Synthesis, Antibacterial and Antifungal Activity of New 3-Aryl-5H-pyrrolo[1,2-a]imidazole and 5H-Imidazo[1,2-a]azepine Quaternary Salts

A series of novel 3-aryl-5H-pyrrolo[1,2-a]imidazole and 5H-imidazo[1,2-a]azepine quaternary salts were synthesized in 58–85% yields via the reaction of 3-aryl-6, 7-dihydro-5H-pyrrolo[1,2-a]imidazoles or 3-aryl-6,7,8,9-tetrahydro-5H-imidazo[1,2-a]azepines and various alkylating reagents. All compounds were characterized by 1H NMR, 13C NMR, and LC-MS. The conducted screening studies of the in vitro antimicrobial activity of the new quaternary salts derivatives established that 15 of the 18 newly synthesized compounds show antibacterial and antifungal activity. Synthesized 3-(3,4-dichlorohenyl)-1-[(4-phenoxyphenylcarbamoyl)-methyl]-6,7-dihydro-5H-pyrrolo[1,2-a]imidazol-1-ium chloride 6c possessed a broad activity spectrum towards Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Cryptococcus neoformans, with a high hemolytic activity against human red blood cells and cytotoxicity against HEK-293. However, compound 6c is characterized by a low in vivo toxicity in mice (LD50 > 2000 mg/kg).


Introduction
Fighting infections has been one of the most pressing problems for mankind throughout its history. Infectious diseases occupy a leading place among the general morbidity of the population. The number of pathologies of infectious origin not only does not decrease, but also tends to increase. Insufficient effectiveness of antibiotic therapy is one of the main causes of mortality in patients with infectious diseases. For example, at least 50,000 people in Europe and the United States alone, and hundreds of thousands of people in other countries, die each year from infections caused by antimicrobial-resistant microorganisms [1].
Bacteria such as ESBL (extended spectrum β-lactamase)-producing Enterobacteriaceae, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant enterococci (VRE) are especially dangerous today, as these pathogens are resistant to most modern antimicrobial drugs. Thus, a study conducted in various countries determined the sensitivity of Escherichia coli to antibiotics and found that the level of resistance to 3rd generation
Earlier, it was shown [21] that the heating of phenacyl salts 10 in an alkaline medium is accompanied by intramolecular condensation of the carbonyl group of the phenacyl fragment and the methylene group in the 9th position of the core heterocyclic system with the formation of 1,4-diaryl-5,6,7,8-tetrahydro-2a,4a-diazacyclopenta[cd]azulene (Scheme 3). This reaction is a logical continuation of the method for the synthesis of 2-phenylindolysine from 2-methylpyridine and phenacyl bromide proposed by A. Tschitschibabin in 1927 [22]. Based on this approach, derivatives 13 and 14 were obtained for the purpose of preliminary SAR analysis (Scheme 3).
Earlier, it was shown [21] that the heating of phenacyl salts 10 in an alkaline medium is accompanied by intramolecular condensation of the carbonyl group of the phenacyl fragment and the methylene group in the 9th position of the core heterocyclic system with the formation of 1,4-diaryl-5,6,7,8-tetrahydro-2a,4a-diazacyclopenta[cd]azulene (Scheme 3). This reaction is a logical continuation of the method for the synthesis of 2-phenylindolysine from 2-methylpyridine and phenacyl bromide proposed by A. Tschitschibabin in 1927 [22]. Based on this approach, derivatives 13 and 14 were obtained for the purpose of preliminary SAR analysis (Scheme 3). The synthesized novel heterocyclic compounds were characterized by 1 H and 13 C NMR (Experimental part, Supplementary Materials). In the 1 H NMR spectra of 3-aryl-5H-pyrrolo[1,2-a]imidazole quaternary salts 6a-e, the signals of the saturated The synthesized novel heterocyclic compounds were characterized by 1 H and 13 C NMR (Experimental part, Supplementary Materials). In the 1 H NMR spectra of 3-aryl-5Hpyrrolo[1,2-a]imidazole quaternary salts 6a-e, the signals of the saturated pyrrole ring were observed at 2.75-4.57 ppm. The two-proton singlet of the methylene group N + CH 2 CO for all compounds appears at 5.24-5.26 ppm. The one-proton singlet of the imidazole 2-CH-group was registered at 7.98-8.24 ppm. It should be noted that the position of this signal depends on the electronic properties of the benzene ring substituents in the third position of core heterocycle. Thus, for compound 6d with an electron-donating group OMe, a single-proton singlet 2-CH was observed at 7.98 ppm. Whereas, in compound 6b, with an electron withdrawing substituent Cl, the mentioned signal was appeared at 8.15 ppm. The introduction of the second chlorine atom into the third position of the benzene ring (compound 6c) led to the appearance of the imidazole 2-CH proton in an even weaker field at 8.24 ppm. The methylene group N + CH 2 COO of menthol substituted compound 5 appeared as two doublets of the characteristic AB-system, at 5.26 and 5.36 ppm with J = 17.9 Hz, instead of the expected two-proton singlet. A similar picture was observed for the 3-(4-methoxyphenyl)-6,7,8,9-tetrahydro-5H-imidazo[1,2-a]azepine 15. For this compound, the methylene group N + CH 2 COO was also registered as two doublets at 5.37 and 5.49 ppm with J = 18.1 Hz. This fact could be explained by the use of optically pure isomer L-menthol for the synthetic procedure.
In the 1 H NMR spectra of 3-aryl-6,7,8,9-tetrahydro-5H-imidazo[1,2-a]azepines quaternary salts 9-12, the signals of the azepine methylene groups appeared at 1.70-4.23 ppm. The two-proton singlet of the methylene group N + CH 2 CO phenacyl substituted 5H-imidazo[1,2a]azepines 10b-f was observed at 6.14-6.17 ppm. Whereas, for 2-difluoromethoxyphenyl derivative 10a this signal was shifted to a higher field, at 5.95 ppm. This fact can be explained by the spatial screening of the methylene group by the difluoromethoxy group in the ortho-position of the phenacyl fragment. It should be noted that the signal of the OCHF 2 group for compound 10a was registered as a triplet at 7.43 ppm with J = 72.9 Hz. In the 13 C NMR spectrum it was also observed as a triplet at 116.4 ppm with J = 259.2 Hz. The signal of the 2-CH-fragment of the imidazole ring for 5H-imidazo[1,2-a]azepines 9-12 was registered as a singlet in a narrow range at 7.69-7.77 ppm and practically does not depend on the electronic character of the substituents of the benzene ring in the third position of the core heterocycle.
In the 1 H NMR spectrum of diazacyclopenta[cd]azulene 14, the protons of the 6-and 7-CH 2 groups correspond to two multiplets at 1.86 and 2.01 ppm, and the signals of the 8-and 5-CH 2 groups were observed at 2.75 and 3.70 ppm, respectively. The singlets of the 2-CH pyrrole and 3-CH imidazole fragments were recorded at 6.80 and 7.39 ppm, respectively.

Biological Activity
Throughout the years of the antibiotic era, more than 1000 antimicrobial drugs have been discovered, of which only about 45 are used in clinical practice, with 6500 names. The cause of this situation is an irreversible biological phenomenon; the resistance of microorganisms. The overuse and misuse of antibiotics were highlighted as major contributing factors to this resistance [23]. Antibiotic resistance is associated with the high adaptive capacity of microbes and has existed since the beginning of the antibiotic era and is progressively increasing. Today, the main task of scientists is to find ways to overcome this. One of them is the search for new compounds with antimicrobial action for further development as new antimicrobial drugs. Novel 5H-pyrrolo[1,2-a]imidazole and 5H-imidazo[1,2-a]azepine quaternary salts are promising in this regard. These derivatives belong to the so-called quaternary ammonium compounds (QACs). QACs are a well-known class of cationic biocides with a broad spectrum of antimicrobial activity. QACs kill bacteria by impairing the permeability of cell membranes and do not contribute to antimicrobial resistance [24].
Assessment of biological activity was performed by the Community for Open Antimicrobial Drug Discovery (CO-ADD) [25,26], by testing the compounds against a range of pathogenic Gram-negative and Gram-positive bacteria, and fungi (yeasts).
The evaluation of antifungal activity showed no, or only weak, activity for the compounds for inhibiting the growth of C. albicans or C. neoformans. A few compounds (6a-c, 12 and 14) displayed MIC values of 32 µg/mL against C. albicans. On the other hand nine compounds displayed MIC values against C. neoformans in the range of 8-16 µg/mL (15.5-34.8 µM). The greatest activity was thereby shown by compound 6c (MIC is 8 µg/mL/15.5 µM), being equipotent with fluconazole, while compounds 6e, 10c and 12 appeared to be two times less potent than 6c.
According to the results of cytotoxicity testing, most compounds displayed a significant level of cytotoxicity against HEK-293 (human embryonic kidney) cells, with most CC 50 values similar to the MIC values or only slightly above.
Among the studied 5H-pyrrolo[1,2-a]imidazole and 5H-imidazo[1,2-a]azepine quaternary salts, compound 6c is of special interest. This derivative had a significant activity (MIC up to 4 µg/mL/7.8 µM), as well as a broad spectrum of action, against most of the tested microorganisms (except C. albicans and P. aeruginosa), unfortunately it also displays a similar activity against mammalian cells, as well as hemolytic activity against human red blood cells, indicating that the antibacterial effect is probably due to cytotoxicity. It is interesting to note that 1,4-diaryl-5,6,7,8-tetrahydro-2a,4a-diazacyclopenta[cd]azulenes 13 and 14 had a low level of hemolytic activity against human red blood cells, so heterocyclization of phenacyl 5H-pyrrolo[1,2-a]imidazole and 5H-imidazo[1,2-a]azepine quaternary salts to the appropriate diazacyclopenta[cd]azulenes may be one option for solving the problem of the cytotoxicity of the studied class of heterocycles.
For a more detailed biological evaluation of active compounds 6c, 12, and 14, their in vivo acute toxicity was studied in mice ( Table 2). In general, the studied compounds possessed satisfactory in vivo toxicity. Thus, the clinical picture of acute intoxication by compound 6c included decreased motor activity, gait disturbance, lethargy, and tremor in animals receiving doses ≥1000 mg/kg. The death of animals was not recorded. The obtained data made it possible to classify compounds 12 and 14 as III toxicity class, while compound 6c was IV toxicity class [27].

General Information
All solvents were purified before use. Ethyl acetates were purchased from Acros Organics (Geel, Belgium) and used without purification. Reactions were monitored by thinlayer chromatography (TLC) using Fluka silica gel (60 F 254) plates (0.25 mm). Visualization was performed with UV light. The melting points of the synthesized compounds were taken on a melting point tube. The 1 H NMR spectra were recorded on a Varian Gemini 400 MHz and 13 C NMR spectra on a Varian Mercury-400 100 MHz in DMSO-d 6 using tetramethylsilane as an internal standard. Chemical shifts are reported in ppm units with use of the d scale. The mass spectra were recorded on an Agilent 1200 LC/MSD SL instrument (Santa Clara, CA, USA).

Compound Preparation
The tests were carried out initially at a single compound concentration of 32 µg/mL in duplicates, to identify any actives, and followed up by a hit confirmation of the active compounds by a dose response test, using 8 concentrations at 1:2 dilution, in duplicates, to determine the minimum inhibitory concentration (MIC) against bacteria and yeasts, CC 50 against mammalian cells and HC 10 against human red blood cells.
All compounds were dissolved in DMSO to give a stock concentration of 10 mg/mL, and aliquots were diluted in water and 5 µL was dispensed into empty 384-well plates in duplicates for each strain and cell assayed. Once cells were added to the plates, this gave a final compound concentration of 32 µg/mL, or in the case of a serial dilution assay compound, concentrations from 32 to 0.25 µg/mL, in both cases with a maximum DMSO concentration of 0.3%.
All bacteria were cultured in Cation-adjusted Mueller Hinton broth (CAMHB) at 37 • C overnight. The resultant mid-log phase cultures were added to each well of the compound containing plates (384-well non-binding surface plates-Corning 3640), giving a cell density of 5 × 10 5 CFU/mL (colony forming units/mL). All plates were covered and incubated at 37 • C for 18 h without shaking. Inhibition of bacterial growth was determined by measuring absorbance at 600 nm (OD600) using a Tecan M1000 Pro monochromator plate reader.
Yeast strains were cultured for 3 days on yeast extract-peptone dextrose (YPD) agar at 30 • C. A yeast suspension of 1 × 10 6 to 5 × 10 6 CFU/mL (as determined by OD 530 ) was prepared from five colonies. These stock suspensions were diluted with yeast nitrogen base (YNB) broth to a final concentration of 2.5 × 103 CFU/mL. Then, 45 µL of the yeast suspension was added to each well of the compound containing plates (384-well nonbinding surface plates; Corning 3640). Plates were covered and incubated at 35 • C for 24 h without shaking. Growth inhibition of C. albicans was determined by measuring the absorbance at 530 nm (OD 530 ), while the growth inhibition of C. neoformans was determined by measuring the difference in absorbance between 600 and 570 nm (OD 600-570 ), after the addition of resazurin (0.001% final concentration) and incubation at 35 • C for an additional 2 h. The absorbance was measured using a Biotek Synergy HTX plate reader.
Growth inhibition was calculated as the percentage difference between untreated cells (positive growth control) and media only (negative growth control). Compounds with ≥80% growth inhibition were selected as actives in the initial screening, and MIC was determined following EUCAST recommendations, using 80% growth inhibition as a threshold for full inhibition.
Colistin sulfate (Sigma Aldrich, St. Louis, MO, USA; Cat# C4461) and vancomycin HCl (Sigma Aldrich, St. Louis, MO, USA; Cat# 861987) were used as positive bacterial inhibitor standards for Gram-negative and Gram-positive bacteria, respectively. Fluconazole (Sigma Aldrich, St. Louis, MO, USA; Cat# F8929) was used as a positive yeast inhibitor standard for C. albicans and C. neoformans.

Cytotoxicity Assay
HEK-293 (human embryonic kidney) ATCC CRL-1573 cells were counted manually in a Neubauer hemocytometer and then plated in 384-well tissue culture treated plates (Corning 3712) containing the compounds to give a density of 5000 cells/well in a final volume of 50 µL. DMEM supplemented with 10% FBS was used as growth media, and the cells were incubated together with the compounds for 20 h at 37 • C in 5% CO 2 . Cytotoxicity (or cell viability) was measured by fluorescence, ex: 560/10 nm, em: 590/10 nm (F 560/590 ), after addition of 5 µL of 25 µg/mL resazurin (2.3 µg/mL final concentration) and after incubation for a further 3 h at 37 • C in 5% CO 2 . The fluorescence intensity was measured using a Tecan M1000 Pro monochromator plate reader, using automatic gain calculation. CC 50 (concentration at 50% cytotoxicity) was calculated by curve fitting the inhibition values vs. log (concentration) using a sigmoidal dose-response function, with variable fitting values for bottom, top, and slope.

Hemolysis Assay
Human whole blood was washed three times with 3 volumes of 0.9% NaCl and then resuspended in the same to a concentration of 0.5 × 108 cells/mL, as determined by manual cell count in a Neubauer hemocytometer. The washed cells were then added to the 384-well compound containing polystyrene plates (Corning 3657) for a final volume of 50 µL. After a 10 min shake on a plate shaker, the plates were incubated for 1 h at 37 • C. After incubation, the plates were centrifuged at 1000× g for 10 min to pellet cells and debris, and 25 µL of the supernatant was then transferred to a polystyrene 384-well assay plate (Corning 3680). Hemolysis was determined by measuring the supernatant absorbance at 405 mm (OD 405 ). The absorbance was measured using a Tecan M1000 Pro monochromator plate reader. HC 10 (concentration causing 10% hemolysis) were calculated by curve fitting the inhibition values vs. log (concentration) using a sigmoidal dose-response function with variable fitting values for top, bottom, and slope. The use of human blood (sourced from the Australian Red Cross Blood Service) for hemolysis assays was approved by The University of Queensland Institutional Human Research Ethics Committee, Approval Number 2014000031.

In Vivo Toxicity Assay
The in vivo acute toxicity of compounds 6c, 12, and 14 was studied by the oral route of administration in white non-linear female mice weighing 20 ± 2 g. The animals were kept on a standard diet and received food and drink ad libitum. The studies were carried out in accordance with the application for a preclinical study approved by the Institute of Pharmacology and Toxicology Bioethical Committee (Approval Number 3.06.19/25.12.2019). Compounds were administered to animals once intragastrically in the form of a wateralcohol emulsion, using Tween-80 as an emulsifier. The volume of the substance did not exceed 0.5 mL. The solvent used was ethyl alcohol 95% (5%) and distilled water. Animals were randomized into groups of 5 animals each. The compounds were administered in doses: 6c-100, 500, 1000, 2000 mg/kg; 12-100, 250, 500, 750, 1000 mg/kg; and 14-100, 500, 1000, 1500, 2000 mg/kg. During the experiment, the state of the animals and the timing of death were monitored. The results were recorded in an alternative form (the number of dead animals) 14 days after a single injection. Toxicity was calculated using the Litchfield-Wilcoxon method [28].

Conclusions
Primary screening of the newly synthesized compounds to assess their antibacterial and antifungal properties showed that derivatives of 3-aryl-5H-pyrrolo[1,2-a]imidazole and 5H-imidazo[1,2-a]azepine quaternary salts have a high inhibitory effect. Among the newly synthesized compounds, a potential hit 6c, with a broad spectrum of action against S. aureus, E. coli, K. pneumoniae, A. baumannii, and C. neoformans, was identified. Notably, compound 6c possess a high hemolytic activity against human red blood cells and cytotoxicity against HEK-293, but possessed a low in vivo toxicity in mice.