RU2708628C1 - Novel derivatives of eremomycin and use thereof for treating bacterial infections - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.SUBSTANCE: invention relates to an eremomycin derivative which can be used in medicine and pharmaceutical industry, corresponding to formula:,where Py – independently denotes pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, n=1–3, as well as its quaternized N-Calkyl derivatives, hydrates, pharmacologically acceptable salts.EFFECT: novel derivatives of eremomycin effective for treating bacterial infections caused by gram-positive bacteria.4 cl, 11 ex, 2 tbl

Description

Technical field

The invention relates to the pharmaceutical industry and relates to the structure of new amides of the glycopeptide antibiotic eremomycin containing a residue of alkylpyridine in the carboxamide fragment, their quaternized derivatives, and also their medical use for the treatment of infections caused by gram-positive bacteria.

State of the art

It is known that vancomycin (1) is the most important glycopeptide antibiotic with a macrocyclic peptide core, glycosylated with a disaccharide, consisting of β-D-glucose and vancosamine [M. Shafer, T.R. Schneider, G.M. Sheldrick. Structure, 1996, 4, 1509]. The antibacterial activity of vancomycin and other glycopeptide antibiotics is associated with inhibition of murein synthesis due to the formation of a complex with terminal peptide groups of the growing chains of peptidoglycan [C. Watanakunakorn. J. Antimicrob. Chemother., 1984, 14, 7].

Vancomycin is highly active against gram-positive bacteria and, until recently, was the drug of choice for the treatment of severe antibiotic-resistant bacterial infections, primarily caused by methicillin-resistant Staphylococcus aureus (MRSA) [C. Watanakunakorn. J. Antimicrob. Chemother, 1984, 14, 7]. The widespread use of vancomycin in recent decades has led to the spread of resistant strains of enterococci and staphylococci with resistance to glycopeptides (VRE and GISA) [M.G. Page, K. Bush. Curr. Opin. Pharmacol., 2014, 18, 91]. In addition, the disadvantages of vancomycin are its nephro- and ototoxicity, as well as pseudo-allergic reactions associated with the release of histamine [S.K. Hubert et al., J Clin Microbiol., 1999, 37, 3590; R.E. Polk. Ann. Pharmacother., 1998, 32, 840].

It has been shown that it is possible to increase the activity, effectiveness and safety of natural glycopeptides, as well as overcome cross-resistance, by chemical modification [D. Kahne et al. Chem. Rev. 2005, 105, 425; E.N. Olsufyeva, A.N. Tevyashova. Curr. Topics in Med. Chem., 2017, 17, 2166]. A promising direction in the study of glycopeptide antibiotics is the synthesis of cationic lipoglycopeptides based on them. For this type of derivative, it was shown that due to an increase in affinity for membranes and building blocks of peptidoglycan, they have significant activity against vancomycin-resistant bacteria [V. Yarlagadda et al. J. Antibiot., 2014, 67, 1]. It should also be noted that cationic agents are highly effective anti-infective drugs that are currently widely used for the treatment and prevention of bacterial diseases [M.S. Butler, M.A.T. Blaskovich, M.A. Cooper. J. Antibiot. 2017, 70, 3].

Eremomycin 2 is a glycopeptide antibiotic that is similar in structure to vancomycin, a distinctive feature of which is a disaccharide attached to the D-ring and consisting of β-D-glucose and eremosamine, as well as eremosamine attached to the 6 amino acid residue [G.F. Gauze et al. Antibiotics and chemotherapy, 1989, 5, 348].

Eremomycin in experiments in vitro and in vivo is 3-5 times more active than vancomycin in relation to most clinically significant gram-positive bacteria, including MRSA, and also has less pronounced side effects [I.V. Malkova. Antibiot. Chemother., 1989, 34, 52]. Semi-synthetic derivatives of eremomycin also have advantages in biological properties over similar derivatives of vancomycin [K.R. Maples et al. J. Med. Chem., 2007, 50, 3681]. Also, for a number of eremomycin derivatives, lower allergenicity was previously noted compared to eremomycin [A.Y. Pavlov et al. J. Antibiotics, 1996, 49, 194; RF patent No. 2641912]. All of the above makes eremomycin a promising base for the creation of highly effective semi-synthetic antibiotics.

Disclosure of invention

The aim of the present invention is the creation of more effective antibacterial agents based on new semi-synthetic eremomycin amides, in particular, on the basis of eremomycin picolylamides and their quaternized derivatives. The found new modification of eremomycin allows to improve the pharmacological properties of eremomycin and, in particular, to increase activity against pathogen strains that are sensitive and resistant to glycopeptides. The present invention includes compounds of Formula 3, their pharmacologically acceptable salts, suitable for the treatment of severe antibiotic-resistant bacterial infections, especially those caused by MRSA, GISA (Glycopeptide-Intermediate S. aureus) and VRE (Vancomycin-resistant enterococci) strains.

where Py is independently pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, and also their quaternized NC _10-20 alkyl derivatives; n = 1-3.

Compounds of formula 3 can be prepared by condensation of eremomycin 2 or its salts and the corresponding picolylamine or its quaternized derivative using a base condensing agent or salts necessary to maintain the optimum pH in a suitable inert aprotic solvent (Scheme 1).

Scheme 1

A comparative study of the antibacterial properties of new eremomycin derivatives of formula 3 and the vancomycin clinic used in the control strain of S. aureus, as well as sensitive and resistant enterococcal strains, showed that a new modification of the eremomycin carboxyl group can significantly increase its activity against glycopeptides resistant strains while maintaining activity against sensitive strains.

Unless otherwise specified, the terms used in the description of the application and the claims have the meanings indicated below.

“Alkyl” means, unless otherwise indicated, a monovalent straight or branched chain saturated hydrocarbon radical comprising only carbon and hydrogen atoms and containing from 10 to 20 carbon atoms.

"Solvates" means solvated forms containing a stoichiometric or non-stoichiometric amount of solvent. Some compounds are able to retain a fixed number of solvent molecules in the crystal lattice, forming a solvate. Hydrates are formed if water is used as the solvent, and alcoholates are formed if the solvent is alcohol.

"Pharmaceutically acceptable salts" of compounds means salts that are pharmaceutically acceptable and possess the necessary pharmacological activity of the parent compound. Such salts include acid addition salts of inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and the like, or organic acids such as acetic acid, benzoic acid, citric acid, fumaric acid, glutamic acid acid, glycolic acid, lactic acid, maleic acid, malic acid, methanesulfonic acid, propionic acid, salicylic acid, succinic acid, tartaric acid, toluenesulfonic acid, and the like. Pharmaceutically acceptable salts are intended to include solvates or crystalline forms (polymorphic formations) of said acid addition salt. Preferred pharmaceutically acceptable salts are those of hydrochloric acid, sulfuric acid, methanesulfonic acid, acetic acid, maleic acid, phosphoric acid, tartaric acid, citric acid, sodium, potassium, calcium, zinc and magnesium salts.

The initial eremomycin (2) was developed at a pilot plant in the NIINA named after G.F. Gauze RuBOP, triethylamine, dimethyl sulfoxide, picolylamines, alkyl halides, solvents and other reagents are commercial chemical compounds supplied by companies such as Aldrich Chemical Co.

Examples showing the possibility of obtaining new derivatives of eremomycin corresponding to formula 3, which are the subject of the present invention, as well as their physico-chemical properties, antibacterial activity and in vivo efficacy described below, are given only to illustrate the present invention, and not to limit the scope of claims.

Example 1. N- (Pyridin-4-ylmethyl) amide of eremomycin (3-1)

With gentle heating and vigorous stirring, eremomycin sulfate 2 (0.5 g, 0.29 mmol) and ethyldiisopropylamine (0.08 ml, 4.2 mmol) are dissolved in anhydrous dimethyl sulfoxide (12.5 ml). To the resulting solution were added 4-picolylamine (0.15 g, 1.3 mmol) and RuBOP (0.19 g, 0.33 mmol). After 10 minutes, 4-picolylamine (0.045 g, 0.4 mmol) and RuBOP (0.06 g, 0.12 mmol) were additionally added to the mixture with stirring and held for 30 minutes. Isopropanol (8.5 ml), acetone (25 ml) and diethyl ether (15 ml) were added to the stirred solution. The precipitate formed is filtered off, washed with acetone (3 × 10 ml), petroleum ether (10 ml) and dried in vacuo. The precipitate was dissolved in water (2 ml), isopropanol (3-4 ml) was added with stirring until the solution became cloudy, and the product was re-precipitated with a mixture of acetone (25 ml) and diethyl ether (15 ml). The precipitate formed is filtered off, washed with acetone (3 × 10 ml), petroleum ether (10 ml) and dried in vacuo. Technical eremomycin picolylamide is obtained in 89% yield as a cream powder.

The technical product is dissolved in water (40-60 ml), and the antibiotic is adsorbed on the ion exchange column on the Dowex 50Wx2 resin (12.5 ml) in the NH ₄ ⁺ form. The resin is washed with water (60-80 ml), and the product is stripped with an aqueous solution (0.25%) of ammonia. Fractions were monitored by TLC (Silicagel 60 F254, Merck, eluent ethyl acetate-n-propanol-25% ammonia, 3: 3: 4). The fractions containing the product are combined, concentrated in vacuo to a volume of ~ 5 ml. The solution is acidified with thorough stirring with an aqueous solution of hydrochloric acid (1%) to a pH of ~ 5, evaporated to a volume of 1-2 ml, after which the product is precipitated with acetone (50 ml). The precipitate formed is filtered off, washed with acetone (2 × 10 ml), diethyl ether (2 × 10 ml), petroleum ether (8 ml) and dried in vacuum. 0.31 g (64.6%) of eremomycin amide N- (pyridin-4-ylmethyl) amide hydrochloride (3-1) is obtained in the form of a cream powder. HRMS (ESI) calcd for C ₇₉ H ₉₆ ClN ₁₂ O ₂₅ [M + H] ⁺ : 1647.6293; found 1647.6290. HPLC (Kromasil-100-5-μm C-18 column 4.6 × 250 mm, LW = 260 nm, eluent: A — H ₃ PO ₄ (0.6%) pH = 7.8, B — MeCN; gradient B, 8 → 70% (40 min): Rt = 25.4 min, purity 95.9%. Found,%: С 51.19, Н 6.11, N 8.85; calculated for C ₇₉ H ₉₆ ClN ₁₂ O ₂₅ * 3HCl * 5Н ₂ О,%: С 51.36, Н 5.89, N 9.10.

Example 2. N- (Pyridin-3-ylmethyl) amide of eremomycin (3-2)

Obtained by a method similar to that described in example 1 from eremomycin (2) and 3-picolylamine. The output of the hydrochloride of N- (pyridin-3-ylmethyl) amide eremomycin (3-2) -0.24 g (51%), in the form of a cream powder. HPLC (Kromasil-100-5-μm C-18 column 4.6 × 250 mm, LW = 260 nm, eluent: A - H ₃ PO ₄ (0.6%) pH = 7.8, B - MeCN; gradient B 8 → 70% ( 40 min): Rt = 26.7 min, purity 97.6%. HRSM (ESI) calcd for C ₈₃ H ₁₀₀ ClN ₁₂ O ₂₅ [M + H] ⁺ : 1647.6293; found 1647.6298.

Example 3. N- (Pyridin-2-ylmethyl) amide of eremomycin (3-3)

Receive by the method similar to that described in example 1 from eremomycin (2) and 2-picolylamine. The yield of N-pyridin-2-ylmethyl) amide of eremomycin hydrochloride (3-3) is 0.30 g (62.5%) as a cream powder. HPLC (Kromasil-100-5-μm C-18 column 4.6 × 250 mm, LW = 260 nm, eluent: A - H ₃ PO ₄ (0.6%) pH = 7.8, V - MeCN; gradient B 8 → 70% ( 40 min): Rt = 25.8 min, purity 95.5%. HRSM (ESI) calcd for C ₈₁ H ₉₉ ClN ₁₁ O ₂₅ [M + H] ⁺ : 1647.6295; found 1647.6293.

Example 4. N-2- (2-pyridyl) ethylamide eremomycin (3-4)

Obtained by a procedure similar to that described in example 1 from eremomycin (2) and 2- (2-pyridyl) ethylamine. The output of N-2- (2-pyridyl) eremomycin ethylamide hydrochloride (3-4) is 0.25 g (47.4%) as a cream powder. HPLC (Kromasil-100-5-μm C-18 column 4.6 × 250 mm, LW = 260 nm, eluent: A - H ₃ PO ₄ (0.6%) pH = 7.8, B - MeCN; gradient B 8 → 70% ( 40 min): Rt = 26.8 min, purity 95.5%. HRSM (ESI) calculated for C ₈₀ H ₉₈ ClN ₁₂ O ₂₅ [M + H] +: 1661.6479; found 1661.6468. Found,%: C 51.80, H 6.05, N 8.91; calculated for C ₈₀ H ₉₈ ClN ₁₂ O ₂ * 3HCl * 4H ₂ O,%: C 52.12, H 5.90, N 9.12.

Example 5. N - ((1-Decylpyridin-1-yum-4-yl) methyl) eremomycin amide (3-5)

With gentle heating and vigorous stirring, dissolve eremomycin sulfate (2, 0.5 g, 0.29 mmol) and triethylamine (0.2 ml, 1.45 mmol) in dimethyl sulfoxide (12.5 ml). To the resulting solution, 4- (aminomethyl) -1-decylpyridium chloride hydrochloride (0.42 g, 1.35 mmol), RuBOP (0.258 g, 0.5 mmol) are added with stirring and incubated for 1 h. Isopropanol (8.5 ml), acetone are added to the stirred reaction mixture. (25 ml) and diethyl ether (15 ml). The precipitate formed is filtered off, washed with acetone (3 × 10 ml), petroleum ether (10 ml) and dried in vacuo. The precipitate was dissolved in water (2.5 ml), isopropanol (3-4 ml) was added with stirring until the solution became cloudy, and the product was reprecipitated with acetone (25 ml) and diethyl ether (15 ml). The precipitate formed is filtered off, washed with acetone (3 × 10 ml), petroleum ether (10 ml) and dried in vacuo. Receive technical N-decylpicolylamide eremomycin (3-5) with a yield of 83% in the form of a cream powder.

The technical product is dissolved in 3-5 ml of water and purified by reverse phase chromatography (eluent water - ethanol). The fractions containing the product are combined, concentrated in vacuo to a volume of 2-3 ml, and the product is precipitated with acetone (50 ml). The precipitate formed is filtered off, washed with acetone (2 × 8 ml), diethyl ether (2 × 8 ml), petroleum ether (8 ml) and dried in vacuum. 0.23 g (40%) of eremomycin N-decyl-4-picolylamide hydrochloride (3-5) is obtained in the form of an almost colorless powder. HPLC (Kromasil-100-5-μm C-18 column 4.6 × 250 mm, LW = 260 nm, eluent: A — H ₃ PO ₄ (0.01 M) pH = 2.6, B — MeCN; gradient B 10 → 90% ( 45 min): Rt = 24.2 min, purity 95.6%. HRSM (ESI) calcd for C ₈₉ H ₉₆ ClN ₁₂ O ₂₅ [M] ⁺ : 1787.7858; found 1787.7856.

Example 6. N - ((1-Dodecylpyridin-1-ium-4-yl) methyl) eremomycin amide (3-6)

Obtained by a method similar to that described in example 5 from eremomycin (2) and 4- (aminomethyl) -1-dodecylpyridinium chloride hydrochloride. The yield of eremomycin 1-dodecyl-4-picolylamide hydrochloride (3-6) is 0.29 g (56%) as a cream powder. HPLC (Kromasil-100-5-μm C-18 column 4.6 × 250 mm, LW = 260 nm, eluent: A — H ₃ PO ₄ (0.01 M) pH = 2.6, B — MeCN; gradient B 10 → 90% ( 53 min): Rt = 27.0 min, purity 95.3%. HRSM (ESI) calculated for C ₉₁ H ₁₂₀ ClN ₁₂ O ₂₅ [M] ⁺ : 1815.8171, found 1815.8179.

Example 7. N - ((1-Tetradecylpyridin-1-yum-4-yl) methyl) eremomycin amide (3-7)

Prepared by a procedure similar to that described in example 5 from eremomycin (2) and 4- (aminomethyl) -1-tetradecylpyridinium chloride hydrochloride. The output of N-tetradecyl-4-picolylamide eremomycin hydrochloride (3-7) is 0.15 g (27%) as a cream powder. HPLC (Kromasil-100-5-μm C-18 column 4.6 × 250 mm, LW = 260 nm, eluent: A — H ₃ PO ₄ (0.01 M) pH = 2.6, B — MeCN; gradient B 10 → 90% ( 53 min): Rt = 28.1 min, purity 96.1%. HRMS (ESI) calcd for C ₉₁ H ₁₂₀ ClN ₁₂ O ₂₅ [M] ⁺ : 1815.8171, found 1815.8179.

Example 8. N - ((1-Hexadecylpyridin-1-yum-4-yl) methyl) eremomycin amide (3-8)

Obtained by a procedure similar to that described in example 5 from eremomycin (2) and 4- (aminomethyl) -1-hexadecylpyridinium chloride hydrochloride. The output of N-hexadecyl-4-picolylamide eremomycin hydrochloride (3-8) is 0.20 g (33%) as a white powder. HPLC (Kromasil-100-5-μm C-18 column 4.6 × 250 mm, LW = 260 nm, eluent: A — H ₃ PO ₄ (0.01 M) pH = 2.6, B — MeCN; gradient B 10 → 90% ( 53 min): Rt = 29.0 min, purity 95.2%. HRSM (ESI) calculated for C ₉₅ H ₁₂₈ ClN ₁₂ O ₂₅ [M] ⁺ : 1871.8797, found 1871.8791.

Example 9. N - ((1-Tetradecylpyridin-1-yum-3-yl) methyl) eremomycin amide (3-9)

Obtained by a procedure similar to that described in example 5 from eremomycin (2) and 3- (aminomethyl) -1-tetradecylpyridinium chloride hydrochloride. The output of N-tetradecyl-3-picolylamide eremomycin hydrochloride (3-9) is 0.27 g (50%) as a colorless powder. HPLC (Kromasil-100-5-μm C-18 column 4.6 × 250 mm, LW = 260 nm, eluent: A — H ₃ PO ₄ (0.01 M) pH = 2.6, B — MeCN; gradient B 10 90% (45 min): Rt = 27.8 min, purity 98.0%. HRSM (ESI) calcd for C ₉₃ H ₁₂₄ ClN ₁₂ O ₂₅ [M] ⁺ : 1843.8480, found 1843.8489.

Example 10. Antibacterial activity of derivatives 3-1-3-9

The antimicrobial activity of the semisynthetic eremomycin derivatives of the present invention was studied in comparison with vancomycin 1 on strains of Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, also a panel of clinical isolates, including vancomycin-sensitive strains of S. epidermidolit 533, S. epidermidol 533 . faecalis 6413 and glycopeptide resistant strains S. aureus 3797, S. aureus 3798, E. faecium 1, E. faecium 2, E. faecium 4, E. faecium 569, E. faecium 3567, E. faecalis 560, E. gallinarum 1308 obtained from the Museum of the Laboratory of Medical Microbiology of the State Scientific Center for Anti iotikam (GNTSA). The minimum inhibitory growth of microorganisms concentration (MIC) for the tested compounds was determined by the method of serial dilutions in broth, in accordance with the recommendations of CLSI.

The results obtained, presented in table 1, indicate that in most cases, in vitro activity, eremomycin amides 3-1 - 3-9, which are the subject of the present invention, superior to the comparison drug - vancomycin (1). Eremomycin pyridinylalkylamides 3-1 - 3-4 are 2-20 times more active than vancomycin (1) in relation to strains that are sensitive and / or resistant to glycopeptides (Table 1). Quaternization of the pyridine nucleus, although it leads to a decrease in the activity of lipoglycopeptides 3-5 - 3-9 in relation to sensitive strains and strains with intermediate resistance (compared with pyridinylalkylamides 3-1 - 3-4), however, these derivatives are much more active than vancomycin ( 1) in relation to strains of E. faecium 569, E. faecalis 560, E. gallinarum 1308 and E. faecium 3567 highly resistant to glycopeptides.

* nt - connection not tested

Example 11. Antibacterial efficacy in vivo

A comparative study of the efficacy of N - ((1-tetradecylpyridin-1-ium-4-yl) methyl) amide eremomycin (3-7, example 7) and vancomycin (1) was carried out on a model of mouse staphylococcal sepsis. In the experiment, female SHK colony mice weighing 20-22 g were used. As an infectious agent, Staphylococcus aureus (strain 10, clinical isolate), adapted for growth in vivo, by five-fold passage in mice was used to increase virulence. The lethal dose (LD ₁₀₀ ) of staphylococcus was initially determined for a given line of mice of a specific weight with the intravenous route of infection. Accounting for the death of mice was carried out daily for 10 days. The lethal dose (LD ₁₀₀ ) was 8 × 10 ⁸ CFU / mouse. Mice were seeded into cells of 10 animals each, infected with a lethal dose of Staphylococcus aureus, and the efficacy of the test drugs was determined by the ED ₅₀ value (i.e. doses at which 50% of the experimental animals survive) 30 minutes after infection, mice were injected intravenously with N- ((1-tetradecylpyridin-1-yum-4-yl) methyl) eremomycin amide (3-7) in doses of 0.25 to 3.5 mg / kg or vancomycin hydrochloride (1) in doses of 2.5 to 7.5 mg / kg. As a dose control, a group of untreated animals infected with a lethal dose of Staphylococcus aureus was present in the experiment. Determination of the ED _{50 of the} tested drugs was performed in one experiment with a single control using the Barenz method (frequency accumulation). The animals were observed for 14 days, daily death was taken into account. The experimental results are presented in table 2.

Based on experimental data on the survival of mice (table 2), the values of the efficacy indicators of the tested drugs (ED ₅₀ ) were calculated:

- N - ((1-tetradecylpyridin-1-yum-4-yl) methyl) eremomycin amide ED ₅₀ = 0.43 mg / kg;

- Vancomycin ED ₅₀ = 4.1 mg / kg.

The results obtained are presented in table 2, and the value of ED ₅₀ indicate that the in vivo effectiveness of N - ((1-tetradecylpyridin-1-ium-4-yl) methyl) eremomycin amide (3-7), which is the subject of the present invention significantly superior in effectiveness (ED ₅₀ ) to the comparison drug vancomycin (1).

Claims (6)

1. A derivative of eremomycin containing a substituted or unsubstituted at the nitrogen atom of the heterocycle aminoalkylpyridine residue linked to eremomycin via an amide bond with a C-terminal amino acid, as well as its pharmacologically acceptable salts, hydrates, corresponding to the formula:

where Py - independently means pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, as well as their quaternized N — C _10-20 alkyl derivatives; n = 1-3. 2. A method for the treatment of bacterial infectious diseases caused by strains of gram-positive pathogens, comprising parenteral administration to a patient in need of a therapeutically effective amount of a substance according to claim 1. 3. A method of treating bacterial infections according to claim 2, wherein the pathogen is selected from the group of Staphylococcus spp. and Enterococcus spp. 4. A method of treating bacterial infections according to claim 2, wherein the pathogen has resistance to other drugs.