Aminopolysaccharide sc-4416, method thereof, an inhibitor of sharedlibrary and antibacterial agent

 

(57) Abstract:

The invention relates to a new aminopolysaccharide SC-4416 formula I, where m is an integer 0 or 1; n = 1-4, m+n is an integer from 1 to 5; R1lowest alkylperoxide and R2is hydrogen or lower alkyl, and pharmaceutically usable non-toxic salts, which have a strong ability of inhibiting sharedlibrary and antibacterial activity. The invention also concerns the method of obtaining this compound and pharmaceutical compositions containing it as active ingredient. In accordance with the invention, the authors identified a new aminopolysaccharide derived from the soil microorganism classified as Streptomyces species, and found that it can be used as a potent inhibitor of sharedlibrary, as well as antibacterial agents. 4 S. p. f-crystals, 4 Il., 6 table.

The present invention relates to new aminopolysaccharide derivative and its pharmaceutically usable non-toxic salts, which have a strong ability of inhibiting sharedlibrary and antibacterial activity. The invention also concerns the method of obtaining this product and farmaceuticas know, in the treatment or prevention of diabetes, hyperlipoproteinemias, hyperlipidemia, obesity, or other related secondary symptoms can be applied such inhibitors sharedlibrary as amylase and saharas.

From this point of view is informed about some aminopolysaccharide derivatives as potent inhibitors sharedlibrary, such as, for example, A-2396, which produces a Streptomyces species A (see: lined the Japan patent (Sho) 54-92909), Oligostatin that produces Streptomyces myxogenes (see: J. Antibiotics, 34:1424-1433 (1981)), Adiposin (see: J. Antibiotics, 35: 1234-1236 (1982), and NS complex, which produces the Streptomyces flavoprotein (see: laid open Patent Japan (Hei) 3-19239).

On the other hand, NS complex chemical structure which is completely equivalent to the structure aminopolysaccharide derivative of the present invention, is described below formula:

< / BR>
In accordance with the present invention applicants have identified a new aminopolysaccharide derived from the soil microorganism, describing it as a Streptomyces species, and found that it can be used as a potent inhibitor for sharedlibrary, and also as an antibacterial which I is the new aminopolysaccharide derived SC-4416, represented by the General formula (I) and salts thereof:

< / BR>
where m is an integer 0 or 1;

n is an integer from 1 to 4;

m + n is an integer from 1 to 5;

R1lowest alkylperoxide; and

R2is hydrogen or lower alkyl.

Another object of the present invention is the use of aminopolysaccharide derived as inhibitors sharedlibrary and antibacterial agents.

The next object of the present invention is a method for aminopolysaccharide derived from Streptomyces species SC-4416.

Brief description of drawings

The above and other objects and distinctive features of the present invention will be clear from the following description given in connection with the drawings, in which:

in Fig. 1 shows the IR spectrum of the substance SC-4416 according to this invention;

in Fig. 2 shows an NMR spectrum1H SC-4416;

in Fig. 3 shows an NMR spectrum13C SC-4416; and

in Fig. 4 shows the spectrum FAB-MS/MS (mass spectrometry bombarded with fast electrons) SC-4416.

Detailed description of the invention

The applicants of the present invention have conducted screening studies of soil microorganisms with the aim of identifying the microbe, the producers who were aziraphale as a species of the genus Streptomyces and named Streptomyces species SC-4416. In addition, it was found that obtained from it aminopolysaccharide derived is new and it has designated as SC-4416.

SC-4416 get by cultivating Streptomyces species SC-4416 on a medium containing sources of carbon and nitrogen under aerobic conditions, using the culture with shaking or culture with regular aerobic mixing. As carbon source, you can use commercially available glucose, glycerol, maltose, starch, sucrose, molasses, and dextrin; as the source of nitrogen can be used commercially available soy flour, liquid for soaking grains, meat extract, flour from the seeds of cotton, peptone, wheat seed, fish meal, inorganic ammonium salts and NaNO3as the inorganic salts can be used CaCO3, NaCl, KCl, MgSO4and phosphates. In addition, the medium for culturing Streptomyces species SC-4416, if necessary, may contain some metal ions, such as Fe, Mn and Zn, in trace quantities and protivovspenivayushchie agent, such as vegetable oils, higher alcohols, including octadecanol, and silicon compounds. The medium may also contain any other compounds that facilitate the culture of Streptomyces species SC-4416 develop Vasastan, using solid and liquid cultures. The cultivation liquid is usually carried out using a stationary culture, culture with constant stirring culture with shaking and culture with aeration, the preferred culture with shaking and submerged culture with aeration. Incubation is carried out at a temperature in the range from 20 to 37oC, more preferably from 25 to 30oC at neutral pH from 6 to 8 during the period from 24 to 192 hours, more preferably from 48 to 120 hours.

SC-4416 obtained from the culture of Streptomyces species SC-4416 in accordance with well-known in the practice of purification methods using ion-exchange, adsorption, separation and gelfiltration chromatography. SC-4416 you can also select the method of high performance liquid chromatography HPLC or thin-layer chromatography (TLC).

CK-4416 can be used in the treatment or prevention of insulin-independent diabetes, hyperlipoproteinemias and obesity caused by hyperlipidemia, or as a regenerative agent immunological failure, and can also be used as an antibacterial agent.

In addition, the present invention proillyustriroval the identification of Streptomyces species CK-4416.

Conducted sampling of soil microorganisms with the aim of identifying a microorganism, specified in the name, and distinguish it from forest soil collected in the area Chochun-Eup, Buk-Kun, Cheju-Do, Korea. The selected microorganism is characterized as follows.

1. Characteristics of growth

Characteristics of the growth of the selected microorganism on a variety of nutrient media are summarized in the following at the end of the table. 1.

2. Morphological characteristics

Microscopic examination of Streptomyces species SC-4416 revealed that they see one or three spiral from more than 20 spores per chain, which grow from the ends of the aerial mycelium, with whorled mycelium and fragmentation is not supervised. See oval spores, which have smooth surfaces and dimensions (0,6 - 0,7) x (0.5 to 1.0) μm, it does not see specific organelles, such as sclerotia and sporangium.

3. The effect of temperature growth (incubation on agar with decoction of oats within 14 days)

4oC: no growth

15oC: weak growth, aerial mycelium none

20oC: poor growth, sparse aerial mycelium

28oC: moderate growth, good aerial mycelium

37oC: moderate growth, good air IIC is ical characteristics

1) Hydrolysis of starch (Agar starch-inorganic salt): (+)

2) the Production of melanin pigment: (-)

5. The ability of the assimilation of the carbon source.

Streptomyces species SC-4416 cultivated at a temperature of 28oC for 14 days in a nutrient medium Pridham-Gottlieb gar medium (ISN N 9), including the appropriate sugar, presented in Table 2 (see end of description) and determine the capacity of absorption of these sugars.

6. The composition of the cell wall of Streptomyces species SC-4416. Define LL-diaminopimelic acid and glycine at a fraction of the cell walls.

From a study of the growth, morphology, physiology, and the ability of the assimilation of carbon sources for microorganisms selected in the present invention, in conclusion, identify the microorganism as the genus Streptomyces, which are not described. In this regard, the new bacterium called Streptomyces species SC-4416 and included in a Collection of Type Cultures, Korea (CSTS) and placed in KRIBB, KIST, P.O. Box 115, Yusong-Ku, Taejon, 305-600, Korea, an international depository authority (IDA) with the incoming number N XTS 0131 BP.

Example 2: Culture of Streptomyces species SC-4416.

Four Erlenmeyer flask 500 ml add 100 ml of medium for seed culture (pH 6.5) containing glucose 1% (weight/volume), dextrose/volume), and sterilized at 120oC for 15 minutes. In each of the four flasks make 1 platinum loop of the culture of Streptomyces species SC-4416 on the sloped agar obtained from subcultures, and incubated with shaking at 27oC for 3 days. Then 200 ml of material in a 500 ml Erlenmeyer flask, add 100 ml of culture medium (pH 6.5) containing soluble starch in an amount of 3% (weight/volume), powder from soybeans to 1.5% (weight/volume), liquid for soaking corn to 1.5% (weight/volume), polypeptid of 0.2% (weight/volume), Na2S2O30,1% (weight/volume), the calcium carbonate of 0.5% (weight/volume), cobalt chloride and 0.0001% (weight/volume), and sterilized at 120oC for 30 minutes. 2% (volume/volume) seed culture is added to the medium and incubated with shaking speed of 240 rpm at 27oC for 3 days. Quantify SC-4416 by determining the antimicrobial activity using the test organism Comamonas terrigena (ATSS 8461) in accordance with the well-known way using paper disks. After 3 days of cultivation define pH and output, which should be 7.2 and 0.06 mg/ml, respectively.

Example 3. The selection of substances SC-4416 (1).

18 l of the culture obtained in Example 2, centrifuged DL is t in column 6 cm x 30 cm with a flow rate of 50 ml/min, filled with activated charcoal (Wako Junyaku, Japan), which is an adsorption resin, washed with 5 l of distilled water, and elute 50% (vol/vol) methanol. Since the eluate fractionary 400 ml, SC-4416 extracted from fractions NN 3-6. These fractions are collected and concentrated under reduced pressure, obtaining 14 g of a yellow oily substance. This substance is dissolved in 50 ml of distilled water and adjust the acidity of the solution to pH of 3.1. The resulting solution contribute in column 6 cm x 30 cm, filled with Sp-Sephadex(H+) from Sigma Chemical Co., USA, at a flow rate of 20 ml/min, washed with 5 l of distilled water and elute 1 N aqueous solution of ammonia. Since the eluate fractionary 400 ml fractions containing the substance SC-4416, suiryudan under NN 3-4. These fractions are collected, concentrated under reduced pressure and dried, obtaining 1 g of yellow powder. Then this powder was dissolved in 30 ml of distilled water. The acidity of the resulting solution adjusted at pH 3.1 solution contribute in column (3 cm x 40 cm) filled Dawex 50 W-X8 (salt of pyridine) from Sigma Chemical Co., USA, at a flow rate of 5 ml/min, washed with 2 l of distilled water and elute 3 l of buffer solution of pyridine-formic acid (pH 3,1) with a gradient from About 0.2 M as the eluate fraction the t concentrated under reduced pressure, receiving, respectively, 500 mg of compound A and 90 mg of substance B in the form of light yellow powder.

Example 4: separation of substances SC-4416 (II).

Substance As obtained in Example 3 was dissolved in 4 ml of distilled water, bring in a column 3 cm x 50 cm, filled with Sephadex. Q-10 from Sigma Chemical Co. , USA, and elute with water. Since the eluate fractionary 5 ml active substance isolated from the fractions NN 39-45. These fractions are concentrated and dried, obtaining 390 mg of a white powder. Similarly treated substance B from Example 3, receiving 50 mg of white powder.

Example 5: Selection of substances SC-4416 (III).

60 mg of compound A obtained in Example 4, making a column for preparative high performance liquid chromatography (column: Tosoh Amid - 80, solvent: 60% (vol/vol) acetonitrile) from Shimadzu, Japan. Among the compounds represented by the General formula (I), consistently suiryudan compound of formula (I) in which m = 1, n = 2, R1= metalhydroxide and R2= hydrogen; the compound of formula (I) in which m = 0; n = 4, R1= metalhydroxide and R2= hydrogen; and the compound of formula (I) in which m = 1, n = 3, R1= metalhydroxide and R2= hydrogen. These three concentrate the eluate under reduced pressure and dried at samarasekera 4, and treated in a similar way under similar conditions. Among the compounds represented by formula (I) are, respectively, 10 mg of the compounds of formula (I) in which m = 0, n = 2, R1= metalhydroxide and R2= hydrogen; 7 mg of the compounds of formula (I) in which m = 1, n = 1, R1= metalhydroxide and R1= hydrogen; and 8 mg of the compounds of formula (I) in which m = 0, n = 3, R1= metalhydroxide and R2= hydrogen.

Example 6: Identification of compounds SC-4416.

Connection SC-4416 identified by determining the physico-chemical characteristics of the oligosaccharide derivative of the formula (I) for compounds in which m = 0, n = 4, R1= metalhydroxide and R2= hydrogen, using physical and chemical methods of analysis.

1. Color and style: white powder

Elemental analysis (see Table 2A)

3. Molecular formula: C37H63NO30< / BR>
4. Molecular weight: 1001, 9

FAB-MS/MS(M+H)+1002 (see Fig. 4)

5. Melting point: 162oC

6. Specific rotation: []D= +146o(C 0.1 in H2O)

7. Range of UV absorption (solvent: water, conc.= 0.1 mg/ml): see final absorption

8. IR spectrum: IR spectrum measured way with KBr (see Fig. 1)

Main2O (see Fig. 2)

10. An NMR spectrum 13C: an NMR Spectrum13C is measured in D2O (see Fig. 3)

11. Solubility: Soluble in water and ethanol, insoluble in benzene and normal hexane

12. Color reaction: positive in the reaction with silver nitrate-sodium hydroxide, the reaction Somoginelson and reaction with permanganate; negative reaction with ninhydrin and Sakaguchi reaction

13. Acidic, neutral and basic properties: weakly basic

14. Thin layer chromatography (TLC). R1:O. 14 [Tokyo Kasei K. K., silica gel f(S201), Japan] ; solvent: ethyl acetate-methanol-water = 5:3:2 (volume/volume/volume).

In addition, it was also established that the compound of formula (I) in which m = 0, n = 2, R1= metalhydroxide and R2= hydrogen, has the molecular formula C25H43NO20and molecular weight 677,6111; a compound of formula (I) in which m = 1, n = 1, R1= metalhydroxide and R2= hydrogen, has the molecular formula C25H43NO20and molecular weight 677,6111; a compound of formula (I) in which m = 0, n = 3, R1= metalhydroxide and R2= hydrogen, has the molecular formula C31H53NO25and molecular weight 839,7514; a compound of formula (I) in which m = 1, n = 2, R1= methylhydroxy is a group of formula (I), in which m = 1, n = 3, R1= metalhydroxide and R2= hydrogen, has the molecular formula C37H63NO30and a molecular weight of 1001, 8934.

The analysis of the above results found that aminopolysaccharide derivative of the present invention is a new connection.

Example 7: the Biological activity of SC-4416. Determine the biological activity of SC-4416 for connecting aminopolysaccharide represented by the formula (I) in which m = 0, n = 4, R1= metalhydroxide and R2= hydrogen.

Example 7-1: Antibacterial activity.

Antibacterial activity for gram-positive and negative bacterial strains was determined on the sample of 1 mg/ml SC-4416, applying the method using paper disks. Judging by the results, not see activity for gram-positive bacterial strain, whereas for gram-negative bacterial strains, namely, Comamonas terrigena (ATSS 8461) received 24.8 mm zone of inhibition.

Example 7-2: Inhibition of amylase activity.

Prepare a solution of enzyme (hereafter designated as "solution A") containing the amylase dissolved in 0.25 M phosphate buffer (pH 7.0), 0.25 M solution of phosphate is n as "solution C"), respectively. Then combine 0.05 ml of solution B containing dissolved SC-4416, 0.1 ml of solution B and 0.05 ml of solution B, incubate the solution at 37oC for 5 minutes and then incubated for 30 minutes after adding 0.5 ml of solution C. After incubation, determine the absorption at 660 nm for sample SC-4416 (T) and control (C), not containing the sample. In the end determine the ratio proinvestirovany (50%) for the amylase activity (IC50), which is calculated by the following equation and which is 1.6 10-6M

The ratio of inhibition = (C-T) 100.

Example 7-3: Reduction of blood sugar levels.

Male SD rats, divided into two groups of 5 animals each, and were not taking food for 12 hours, administered orally starch, 1.5 g/kg, together with the sample, 40 mg/kg and 80 mg/kg 1 h after injection in rats take blood to determine the level of glucose in the blood by means of glucose oxidase and compared with the control group, animals which did not give a sample SC-4416. The results are shown below in Table 3 (see the end of the description). As can be seen from Table 3, is clearly established that SC-4416 of the present invention can reduce the level of sugar in the blood, more

Example 7-4: Toxicity.

Tests on acute toxicity of SC-4416 performed after oral administration of 1 g/kg SC-4416 five ICR mice monitored for 14 days. The results showed that mice survive.

Pharmaceutically acceptable salts receive by conventional means, including interaction of the compounds of formula (I) with bases of alkali (Na, K) and alkaline earth (Ca, Ba, Zn, Mn) metals, more preferably, with such bases of alkali metals, such as, for example, dilute solutions of sodium hydroxide and potassium carbonate. In addition, pharmaceutically suitable salt get in the usual ways, including reaction with amines, such as, for example, triethylamine, dibenzylamine, triethanolamine, ethanolamine, N,N'-dibenziletilendiaminom, procaine and the equivalent amines.

Of the compounds of the present invention can be obtained preparative form by any known appropriate means with pharmaceutically suitable carrier and, if necessary, with diluent. For example, for oral administration the compounds of the present invention can include solid preparations such as tablets, pills, granules, powders, capsules and the like, or liquid preparations, such as solution, suspension, emulsion is of Nyenzi, preparations for intravenous drip infusion and the like. To prepare drugs for injection, the compound is preferably dissolved in distilled water or aqueous salt solution, such as sodium chloride. To prepare the drug for intravenous drip infusion, the compound can be dissolved in a suitable liquid medium, such as a physiological salt solution, a glucose solution of sodium chloride and the like.

The number of active component, that is the compounds of formula (I) of the present invention, in the pharmaceutical composition and its standard dosage form can be varied or adjusted widely depending on the particular application, the potential of specific compounds, the desired concentration. Usually the amount of active ingredient varies in the range from 0.5% to 90% by weight of the composition.

An effective dose of a compound of the present invention can be varied depending on the physical condition of the patients. Basically, it is shown that to achieve the desired result mainly enter the active compounds in an amount of about 50 to 1000 mg per 1 m2the surface area of the body.

Example 8: Biological the native compounds, represented by formula (I) in which m, n, R1and R2presented in Table 4 at the end of the description.

Example 8-1: Antibacterial activity.

To determine antibacterial activity of these five aminopolysaccharide compounds of General formula (I), antibacterial activity against gram-negative and gram-positive bacterial strains was determined by the method as described in Example 7-1, except that used the five compounds, such as SC-4416. The results show that for compounds a1, b, b1, c, and d were not observed no activity against gram-positive bacteria. However, the zone of inhibition diameter 25.0 mm, 23,3 mm 14,8 mm and 13.2 mm, respectively, were shown to gram-negative bacterial strain. In this regard, Comamonas terrigena (ATCC 8461), who were known as the causative agents of diseases such as sepsis and cholera, were used as gram-negative bacteria.

Example 8-2: Inhibition action Maltese'll get and in vitro.

It is well known that maltase and Saharsa are two of the main sharidny hydrolases in humans (see: W. F. Caspary et al., Res. Exp. Med. (Berl), 175: 1-6(1979)). To study the inhibitory asset is th analysis similar to as described in Example 7-2, except that the solution a was replaced with the enzyme solution containing Maltese or saharso, and the solution was replaced with a sugar solution containing maltose or sucrose. The results, summarized in Table 5 (see below) show that the compounds a1, b, b1, c, and d effectively inhibit the activity'll get and maltase.

Example 8-3: Reduction of blood sugar levels.

Inhibitory activity in vivo these five compounds against sharidny hydrolases investigated as follows: male Wistar rats (body weight 180-230 g), supplied by the firm Charles River Labs., Japan, were used as experimental animals. Three groups of 7 hungry rats administered orally (a) 1.5 g/kg body weight of cooked starch, (b) 2.0 g/kg body weight of maltose or (C) 2.0 g/kg body weight of sucrose. Fifteen other groups of 7 hungry rats received the same carbohydrates, in the same amounts as described above, simultaneously with the connection a1, b, b1, c or d, respectively, in quantities of from 1 to 50 mg/kg body weight. As a control group of seven rats received suitable volume of saline. Blood samples were collected by cardiac puncture under ether anesthesia. Levels CLASS="ptx2">

The results presented in Table 6 at the end of the descriptions, show that the compounds a1, b, b1, C and d significantly inhibit the increase of glucose in the blood depending on put sugar, starch, maltose or sucrose.

The results presented in Table 6 clearly show that the compounds a1, b, b1, c, and d can be used for the treatment or prevention of diabetes with high efficiency.

1. Aminopolysaccharide SC-4416 represented by the General formula I, and pharmaceutically usable salts

< / BR>
characterized in that:

m is an integer 0 or 1;

n is an integer from 1 to 4;

m + n is an integer from 1 to 5;

R1indicates the lowest alkylperoxide;

R2denotes hydrogen or lower alkyl.

2. The inhibitor sharedlibrary containing as active ingredients aminopolysaccharide SC-4416 or its pharmaceutically suitable salts under item 1 and a pharmaceutically acceptable carrier.

3. Antibacterial agent containing as active ingredients aminopolysaccharide SC-4416 or its pharmaceutically suitable salts under item 1 and a pharmaceutically acceptable carrier.

4. The method of producing aminopolysaccharide SVR) on the environment, containing sources of carbon and nitrogen under aerobic conditions and selection SC-4416 chromatography.

 

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