EA037888B1 - (2-hydroxy-3-allyl-5-isodecylbenzyl)diethylammonium chloride as a specific bactericide against dangerous pathogen staphylococcus aureus - Google Patents

Abstract

As a specific bactericide against dangerous pathogen Staphylococcus aureus, a new organic compound (2-hydroxy-3-allyl-5-isodecylbenzyl)diethylammonium chloride of the following formula is proposed to be used:Using the new compound at a concentration of 62.5 g/ml has a specific effect on the described pathogenic bacteria Staphylococcus aureus by inhibiting their reproduction, without suppressing the growth of bacteria of the normal intestinal biota, which can be used in medicine.

Description

The invention relates to the specific suppression of the growth of a dangerous pathogen Staphylococcus aureus using a special chemical. It can find application in the chemical and pharmaceutical industry.

One of the urgent problems of modern biological and medical science is the development of effective methods for suppressing the growth and destruction of numerous dangerous pathogenic bacteria that are a threat to humans and the environment. Among the methods most frequently used in practice to combat them is the use of antibiotics, including special chemicals from various classes of organic compounds containing nitrogen, oxygen, chlorine, and other elements in their structures [1]. As a result of prolonged use of these substances, pathogenic bacteria acquire resistance to them. Therefore, the search for new antibacterial substances is relevant at the present time.

In recent years, in world practice, special attention is paid to the use of water-soluble ammonium salts of various structures and compositions based on them as bactericides [2, 3].

Described is a disinfectant that has both bactericidal, virucidal and fungicidal properties, obtained on the basis of poly-N, N-dimethylammonium chloride [4].

However, it should be noted that research on the creation of new, even more effective bactericides is constantly growing, especially if we take into account the possibility of their use at very low concentrations, at which complete destruction of dangerous pathogenic bacteria is observed.

Considering the above, the development of bactericides based on substituted phenols is of great interest.

We have developed an effective bactericide of phenolic structure, which is (2-hydroxy-3-allyl-5-isodecylbenzyl) diethylammonium chloride of the following structure:

The chemical compound proposed as a bactericide is not described in the scientific and technical literature and was synthesized by us for the first time. Its synthesis is carried out as follows.

1. Alkylation of phenol with decene-1 in the presence of a sulfonic cationite catalyst (KU-23; KU2) (obtaining 4-isodecylphenol)

Alkylation conditions:

temperature 80 ° C;

the ratio of phenol: decene-1 2: 1 mol;

the amount of catalyst is 10% (for phenol);

the reaction time is 4 hours.

2. Condensation of 4-isooctylphenol with allyl bromide (in the presence of potash) at a temperature of 5055 ° C and subsequent Kleisen rearrangement of the resulting ether under microwave irradiation

3. Condensation of 2-allyl-4-isodecylphenol with formaldehyde and diethylamine (at a ratio of 1: 1: 1 mol), temperature 70-75 ° C and duration 4-5 hours. Solvent - benzene (2-3: 1 by weight ).

4. Treatment of 2-allyl-4-isodecyl-6-diethylaminomethylphenol with hydrogen chloride (at room temperature for 4-5 hours).

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The structure of the proposed compound is proved by the data of NMR spectroscopy:

1H spectrum (ppm): 0.98 t (3H, CH3); 1.2 t (3H, CH3); 1.3 d (3H, CH3); 1.4 m (8H, 4CH2); 1.6 m (2H, CH2); 3.1 m (1H, CH); 3.2 m (2H, CH ₂ Ar); 4.2 s (2H, CH ₂ Ar); 4.9 d-d (2H, = CH ₂ ); 5.9 m (1H, = CH); 6.8 m (1H, Ar); 7.2 m (1H, Ar); 10.1 s (1H, OH).

¹³ C spectrum: 9.7 (CH3); 14.7 (CH3); 19.2 (CH3); 21.3 (CH ₂ ); 24.7 (CH2); 26.2 (CH); 27.3 (CH ₂ ); 31.4 (CH2); 37.4 (CH); 47.3 (CH ₂ ); 73.4 (CH ₂ ); 117.4 (= CH); 121.3 (CH); 122.4 (C); 129.7 (CH); 130.4 (= CH); 137.4 (C); 139.2 (c); 158.4 (C).

To identify the bactericidal properties of the new compound, comprehensive laboratory studies were carried out using the following pathogenic bacteria: Acinetobacter baumanii (BDU 32), Escherichia coli (BDU 12), Klebsiella pneumoniac (BDU 44), Pseudomonas aeruginosa (BDU 49) and gram-negative Stureus DU 23).

Experiments to identify the antibacterial properties of the proposed compound were carried out by the method of serial dilutions and determined the minimum inhibitory concentration [5].

For the growth of bacteria used a nutrient medium Muller Hinton of the following composition (g / l): starch - 1.5; casein hydrolyzate - 17.5; meat extract - 2.0 and agar - 17.0; pH 7.3.

To prepare samples with different concentrations of the substance, an initial solution of 1000 μg / ml (or 1 mg / ml) was first prepared. Samples with the following concentrations were obtained by serial dilution: 500 μg / ml; 250 μg / ml; 125 μg / ml; 62.5 μg / ml and 31.25 μg / ml. Media containing the above concentrations of the test substance were inoculated with 100 μl of the bacterial suspension (containing 10 ⁴ CFU / ml cells) and incubated at 37 ° C for 48 h.

The results of tests to identify the minimum inhibitory concentration of the compound in relation to various bacteria are given in the table.

The minimum inhibitory concentration of (2-hydroxy-3-allyl-5-isodecylbenzyl) diethylammonium chloride against various bacteria

No. Bacteria Minimum inhibitory concentration, μg / ml views Strains sickness one Acinetobacter baumanii BDU 32 Pathogen 500 2 Escherichia coli BDU 12 Conditionally pathogen 1000 3 Klebsiella pneumoniae BDU 44 Conditionally pathogen 2000 4 Pseudomonas aeroginosa BDU 49 Conditionally pathogen 2000 five Staphylococcus aureus BDU 32 Pathogen 62.5

As can be seen from the data in the table, the bacteria Staphylococcus aureus are highly sensitive to the compound used. The minimum concentration of the inhibitory activity of the test substance in relation to this bacterium is only 62.5 μg / ml. To suppress the growth of other types of test bacteria used, it is necessary to use the substance in 8-32 times higher concentrations.

The lysis zone of Staphylococcus aureus BDU32 under the action of the test substance was determined by diffusion in agar [5]. For the growth of bacteria, a nutrient medium of the above composition was used with the addition of agar (17.0 g / l). The nutrient medium was poured into Petri dishes, 20 ml each, and after solidification, bacteria were inoculated over the entire surface of the medium. Then, wells with a diameter of 6 mm were made, into which the solution of the test compound was injected. All experiments in 5 replicates were incubated at 37 ° C for 24 hours. The lysis zone around the well was measured with a ruler, which was 17x19 mm (figure). Distilled water was used as a control.

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Drawing. Lysis zone of Staphylococcus aureus bacteria created by 2-allyl-4-isodecyl-6-diethylaminomethylphenol

The bacteria Escherichia coli and Klebsiella pneumoniae are known to be normal human intestinal microbiota. Many antibacterial substances, along with harmful bacteria, inhibit the growth and bacteria of normal intestinal biota, which often leads to dysbiosis [6].

The results of the experiments show that the compound proposed as a bactericide at a concentration of 62.5 μg / ml has a specific effect on the dangerous pathogenic bacterium Staphylococcus aureus, inhibiting its reproduction.

Therefore, (2-hydroxy-3-allyl-5-isodecylbenzyl) diethylammonium chloride as an antibacterial substance has the following advantages:

a high antibacterial effect is achieved by a very low concentration of the substance (62.5 μg / ml);

low concentration of the substance has a specific antimicrobial effect on the pathogen Staphylococcus aureus, without inhibiting the growth of bacteria in the normal intestinal biota;

the high bactericidal effect of the minimum concentration of the proposed substance can be explained by the presence in its structure, along with phenolic hydroxyl and allyl group, also of a quaternary ammonium group and Cl ^- anion.

Let us consider on specific examples both the method of synthesis of the proposed compound and its antimicrobial properties in relation to various types of bacteria (under the same test conditions).

Example 1.

a) Alkylation of phenol with n-decene-1.

In a three-necked reaction flask equipped with a thermometer, stirrer and reflux condenser, phenol 18.8 g (0.2 mol), catalyst KU-23 1.88 g (10% based on phenol) are loaded. The mass is heated until the phenol melts (T _nl. = 43 ° C), after which 14.0 g (0.1 mol) of n-decene-1 are fed from the dropping funnel with stirring to the flask. Next, the reaction mixture is stirred for 4 hours. At the end of the process, the alkylate is separated from the catalyst and subjected to distillation in a rotary evaporator (in order to separate the unreacted part of n-decene-1. The yield of 4-isodecylphenol is 14.6 g (62% of theory). .).

b) Obtaining 2-allyl-4-isodecylphenol.

A three-necked flask is charged with 23.4 g (0.1 mol) of 4-isodecylphenol, 13.8 g (0.1 mol) of potash, 12.1 g (0.1 mol) of allyl bromide, 30 ml of acetone and the ether formation reaction is carried out at temperature of 5055 ° C for 6 hours. The formed allyl ester of 4-isodecylphenol is extracted from the reaction mixture by vacuum distillation and subjected to Kleisen rearrangement under the influence of microwave irradiation for 7-10 minutes. The yield of 2-allyl-4-isodecylphenol 20.7 g (90% of theory). It is extracted from the reaction mass by vacuum distillation, it is a viscous substance of light yellow color, readily soluble in alcohols, acetone, aromatic hydrocarbons.

c) Synthesis of 2-allyl-4-isodecyl-6-diethylaminomethylphenol.

A three-necked reaction flask is charged with 27.4 g (0.1 mol) of 2-allyl-4-isodecylphenol, 8.1 ml of a 37% aqueous solution of formaldehyde (0.1 mol) and 7.2 g (0.1 mol ) diethylamine and solvent, 50-55 ml of benzene and the condensation reaction is carried out for 4-5 hours at a temperature of 70-75 ° C. Upon completion of the reaction, 2-allyl-4-isodecyl-6-diethylaminomethylphenol is separated from the solvent (using a rotary evaporator) and unreacted reagents in a high vacuum (residual pressure 3 mm Hg). Its yield is 25.0 g (70% of theory).

d) Obtaining the target compound (2-hydroxy-3-allyl-6-isodecylbenzyl) ammonium chloride by treating 2-allyl-4-isodecyl-6-diethylaminomethylphenol with hydrogen chloride at room temperature for 4-5 hours. Yield of the target compound 28.5 g (92%). It dissolves well in water, has surface-active and bactericidal properties, as evidenced by comprehensive studies.

The presence in its structure of a 4-allyl-substituted fragment (forming a bulky cation), as well as a Cl ^- anion, gives it not only antimicrobial, but also protective inhibiting properties in systems consisting of aqueous solutions of salts, hydrocarbons, and other compounds.

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Revealed high anticorrosive properties in systems saturated with hydrogen sulfide, consisting of 2-3% solutions of NaCl and hydrocarbons (petroleum fractions). The degree of protection against corrosion of steel Art. 3 is 90-92%, conc. compounds 50-100 mg / l.

Thus, the studies carried out show that 2-hydroxy-3-allyl-5isodecylbenzyl) diethylammonium chloride can be useful in medicine as a bactericide against Staphylococcus aureus, without suppressing the development of normal human intestinal biota, without impairing the anticorrosive properties of the used structural material.

Literature.

1. Gutenev V.V., Denisov I.A., Chumakova V.N. Study of bactericidal activity and the choice of ionic disinfectants // Ekol. systems and devices, 2005, No. 6, p. 27-29.

2. Pat. 2277906 Russia, IPC ⁷ A61K 31/13, A61K 31/185, 2006.

3. Pat. 2583140 Russia. Disinfectant, IPC A61L 2/16, 2016.

4. Misin V.M., Fedorova L.S., Stoyanov O.V. A new disinfectant based on quaternary ammonium salts // Vestnik Kazan. technol. University, 2016, 19, No. 3, p. 28-31.

5. Dudka I.A., Wasser S.P., Ellonskaya I.F. and other Methods of experimental mycology, Moscow, Nauka, 1982, 274 pp.

6. Albert Balows (Editor in chief) Manual of Chemical microbiology (fifth edition), American society for microbiology, Washington D.C., 1991, 1364 p.

Claims (1)

CLAIM Application of (2-hydroxy-3-allyl-5-isodecylbenzyl) diethylammonium chloride of the following formula: in medicine as a specific bactericide against the dangerous pathogen Staphylococcus aureus.