RU2762182C1 - Gausemycins a and b - glycolipopeptide antibacterial antibiotics and method for production thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a compound, namely to gausemycins A and B by the formula:

The invention also relates to a method for isolating said gausemycins A and B.

EFFECT: production of new compounds exhibiting antibacterial activity against gram-positive bacteria including methicillin-resistant Staphylococcus aureus, potentially applicable in medicine.

6 cl, 1 dwg, 2 tbl, 11 ex

Description

The technical field to which the invention relates

The invention relates to the field of chemical technology, chemistry of natural compounds and medicine, namely to a multistage method for producing biologically active compounds exhibiting antibacterial activity and applicable as a drug in medical practice.

Prior art

In recent years, the effectiveness of existing antibiotics in the treatment of common infections has been significantly reduced. In this regard, in 2017, the world health organization compiled a list of deadly bacteria for which new therapy is urgently required [Tacconelli E, Carrara E, Savoldi A, et al., Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. The Lancet Infectious Diseases. 2018; 18 (3): 318-327]. This list includes such a pathogen as Staphylococcus aureus (Staphylococcus aureus), which is one of the most common and dangerous pathogens in the human population [Mulani MS, Kamble EE, Kumkar SN, Tawre MS, Pardesi KR. Emerging Strategies to Combat ESKAPE Pathogens in the Era of Antimicrobial Resistance: A Review. Frontiers in Microbiology. 2019; 10: 539]. Natural antibiotics of a peptide nature have significant potential for use in clinical practice to combat resistant pathogenic microorganisms, striking examples of commercially successful drugs are glycopeptide antibiotics and their derivatives [Blaskovich MAT, Hansford KA, Butler MS, Jia Z, Mark AE, Cooper MA. Developments in Glycopeptide Antibiotics. ACS Infect Dis. 2018; 4 (5): 715-735] and the lipopeptide antibiotic daptomycin [Raja A, LaBonte J, Lebbos J, Kirkpatrick P. Daptomycin. Nature Reviews Drug Discovery. 2003; 2 (12): 943-944].

At the Research Institute for the Research of New Antibiotics. G.F. Gauze found a strain producing the antibiotic complex INA-5812, in which more than 20 chemically related substances [Lapchinskaya O.A., Katrukha GS, Gladkikh EG and others. Study of the antibiotic complex INA-5812. Bioorganic chemistry. 2016; 42 (6): 732-740]. It is active against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. Was described the producer strain of Streptomyces roseoflavus INA-Ac-5812, a method of obtaining a crude antibiotic from the culture fluid of the strain and a component with a molar mass of 1917.8 Da [Patent RU 2572341]. The productivity of the antibiotic complex producer strain was significantly increased and a new producer strain of Streptomyces tendae VKPM Ac-1980 was obtained, which accumulates a greater amount of the antibiotic complex in the culture liquid during cultivation [Patent RU 2710733].

The structure, the method for determining the structure, the method of isolation, the gross formula, the complete amino acid composition, the biological activity of the claimed compounds are not described in the patent and scientific and technical literature.

The essence of the invention

The essence of the invention lies in the development of technology for the isolation of individual components of the antibiotic complex of gauzemycins A and B, the establishment of their structure, physicochemical properties and antibacterial activity.

The method for isolating biologically active compounds in an individual form is a multistage method. It can be roughly divided into three main stages.

1. Extraction of the culture of the producer strain.

2. Obtaining an antibiotic concentrate from an extract using a combination of methods (reprecipitation, selective sorption / desorption, chromatography).

3. Isolation of individual compounds from antibiotic concentrate using preparative chromatography of hydrophilic interactions.

Extraction of the culture of the producer strain consists in the separation of the mycelium by centrifugation or filtration; subsequent liquid or solid phase extraction of the aqueous phase; liquid extraction of mycelium; defatting the obtained extracts.

Obtaining an antibiotic concentrate from an extract can be carried out in two ways.

1. Sequential chromatographic enrichment of the extract at low pressure on gel sorbents of the Sephadex G15 type, reverse phase sorbent (octadecyl silica gel) under neutral conditions and the final stage of precipitation in an acidic medium.

2. Sorption of the obtained extract with a solid-phase sorbent LPS500-H, selective desorption, chromatographic separation of the resulting concentrate by reversed phase chromatography.

The subsequent isolation of individual components is carried out by chromatography of hydrophilic interactions under HPLC conditions. The resulting components are characterized by physicochemical and biological methods.

Compared with the earlier developed inventions [Patent RU 2572341, Patent RU 2710733], the described method makes it possible to obtain several individual components from a complex mixture in an amount and with a purity sufficient for carrying out physicochemical and biological studies.

Implementation of the invention

Example 1. Extraction of a culture of Streptomyces tendae VKPM Ac-1980

The culture liquid (271 ml) is centrifuged for 20 min at 5000g. The aqueous phase is decanted, acidified with HCl to pH 4 and extracted with n-butanol (2 × 52 ml). The organic extracts are combined and evaporated to dryness. The dry residue is defatted: 19 ml of ethyl acetate is poured in and sonicated in an ultrasonic bath (40 kHz, 60 W, 10 min at a temperature of 25 ° C), then the solution is filtered. The solution is discarded, and further work is carried out with the extract (1.3 g) remaining on the filter.

The mycelium is suspended in 50 ml of ethanol, sonicated in an ultrasonic bath (40 kHz, 60 W, 20 min at 25 ° C) and filtered. The filtrate is concentrated in vacuo. The dry residue is degreased as described above. The mass of the mycelium extract is 0.8 g. The mycelium extract, along with the extract of the aqueous phase, can be used to obtain hausemycins.

Example 2. Extraction of a culture of Streptomyces roseoflavus INA-Ac-5812

The culture liquid (7.0 l) is filtered from the mycelium. The XAD-4 sorbent is added to the aqueous phase (at the rate of 100 g of air-dry sorbent per 1 L of CL). Stirred for 4-12 hours at room temperature until the sorbent is saturated. The sorbent is filtered off, the filtrate is discarded and the sorbent is washed with one volume of 1% acetic acid. The active substances are eluted with 3-fold volume of methanol.

The mycelium is suspended in 500 ml of methanol, sonicated in an ultrasonic bath (40 kHz, 60 W, 20 min at 25 ° C) and filtered. The methanol filtrates are combined and evaporated. The dry residue is defatted with ethyl acetate (2 × 100 ml) by the method described in example 1.

Further work is carried out with the obtained extract (17.3 g).

Example 3. Extraction of the culture of Streptomyces tendae VKPM Ac-1980

The culture liquid (6.4 l) is centrifuged for 15 min at 5000g. The aqueous phase is decanted, acidified with HCl to pH 4 and extracted with isobutanol (2 × 900 ml). The organic extracts are combined and evaporated to dryness. The dry residue is defatted with ethyl acetate (100 ml) as described in example 1 by the method. 10.7 g of extract are obtained.

The mycelium is suspended in 500 ml of ethanol, sonicated in an ultrasonic bath (40 kHz, 60 W, 20 min at 25 ° C) and filtered. The filter cake is additionally washed with 75 ml of ethanol. The filtrate was evaporated to dryness, the residue was defatted with ethyl acetate (2 × 100 ml) as described in Example 1. 12.3 g of extract are obtained.

Example 4. Extraction of the culture of Streptomyces roseoflavus INA-Ac-5812

The culture liquid (5.0 L) is filtered from the mycelium. Diaion HP-20 sorbent is added to the aqueous phase (at the rate of 100 g of air-dry sorbent per 1 L of CL). Stirred for 4-12 hours at room temperature until the sorbent is saturated. The sorbent is filtered off, the filtrate is discarded and the sorbent is washed with one volume of 1% acetic acid. The active substances are eluted with 3-fold volume of isopropanol.

The mycelium is suspended in 450 ml of isopropanol, sonicated in an ultrasonic bath (40 kHz, 60 W, 20 min at 25 ° C) and filtered. Isopropanol extracts are combined and evaporated. The dry residue is defatted with ethyl acetate (3 × 100 ml) by the method described in example 1. 16.9 g of extract are obtained.

Example 5. Obtaining an antibiotic concentrate from an extract

The extract (1.3 g) is purified from low-molecular impurities by gel filtration on a column (22 × 70 mm) with a Sephadex G15 sorbent using isocratic elution (20% methanol in water, flow rate 7 ml / min, absorption detection at 210 , 260, 360, 410 nm). Fraction 1 (0.44 g) was collected with a retention time of 0-10 minutes.

Fraction 1 is dissolved in 4 ml of 50% acetonitrile and chromatographed on a column (25 × 130 mm) with a reversed phase sorbent (octadecyl silica gel) using step elution (flow rate 14 ml / min, 5 minute steps: 20%, 30% , 40%, 50%, 60%, 80% acetonitrile in water; absorption detection at 210, 260, 360, 410 nm). Column capacity - 0.2-0.3 g of the mixture. The fraction with a retention time of 16.0-17.0 min, according to chromatomass-spectrometry data, contains mainly hausemycins A and B, and impurities. Separation of 0.440 g of the mixture yields 0.062 g of this fraction.

A sample of the resulting mixture of hausemycins (0.12 g) is dissolved in 2.4 ml of an aqueous ammonia solution with pH 10. The resulting solution is acidified with an aqueous solution of hydrochloric acid to pH 5 and the formation of a dark brown precipitate is observed. The solution is filtered, the resulting precipitate is dissolved in 1.1 ml of an aqueous ammonia solution with pH 10 and acidified with an aqueous solution of hydrochloric acid to pH 5; the formation of a dark brown precipitate is again observed. As a result, a combined solution is obtained after two precipitations and a precipitate with a mass of 0.053 g. The solution after two precipitations (3 ml) is extracted with butanol (3 × 3.85 ml). The organic extracts are combined, washed with acidified water at pH 5 and evaporated to dryness. The result is a powder (0.036 g) with a yellowish tinge. This antibiotic concentrate mainly contains gauzemycins A and B, and is used to isolate individual compounds.

Example 6. Obtaining an antibiotic concentrate from an extract

The extract (1 g) is dissolved in 0.25 l of water with the addition of 10 ml of concentrated aqueous ammonia. The resulting solution is passed through a column with 5 ml of LPS500-H air-dry sorbent. Collect fraction "0" containing impurities that have not bound to the sorbent. Then, chromatography is carried out using stepwise elution with an increasing concentration of acetonitrile in water (steps of 0%, 10%, 20%, 30%, 40%, 50%, 100%, 50 ml each). Seven fractions are obtained. Fractions 20-50% contain mainly hausemycins A, B. As a result of the separation of 1 g of the extract, 0.430 g of fraction "0", 0.051 g of fraction "0%", 0.044 g of fraction "10%", 0.061 g of fraction "20%" , 0.085 g of the "30%" fraction, 0.043 g of the "40%" fraction, 0.072 g of the "50%" fraction, 0.054 g of the "100%" fraction.

The fraction "20%" (61 mg) is dissolved in 2 ml of 50% acetonitrile and applied to a preparative ZORBAX SB-C18 column (250 × 21.2 mm, octadecyl silica gel, 7 μm). Separation is carried out by isocratic elution with solution B (0.1% trifluoroacetic acid in acetonitrile, eluent A - 0.1% trifluoroacetic acid in water) 33% for 30 min, then elution with 50% B for 10 min, at a flow rate of 20 ml / min. Detected by UV absorption at 210, 260, 360, 410 nm or using a diode array detector. The fraction with a retention time of 9-13 min, according to chromatomass spectrometry data, contains mainly hausemycins A and B. It is dried in a vacuum. The result is 25 mg of the antibiotic concentrate in the form of a white powder.

Example 7. Preparative liquid chromatography of hydrophilic interactions

The antibiotic concentrate (2 mg in 500 μL of 50% acetonitrile) is chromatographed on a VDSpher PUR 100 SIL column (250 × 10 mm, 5 μm) using a solvent mixture of 0.1% trifluoroacetic acid, 4% water, 24% as eluent propanol-1 and 71.9% acetonitrile by volume. Flow rate 6 ml / min, detection by UV absorbance at 205 nm. The fraction with a retention time of 8.0-10.0 min contains gausemycin A, the fraction with a retention time of 10.0-13.0 min contains gausemycin B. Organic solvents are removed from the obtained fractions under reduced pressure, the resulting aqueous solution is frozen and lyophilized. The content of the main components in the fractions is controlled chromatographically and mass spectrometrically, the stage of hydrophilic interaction chromatography is repeated until the complete removal of impurities.

Separation of 36 mg of the antibiotic concentrate (prepared according to the method described in Example 3) gives 0.8 mg of gauzemycin A and 1.1 mg of gauzemycin B in the form of white powders.

Separation of 25 mg of the antibiotic concentrate (prepared according to the method described in Example 4) gives 1.9 mg of gauzemycin A and 5.3 mg of gauzemycin B in the form of white powders.

Example 8. Analytical chromatography to control the content of hausemycins A and B

To analyze the content of minor components of the antibiotic complex and low molecular weight impurities, analytical chromatography is performed. For this, a solution of the analyzed sample with a concentration of 4 mg / ml in methanol is prepared, 20 μl of the analyzed solution is applied to a Waters Sunfire column (C 18; 5 μm, 4.6 × 250 mm), and a gradient elution is carried out (20-80% solution B for 25 min, where solution A is 0.1% trifluoroacetic acid in water, solution B is 0.1% trifluoroacetic acid in acetonitrile) at a flow rate of 1 ml / min. The retention time of a group of compounds, including gauzemycins A, B and a component with a weight of 1930 mg, is 10.2 minutes.

Chromatographic control of the content of gauzemycins A, B and a component with a mass of 1930 Da is carried out under normal phase analytical HPLC. Prepare a solution of the analyzed sample with a concentration of 4 mg / ml in methanol, applied to a Waters Spherisorb Silica column (Silica gel, 5 μm, 4.6 × 250 mm) 10 μl of the solution. Gradient elution is carried out (5-17% solution B in 20 min, where solution A is 0.1% TFA, 25% PrOH, 74.9% CH ₃ CN, solution B is 0.1% TFA in HrO, also during all chromatography was additive) at a flow rate of 1 ml / min. The retention time of gausemicin A is 13.8 min, gausemicin B is 14.7 min, and the component with a mass of 1930 Da is 15.5 min. To ensure reproducibility of the retention time, the normal phase column is constantly saturated with water and the retention times are periodically calibrated against standard mixtures.

Mass spectrometric control of the obtained samples is carried out using an Agilent 6340 Ion Trap detector with an electrospray ionization source connected to an Agilent 1100 HPLC system. Sample separation was carried out on a YMC-Triart С18 (YMC) column (50 × 2.1 mm, 1.9 μ.m) by elution with a linear gradient (2-98% acetonitrile in water with the addition of 0.1% formic acid in both eluents (when analyzing in chromatomass The content of gauzemycins A and B in the samples is monitored by the total ionic current of doubly charged ions with masses of 924 Da and 959 Da, respectively, the content of a component with a mass of 1930 is monitored by the ion current of a doubly charged ion with a mass of 967 Da. -spectrometrically for chromatographically pure samples due to the noticeable association of gauzemycins with low molecular weight impurities.

Example 9. Determination of the main physicochemical and spectral characteristics of hausemycins A, B

The resulting samples of gauzemycins A and B are amorphous solids.

Solutions of substances have optical activity. The measured specific rotation: gausemicin A [α] ²² _D -6.9 (c 0.3, methanol); hausemycin B [α] ²⁵ _D = -9.7 (c 0.4, methanol).

Gausemycins have a characteristic UV absorption spectrum:

hausemycin A (methanol) λ _max , nm (log ε) 203 (4.7), 229 (4.5), 262 (4.4), 363 (3.7);

hausemycin B (methanol) λ _max , nm (log ε) 204 (4.7), 229 (4.5), 262 (4.4), 363 (3.7).

When irradiated in the UV range for both substances, fluorescence is observed with an emission maximum at 448 nm (96% ethanol, C = 1 μM, λ _exc 350 nm).

The absorption spectra in the IR range have characteristic maxima:

hausemycin A (in KBr) ν _max , cm ^-1 : 3330, 3070, 2960, 2930, 1660, 1540, 1450, 1200, 1180, 1070;

hausemycin B (in KBr) ν _max , cm ^-1 : 3330, 3070, 2960, 2930, 1660, 1540, 1450, 1230, 1200, 1180, 1070.

Example 10. Establishment of the composition and structure of hausemycins A, B

The gross formulas of gauzemycins A and B are established on the basis of high-resolution mass spectra. The high-resolution mass spectrum of hausemycin A contains an ion with m / z 1845.788, which is consistent with the gross formula C ₈₄ H ₁₁₆ ClN ₁₇ O ₂₈ (calculated value m / z = 1845.786 for [M + H] ⁺ ), mass spectrum high-resolution gauzemycin B contains a doubly charged ion with m / z 959.4187, which is consistent with the gross formula C ₈₇ H ₁₂₁ ClN ₁₈ O ₂₉ (calculated value m / z = 959.4191 for [M + 2H] ²⁺ ).

The structure of hausemycins A and B shown in Figure 1 was established based on NMR data (Table 1). The assignment of signals in the ¹ H, ¹⁴ N and ¹³ C NMR spectra was carried out on the basis of two-dimensional spectra, including both homonuclear and heteronuclear correlations. The spectra were recorded for solutions of individual substances in perdeuterodimethyl sulfoxide at an elevated temperature (45 ° C) to simplify the complex conformational dynamics observed at lower temperatures. At higher temperatures, partial destruction of the sample is observed.

Example 11. Evaluation of the antibacterial properties of hausemycins A, B

Preliminary experiments have shown that hausemycins have a marked antibacterial activity against gram-positive bacteria, primarily staphylococci. The antibacterial properties of the isolated hausemycins A and B were assessed by the method of serial microdilutions in a liquid nutrient medium in accordance with the clinical guidelines "Determination of the sensitivity of microorganisms to antimicrobial drugs", version 2018-03, approved on October 18, 2017 by an expert meeting of the profile commission in the specialty "Clinical microbiology and antimicrobial resistance. "At the Ministry of Health of the Russian Federation. Standard samples of peptide antibiotics (daptomycin, vancomycin) were used as a control. The obtained values of the minimum inhibitory concentration are presented in table. 2.

Claims (7)

1. Gausemycins A and B, which have antibacterial activity against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, and corresponding to the formula:

2. A method of isolating peptide antibiotics hausemycins A and B from claim 1, which consists in isolating the extract by extracting the culture fluid of the producer strain; obtaining an antibiotic concentrate using a combination of methods: reprecipitation, selective sorption / desorption, chromatography, preparative separation of the resulting concentrate by chromatography of hydrophilic interactions under HPLC conditions and physicochemical and biological characteristics of the obtained samples. 3. A method of isolating peptide antibiotics hausemycins A and B from p. 1 in the form of an extract according to p. 2, in which a liquid-liquid extraction of the culture liquid of the producer strain with an organic solvent selected from n-butanol or isobutanol is carried out; the antibiotic concentrate is obtained using sequential chromatographic separations at low pressure: gel filtration on Sephadex G15 sorbent, reversed phase sorbent under neutral conditions; and subsequent precipitation upon acidification. 4. A method for isolating peptide antibiotics hausemycins A and B from claim 1 in the form of an extract according to claim 2, in which the culture liquid is extracted with a solid-phase sorbent based on polystyrene. 5. A method for isolating peptide antibiotics hausemycins A and B from claim 1 in the form of an extract according to claim 2, in which the extraction of the mycelium of the producer strain is carried out with an organic solvent selected from methanol, ethanol, propanol-2 or mixtures thereof. 6. A method for isolating peptide antibiotics hausemycins A and B from claim 1 in the form of a concentrate according to claim 2, which uses extract sorption with a solid-phase sorbent LPS500-H, followed by selective desorption and chromatographic separation by reversed-phase chromatography under HPLC conditions.

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