RU2578604C1 - Chimeric antibiotics based on azithromycin and glycopeptide antibiotics, having antibacterial activity and synthesis method thereof - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.

SUBSTANCE: disclosed are compounds of formula 2

, where n=1-10, and R is a fragment of vancomycin, eremomycin or teicoplanin aglycone. Method for preparing compounds of formula 2 comprises conducting acylation reaction 11-O-(ω-aminoalkylcarbamoyl)azithromycin (6) with glycopeptide antibiotic (eremomycin or vancomycin, or teicoplanin aglycone) in presence of a condensing agent.

EFFECT: invention relates to novel derivatives of azithromycin and a method for production thereof, which can be used in pharmaceutical industry and medicine.

2 cl, 4 dwg, 2 ex, 2 tbl

Description

The invention relates to the pharmaceutical industry and relates to new derivatives based on the antibiotic azithromycin and glycopeptide antibiotics, and a method for their preparation.

Azithromycin (1) (Figure 1) is a semi-synthetic antibiotic, the first representative of a subclass of azalides that are slightly different in structure from classical macrolides, the basis of the chemical structure of which is a macrocyclic lactone ring. Obtained by the modification of 14-membered macrolides by the inclusion of a nitrogen atom in the lactone ring between 9 and 10 carbon atoms, while the ring turns into a 15-membered one. This structural adjustment leads to a significant increase in the acid resistance of the drug - 300 times compared with erythromycin. A broad-spectrum antibiotic acts bacteriostatically. By binding to the 50S subunit of ribosomes, it inhibits the peptide translocase at the translation stage, inhibits protein synthesis, slows the growth and reproduction of bacteria, and has a bactericidal effect in high concentrations. It acts on extra- and intracellular pathogens. Inactive against gram-positive bacteria resistant to erythromycin.

It has a unique ability to accumulate in organs and tissues in comparison with other macrolides. Azithromycin is actively absorbed by various cells, including leukocytes, fibroblasts, macrophages and phagocytes, and is transported with them to the site of infection (inflammation) [Lalak NJ, Morris DL, Azithromycin Clinical Pharmacokinetics, Clinical Pharmacokinetics, 1993, V. 25, P. 370- P. 370- 374; Gladue R.P., Bright G.M., Isaacson R.E., Newborg M.F. In vitro and in vivo uptake of azithromycin (CP-62,993) by phagocytic cells: possible mechanism of delivery and release at sites of infection. Antimicrobial Agents Chemotherapy, 1990, V. 34, P. 1056-1060]. Azithromycin refers to tissue antibiotics, since its concentration in the blood serum is much lower than tissue. It is well distributed in the body, creating high concentrations in various tissues and organs (including the prostate gland), especially with inflammation. In this case, azithromycin penetrates into the cells and creates high intracellular concentrations. It is metabolized in the liver with the participation of the microsomal system of cytochrome P-450, metabolites are excreted mainly with bile. The half-life of azithromycin is 55 hours. With renal failure, this parameter does not change [Practical Guide to Anti-Infectious Chemotherapy. NIIAH SGMA. 2007. Edited by L.S. Strachunsky, Yu.B. Belousova, C.H. Kozlova].

Vancomycin (2) is a tricyclic heptapeptide to which a disaccharide consisting of aminodeoxysugar (vancosamine) and D-glucose (Formula 1) is attached [Sztaricskai F., Pelyva′s-Ferenczik I. Chemistry of carbohydrate components. In Glycopeptide Antibiotics, 1st edn (Ed. Nagarajan R.), 1994, Marcel Dekker, New York, NY, USA.].

Eremomycin (3) is an original domestic antibiotic whose aglycon differs from vancomycin aglycon by the absence of a chlorine atom in the lateral aromatic radical of amino acid 6, the structure of the disaccharide chain (2- (O- (α-L-eremosaminyl) -β-D-glucopyranosyl) and the presence of third carbohydrate residue (L-eremosaminyl) in the side radical of amino acid 6 (formula 1) [Gause GF, Brazhnikova MG, Lomakina NN, Berdnikova TF, Fedorova GB, Tokareva NL, Borisova VN, Batta G. Eremomycin - new glycopeptide antibiotics. Chemical properties and structure. J. Antibiotics, 1989, V. 42, P. 1790-1799].

Teicoplanin aglycone (4) is a heptapeptide in which, unlike vancomycin and eremomycin aglycones, the side radicals of amino acids 1 and 3 are aromatic and are interconnected by an ether bond (formula 1).

Vancomycin has been used in clinical practice since 1958, teicoplanin - since the mid-80s. Recently, interest in glycopeptides has increased due to an increase in the frequency of nosocomial infections with gram-positive microorganisms. Glycopeptides are active against gram-positive aerobic and anaerobic microorganisms, including methicillin-resistant Staphylococcus aureus (MSRA), methicillin-resistant Staphylococcus epidermidis (MRSE), streptococci, pneumococci (including ARP), enterococci, ampecoccus tuberculosis, pepticaria, resistant to corynebacteria, clostridia (including C. difficile). Gram-negative microorganisms are resistant to glycopeptides.

Glycopeptides disrupt the synthesis of the bacterial cell wall. They have a bactericidal effect, however, they act bacteriostatically against enterococci, some streptococci and coagulase-negative staphylococci. Glycopeptides are practically not absorbed when taken orally. The bioavailability of teicoplanin with intramuscular injection is about 90%. Glycopeptides are not metabolized, excreted by the kidneys unchanged, so dose adjustment is required for renal failure. Drugs are not removed during hemodialysis. The half-life of vancomycin with normal kidney function is 6-8 hours, teicoplanin is from 40 hours to 70 hours. The long half-life of teicoplanin makes it possible to prescribe it once a day [Practical Guide to Anti-Infectious Chemotherapy. NIIAH SGMA. 2007. Edited by L.S. Strachunsky, Yu.B. Belousova, C.H. Kozlova].

Vancomycin and teicoplanin are similar in the spectrum of antimicrobial activity, however, there are some differences in the level of natural activity and acquired resistance. Their successful use in the clinic is jeopardized by the spread of vancomycin-resistant gram-positive bacteria: enterococci (VRE) and vancomycin-resistant Staphilococcus aureus (VRSA) [Mendez-Alvarez S., Perez-Hernandez X., Claverie-Martin F. Glycopeptide resistance in enterocococ // Internat. Microbiol. - 2000. - V. 3. - P. 71-80; Cetinkaya Y., Falk P., Mayhall C.G. Vancomycin-resistant Enterococci // Clin. Microboil. Rev. - 2000. - V. 13. - P. 686-707]. VRE strains, and in particular VRSA, are resistant to the vast majority of antibiotics used, and therefore the spread of such microorganisms is a very serious problem.

The wide spread of antibiotic resistance among pathogens has led to the loss of the clinical significance of a number of drugs and served as an incentive for the search for new effective antimicrobial agents. One of the promising strategies aimed at creating drugs that are active against resistant microorganisms is the creation of double-acting antibiotics - “chimeric” antibiotics. They consist of molecules of different antibiotics, interconnected in various ways through the so-called "spacers". They have an extended spectrum of action compared to the original antibiotics and slow down the development of antibiotic resistance.

The present invention is intended to obtain dual-action antibiotics based on the macrolide antibiotic azithromycin and glycopeptide antibiotics with antibacterial activity.

The combination of azithromycin in one molecule with another antibacterial agent can potentially expand the spectrum of action of the obtained chimeric antibiotic in comparison with the spectrum of action of the original antibiotics. The pharmacokinetic properties of such a chimeric antibiotic may be superior to the pharmacokinetic properties of the second antibacterial agent used for its synthesis.

Glycopeptide antibiotics were chosen as the second antibacterial agent in the synthesis of chimeric azithromycin-based antibiotics, since they are active against gram-positive aerobic and anaerobic microorganisms and are the drugs of choice for infections caused by resistant gram-positive bacteria that are widespread in clinics.

The invention includes compounds corresponding to the structural formula depicted in Figure 2, where n = 1-10, and R is the remainder of a glycopeptide antibiotic such as vancomycin, or eremomycin, or teicoplanin aglycon.

The invention also includes a method for producing antibiotics based on azithromycin and glycopeptide antibiotics (Figure 1), which consists in carrying out the acylation reaction of 11-O- (ω-aminoalkylcarbamoyl) azithromycin (6) with a glycopeptide antibiotic (eremomycin (2), or vancomycin (3) , or teicoplanin aglycon (4)) in the presence of a condensing agent (Figure 3).

11-O- (ω-aminoalkylcarbamoyl) azithromycin (6) was obtained from 2′-O-acetyl-11,12-cyclic azithromycin carbonate (5) by a method similar to that described in the literature [X. Li, S. Ma, M. Yan, Y. Wang, S Ma. Synthesis and antibacterial evaluation of novel 11.4 ′ ′ - disubstituted azithromycin analogs with greatly improved activity against erythromycin-resistant bacteria. European Journal of Medicinal Chemistry, 2013, V. 59, pp 209-217].

This work describes the preparation of 11,4 ″ disubstituted derivatives of azithromycin, derivatives of 11-O- (aminophenylcarbamoyl) -4 ″ O O- (amino) carbamoylazitromycin, which showed high activity against erythromycin-sensitive S. pneumoniae strains and significantly improved activity against erythromycin-resistant strains of S. pneumonia (MIC 2 ÷ 16 μg / ml vs 256 μg / ml).

The acylation reaction of 11-O- (ω-aminoalkylcarbamoyl) azithromycin (6) (Fig. 3, Scheme 1) with a glycopeptide antibiotic to produce a compound of formula 2 is carried out in the presence of condensing agents known from the prior art and used to form an amide bond, for example benzotriazole- 1-yl-hydroxy-trispyrrolidinophosphonium hexafluorophosphate (RuBOP) or O- (benzotriazol-1-yl) -N, N, N ′, N′-bis (tetramethylene)) urea hexafluorophosphate (HBPyU). The acylation reaction of 11-O- (ω-aminoalkylcarbamoyl) azithromycin (6) (Figure 3) with a glycopeptide antibiotic to produce a compound of the structural formula shown in Figure 2 and shown in Figure 4, is carried out in a solvent selected from methanol, ethanol, Ν, Ν-dimethylformamide or dimethyl sulfoxide.

Compounds of the structural formula depicted in figure 2 have pronounced antibacterial activity, including against strains resistant to vancomycin (see Example 2) and can be used to treat infectious diseases.

Aids

Eremomycin sulfate was obtained at a pilot plant of the Research Institute for the search for new antibiotics named after G.F. Gause RAMS. Vancomycin hydrochloride was a commercial product of Aldrich (USA). Teicoplanin aglycon was obtained from Lepetit Research Center (Gerenzano (Varese), Italy). Benzotriazol-1-yl-hydroxy-trispyrrolidinophosphonium hexafluorophosphate (RuBOP), O- (benzotriazol-1-yl) -N, N, N ′, N′-bis (tetramethylene)) urea hexafluorophosphate (HBPyU) were commercial products of Acros. All solutions were dried over sodium sulfate and evaporated at a temperature not exceeding 40 ° C.

Thin layer chromatography was performed on G60 silica gel plates (Merck) in a solvent mixture: system (A) AcOEt-nPrOH-NH ₄ OH, 1: 1: 2. For preparative purification, column chromatography on Merck silanized silica gel with a particle size of 0.040-0.063 μm was used.

Analytical HPLC was carried out on an LC-10 chromatograph (Shimadzu, Japan) using a UV detector and a Kromasil 100-C18 4 × 250 mm column, particle size 6 μm (BioChemMak ST, RF). The mobile phase was a system consisting of two components A and B:

System (C): A (0.2% HCOONH ₄ pH 4.5) and B (MeCN), isocratic regime of 8% acetonitrile from 0 to 5 minutes, then a linear gradient of the concentration of acetonitrile 8 → 70% from 5 to 40 min, flow rate 1.0 ml / min

IR spectra were recorded in a KBr tablet on a DTGS spectrophotometer. Melting points were obtained on a Buchi SMP-20 device.

Mass spectra during electrospray ionization (ESI) were obtained on a Finnigan MAT 900S instrument (Germany).

Examples of the preparation of derivatives of azithromycin and glycopeptide antibiotics of the present invention and the study of their antibacterial activity:

Example 1. General method for producing chimeric antibiotics based on residues of azithromycin and a glycopeptide antibiotic.

2′-O-acetyl-11,12-cyclic azithromycin carbonate (5).

To a solution of azithromycin (5 g, 3.27 mmol) in 24 ml of ethyl acetate was added K ₂ CO ₃ (0.64 g, 4.63 mmol), the mixture was heated to boiling, and then 1.6 g (18.2 mmol) of ethylene carbonate was added slowly over 20 minutes while boiling. The mixture was boiled for 24 hours, then ethyl acetate was evaporated. The residue was dissolved in dichloromethane (30 ml) at room temperature, then acetic anhydride (0.61 ml, 4.37 mmol) and triethylamine (1.8 ml, 13 mmol) were added, and the reaction mixture was stirred for 24 h at room temperature. A 5% aqueous solution of NaHCO ₃ (30 ml) was added to the reaction mixture, and the aqueous solution was extracted with dichloromethane (3 × 10 ml). The combined dichloromethane layers were dried with anhydrous Na ₂ SO ₄ and dried in vacuo. Next, the residue was dissolved in chloroform and applied onto a chromatographic column (45 × 1.5 cm) with Merck silica gel equilibrated with chloroform, then eluted with chloroform (40 ml), then with a mixture of chloroform-methanol (10: 1). Fractions containing the target substance were evaporated and dried in vacuo.

11-O- (ω-aminoalkylcarbamoyl) azithromycin (6).

2′-O-acetyl-11,12-cyclic azithromycin carbonate (5) was dissolved in a minimal volume of Να, Νω-diaminoalkane and stirred at room temperature for 48 hours. Then, CHCl ₃ (20 ml) and water (20 ml) were added to the reaction mixture. ), the mixture was shaken. The organic layer was separated and washed with water 6 times. The organic layers were combined and water and 0.5 n HCl were added, shaking the layers so that the pH of the aqueous layer was 8. The organic layer was separated, Na ₂ SO _{4 was} added, held for 1 h, the precipitate Na ₂ SO _{4 was} filtered off, washing with chloroform. The organic layer was evaporated and dried in vacuo.

The general procedure for carrying out the acylation reaction of 11-O- (ω-aminoalkylcarbamoyl) azithromycin (6) with glycopeptide antibiotics (vancomycin or eremomycin or teicoplanin agglone), acetyl group cleavage and purification.

To a solution of the glycopeptide antibiotic (0.47 mmol) in DMSO (7 ml) was added 11-O- (ω-aminoalkylcarbamoyl) azithromycin (6) (0.5 eq., 0.235 mmol), the pH of the reaction mixture was adjusted to ~ 7.5 by adding Et ₃ N. RuBOP (1.1 eq., 0.26 mmol) was added in portions over 1 h, maintaining the reaction mixture at a pH of ~ 7.5 by adding Et ₃ N. The reaction mixture was stirred for 20 hours at room temperature, then a five-fold volume of diethyl ether was added. The resulting mixture was stirred vigorously, then the ether layer was removed. The procedure was repeated twice until a viscous oil was obtained, then methanol (0.5 ml), acetone (2 ml) and an excess of diethyl ether were added, the precipitate formed was filtered off, washed with diethyl ether and dried. The product was further purified by column chromatography on silicinized silica gel. The substance was dissolved in a 30% aqueous solution of MeOH, 1 cm ^{3 of} silanized silica gel was added and the mixture was dried in vacuo, then it was applied to a silanized silica gel column equilibrated with water. The elution was carried out first with water (100 ml), then with a 0.05 M solution of CH ₃ COOH, then with a MeOH-0.05 M CH ₃ COOH system (30:70) (100 ml) for compounds 7-9 or with a MeOH-0.05 M CH ₃ COOH system ( 30:70) (100 ml) for compounds 10-11. The fractions containing the target substance were combined, evaporated in a rotary evaporator with the addition of n-BuOH, acetone and diethyl ether were added to the residue. The precipitate was filtered off, washed with acetone and dried in vacuo. Physico-chemical data for derivatives 7-11 (Figure 4) are presented in Table 1.

Example 2. The study of the antibacterial activity of antibiotics based on azithromycin and glycopeptide antibiotics

The antibacterial activity of derivatives 7-11 in comparison with azithromycin (1) and vancomycin (2) was studied on the panel of strains of gram-positive and gram-negative bacteria (Table 2).

The data obtained indicate that all synthesized chimeric antibiotics based on azithromycin and glycopeptides 7-11 were not inferior or superior to azithromycin and vancomycin in antibacterial activity against all studied strains of gram-positive bacteria. The high activity of derivatives (higher than that of azithromycin and vancomycin) against pneumococci, which are the cause of most cases of meningitis, community-acquired pneumonia, and a number of purulent-septic infections, is valuable. Derivatives based on eremomycin and azithromycin were active against vancomycin resistant strains of Enterococcus faecium and Enterococcus faecalis.

Thus, the proposed method for producing antibiotics based on azithromycin and glycopeptide antibiotics allows to obtain new compounds with high antibacterial activity, including against a number of Enterococcus strains resistant to vancomycin.

Claims (2)

1. Compounds corresponding to formula 2

,
where n = 1-10, a R represents the remainder of a glycopeptide antibiotic, such as vancomycin, or eremomycin, or teicoplanin aglycon. 2. A method of producing antibiotics based on azithromycin and glycopeptide antibiotics according to claim 1, which consists in conducting an acylation reaction of 11-O- (ω-aminoalkylcarbamoyl) azithromycin (6) with a glycopeptide antibiotic (eremomycin, or vancomycin, or teicoplanin aglycon) in the presence of .

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