RU2851119C1 - Method for obtaining antibacterial preparation - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: method for producing an antibacterial drug is proposed, which involves bringing epoxidised soybean oil into contact with 3-dimethylaminopropylamine at a molar ratio of 1:4, respectively, at a temperature of 130-140°C for 7 hours.

EFFECT: expansion of the arsenal of veterinary medicinal products with high bacteriostatic and bactericidal activity, as well as antiprotozoal activity.

1 cl, 2 tbl

Description

Field of technology to which the invention relates. The invention relates to agriculture and veterinary medicine, specifically to a method for producing an antibacterial drug.

State of the art

Quaternary ammonium compounds (QACs), a type of surfactant, are a class of compounds with powerful, broad-spectrum antibacterial properties. They are widely used as disinfectants and biocides in human and veterinary medicine. The need to develop new QACs and expand their range of applicability is constantly noted in Russian and international literature.

It's worth noting the terminology used in the literature for surfactants with biological activity. Compounds containing a quaternized nitrogen atom, that is, one bonded to four carbon atoms, are defined as quaternary ammonium compounds (QACs). Their precursors are tertiary ammonium compounds (TACs), in which the nitrogen atom is bonded to three carbon atoms and therefore lacks a positive charge. However, they readily form salts with organic and inorganic acids, acquiring properties similar to those of QACs with respect to their effects on living organisms.

One of the reasons for the development of microbial resistance is the unnecessary use of antibiotics in cases where other medications are adequate. This trend is particularly noticeable in veterinary medicine. One potential alternative to antibiotics in veterinary medicine is surfactants, which have a broad spectrum of antibacterial activity and, most importantly, are very slow in inducing the emergence of resistant strains.

The search for QACs has not diminished in recent years. New QACs are being synthesized, and their range of applications is expanding. The title of the article, "From Disinfectants to Antibiotics," eloquently demonstrates the growing need to expand the applicability of QACs. The authors presented a new strategy for enhancing the antimicrobial efficacy and biosafety of QACs, thereby expanding their application in the treatment of systemic infections [1].

The development of compositions containing QACs both as the primary active ingredient and as a component that enhances the composition's action is highly attractive. For example, article [2] proposes a composition consisting of QACs and silver nanoparticles, which has demonstrated good results in the disinfection of veterinary facilities.

In works [3] and [4] disinfectants are described that consist of QACs and simple aldehydes, which ensures high efficiency of the preparations.

Researchers [5] from Tatarstan have developed an effective disinfectant composition based on a quaternary ammonium compound, which is distinguished by the fact that in addition to glutaraldehyde, water, and isopropyl alcohol, it also contains a quaternary ammonium compound based on N-methyl isocyanurate - 1,3-bis(5-decyldiethylammoniopentyl)-5-methylisocyanurate dibromide. The authors conducted an extensive test of the antibacterial properties against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus cereus, Salmonella typhimurium, Klebsiella pneumonia, Enterobacter cloacae, and Enterococcus fecalis.

For all tested cultures, the minimum inhibitory concentration ranged from 7.8 to 15.6 μg/ml.

Foreign researchers are also intensively developing new QACs for various applications in human and veterinary medicine [6]. The authors note that quaternary ammonium compounds are a well-known class of antibacterial agents. They synthesized tertiary amines with different structures to produce several different classes of QACs. Many of the 200 compounds exhibit potent antibacterial activity. Analysis of the structure-activity relationship led the authors to develop inexpensive QACs that exhibit activity against a range of bacteria at low concentrations and, presumably, do not trigger bacterial resistance systems in methicillin-resistant Staphylococcus aureus.

The authors of the study [7] note that the need to develop new QACs is dictated by the fact that bacterial strains with a long history of use in medical practice are being found that are resistant to QACs, in particular, to benzalkonium chloride (BACL). This study determined the prevalence of resistance genes in Staphylococcus aureus from nosocomial infections and their susceptibility to quaternary ammonium compounds. The susceptibility of 150 unique clinical isolates of St. aureus to antimicrobials and benzalkonium chloride was determined by the agar diffusion method and the method of serial dilutions in a liquid nutrient medium. Reduced susceptibility to BACL was found in 30% of the isolates (minimum inhibitory concentration threshold >8 mg/L). Resistance genes were found in 13 isolates with reduced susceptibility to BACL. The authors observed a statistically significant prevalence of antiseptic resistance genes among isolates resistant to cefoxitin, ciprofloxacin, clindamycin, oxacillin, tetracycline, and erythromycin.

Synthetic fatty acids, fatty alcohols, various oils and fatty amines are used as starting materials in the development of QACs.

The aim of the invention is a method for obtaining a new antibacterial drug to expand the arsenal of veterinary medicinal products.

The stated goal is achieved by using epoxidized soybean oil (ESO), shown in Figure 1, and 3-dimethylaminopropylamine (Figure 2) as the starting material for the synthesis of the antibacterial quaternary ammonium compound.

The structural formula of epoxidized soybean oil (ESO) is shown in Figure 1.

Figure 1. Gross formula: C ₅₇ H ₁₀₂ O ₁₀ , average molecular weight is 947.59.

Figure 2. Formula of 3-dimethylaminopropylamine

It should be noted that the composition of unsaturated fatty acids in soybean oil, and naturally in epoxidized soybean oil, is not constant, which is why the term “average molecular weight” is used.

We chose 3-dimethylaminopropylamine, an inexpensive, readily available, and low-toxic amine, as the amine component for introduction into the epoxidized soybean oil (ESO) molecule. The ESO molecule contains four epoxide rings. Therefore, we chose a 1:4 ESO:amine molar ratio for the reaction, ensuring the opening of all four rings to form 2-hydroxyamines. Scheme 1 shows the general ring-opening reaction of one of the epoxide rings.

Scheme 1

It should be noted that the resulting product contains a secondary amino group, which could, during the reaction, attach to another epoxide ring, ultimately resulting in cross-linked polymer products with low solubility. Such processes occur during the production of epoxy resins. However, in our case, and this is the essence of the invention, this does not occur, and the resulting product is a light-brown viscous oil that readily dissolves in dilute mineral and organic acids, forming clear, foaming solutions.

Synthesis method. Option 1: 9.47 g (0.01 mol) of epoxidized soybean oil, 4.09 g (0.04 mol) of 3-dimethylaminopropylamine, 10 ml of dimethylformamide were mixed and boiled for 7 hours.

The solvent was distilled off under vacuum on a boiling water bath until constant weight was reached. Yield 90.3%.

Option 2: 9.47 g (0.01 mol) of epoxidized soybean oil and 4.09 g (0.04 mol) of 3-dimethylaminopropylamine were mixed and heated without solvent under reflux at a temperature of 130-140 °C for 7 hours. Yield 92.5%.

The general procedure for determining the amount of amino groups introduced into the ESM molecule was as follows: a sample of approximately 2 g of the drug, weighed on an analytical balance, was converted to the hydrochloride by treatment with a slight excess of hydrochloric acid, stirred for 10 minutes, and then evaporated under vacuum in a boiling water bath to dryness to constant weight. The sample was then treated with warm distilled water, acetic acid was added, and the chloride ion was precipitated using silver nitrate. The precipitate was filtered and washed with dilute acetic acid and water. The silver chloride precipitate was dried, treated with boiling ethanol, filtered, washed on the filter with ethanol (2 x 10 ml), dried at 100°C to constant weight, and then calculated.

The results of the experiment showed that one molecule of the drug contains 7.2 amino groups (secondary plus tertiary), that is, according to this synthesis method, on average 90% of the epoxy groups reacted with 3-dimethylaminopropylamine.

The biological activity of the synthesized QAC was studied in the form of a salt with citric acid. The salt was prepared using 1 mole of acid per 3 amino groups. The results are presented in Table 1.

Biological activity was studied using two methods: disk diffusion and two-fold serial dilutions to determine both bacteriostatic and bactericidal activity. The research methods are described in our papers [9].

The results of the study using the disk diffusion method for Penicillium italicum, St. aureus, E. coli, as well as the serial dilution method for Colpoda steinii, are presented in Table 1.

The table data demonstrates that the product obtained by reacting epoxidized soybean oil with 3-dimethylaminopropylamine exhibits high biological activity. At concentrations of 1% and above, the product is comparable to furazolidone against both gram-positive and gram-negative bacteria. Even at a concentration of 0.01%, it exhibits a significant growth inhibition effect.

It is necessary to note the high antiprotozoal activity of the drug; even a 0.01% solution is tens of times more active than toltrazuzil.

The results of studies using the serial dilution method are presented in Table 2.

The data in Table 2 show that a 1% solution of the claimed quaternary ammonium compound has significant bacteriostatic and bactericidal activity.

Bibliography

1. Mai Y, Wang Z, Zhou Y, Wang G, Chen J, Lin Y, Ji P, Zhang W, Jing Q, Chen L, Chen Z, Lin H, Jiang L, Yuan C, Xu P, Huang M. From disinfectants to antibiotics: Enhanced biosafety of quaternary ammonium compounds by chemical modification. J Hazard Mater. 2023 Oct 15; 460:132454. doi: 10.1016/j.jhazmat.2023.132454. Epub 2023 Sep 3. PMID: 37703742.

2. Miroshnikova, A.I. Application of a new biocide based on a quaternary ammonium compound and silver nanoparticles for disinfection of veterinary surveillance facilities / A.I. Miroshnikova, I.V. Kireev, V.A. Orobets // Bird and poultry products. - 2015. - No. 1. - P. 53-54. -EDN TOBXMZ.

3. Kulitsa, M.M. Prospects for the use of disinfectants based on glutaraldehyde and quaternary ammonium compounds for aerosol disinfection in poultry farming / M.M. Kulitsa, V.I. Dorozhkin // Poultry farming.-2021.- No. 11. - P. 72-75.-DOI 10.33845/0033-3239-2021-70-11-72-75. - EDN MHFTLV.

4. New effective disinfectants based on quaternary ammonium compounds / R.Z. Raskil'din, A.I. Nizamova, A.F. Abdullin [et al.] // Actual problems and directions of development of technologies of organic and inorganic synthesis in the context of import substitution: Collection of works of the II International scientific and practical conference, Sterlitamak, May 17-19, 2022. - Ufa: Pero Publishing House, 2022. - P. 58-60. - EDN ICNQLP.

5. Patent No. 2793332 C2 Russian Federation, IPC A61L 2/18, A61L 101/34. Disinfectant based on the quaternary ammonium compound "Izobak": No. 2022107980: declared 25.03.2022: published 31.03.2023 / F.M. Nekhaychik, M.M. Shulaeva, D.N. Mingaleev [et al.]; applicant Federal State Budgetary Educational Institution of Higher Education "Kazan State Academy of Veterinary Medicine named after N.E. Bauman". - EDN ENOYLO.

6. Kevin R.S. Minbiole, Megan S. Jennings, Laura E. Ator, Jacob W. Black, Melissa C. Grenier, Jade E. LaDow, Kevin L. Caran, Kyle Seifert, William M. Wuest / From antimicrobial activity to mechanism of resistance: the multifaceted role of simple quaternary ammonium compounds in bacterial eradication // Tetrahedron 2016 June 23, Pages 3559-3566 https://doi.org/10.1016/j.tet.2016.01.014.

7. El Sayed Zaki M, Bastawy S, Montasser K. Molecular study of resistance of Staphylococcus aureus to antiseptic quaternary ammonium compounds. J Glob Antimicrob Resist.2019 Jun; 17:94-97. doi: 10.1016/j.jgar.2018.11.022. Epub 2018 Dec 12. PMID: 30553116 https://doi.org/10.1016/j.jgar.2018.11.022.

8. Burlov A.S., Koshchienko Y.V., Makarova N.I., Kolodina A.A., Vlasenko V.G., Zubenko A.A., Drobin Y.D., Fetisov L.N., Zubavichus Y.V., Trigub A.L., Levchenkov S.I., Garnovskii D.A. / Synthesis, characterization, luminescent properties and biological activities of zinc complexes with bidentate azomethine schiff-base ligands // Polyhedron. 2018. T. 154. pp. 65-76. DOI: 10.1016/j.poly.2018.07.034.

Claims (1)

A method for producing an antibacterial drug, characterized by the fact that epoxidized soybean oil is introduced into interaction with 3-dimethylaminopropylamine at their molar ratio equal to 1:4, respectively, at a temperature of 130-140°C for 7 hours.