CN1034580A - The manufacture method of a kind of antiparasitic new antibiotic and its esters - Google Patents

Abstract

A kind of manufacture method of antiparasitic novel antibiotic and its salt, its It is characterized in that, from the KP-197 strain culture, it is separated by solvent method, Two substances with antiparasitic activity are extracted and refined. The special The qualitative antiparasitic activity is significantly higher than that of known drugs, while its toxicity is low Because of known drugs, it can be used as a treatment for human, animal and plant parasites effective drug.

Description

The present invention relates to a method for producing new antiparasitic substance, JEITACINS (JEITACINS) and its salts, which can be extracted from microbial culture. The chemical structural formula of the antibiotic and its pharmaceutically acceptable salts is:

In the formula: R = isopropyl or isobutyl

It is already known that as anti-parasitic drugs, among antibiotics, there are AVERMECTIN (AVERMECTIN) and paromomycin (PAROMOMYCIN), and among synthetic drugs, there are levamisole (LEVAMISOLE), niclosamide (NICLOSAMIDE) )wait. These medicines are used as medicines and animal medicines to treat nematodes, filariasis, etc., and their effects are certain. However, known antiparasitic drugs have problems of toxicity, side effects, or drug resistance.

The object of the present invention is to search for antiparasitic drugs with less toxic and side effects, in order to provide substances with antiparasitic activity and solve the needs in human and animal treatment.

The gist of the present invention is to isolate strains from different soil samples and to study their products with the aim of exploring new biologically active substances. As a result, the KP-197 strain was found, and its culture contained substances that inhibited the motility activity of the parasite. Two pure substances inhibiting the locomotor activity of the parasite were isolated and refined from the culture of this strain. The physical and chemical properties of these two substances were studied and their chemical structures were determined. Substances with this chemical structure have not been reported yet. These substances are collectively referred to as JEITACINS. The compound in which R is isopropyl in the structural formula is called JEITACINS A (JEITACINS A), and the compound in which R is isobutyl is called JEITACINS B (JEITACINS B). The physical and chemical properties of Tymycin A and Tymycin B are as follows:

Withdrawal from Tymycin A:

(1) white powder

(2) Elemental analysis

C ₁₈ H ₃₄ N ₂ O ₂

Calculated value: C69.57; H11.04; N9.02%

Experimental value: C70.40; H11.09; N7.68%

(3) Molecular weight and molecular formula

In the mass spectrum of Taimycin A, there is a (M+H) ⁺ molecular ion peak at m/z311; in high-resolution mass spectrometry, there is a (M-OH) ⁺ ion peak at m/z293. From this, it was determined that its molecular formula was C ₁₈ H ₃₄ N ₂ O ₂ , and its molecular weight was 310.

(4) UV absorption spectrum

As shown in Figure 1, there is a characteristic absorption peak at 228 nm; a shoulder peak appears at 250 nm.

(5) Infrared absorption spectrum

Measured by KBr solvent method, as shown in Figure 2, the main absorption maximum wave numbers are as follows: 2950, 2870, 1705, 1475, 1415, 1380, 1270, 1090, 960 (cm ^-1 ).

(6) NMR spectrum

Using Varian XL-400, 400MH nuclear magnetic resonance instrument, the ¹ H nuclear magnetic resonance spectrum measured in heavy chloroform is shown in FIG. 3 .

(7) Color reaction

p-methoxybenzaldehyde-sulfuric acid Positive

Potassium permanganate Positive

sulfuric acid negative

Dragendorff test negative

(8) Solubility

Soluble in acetone, ethyl acetate, chloroform, hexane, dimethyl sulfoxide. Insoluble in methanol and water.

(9) Chemical structure

Among the structural formulas elucidated in the above technical field, R is the structural formula of isopropyl, which is obtained by comparing the physical and chemical properties listed in (1)-(9) with known similar compounds. It is 14-methyl-1-vinyl diisopropyl Nitroxide 15-alkan-8-one.

Withdrawal from Tymycin B:

(1) Colorless oil

(2) Molecular formula. In the high-resolution mass spectrometry analysis, there is (MN) ion peak at m/z307, so its molecular formula is determined to be C ₁₉ H ₂₆ N ₂ O ₂ , and its molecular weight is 324.

(3) UV absorption spectrum

Tymycin A

(4) Infrared absorption spectrum

Tymycin A

(5) Color reaction

Tymycin A

(6) Solubility

Tymycin A

(7) Chemical structure

Tetramycin B is an analog of tetramycin A, and the number of carbon atoms on the alkyl side chain is different.

Among the structural formulas elucidated in the above technical field, R is the structural formula of isobutyl, which is the result of comparing the physical and chemical properties listed in (1)-(7) with known compounds. It is 15-methyl-1-vinyl Nitroxide 16-alkan-8-one.

Microorganisms capable of producing tinomycin belong to the genus Streptomyces, for example, the KP-197 strain of Streptomyces isolated in the present invention is one of the most effective strains used in the present invention. The taxonomic characteristics of this strain are as follows:

(1) The vegetative hyphae developed on various media, no hyphae breakage was observed. On the starch inorganic salt medium, the aerial hyphae are very rare and white. Microscopic observation shows that the spore filaments are linear, and the spore chain is composed of more than 20 spores. The spore size is 0.7-1.0×0.7μm, cylindrical, spore The surface is smooth, no sclerotia, sporangia and zoospores were found.

(2) Characteristics on various media:

The cultural characteristics of the producing bacteria were studied by the method of Segova (E.B.SHIRLING and D.G.GOTTLIEB-International Journal of Systematic Bacteriology, Volume 16, Page 313, 1966), and the results are shown in the table: (see the table on page 3)

(3) Physiological characteristics

melanin formation positive

Tyrosine agar Positive

Peptone Yeast Extract Iron Agar Positive

Glucose peptone gelatin medium (21-23°C) Positive

Tryptone Yeast Extract Liquid Medium Positive

Tyrosinase reaction positive

Hydrogen sulfide production Negative

Nitrate reduction Positive

Gelatin liquefaction (glucose, peptone, gelatin medium) (21-23°C) Positive

Starch hydrolysis Positive

Milk coagulation (37°C) Negative

Milk peptonization (37°C) Positive

Card temperature range 15-38℃

Carbon source utilization (using Pugova PRIDHOM-GOTTLIEB agar as basal medium):

Utilize: D-glucose, D-fructose, mannose

Slight use: D-mannitol, (-arabinose, D-xylose, sucrose,

Do not use: L-rhamnose, I-inositol, raffinose, mebiose, salicylic acid.

Cellulolysis Negative

(4) Cell wall composition

The diaminopimelic acid (DAP) of the cell wall is LL-DAP.

In summary, the taxonomic features of this strain can be summarized as follows: the diaminopimelic acid in the cell wall is LL-DAP, the shape of the sporocystosis is linear, the spore chain, the spore surface is smooth, the vegetative hyphae are beige, aerial The hyphae are white and form melanin. It can be determined that the strain belongs to the genus Streptomyces (STREPTOMYCES). According to Puchai's classification method (PRIDHAM AND TRESNER) (Pg. 748-829, eighth edition of the Handbook of Bacteria Classification of Bethwa, 1974), it was confirmed that the produced strain belonged to the white series.

In the present invention, as an example of the microorganisms in the culture medium, first, microorganisms belonging to the genus Streptomyces capable of producing streptomycin were used. However, as long as a mutant of the strain can produce orthotymycin, it can also be used in the present invention.

As the medium, a synthetic medium or a natural medium can be used. These mediums generally contain carbon sources, nitrogen sources and inorganic salts suitable for cultivating actinomycetes, as well as appropriate and necessary other nutrients.

Various carbohydrates such as glucose, glycerol, fructose, maltose, mannose, xylose, galactose, ribose, starch and its hydrolyzate can be used as the carbon source in the medium, and the use concentration is usually 0.1-5%. In addition, there are organic acids such as gluconic acid, pyruvic acid, lactic acid and acetic acid, amino acids such as glycine, glutamic acid and alanine, alcohol compounds such as methanol and ethanol, and non-aromatic hydrocarbons such as n-paraffins or animal and plant All kinds of oils can be used.

As a nitrogen source, ammonium, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium nitrate and other inorganic or organic acid ammonium salts, urea, peptone, NZ-amine, meat extract, yeast extract, dry yeast, corn steep liquor, cheese Nitrogen-containing organic substances such as protein hydrolyzate, fish meal or its hydrolyzate, soybean meal or its hydrolyzate, defatted soybean or its hydrolyzate, etc. There are also amino acids such as glycine, glutamic acid, and alanine. Inorganic salts use various phosphates, sulfates, table salts and trace amounts of heavy metal salts.

When using mutant strains with special nutritional requirements, substances that can meet their nutritional requirements must be added to the medium. Such nutrients do not need to be specially supplemented when using a medium containing natural substances.

Fermentation culture is usually carried out under aerobic conditions such as vibration or aeration and stirring. The actual production is to adopt deep aeration and stirring culture. The pH value of the medium can be 5-8, but generally neutral pH is better. The culture temperature can be 20-40°C, but generally 26-32°C is better (the ideal temperature is around 27°C). The time of liquid culture medium is usually 3-6 days, and the culture is stopped when the titer of ortamycin reaches the highest peak. Culture conditions such as culture medium composition, culture temperature, stirring speed, and air flow can be adjusted and selected according to the strains used and external conditions. When foam occurs in liquid culture, surfactants such as silicone resin and vegetable oil can be used to eliminate foam.

Tetramycin accumulated in culture by the method described above is usually present in the culture medium. In order to extract tyramycin from the culture filtrate, the method of extracting metabolites from microbial cultures is generally used. These methods can be used alone or in combination or repeatedly. For example: filtration, centrifugation, dialysis, concentration, drying, freezing, adsorption, elution; methods with different solubility of various solvents (such as precipitation, crystallization, recrystallization, transfer, countercurrent distribution), chromatography and other methods.

Since tatyramycin is mainly produced in the culture fluid, in order to separate and extract the compound, first remove the bacteria from the culture fluid, and then extract the substance from the culture filtrate.

In order to extract Tyramycin from the culture filtrate, use non-hydrophilic organic solvents such as ethyl acetate to extract, or use activated carbon, aluminum oxide, porous polymer synthetic resin, ion exchange resin, etc. to adsorb, and then use Elute with a solvent such as ethyl acetate, and the obtained extract or eluate is concentrated under reduced pressure, and then extracted with hexane or the like. The crude extract thus obtained is purified by a usual method for refining fat-soluble substances, such as silica gel or alumina column chromatography.

The compound can be used as pharmaceutically acceptable salts such as hydrochloride, sulfate, nitrate, phosphate, carbonate, tartrate, citric acid, succinate, etc., which can be prepared by conventional methods.

The advantages and positive effects of the present invention are that the active substance prepared by the above-mentioned method has anti-parasitic properties. XI Gui˙URSAPHLENCHUS LIGNICLOUS) was used as the treatment object, and the method introduced by Kimura et al. (AGRICULTURE BIOLOGICAI CHEMISRY 45, 249, 1981) was used to measure the locomotor activity of Tymycin in inhibiting pine nematodes, and the results were as follows:

The lethality rate of nematodes from Tymycin A (%)

0.5ug/ml 100%

0.25ug/ml 100%

0.125ug/ml 99%

0.063ug/ml 86%

The lethality rate of nematodes caused by Tymycin B (%)

0.5ug/ml 100%

0.25ug/ml 100%

0.125ug/ml 93%

0.063ug/ml 80%

Lethality of the known drug AVERMECTIN BLA to nematodes (%)

5ug/ml 100%

2.5ug/ml 100%

1.25ug/ml 64%

0.63ug/ml 58%

From the above comparative tests, it can be seen that the antiparasitic activity of Tymycin is more than ten times stronger than that of known drugs, and the curative effect of the former is obviously better than that of the latter.

In the acute toxicity test, mice were used as experimental animals. Tymycin 10 mg/kg was not lethal by intraperitoneal injection. The acute toxicity of the known drug AVERMECTIN BLA was observed in the same way, and the mice died as a result. It can be seen that the acuteness of quitting Tyromycin is lower than that of known antiparasitic drugs.

The above activity and toxicity tests show that the tatyramycin obtained in the present invention is useful as an antiparasitic drug for humans, animals and plants.

Example:

Put 100ml of culture medium in a 500ml plate mouth bottle (medium composition: glucose 0.1%, potato starch 0.4%, peptone 0.5%, meat extract 0.3%, yeast extract 0.5%, calcium carbonate 0.4%, pH7.0), 121 ℃, 15 minutes after steam sterilization, a slant culture of white gold ear KP-197 (composition: 1.0% glycerol, 1.0% calcium malate, 0.1% yeast extract, 1.5% agar) was planted into the above-mentioned sterilized The medium was cultured on a rotary shaker at 27° C. for three days, and the culture was used as a seed. Prepare two 30-liter fermentation tanks, each filled with 15 liters of medium (composition of fermentation medium: glycerin 2.0%, soybean powder 2.0%, NaCl 0.3%, citrulline 0.01%, CoCl 0.002%, pH7. 0), after steam sterilization at 121°C for 30 minutes, 6 bottles of the above-mentioned seeds were planted into each fermenter, the stirring speed was 250r.p.m, the ventilation rate was 15 liters/min, and the culture was carried out at 27°C for 120 hours.

Centrifuge the culture (10,000r.p.m), separate the bacteria and the culture medium, obtain 30 liters of supernatant, adjust the pH value of the above supernatant to 3.0 with 6N HCl, and then centrifuge to obtain a precipitate, add 15 liters of 50% acetone solution, stirred, extracted, and filtered to obtain the extract, which was concentrated under reduced pressure to 6 liters, added with 3 liters of ethyl acetate, extracted twice by vibration, and the obtained ethyl acetate extract was concentrated under reduced pressure, Obtain oily crude extract, add 750ml hexane to the crude extract, extract twice to obtain hexane extract, concentrate under reduced pressure to obtain 4 grams of crude product, crude product is packed into silica gel column (manufactured by Mark Company, ART7734, 160 grams ) chromatography, eluted with hexane-ethyl acetate solution (30:1), collected in fractions, each part was used for biological assay with pine nematode, the active parts were combined, concentrated under reduced pressure to obtain 30 mg of crude product, dissolved in In a small amount of chloroform, 1/20 of the amount was injected each time, and high-pressure liquid chromatography was performed on a reverse separation column (manufactured by Yamamura Chemical Research Institute, AM324 (ODS, 10×300mm), washed with 60% acetonitrile water, and the flow rate 2 milliliters per minute, collect the active parts of the peaks showing retention times of 18.5 minutes and 19.6 minutes, concentrate and dry under reduced pressure to obtain 7.2 mg of Tetramycin A and 5 mg of Tetramycin B respectively.

Description of drawings:

Figure 1. The UV absorption spectrum of Tetramycin A (determined in cyclohexane)

Figure 2, Infrared Absorption Spectrum of Tetramycin A (measured in KBr solution)

Figure 3. The NMR spectrum of the protons of Tymycin A (determined in heavy chloroform)

Claims (7)

1, a kind of manufacture method that can extract anti-parasitic new antibiotic substance or typmycin and its salts from microbial culture, it is characterized in that it is by from the KP-197 bacterial strain of Streptomyces Prepared by aqueous organic solvent extraction. 2. The manufacturing method according to claim 1, characterized in that said KP-197 bacterial strain, the diaminopimelic acid of its cell wall is LL-DAP, the shape of the spore filament is linear, the spore chain is long, and the surface of the spore is Smooth, vegetative hyphae are beige, aerial hyphae are white, and can form melanin. 3. The production method according to claim 2, characterized in that said KP-197 strain can be cultivated in synthetic medium or natural medium. These mediums generally contain carbon sources, nitrogen sources and inorganic salts suitable for cultivating actinomycetes, as well as other nutrients necessary in appropriate amounts. Fermentation culture is usually carried out under aerobic conditions such as vibration or aeration and stirring. The temperature of deep aeration and agitation culture is 20-40°C, pH value is 5-8, and the culture time is 3-6 days. 4, by the described manufacturing method of claim 1, it is characterized in that for extracting tyramycin from culture filtrate, use non-hydrophilic organic solvent ethyl acetate to extract, or use active carbon, aluminum oxide, porosity Polymer synthetic resin, ion exchange resin for adsorption, and then eluted with ethyl acetate solvent, the obtained extract or eluate is concentrated under reduced pressure, and then extracted with ethane, the refined extract obtained in this way is usually refined Methods for ester-soluble substances such as silica gel or alumina column chromatography for purification. 5. The production method according to claims 1 and 4, characterized in that the compound can be used as pharmaceutically acceptable salts such as refined hydrochloride, sulfate, nitrate, phosphate, carbonate, tartrate, citrate, Succinate, etc., can be prepared using conventional methods. 6. The manufacturing method according to claim 4, characterized in that the molecular formula of the Tyramycin A prepared by extracting and preparing from this recipe is C ₁₈ H ₃₄ N ₂ O ₂ , the molecular weight is 310, and the molecular formula of the Tyramycin B is C ₁₉ H ₃₄ N ₂ O ₂ , molecular weight 324. 7. The manufacturing method according to claim 4, characterized in that the compound prepared by this method uses the squirrel nematode as the treatment object, and carries out a control test with the known drug AVERMECTIN BLA. The results show that the antiparasitic effect of Tymycin The activity is more than ten times stronger than that of the control group, but its toxicity is lower than that of the control group. Therefore, the tatyramycin obtained in the present invention is useful as an antiparasitic drug for humans, animals and plants.

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