RU2757741C1 - Ciprofloxacin derivative with antibacterial activity against antibiotic-resistant strains of microorganisms - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a ciprofloxacin derivative of general formula (I) with antibacterial activity, which can be used in veterinary medicine for the treatment of bacterial infections caused by gram-positive and gram-negative bacteria, including those resistant to ciprofloxacin.

EFFECT: improvement of ciprofloxacin derivative.

(I)

1 cl, 2 dwg, 2 tbl, 4 ex

Description

The invention relates to the chemistry of organic heterocyclic compounds, namely to a new derivative of ciprofloxacin of general formula (I)

possessing antibacterial activity against antibiotic-resistant strains of microorganisms. The claimed derivative can be used in medicine and veterinary medicine.

Currently, antibiotics are widely used in agriculture. This is due to the fact that, firstly, in industrial production, a large number of animals are kept on relatively small areas, which predetermines the spread of various infections. Secondly, the raising and keeping of animals provides for drug treatment. Third, large-scale antibiotic prophylaxis is also needed when transporting animals to relieve stress. The use of antibiotics as an additive to feed to increase the rate of weight gain in animals has also become widespread.

The widespread introduction of antibiotics into medical and veterinary practice leads to an increase in the number of infectious diseases caused by antibiotic-resistant bacteria throughout the world. In recent years, gram-positive microflora, including bacteria of the genus Staphylococcus, again occupies a dominant place in the etiology of infectious diseases. Microorganisms with multidrug-resistant organisms (MDRO), such as methicillin-resistant Staphylococcus aureus, vancomycin-insensitive enterococci or third-generation cephalosporin-resistant Gram-negative bacteria, may become more common causes of death than cancer in the coming decades [Zipperer ., et al., Human commensals producing a novel antibiotic impair pathogen colonization. // Nature. 2016, V. 535, P. 511-516].

One of the most effective antibacterial drugs used in modern practice to suppress methicillin-resistant strains of Staphylococcus aureus is fluoroquinolone drugs, in particular ciprofloxacin. According to its chemical structure, ciprofloxacin belongs to the derivatives of the fluoroquinolone series. However, for this antibiotic, there is a significant increase in the resistance of microorganisms. [Norrby S.R. Side-effects of quinolones: comparisons between quinolones and other antibiotics // Eur. J. Clin. Microbiol. Infect. Dis. - 1991 - V. 10 - P. 378-383].

At the date of filing the proposed technical solution, it is known that most staphylococci resistant to methicillin are resistant to ciprofloxacin [Lowy F.D. Antimicrobial resistance: the example of Staphylococcus aureus // J. Clin. Invest. - 2003 -V. 111. - P. 1265-1273].

The study of the relationship of structure-biological activity in a series of ciprofloxacin derivatives revealed that the nature of the substituent at the C-7 atom has the greatest influence on their biological effect [Emamia S., Shafiee A., Foroumadi A. Quinolones: Recent Structural and Clinical Developments // Iran. J. Pharm. Res. - 2005. - V. - P. 123-136; application WO 2011034971 Al, publ. March 24, 2011. Modified fluoroquinolone compounds and methods of using the samee]. Most often, such substituents are five- and six-membered nitrogen-containing heterocycles, such as piperazine, pyrimidine, 1,2,3-triazole, pyrrolidine and their substituted derivatives. In the source [N. German, et al., Synthesis and evaluation of fluoroquinolone derivatives as substrate-based inhibitors of bacterial efflux pumps // Eur. J. Med. Chem. - 2008 - V. 43. - P. 2453-2463] describes ciprofloxacin derivatives modified at the C-7 atom of the piperazine ring with fragments of various peptides and substituted diarylureas. These compounds showed slightly less antibacterial activity compared to ciprofloxacin.

One of the approaches to the creation of antibacterial drugs with a wide spectrum of action and with a reduced likelihood of the emergence of resistant strains of microorganisms is to obtain hybrid structures that combine molecules with different mechanisms of action.

Thus, the modification of ciprofloxacin at the C-7 atom of the piperazine ring with the aminoglycoside antibiotic neomycin through a bridge containing a 1,2,3-triazole fragment led to the production of hybrid structures exhibiting high antibacterial activity against strains of gram-negative and gram-positive bacteria [Pokrovskaya V., et al., Synthesis, and Evaluation of Novel Fluoroquinolone-Aminoglycoside Hybrid Antibiotics // J. Med. Chem. - 2009 - V. 5. - P. 2243-2254].

The disadvantage of these compounds is a complex synthesis, which makes the use of these compounds in veterinary medicine unprofitable. In addition, these compounds are not effective against microorganisms resistant to both antibiotics used.

An alternative to antibiotics when creating hybrid structures can be cationic amphiphiles - molecules with one or more positively charged groups and lipophilic fragments [Molchanova N., et al., Advances in development of antimicrobial peptidomimetics as potential drugs // Molecules. 2017. Vol. 22, P. 1430]. These compounds are synthetic analogs of cationic natural antimicrobial peptides and can cause disruption of the transmembrane potential, leakage of cytoplasmic contents and, ultimately, cell death [Ghosh C, Haldar J. Membrane-active small molecules: Designs inspired by antimicrobial peptides // ChemMedChem. 2015. Vol. 10, P. 1606-1624].

So, in the works [Zimmermann L., et al., New broad-spectrum antibacterial amphiphilic aminoglycosides active against resistant bacteria: From neamine derivatives to smaller neosamine analogues // J. Med. Chem. 2016. Vol. 59, P. 9350-9369; Zimmermann L., et al. Broad-spectrum antibacterial amphiphilic aminoglycosides: A new focus on the structure of the lipophilic groups extends the series of active dialkyl neamines // Eur. J. Med. Chem. 2018. Vol. 157, P. 1512-1525] describes the synthesis and antibacterial activity of a number of aminoglycazide antibiotics (neomycin, kanamycin, amiksin), in which the cationic part of the antibiotic is simultaneously the cationic fragment of the amphiphile.

The closest structural analogue to the claimed compound, the prototype, is a compound with a hybrid structure, including the antibiotic linezolid and cationic amphiphile based on quaternary ammonium salts, which is obtained from deacylated linezolid by acylation with chloroacetic acid chloride followed by alkylation with tritic amines. [Bai P.Y. et al., Synthesis and antibacterial bioactivities of cationic deacetyl linezolid amphiphiles // Eur J Med Chem. 2018. Vol. 155, P. 925-945].

The disadvantages of the known structural analogs are significantly lower activity against gram-negative microorganisms and incomplete restoration of the activity of the conjugate against strains of microorganisms that have developed resistance to linezalid.

The objective of the invention is to obtain a new derivative of ciprofloxacin, which has antibacterial activity against antibiotic-resistant strains of microorganisms.

The technical result of the claimed technical solution is to expand the spectrum of antibacterial activity of the new derivative of ciprofloxacin, as well as to expand the range of derivatives of ciprofloxacin, which have antibacterial activity against antibiotic-resistant strains of microorganisms.

The task is achieved by the proposed compound, which is a conjugate of ciprofloxacin with 1 - [(4-dodecyl-1,4-diazoniabicyclo [2.2.2] octan) -1-yl)] - 5 - [(4-pentyl-1,4- diazoniabicyclo [2.2.2] octan) -1-yl)] pentane tetrabromide with the structural formula (I).

FIG. 1 shows a general scheme for the synthesis of the claimed ciprofloxacin derivative.

The proposed compound is prepared as follows: the starting reagent bromide 1-tetradecyl-4-aza-1-azoniabicyclo [2.2.2] octane (1) is treated in a dilute solution with 1,5-dibromopentane to form intermediate (2). In parallel, ciprofloxacin (3) acylated with 6-bromocaproic acid at the nitrogen atom of the piperazine ring is treated with an excess of 1,4-diazabicyclo [2.2.2] octane. The resulting intermediate (4) is reacted with the previously prepared bis-quaternary salt (2) to form the target compound (5)

The invention is illustrated by the following examples.

Example 1. Obtaining a new derivative of ciprofloxacin.

The starting reagent bromide 1-tetradecyl-4-aza-1-azoniabicyclo [2.2.2] octane (1) is dissolved in acetonitrile. The resulting solution is added dropwise with stirring over 5 hours to a solution of 1,5-dibromopentane in acetonitrile at 44 ° C with a final ratio of the components of 1: 1. Without cooling the reaction mixture, the precipitate that formed was filtered off and dried in a vacuum. The yield of compound (2) is 57.4% -59.2%.

The original nitrogen-acylated ciprofloxacin (3) is dissolved in methanol. The resulting solution was added to a solution of 1,4-diazabicyclo [2.2.2] octane in acetonitrile (reagent ratio 1: 2) and the reaction mixture was stirred at room temperature for 48 h. The precipitate that formed was filtered off and dried in vacuum. The yield of compound (4) is 86.5% -87.3%. A solution of compounds (2) and (4) in methanol, taken in a 1: 1 ratio, is refluxed for 6 hours. The reaction mixture is cooled and an equivalent volume of acetonitrile is added to the reaction mixture. The precipitate that formed is recrystallized twice from a mixture of methanol with acetonitrile (1: 1). The yield of the target compound (5) is 24.3-25.6%.

1H NMR spectrum, δ, ppm (J, Hz): 0.93 (br. T, 3H, CH ₃ (CH ₂ ) ₁₃ N +, J 6.6), 1.14-1.28 (m, 6H, H-5 _cip , H-6 _cip , CH ₂ CH ₂ CH ₂ CO-,); 1.32-1.45 (m, 26H, CH ₃ (CH ₂ ) ₁₁ CH ₂ CH ₂ N +, CH ₂ CH ₂ CH ₂ CH ₂ CO-); 1.32 (m, 2H, + NCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N +); 1.88 (m, 2H, CH ₃ (CH ₂ ) ₁₁ CH ₂ CH ₂ N ++); 1.98 (m, 4H, + NCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N +); 2.38 (t, 2H, J = 7.3, —CH ₂ CO—); 3.12 (m, 4H, H-8 _cip , H-10 _cip ,), 3.62 (m, 6H, CH ₃ (CH ₂ ) ₁₂ CH ₂ N +. H-7 _cip , H-9 _cip ); 3.75 (m, 5H, + NCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N +. H-4 _cip ); 4.07-4.12 (m, 24H, + N (CH ₂ CH ₂ ) 3N +); 7.53 (d, J = 7.4Hz, 1H, H-2 _cip ); 7.86 (d, J = 13.0Hz, 1H, H-3 _cip ); 8.61 (s, 1H, Hl _cip ); 15.16 (s, 1H, COOH)

ESI-MS m / z: [M + H] ⁺ 1240.53 (calcd for C ₅₄ H ₉₁ Br ₄ FN ₇ O ₄ ⁺ : 1240.38).

Example 2. Evaluation of the antibacterial activity of compound (5) on pathogenic test strains: Salmonella typhimurium, Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa.

Solutions of compound (5) were introduced into the nutrient medium simultaneously with the inoculum concentration of the test strain, incubated stationary in test tubes (1 ml each) at 37 ° C for 24 hours. The results were taken into account by the method of biological titration, determining the number of microorganisms (titer) before and after incubation (in the colony forming units per 1 ml, CFU / ml). As a control, we used a medium with a test strain without adding a test preparation. Number of repetitions 4.

Bactericidal activity (BA) was assessed conditionally:

+ the presence of bactericidal activity (a sharp decrease in the number of microorganisms);

+ \ - the presence of bacteriostatic activity (no increase or a slight increase in the number of microorganisms);

- lack of activity (an increase in the number of microorganisms, comparable to the control).

Table 1 shows the average indicators of the activity of various concentrations of the test drug on pathogenic strains of bacteria, where C is the concentration of compound (5), and the value <10 ² shows that not a single colony of pathogenic microorganisms has grown on Petri dishes.

Table 1 shows that the test compound (5) has a pronounced antimicrobial effect against pathogenic strains of both gram-negative and gram-positive microorganisms. At a drug concentration of 100 μg / ml, all the studied microorganisms are completely inactivated. When the concentration is reduced to 10 μg / ml, the drug completely inactivates the bacteria strains Salmonella typhimurium, Staphylococcus aureus, Proteus vulgaris and reduces the titer of Pseudomonas aeruginosa by 10 thousand times in relation to the control. The test compound is most active against Staphylococcus aureus.

Example 3. Evaluation of the antibacterial activity of compound (5) on pathogenic strains of Staphylococcus aureus resistant to the action of ciprofloxacin.

Clinical isolates of methyl-resistant strains of Staphylococcus aureus microorganisms were used for testing: EMTK 675, EMTK 707, EMTK 1695, EMTK 1732, EMTK 1733, EMTK 1850. As a comparison drug, we used the dosage form of the antibiotic ciprofloxacin (solution for intravenous administration, C = 2 mg / ml ).

For work, a suspension of overnight broth cultures grown on standard nutrient media was used. The number of microorganisms (titer) in the suspension was determined by the optical density of the cell suspension (OD) at a wavelength of 595 nm.

To assess the antibacterial effect of the preparations, the cell cultures were co-incubated with the test compounds in 96-well culture plates. Overnight broth cultures were resuspended in Mueller-Hinton's medium, bringing the number of microorganisms to a seed dose of 5 × 10 ⁵ CFU / ml. Cell suspension and test preparations were simultaneously introduced into the wells to the final concentration of the latter 8, 4, 2, 1, 0.5 and 0.25 μg / ml. The number of repetitions is 4. Incubated at 37 ° C for 24 hours, OD measurement was performed after 1, 2, 4, 8, 12, 24 hours on a multi-reader at a wavelength of 595 nm. The presence of bactericidal activity was assessed by the change in OD in comparison with the control.

The test results are summarized in Table 2, where C is the concentration of compound (5); "+" - active growth of microorganisms, comparable to the control (water), "-" - no cell growth (bactericidal effect), "+/-" - weak cell growth (bacteriostatic effect).

From the presented data, it can be seen that the test compound completely inhibits the growth of all strains at a concentration of 1 μg / ml and above. With a decrease in concentration to 0.5 μg / ml, a significant delay in the growth of microorganisms occurs (bacteriostatic effect).

The original antibiotic is much less effective against these microorganisms. Ciprofloxacin exhibits only bacteriostatic properties in relation to 3 strains out of 6 at the maximum of the studied concentrations (8 μg / ml), does not act at all on 2 strains at the tested concentrations, and only for strain 1732 exhibits bactericidal activity at a concentration of 2 μg / ml and bacteriostatic activity at a concentration of 1 μg / ml.

Thus, the new derivative of ciprofloxacin (compound 5) has a pronounced antibacterial effect against antibiotic-resistant strains of microorganisms.

Example 4. Comparison of the kinetics of inhibition of Staphylococcus aureus with compound (5) and ciprofloxacin.

Investigated the antibacterial activity of the claimed compound (5), depending on the time of exposure for the example of suppression of Staphylococcus aureus ATCC 25923. Measurements were carried out analogously to example 3. The results are shown in Fig. 2.

As can be seen from the presented results, compound (5) begins to show activity almost immediately after administration. Already after 1 hour, the concentration of viable microorganisms decreases by more than ten times. A similar effect for ciprofloxacin is observed closer to 8 hours. While after 8 hours for compound (5) 100% death of microorganisms is observed. Since cationic amphiphiles are capable of disrupting bacterial cell membranes, it can be assumed that the significantly higher rate of death of Staphylococcus aureus under the influence of compound (5) may be due to the more rapid penetration of the conjugate into microorganisms.

Thus, the claimed new derivative of ciprofloxacin has the following advantages over the known ones:

- has a pronounced antibacterial effect against antibiotic-resistant strains of microorganisms;

- has a higher rate of inactivation of microorganisms, compared to the base antibiotic.

Claims (3)

Ciprofloxacin derivative of general formula (I)

, possessing antibacterial activity against antibiotic-resistant strains of microorganisms.

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