KR810001325B1 - Process for the preparation of a new cyclopeptide antibiotic - Google Patents

Abstract

The antibiotics I (R = -CONH-C(CH2)-CONH2), designated 35665 RP was prepd. by the partial hydrolysis of the antibiotic nosiheptide in an acid medium. I is effective against Gram (T) bacteria and is useful as a growth promotor for animals esp., poultry by incorporation into feeds at 1-50 g per tonne. In mice it caused no toxic manifestations at 2.5 g/kg orally. I is compatible with all standard feed components.

Description

Method for preparing cyclopeptide antibiotic

1 is an infrared absorption spectrum of a compound of the present invention.

2 is a nuclear magnetic resonance spectrum of a compound of the present invention.

3 is the ¹³ C nuclear magnetic resonance spectrum of the compounds of the present invention.

The present invention relates to a method for producing a novel cyclopeptide antibiotic represented by 35665RP.

The new antibiotic 35665RP is of low toxicity and is particularly valuable as a growth factor for use in animal feed. The structural formula of the antibiotic 35665RP represented by the formula C ₄₈ H ₄₀ O ₁₁ N ₁₂ S ₆ is as follows.

In which R is a group-CONH ₂ .

35665RP has the following physical-chemical properties.

Appearance: Yellowish amorphous powder.

Solubility: Soluble in dimethylformamide, pyridine and acetic acid; slightly soluble or insoluble in water, ethanol, acetone, chloroform and n-hexane.

Melting point measurement (in capillary tube): Burn at 270 ° C without melting to 300 ° C.

Elemental Analysis (for obtained compounds):

Found: C48.3%, H3.6%, 0 16.0%, N13.5%, S15.35%

Theoretic: C49.99%, H3.50%, 0 15.26% N14.57%, S16.68%

Ignition residue: 0.6%

Weight loss under reduced pressure at 100 ℃: 5.9%

35665RP is negative in the ninhydrin test and the product after hydrolysis in 6N hydrochloric acid for 20 hours under reflux under nitrogen atmosphere shows positive ninhydrin reaction according to its polytetided nature.

UV spectrum (measured using methanol solution containing 10.5 mg / l):

=313(약 295nm에서 쇼울더);λ max 320 nm,

= 313 (shoulder at about 295 nm);

Visible spectrum (measured using methanol solution containing 21 mg / l);

=107(약 372nm에서 쇼울더);λ max = 405 nm,

107 (a shoulder at about 372 nm);

Infrared spectrum (measured for tablets in admixture with KBr): The major infrared absorption band of 35665 RP is shown in wavenumber (cm ⁻¹ ) in Table 1 below.

TABLE 1

The spectrum is shown in FIG. 1, and in FIG. 1, the upper scale of the abscissa represents the wavelength (in microns), the lower scale represents the wave number (cm ⁻¹ ), and the ordinate represents the optical density.

=61±1.30(C=0.88; 피리딘)Radiance:

= 61 ± 1.30 (C = 0.88; pyridine)

Other physico-chemical properties of 35665RP are:

Quantum Nuclear Magnetic Resonance Spectrum in hexaderated dimethylsulfoxide: This spectrum is shown in FIG. 2 and recorded at the frequency 250MHZ on a Cameca TSN250 machine.

Chemical shifts are shown in Table 2 below. The chemical shift δ is calculated in ppm with the positive side towards the bottom from the internal standard TMS (tetramethylsilane).

TABLE 2

13 C Nuclear Magnetic Resonance Spectrum in hexaderated dimethylsulfoxide: This spectrum is shown in FIG. 3 and is recorded at the Kameka TSN 250 mechanical phase frequency 62.86MHZ.

Chemical shifts are measured for TMS using hexadeuterated dimethylsulfoxide as internal standard at 39.5 ppm (ppm).

TABLE 3

In both proton and 13C nuclear magnetic resonance spectra, 35665RP has 48 C atoms, of which 11 are SP ³ carbon atoms and 37 are SP ² carbon atoms.

Thin layer chromatography: 35665RP has an Rf value of 0.16 when chromatographed using a benzene / methanol / acetic acid / pyridine mixture (75/10/2/15 volume ratio) as the developing solvent on a thin layer of silica gel.

The location of 35665RP is observed using its yellow fluorescence under ultraviolet light of 366 nm.

Average Activity: The average activity of 35665RP against a number of microorganisms was determined by the dilution method commonly used for this purpose. For each microorganism, the minimum concentration of a substance that prevented the growth of bacteria in the appropriate nutrient medium under certain conditions was measured. The results of the various measurements are shown in Table 4 as the minimum inhibition concentration (mcg of 35665 RP per cc of test medium).

TABLE 4

Toxicity of 35665RP was tested in mice.

35665RP is nontoxic when administered orally at a dose of 2.5g / kg (weight of animal).

The antibiotic 35665RP can be obtained by hydrolysis of the antibiotic 9671RP known as Nosiheptide.

The preparation thereof is known by culturing nociheptide and Streptomyces actousus (NRRL2954).

인 구조식Ⅰ에 상응한다.The structure of the material is Tetrahedron Letters, No. 16, P1395 1046 (1977), whose structure is R

Corresponds to Phosphorus I.

인 일반식(Ⅰ)의 노시헵티드를 가수분해시켜 분자의 나머지는 영향을 주지 않고 기

를 기 -CONH₂로 전환시킴으로써 항생제 35665RP를 제조하는 방법에 관한 것이다.In the present invention, R is a group under acidic conditions.

Hydrolyze the nociheptide of the general formula (I), leaving the rest of the molecule intact

To a group -CONH ₂ to prepare antibiotic 35665RP.

In particular, hydrolysis conditions should not cause breakage of peptides or lactone bonds. Hydrolysis is preferably carried out by heating the nociheptide in an inorganic acid solution in a neutral organic solvent or in an aqueous solution of an organic acid of C ₃ or less at a temperature of 40 to 120 ° C. The reaction time is generally 10 minutes to 4 hours, depending on the temperature and the acidity of the reaction medium. For example, a 1-5N anhydrous hydrogen chloride solution in a mixture of methylene chloride and methanol may be regarded as a suitable reaction medium.

After hydrolysis, a Poor solvent was added to the reaction mixture to precipitate, and the obtained 35665RP was separated from the reaction medium by filtration. The obtained 35665RP can be purified by physico-chemical methods such as chromatography,

The following example shows a method for preparing 35665RP.

Example 1

The suspension in which nociheptide (100 mg) was suspended in an acetic acid / water mixture (90/10 volume ratio; 10 cc) was heated under reflux for 1 hour, and after cooling, the resulting solution was poured into diethyl ether (100 cc).

The precipitate formed is filtered off, washed with diethyl ether (50 cc) and dried at 35 ° C. under reduced pressure (5 mmHg).

The resulting product (100 mg) was purified by HPLC on a stainless steel column 60 cm long, 0.75 cm in diameter, containing silica gel (about 15 g; particle size 10-40 mm, charged under pressure of 300 bars). In each operation, a solution of 50 mg of the product in a chloroform / ethanol / water mixture (80/20 / 0.25 volume ratio; 1 cc) is injected into the column.

Eluent consists of chloroform / ethanol / water (95/5 / 0.25 volume ratio).

Elution under 50 bar pressure (flow rate of 2.5 cc / min) and continuous measurement of the optical density of the eluate at wavelength 360 nm The fractions containing 35665RP were concentrated to dryness under shading under reduced pressure to obtain 13 mg 35665RP.

Example 2

A suspension in which nosiheptide (100 mg) was suspended in a mixture of methylene chloride and 8N methanol solution of hydrogen chloride (70/30 volume ratio; 10 cc) was heated under reflux for 1 hour, and the mixture was cooled to 20-25 ° C.

The resulting solution was poured into diethyl ether (100 cc), the resulting precipitate was filtered off, washed with diethyl ether (50 cc) and dried at 35 ° C. under reduced pressure (5 mmHg).

The resulting product (100 mg) was purified as in Example 1 to give 27 mg of 35665RP.

The antibiotic 35665RP, when added to animal feed, gains weight more rapidly than in animals fed non-augmented feed. The antibiotic also has the advantage of being very stable on storage and thus can be mixed with all foods supplied to animals without risk of deterioration during storage.

Animal feed consisting of animal feed material and antibiotic 35665RP is also included within the scope of the present invention.

Enriched mixtures for animal feed are also included in the present invention. The dosage required to achieve a moderate effect varies widely depending on the animal's condition and the nutritional value of the feed itself. In general, a dose of 1-50 g of antibiotic 35665RP per tonne of feed is sufficient for raising animals.

The antibiotic 35665RP may be present as a uniform dispersion in the complete feed composition at this dose. The compound may also be included in supplementary feeds, with other additives such as vitamins and mineral salts, in a proportion of 0.1 to 0.001% by weight of the feed.

These supplements are either mixed with or consumed with aquaculture and typically make up 5-20% of aquaculture.

The premixes according to the invention used to prepare complete aquaculture or supplemental feeds generally contain the antibiotic 35665RP, which corresponds to 0.05-20% by weight of the edible weight. These are convenient intermediates that facilitate the uniform distribution of the antibiotic 35665RP in the feed.

The premix itself is generally obtained from concentrates containing antibiotics 35665RP in concentrations of 9.9 to 20% by weight relative to edible denaturants such as edible dyes, edible antipigments, dispersants or coagulants and food weight agents. For example, this concentrate may contain 99% by weight of antibiotic 35665RP, 0.1% by weight of dye and 0.9% by weight of anticoagulant. Concentrates and premixes are generally powders, and supplemental feeds and complete compositional feeds may be in powder form or granules (prepared according to conventional methods).

The antibiotic 35665RP in various compositions may be in the form of particles coated or uncoated.

The antibiotic 35665RP can be properly administered to all livestock, especially in poultry.

The following examples show the compositions of the present invention.

Example 3

To prepare a feed for chicks having the following composition (% by weight).

The antibiotic 35665RP is evenly distributed in the feed at a rate of 5 g to 10 g per tonne of feed.

Six 84 chicks (STUD 160 whites, 42 male and 42 female chicks) are blocked and placed in a cage for 14 chickens per cubicle.

Place chicks 1 to 4 weeks old in hot coops and 4 to 8 weeks old in one coop. Feed from day 1 to weigh 4 to 8 weeks old hens and roosters.

Measured as follows.

1) The weight gain of chickens fed with 35665RP of antibiotics was compared with the control bacteria fed with 35665RP.

를 대조사료로 사육된 닭의 전환지수와 비교했다.2) The conversion index for chickens raised with feed containing 35665 RP of antibiotics

Was compared with the conversion index of the chickens raised for the survey.

The results are summarized in the following table.

Claims (1)

R is

Which hydrolyze the heptane-shi lactide of formula (Ⅰ) under acidic conditions the R having the following physico-chemical properties such as the group does not affect the rest of the molecule described above into the body by conversion to the group -CONH ₂ -CONH ₂ A process for preparing the novel antibiotic 35665RP of formula (I).

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