosaminidate inhibitor and a preparation method thereof. More specifically, the bioactive substance having N-acetyl-β-D-glycosaminidate inhibitory activity is Gemstatin and a method for preparing the same from a culture of microorganisms.

Until now, many bioactive substances have been found in microbial cultures. Among them, inhibitors of enzymes present in the cell membrane and enzymes that cut the sugar chains bound to the cell membrane have been recognized to have an immunomodulatory effect. For example, the enzyme hydrolysing agent Swainsonin (Journal of Antibiotics, 38, 925-935 (1985)), Kifunesin (Journal of Antibiotics, 40, 612-622 (1987)), FR-900483 (Journal of Antibiotics, 41, 296-301 (1988)), and others. It is reported to enhance the response. Since immunopotentiators are known to be effective in treating cancer or preventing aging, there is an increasing demand for more powerful and effective immunopotentiators.

Therefore, the present inventors are searching for an inhibitor of N-acetylβ-D-glycosaminidate, an enzyme that cleaves sugar chains bound on the cell membrane at the N-acetylglucosamine position in order to develop more effective immunomodulators. In the culture of Streptomyces genus strains, a substance showing a strong inhibitory effect on N-acetyl-β-D-glycosaminidates was produced, which isolated the active substance, grammestatin, and its physicochemical properties. The present invention has been completed by confirming the characteristics.

Accordingly, it is an object of the present invention to provide novel bioactive substances which act as N-acetyl-β-D-glycosaminidate inhibitors and methods for their preparation.

In order to achieve the above object, the present invention provides a novel bioactive substance grammestatin having the following structural formula (I):

The present invention also provides a method for preparing Nagmestatin of the above formula (I) comprising culturing Streptomyces sp. Strain and separating Nagmestatin from the culture.

Hereinafter, the present invention will be described in detail.

In the present invention, as a microbial strain producing Nagmestatin, which is an N-acetyl-β-D-glucosaminidate inhibitor, for example, MR970 strain of Streptomyces genus isolated from the soil by the present inventors There is.

The form of the MR970 strain is as follows. The MR970 strain forms a hooked mycelium in the nutrient mycelium branched under the microscope, and the mature spore chains show 20 to 40 or more natural chains and the size of the spores is 0.5 to 0.6 x 1.2 to 1.5 μm. The surface was smooth.

The growth state in various media of the strain is as follows. (1) In Trypton's yeast extract agar medium (ISP medium, 1, 28 ℃ culture), the growth condition was good, the color of the mycelia was pale orange and no soluble pigment was formed. (2) In yeast extract-malt extract agar medium (ISP medium 2, 28 ° C. culture), very pale orange mycelia were formed in light off-white growth, and no soluble pigment was formed. (3) In oatmeal agar medium (ISP medium 3, 28 ° C. incubation), pale orange mycelia were formed in pale orange growth, and no soluble pigment was formed. (4) In starch inorganic salt agar medium (ISP medium 4, 28 ° C. culture), very pale orange mycelia were formed in off-white growth and no soluble pigment was produced. (5) In glycerol asparagine agar medium (ISP medium 5, 28 ° C. culture), very pale orange mycelia were formed in off-white growth, and no soluble pigment was produced. (6) In tyrosine agar medium (ISP medium 7, 28 ° C. culture), very pale orange mycelia were formed in off-white growth, and no soluble pigment was produced.

Physiological properties of the strain are as follows. First, in relation to the growth temperature range, using Bennett medium (Bennet, glucose 1.0%, peptone 0.2%, beef extract 0.1%, yeast extract 0.1%) 10 ℃, 20 ℃, 24 ℃, 27 ℃, 30 ℃, 37 As a result of growing test at each temperature of ℃ and 45 ℃, it was grown at all temperatures except 45 ℃ and 10 ℃, but the optimum growth temperature is considered to be in the range of 24 ℃ to 30 ℃. It was positive in the liquefaction of gelatin, liquefaction of soluble starch and peptonization of skim milk, and tyrosine agar in peptone-yeast extract-iron-agar medium (ISP medium 6) in melanin formation reaction. All of the medium (ISP medium 7) did not form a dark brown pigment. On the other hand, in the availability of the carbon source, it is grown by using D-glucose, D-fructose, D-galactose, D-xylose, L-arabinose, D-rapinose, mannitol, salicycin, cellobiose, melibiose, and sucrose. However, inulin, inositol, adonitol, and lactose did not grow by using. Negative nitrate and hydrogen sulfide reactions were negative.

The 2,6-diaminopimeline acid contained in the cell walls of the whole cells was found to be LL type. Based on the above characteristics, the MR970 strain was identified as actinomycetes belonging to the genus Streptomyces. This strain has been deposited with KCTC 8652P, accession no. As the strain is easy to change its appearance as shown in other actinomycetes, mutants, transformants and genetic recombinants derived from the strain can all be used in the present invention.

According to the present invention, the MR970 strain is first cultured in a medium containing nutrients available to ordinary microorganisms. As a carbon source, glucose, starch syrup, sugar, starch, molasses, etc. can be used, and as a nitrogen source, soybean meal, soyton, soybean meal, peptone, yeast extract, etc. are used. In addition, inorganic salts which can generate sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other ions may be added as necessary. In addition, it is possible to appropriately add an organic or inorganic substance that aids in the growth of bacteria and promotes the production of nagmestatin.

It is preferable that the strain is cultured under aerobic conditions, and in particular, it is most suitable to culture by liquid deep culture. The culture temperature is 20 to 40 ° C., preferably 26 to 30 ° C., and the pH suitable for the culture is 6 to 12, preferably 6 to 8. The production of nagmestatin varies depending on the medium or culture conditions, but the accumulation amount reaches the highest in 2 to 10 days in shaking culture or tank culture. When the accumulation amount of nagmestatin in the culture is the highest, the culture is stopped and the desired substance nagmestatin is isolated and purified from the culture solution.

Nagmatstatin can be extracted and purified in various ways according to the physicochemical properties described below, and in particular, it can be effectively extracted and purified from the culture by the following method: Activated charcoal in the filtrate from which the solid is removed from the culture containing the active ingredient. Alternatively, after adsorbing by adding a synthetic adsorbent, the effective substance adsorbed by extracting the adsorbed solvent alone or mixed with water, such as methanol. Subsequently, the oily substance obtained by removing the solvent of the extract is dissolved by adding a small amount of water, and grammestatin is isolated by appropriately performing chromatography using a carrier such as an ion exchange resin, a silica gel, a gel filter, or the like.

The physicochemical properties of Nagmestatin isolated from the culture medium of strain MR970 were as follows:

. Shape: Colorless Powder

. Molecular Weight: 313

. Molecular Formula: C ₁₃ H ₉ N ₃ O ₆

. Ultraviolet absorption: maximum absorption at 223nm

. Infrared absorption: see Figure 1

. Hydrogen Nuclear Magnetic Resonance Spectrum: See Figure 2.

. Carbon Nuclear Magnetic Resonance Spectrum: See Figure 3.

Nagmestatin having the above characteristics was found to have the structural formula (I) by examining various spectra:

As a result of comparing the present known substance with the same structure with that of the conventional hydrolysis inhibitor, it was found that the same substance in molecular weight, molecular formula, infrared absorption spectrum, nuclear magnetic resonance spectrum, etc. It was determined that nagmestatin of the present invention was a novel bioactive substance.

The inhibitory activity of Negacetylstatin against N-acetyl-β-D-glycososaminis was determined according to the method described in Tarentino et al., Method in Enzymology, 28, 772-776 (1952). The absorbance at 400 nm of p-nitrophenol liberated from -N-acetyl-β-D-glucosamide is measured by quantifying the inhibitor.

Hereinafter, the present invention will be described in more detail based on the following examples. However, these Examples are only for illustrating the present invention, the present invention is not limited to these.

Example 1 Culture of Microbial Strains

As the medium and production medium, a medium containing 2% glucose, 1% soyton, 0.4% yeast extract, 0.1% meat extract and 0.2% sodium chloride was used. Dispense 200 ml of seed medium into 1 liter Erlenmeyer flask and sterilize at 120 ℃ for 20 minutes, inoculate 1 to 2 platinum cells of MR970 strain of Streptomyces, and incubate at 28 ℃ for 2 days to incubate Species culture.

Subsequently, 200 ml of the seed culture solution was inoculated in a 5 L fermenter containing 3 L of the production medium sterilized at 120 ° C. for 30 minutes, and incubated at 28 rpm for 4 days at 180 rpm under aeration (3 L / min). After completion of the culture, the filter aid diatomaceous earth was added to remove the solids containing the cells, and then the culture filtrate was 2500ml.

Example 2 Separation and Purification of N-Acetyl-β-D-Glucosaminidate Inhibitor

10 L of the culture filtrate obtained in Example 1 was adsorbed on activated carbon, washed with water, desorbed with a water-methanol (1: 4) solvent, and concentrated under reduced pressure with an aqueous solution of 500 ml. Butanol was added to this aqueous solution and extracted three times, and the butanol layer was reconcentrated. The resulting concentrate was dissolved in a small amount of methanol-butanol (1: 4), and charged into a Higgs 50 column (Sigma), washed with water, and eluted with a linear gradient of ammonia from ON to 0.5N. . The active fractions were collected, concentrated under reduced pressure, and passed through DEAE-Sefadex A-25 (manufactured by Pharmacia) and MCI Gel B (manufactured by Mitsubishi Corporation) to remove impurities and concentrated, followed by Sepha X LH20 (manufactured by Pharmacia). The active fractions were collected by gel chromatography using a 30% methanol solution. The obtained fractions were subjected to high pressure liquid chromatography (Phenomenex Bondclone C18, column diameter 7.6 mm, column length 300 mm, solvent: 30% methanol, flow rate 1.5 ml / min, detection at 220 nm), and the active peaks of about 9 components were collected and lyophilized. Finally, 8 mg of white powder was obtained.

Example 3 Identification of N-Acetyl-β-D-Glucosaminidate Inhibitors

N-acetyl-β-D-glycososaminidase inhibitor Nagmestatin, purified in Example 2, was methylated from 2.06 ppm to singlet, O-methyl from 3.68 ppm to singlet in proton NMR spectra It was found that two methyl groups existed. The methylene group appeared as singlet at 3.62ppm and multipled at 4.01 and 4.09ppm, resulting in two methylene groups. The methine group appeared as a singlet at 7.18ppm, a doublet at 4.99ppm, a doublet, a doublet at 4.42ppm, a multiplet at 4.24ppm, and a doublet, a doublet at 4.08ppm. The methine group confirmed that four methine groups were connected except for the methine group present at 7.18 ppm, and that the 4.24 ppm methine group was connected to a multi-line methylene group on the proton and proton homozygoscopy spectra. In the carbon NMR spectrum, two signals of 175 and 175 ppm are due to carbonyl carbon and quaternary carbon signals appear at 144, 134 and 117 ppm, and signals at 71, 69 and 61 ppm are oxygen-containing carbons and 48 ppm are nitrogen It's a carbon signal. Based on the above NMR analysis results and MS, IR, HMBC analysis, the structure of the nagmestatin of the present invention was determined.

Example 4 Determination of Acetyl-β-D-Glucosaminidate Inhibitory Activity of Nagmestatin

80 μl of 0.1 M acetic acid buffer (pH 4.2), 80 μl of 10 mg / ml p-nitrophenyl-N-acetyl-β-D-glycocosamide solution, 20 μl of enzyme solution of 1mU concentration, and various concentrations The reaction solution consisting of 20 μl Nagmestatin solution was placed in a 96-well microplate and reacted at 37 ° C. for 30 minutes, and 80 μl of 0.4M glycine-caustic soda buffer (pH 10.5) was added to stop the reaction. After 10 minutes at room temperature, the absorbance (a) was measured at 400 nm using a Biorad microplate reader, and the absorbance (b) of the control using only buffer containing no nagmestatin was measured. -β-D-glycosaminidate inhibition was calculated by the formula of () ba) × 100. As a result, nagmestatin inhibited 50% (IC ₅₀ ) of N-acetyl-β-D-glucosaminidate at a concentration of 0.0025 µg / ml.

As described above, nagmestatin, a novel physiologically active substance isolated and purified from the culture medium of Streptomyces spp. According to the present invention, has a strong inhibitory activity against N-acetyl-β-D-glucosaminidates and thus is immunomodulatory. As a substance, there is a possibility to be usefully used for the treatment of cancer and the like.

Claims (2)

Compound of formula (I) A method for preparing a compound of formula (I) according to claim 1, comprising culturing the strain Streptomyces MR970 (KCTC 8652P).