Bacterial infectious diseases remain among the leading causes of death throughout the world [1]. The antibiotics used for treating them lose efficacy over time because of the development of resistance in bacteria [2, 3]. The result is that treatment of resistant microorganisms becomes more problematic and requires the use of new drugs. Studies of the synthesis of novel biologically active compounds and studies of their antibacterial activity are constantly ongoing.

Many researchers seeking novel antibiotics have in recent years been attracted to various condensed derivatives of benzimidazole with nodal nitrogen atoms, such as pyrido-[1,2-a]benzimidazoles. This is associated with the fact that such substances have high antimicrobial activity against a variety of bacterial species [4,5,6,7,8,9, – 10].

Thus, series of compounds have been found to be active against Gram-negative Escherichia coli [4, 7,8,9, – 10], Salmonella typhi [7,8, – 9], and Vibrio cholerae [7, 8] and Gram-positive Bacillus subtilis [6,7, – 8], Clostridium tetani [6, 8], Streptococcus pneumoniae [6], Staphylococcus aureus [4, 9, 10], and Mycobacterium tuberculosis [5]. At the same time, the literature lacks data on the antimicrobial activity of tetrahydro derivatives of pyrido[1,2-a]benzimidazole and the effects of substituents in the benzene ring of this condensed heterocycle on their activity have received virtually no study.

Thus, we report here the synthesis of a wide range of condensed benzimidazole derivatives containing pyridine (I-XVII) or piperidine (XVIII-XXI) rings annealed to the imidazole moiety; we also report the first studies of their antibacterial activity. The mitosis-modifying and mutagenic actions of the substances with the greatest antibacterial effects were also studied.

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EXPERIMENTAL CHEMICAL SECTION

Melting temperatures were measured on a PolyTherm A apparatus with a heating rate of 3°C/min and were not corrected. 1H and 13C NMR spectra were recorded on a Bruker DRX 400 spectrometer, SF = 400 MHz, solvent DMSO-d6, temperature 25°C. The standard for determining chemical shifts consisted of residual proton signals from the solvent in proton spectra (2.5 ppm) or DMSO-d6 signals in carbon spectra (39.5 ppm. High-resolution mass spectra were recorded on a Bruker micrOTOF II apparatus (Bruker Deltronics) with electrospray ionization (ESI) and a mass scanning range (m/z) of 3000 Da, with samples injected by syringe. The solvent was MeCN and the solvent flow rate was 3 μl/min. The interface temperature was 180°C and the carrier gas was nitrogen (4.0 liters/min).

Pyrido[1,2-a]benzimidazoles (I-VIII) and 1,2,3,4-tetrahydropyrido[1,2-a]benzimidazoles (XVIII-XXI) were synthesized as described in [11].

Nitro derivatives of pyrido[1,2-a]benzimidazole (XI-XIV) were synthesized as described in [12].

Amino derivatives of pyrido[1,2-a]benzimidazole (IX, X, XV-XVII) were synthesized as described in [13].

EXPERIMENTAL BIOLOGICAL SECTION

The antibacterial activity of pyrido[1,2-a]benzimidazoles (I-XVII) and 1,2,3,4-tetrahydropyrido[1,2-a]benzimidazoles (XVIII-XXI) was studied using a standard method [14] on microorganisms widely used for developing antibiotics. Tests used two Gram-negative strains (Escherichia coli AB1157 and Pseudomonas aeruginosa PAO1, from the collection of the Institute of Molecular Genetics, provided by the Kurchatov Institute, Moscow) and one Gram-positive strain (Bacillus cereus, from the collection of the Department of Botany and Microbiology, Yaroslavl State University).

The antibacterial activity of pyrido[1,2-a]benzimidazole derivatives was assessed using commercial antibiotics used against Gram-negative and Gram-positive bacteria: gentamicin, tetracycline, kanamycin, levomycetin, and erythromycin.

Testing by the serial dilutions method was run in sterile 96-well immunology plates in volumes of 0.2 ml with final microorganism concentrations of 106 cfu/ml. Minimal inhibitory concentration (MIC) were determined for test compounds, at which they completely suppressed bacterial growth at 24 h of incubation at 37°C. Bacterial growth was assessed by measuring absorption at 600 nm with a Bio-Rad iMark photometric plate reader.

Mitosis-modifying and mutagenic properties of the compounds with the greatest antibacterial activity were studied using a genotoxicity test (the Allium test) on meristem cells from Allium cepa sprout root cells as described in [15]. Commercial antibiotics – tetracycline and gentamycin – were used as reference compounds.

Mutagenic activity was evaluated in terms of the ability of substances to induce chromosomal aberrations at the anaphase-telophase stage. Factors assessed were: the total number of cells (≈600), the total number of dividing cells at the anaphase-telophase stage, and the number of cells with chromosomal aberrations.

Mitotoxic activity and mitosis-modifying actions were assessed in the same time-based preparations as anaphasetelophase analysis. The mitosis-modifying actions of compounds were evaluated in terms of the level of mitotic activity in tissues – the mitotic index (MI, %) – and phase duration indexes. The total number of cells (≈600), the number of nondividing cells, and the numbers of cells at different phases of mitosis were determined.

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Significant differences would experiments and controls were identified by two-factor analysis of variance and Student’s test. Differences were taken as significant at p < 0.05.

RESULTS AND DISCUSSION

Pyrido[1,2-a]benzimidazoles (I-VIII) were synthesized by reductive intramolecular heterocyclization of quaternary N-(2-nitro(phenyl)aryl) pyridinium salts (Scheme 1). The electron source was an electric current, use of which significantly reduced the cost of synthesis and excluded formation of toxic wastes. The extraction procedure was also simplified, as the reaction material at the end of the reaction contained only the reaction product, ethanol, and the background electrolyte, which was 6% HCl. Syntheses were run at a temperature of 40°C by passage of 4.5 F/mol of electricity through an electrolytic cell. The yields of products I-VIII were 85 – 96%.

Nitration and reduction reactions yielded pyrido-[1,2-a]benzimidazoles (XI-XVII), which contained both an electron-donor or electron-acceptor substituent or substituents of different electronic nature. The nitrating agent was a mixture of KNO3 and H2SO4. Reactions were run at 30°C for 3 h. Nitro products XI-XIV were extracted with yields of 89 – 94%. Reduction of nitro compounds V, VIII, and XI-XIV was with titanium (III) chloride, which produced contaminant-free amines IX, X, XV-XVII with yields of 91 – 97%.

1,2,3,4-Tetrahydropyrido[1,2-a]benzimidazoles (XVIIIXXI) were also synthesized by reductive intramolecular cyclization (Scheme 2). By comparison with pyrido[1,2-a]-benzimidazoles (I-VIII), these reactions required passage of half the quantity of electricity. Electrolyte was 8% HCl containing N-(2-nitro-4-R1-phenyl)piperidine. Heterocycles XVIII-XXI were obtained with yields of 89 – 97%.

This yielded a large series of condensed benzimidazole derivatives with a nodular nitrogen atom (structures I-XXI, Table 1), some being compounds not previously described in the literature.

TABLE 1. Characteristics of Pyrido[1,2-a]benzimidazoles (I-XVII) and 1,2,3,4-Tetrahydropyrido[1,2-a]benzimidazoles (XVIII-XXI)
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The structures of pyrido[1,2-a]benzimidazole derivatives (I-XXI) were confirmed by 1H and 13C NMR spectroscopy and high-resolution mass spectrometry (HRMS) (Tables 1 and 2).

TABLE 2. 1H and 13C NMR Spectral Data for Pyrido[1,2-a]benzimidazoles (I-XVII) and 1,2,3,4-Tetrahydropyrido[1,2-a]benzimidazoles (XVIII-XXI)*
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Data on the antibacterial activity of substances I-XXI are presented in Table 3.

TABLE 3. Antimicrobial Activity of Pyrido[1,2-a]benzimidazoles (I-XVII) and 1,2,3,4-Tetrahydropyrido[1,2-a]benzimidazoles (XVIII-XXI)
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As shown by the data in Table 3, MIC for most pyrido- [1,2-a]benzimidazoles (I-XVII) was in the range 500 – 1000 μg/ml. Introduction of acceptor (compounds II-V, XI, XII) and donor (IX, XVII) substituents and substituents with different electronic natures (XV, XVI) into the benzene ring of unsubstituted heterocycle I did not in most cases increase antimicrobial activity. Only compounds containing a chlorine atom (VI) and two nitro groups (XIII) produced 100% inhibition of bacterial growth of P. aeruginosa and Bacillus sp. Respectively, at 62.5 μg/ml. The presence of two methyl groups in the pyridine ring of study compounds had greater influences. Thus, compounds VII, VIII, XIII, and XIV inhibited the growth of Bacillus sp. at significantly lower concentrations than structures IV, V, X, and XI. The antibacterial activity of 2,4-dimethyl-7,8-dinitropyrido- [1,2-a]benzimidazole (XIV) against Bacillus sp. was comparable with or greater than that of the efficacy of commercial tetracycline, kanamycin, levomycetin, and erythromycin formulations. Comparison of pyrido[1,2-a]benzimidazoles (I, II, IV, and VI) and 1,2,3,4-tetrahydropyrido[1,2-a]benzimidazoles (XVIII-XXI) showed that the latter had more marked antibacterial activity, especially against E. coli.

Thus, this study established that the nature of the substituent in the benzene ring had no significant effects on the antimicrobial activity of compounds. MIC was more dependent on the structure of the heterocycle annealed to the imidazole. Pyrido[1,2-a]benzimidazoles produced greater inhibition of the growth of Gram-positive Bacillus cereus bacteria. The presence of methyl groups in the pyridine fragment increased the antimicrobial action of compounds. 1,2,3,4-Tetrahydropyrido[1,2-a]benzimidazoles were more effective against Gram-negative Escherichia coli AB1157.

The mitosis-modifying and mutagenic properties of compounds VI, VII, XIII, XIV, and XVIII, which had the greatest antibacterial activity among the study compounds, were studied using a genotoxicity test (the Allium test).

Study compounds VI, VII, XIII, XIV, and XVIII were shown, like tetracycline and gentamicin, to decrease the number of dividing cells as compared with controls (6.1%) at all concentrations tested. Mitotic index was 5.6%, 5.7%, 3.9%, 3.1%, and 5.8% for substances VI, VII, XIII, XIV, and XVIII respectively. Nitrogen-containing compounds XIII and XIV were found to have the greatest effects on mitotic index. The cytotoxicity of these compounds was comparable with that of tetracycline (MI 3.3%, C = 0.01 mg/ml) and was greater than that of gentamicin (MI 6.0%, C = 0.01 mg/ml). Compounds VI, VII, and XVIII had relatively low toxicity at a concentration of 0.01 mg/ml. All study compounds produced some degree of change in the durations of mitosis phases as compared with controls, and there was an overall tendency to decreases in the numbers of cells at the prophase and anaphase-telophase stages and an increase in the numbers of cells in metaphase (Table 4).

TABLE 4. Phase Indexes in the Meristem of Allium cepa Root Shoots Treated with Study Compounds
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Anaphase-telophase analysis showed that study azaheterocycles induced genetic lesions in Allium cepa cells (Table 5). Compound XVIII, containing an unsubstituted heterocycle, had the smallest effect. The largest numbers of chromosome aberration as compared with controls were obtained with compounds XIII and XIV, containing two nitro groups. At a concentration of 0.01 mg/ml, these compounds increased the number of genetic lesions by factors of 4.1 and 4.6 as compared with controls; at 0.001 mg/ml, increases were by factors of 2.7 and 3.4 respectively. The remaining study compounds displayed no significant mutagenic effects. Positive control substance tetracycline also increased mutation frequency at C = 0.001 mg/ml. Gentamicin produced no increase in the number of chromosome aberrations.

TABLE 5. Frequencies of Chromosome Mutations in Cells of Allium cepa Root Meristem on Exposure to Study Substances (Number of Anaphase-Telophases Examined = 200)
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Thus, compounds VI, VII, XIII, XIV, and XVIII had mutagenicity and mitosis-modifying activity comparable to those of other members of this class of condensed heterocycles.

The antibacterial activity of several of the benzimidazole derivatives with nodular nitrogen atoms studied here (VI, VII, XIII, XIV, and XVIII), along with the low toxicity of thee azaheterocycles, leads to the conclusion that there is potential for further searches for novel antibiotics among compounds of this class.

This study was carried out in the framework of the Yaroslavl State University Development Program, project No. P2-K-1-G-1/2021.