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Article

Fluorinated Analogues of Lepidilines A and C: Synthesis and Screening of Their Anticancer and Antiviral Activity

by
Grzegorz Mlostoń
1,*,
Mateusz Kowalczyk
1,
Małgorzata Celeda
1,
Marcin Jasiński
1,*,
Marta Denel-Bobrowska
2 and
Agnieszka B. Olejniczak
2,*
1
Faculty of Chemistry, University of Lodz, Tamka 12, 91403 Łódź, Poland
2
Institute of Medical Biology, Polish Academy of Sciences, 106 Lodowa St., 93232 Łódź, Poland
*
Authors to whom correspondence should be addressed.
Molecules 2022, 27(11), 3524; https://doi.org/10.3390/molecules27113524
Submission received: 10 May 2022 / Revised: 24 May 2022 / Accepted: 27 May 2022 / Published: 30 May 2022
(This article belongs to the Special Issue Biosynthesis and Biological Activities of Natural Products)
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Abstract

:
Starting with fluorinated benzylamines, a series of 2-unsubstituted imidazole N-oxides was prepared and subsequently deoxygenated in order to prepare the corresponding imidazoles. The latter were treated with benzyl halides yielding imidazolium salts, which are considered fluorinated analogues of naturally occurring imidazolium alkaloids known as lepidilines A and C. A second series of oxa-lepidiline analogues was obtained by O-benzylation of the initially synthetized imidazole N-oxides. Both series of imidazolium salts were tested as anticancer and antiviral agents. The obtained results demonstrated that the introduction of a fluorine atom, fluoroalkyl or fluoroalkoxy substituents (F, CF3 or OCF3) amplifies cytotoxic properties, whereas the cytotoxicity of some fluorinated lepidilines is promising in the context of drug discovery. All studied compounds revealed a lack of antiviral activity against the investigated viruses in the nontoxic concentrations.

Graphical Abstract

1. Introduction

Dried roots of Maca plant (Lepidium meyenii) are well-known and widely applied in traditional folk medicine of the South American region for centuries [1]. They have been known to act not only as a natural drug but also as nutritional ingredients. Nowadays, numerous preparations containing powdered Maca roots or extracts prepared therefrom are commercially available as food additives and valuable dietary supplements [2,3,4]. Some time ago, imidazolium alkaloids known as lepidilines A–D (Figure 1, 1a–1d) were isolated and identified as significant, biologically active components of Maca extracts, and their anticancer activity was reported for the first time in 2003 [5,6]. In a recent publication, resulting from our continuing interest in imidazole chemistry, the synthesis of all lepidilines, as well as their bioactivity, were described. The anticancer activity was checked and compared with the earlier reported results, demonstrating the remarkable cytotoxicity of lepidiline D against the promyelocytic leukemia HL-60 cell lines [7]. In addition, oxa- analogues of lepidilines A and C (alkoxyimidazolium salts) were also synthesized, and the presence of benzyloxy-type substituents was found beneficial in terms of anticancer activity [7,8]. On the other hand, the antiviral activity of isolated lepidilines has not yet been reported. Nevertheless, the antiviral activity of complex Maca extracts against the human influenza virus was studied, and remarkable therapeutic effects in the treatment of Flu-A and Flu-B were demonstrated [9]. Prompted by this observation, and taking into account the reported antiviral properties of some imidazole-based compounds [10,11,12,13], we decided to check the antiviral activity of lepidilines A and C and a series of oxy-imidazolium salts that can be considered close structural analogues of these alkaloids.
In general, introducing a fluorine atom or fluoroalkyl groups into the structure of the heterocyclic core of an organic compound substantially increases its bioactivity [14,15]. Therefore, more than 20% of commercially available medicaments constitute hetero-organic compounds functionalized with fluorine-containing substituents [16]. In spite of this fact, fluorine-containing lepidilines are still unknown, and it seemed reasonable to fill this gap. For this reason, we decided to involve a series of fluorinated lepidilines in the present study to check the anticipated beneficial effects of an F atom, as well as CF3 and OCF3 groups incorporated in their structures, analogous to lepidiline C, at the meta-position of the N(1) benzyl group.
Thus, the main goal of the present study was the synthesis of fluorinated imidazolium salts derived from lepidilines A and C and the examination of their anticancer, as well as antiviral, activity against the selected cell lines (cancer: A549, HepG2, and HeLa and normal: Vero, LLC-MK2, MRC-5, and NCTC clone 929) and model viruses (HSV-1, HCMV, AdV5, HPIV-3, and EMCV), respectively. In addition, taking into account the general importance of 1,3-diadamantyl imidazolium bromide (2a, known as ‘Arduengo salt’ [17]), its bis-oxidized analogue 2b and other structurally related imidazolium salts not only in the chemistry of nucleophilic heterocyclic carbenes [18,19,20] but also in medicinal chemistry [21,22], they were also involved in the present study aimed at the comparison of their antiviral activity with lepidiline analogues (Figure 2).

2. Results and Discussion

2.1. Chemistry

The preparation of lepidilines 1a and 1c was performed following the general procedure described previously employing the respective 2-unsubstituted 4,5-dimethylimidazole N-oxides, which, after deoxygenation and quaternization using benzyl chloride, were converted into final products [7,23]. Similarly, Arduengo salt 2a and its bis-oxy-analogue 2b were obtained based on published methods via the cyclocondensation of glyoxal with 1-aminoadamantane or adamantyl-1-oxyamine, respectively, in the presence of HBr [24].
The synthesis of fluorinated derivatives of lepidilines 1a and 1c, i.e., imidazolium salts 1e–1g, started with the preparation of hitherto unknown fluorinated formaldimines 3a–3c, which are available by treatment of the corresponding benzylamines 4a–4c with formaldehyde (Scheme 1). Crude oily products of type 3 were treated with diacetyl monoxime (5a) in acetic acid at room temperature, yielding the desired imidazole N-oxides 6a–6c in high overall yields (66–86%). In addition, two isomeric benzylamines 4d and 4e bearing the CF3 group located either at the ortho or para position of the phenyl ring, respectively, were involved in the study, and the expected imidazole N-oxides 6d and 6e were obtained (50% and 76% for two steps). In the next step, the N-oxides 6a–6c were deoxygenated using freshly prepared Raney-Ni to afford the corresponding 1-benzyl-4,5-dimethylimidazoles 7a–7c. Finally, N-benzylation performed with benzyl chloride under microwave irradiation in MeCN led to the desired fluorinated analogues of lepidilines 1e–1g in an acceptable overall yield of 30%, 54%, and 22% (from amines 4), respectively.
Prompted by our earlier study focused on the preparation and anticancer activity screening of oxidized analogues of lepidilines [7,8], the starting imidazole N-oxides 6a–6c were also subjected to benzylation reactions under standard conditions, in CHCl3 at room temperature, and in these reactions, no MW activation was necessary to perform O-alkylation. In the case of 6a and 6b, the expected benzyloxy salts 8a and 8b were formed as sole products and isolated as crystalline materials. Analogous results, leading to the formation of imidazolium salts 8c and 8d as single products, were obtained using trifluoromethylbenzyl-functionalized imidazole N-oxides 6d and 6e and benzyl bromide as an alkylating agent (Scheme 2).
In extension of the study, collection of lepidiline analogues with a 4,5-dimethyl-substituted imidazolium core was supplemented by 4,5-diphenyl derivative functionalized with the m-CF3-benzyl group located at N(1) of the imidazolium ring. The key imidazole N-oxide 6f was prepared analogously, starting with benzil monoxime (5b) and trimeric formaldimine 3c. The deoxygenation of 6f with Raney nickel afforded the desired 1,4,5-trisubstituted imidazole 7d in 68% yield. Following the general procedure, N-benzylation of imidazole 7d with benzyl bromide provided the expected salt 9a (Scheme 3) in a 27% overall yield. Similarly, N-oxide 6f was treated with selected benzyl bromides, and the expected benzyloxy-imidazolium salts 8e–8g were obtained as exclusive products.
In a recent publication, the anticancer activity of imidazolium salts, considered as lepidiline analogues with no fluorinated benzyl substituents at N(1), was reported [7]. For comparison of the antiviral activity of both series of lepidiline analogues, i.e., fluorinated and non-fluorinated representatives, they were also involved in the present study. In order to check the influence of the counterion present in imidazolium salts 1 and 9 on the biological activity, selected chlorides were converted into the corresponding hexafluorophosphates 1[PF6] and 9[PF6] by counterion exchange via treatment with ammonium hexafluorophosphate in water/ethanol solution (Figure 3). Furthermore, it is well-known that imidazolium salts are the perfect substrates for the preparation of the corresponding imidazole-2-thiones via nucleophilic carbenes (NHCs) as the in situ generated intermediates [7,18,25,26]. For that reason, some imidazole-2-thiones (10) shown in Figure 3 were also involved in the study focused on antiviral and anticancer activity screening presented in this work (see Supplementary Materials).

2.2. In Vitro Cytotoxicity on Cancer and Normal Cell Lines

Despite the continuous development of modern medicine, finding an effective cure for neoplastic diseases, especially those diagnosed in an advanced stage, is still a challenge. Screening studies for potential anticancer agents is a crucial step in cancer drug discovery. An ideal situation is when the drug can kill the cancer cells but, at the same time, not affect the normal cells [27]. Therefore, it is advantageous to include normal (noncancer) cells in research on the cytotoxicity of potential drugs.
The initial step of our studies was to test cytotoxic properties of the series compounds 1a, 1c, 1a[PF6], 1c[PF6], 9b[PF6], 9c[PF6], 2a, 2b, and 10a–10d during a screening assay at 10 µM on Cercopithecus aethiops normal kidney cells (Vero), Macaca mulatta normal kidney cells (LLC-MK2), Human lung normal fibroblasts (MRC-5), Mus musculus normal subcutaneous connective tissue cells (NCTC clone 929), and Human cervix adenocarcinoma cells (HeLa). Compounds demonstrating cell viability ≥ 50% (in both cytotoxicity and antiviral activity studies mentioned below) were selected for further, extended studies resulting in CC50 (50% cytotoxic concentration, the parameter used for cytotoxicity results) and IC50 (50% inhibitory concentrations, the parameter used for antiviral activity results) (Tables S1–S4, Supplementary Materials).
Screening cytotoxicity studies revealed that the most promising results were observed for compounds 1a; 1c; 1a[PF6]; 1c[PF6] (LLC-MK2 cell line); 10a (LLC-MK2, NCTC clone 929, and HeLa cell lines); and 10d (HeLa cell line). Compound 10a showed the highest cytotoxicity (CC50 < 20 µM) on the LLC-MK2 and HeLa cell lines, whereas compounds: 1a, 1c, 1a[PF6], and 1c[PF6] were cytotoxic in the concentration of CC50 < 80 µM on LLC-MK2 cells. Compound 10d was nontoxic against the tested HeLa cells (CC50 < 400 µM) (Tables S1 and S2, Supplementary Materials).
The fluorine atom is a key part of the medicinal chemist’s repertoire of substitutions used to modulate all aspects of molecular properties, including potency, physical chemistry, and pharmacokinetics [15,28]. Fluorinated compounds are an important class of anticancer and antiviral drugs [29,30].
The in vitro cytotoxic activities of the target compounds 1e–1g, 1e[PF6], 8a–8d, and 9a were investigated in two types of human cell lines—four normal cell lines: Vero, LLC-MK2, MRC-5, and NCTC clone 929, as well as three cancer cell lines: HeLa, Human lung carcinoma cells (A549), and Human hepatocellular carcinoma cells (HepG2). Cytotoxicity of the investigated compounds was established by the measurement of 50% inhibition of cell growth by the MTT assay and expressed as the CC50 parameter (50% cytotoxic concentration). All results are presented in Table 1.
Individual cell lines were characterized by different sensitivities to the tested compounds. The HeLa cell line showed the highest sensitivity among all the tested cell lines, but for some of the investigated compounds, the results obtained for A549 were similar to those observed in HeLa cells. The lowest sensitivity towards the tested compounds was obtained for the HepG2 cells.
Generally, fluorinated lepidilines 1e–1g, 1e[PF6], and 9a were found to be the most cytotoxic against the HeLa cell line, with CC50 values significantly below 1 µM. 4,5-Diphenyl derivative 9a was the most cytotoxic at a concentration as low as 0.019 µM. Its analogues bearing dimethyl groups attached at positions 4 and 5 (1e–1g and 1e[PF6]) were less active (CC50 = 0.039–0.080 µM), and for compound 1g, this activity was almost three times lower compared to 9a. Recently, we published that 4,5-diphenyl analogues of lepidilines A, C, and D showed also increased cytotoxicity against the MCF-7 cell line compared to the corresponding lepidilines bearing methyl groups at C(4) and C(5) of the imidazole ring [7]. The presence of an F atom in compound 1e or OCF3 group in compound 1f resulted in the increase of their activity for the HeLa cell line compared to 1g. Interestingly, the replacement of Cl− (compound 1e) with PF6− (compound 1e[PF6]) resulted in a two-fold decrease in the cytotoxicity. Comparison activities of fluorinated lepidilines in the series containing 4,5-dimethyl groups (1e–1g, 1e[PF6], and 8a–8d) revealed that oxidized analogues 8a–8d were much less active against HeLa cells, with CC50 values in the range 5.500–20.000 µM, which were two (8b, 8d) or three (8a, 8c) orders of magnitude lower than unoxidized 1e–1g and 1e[PF6].
Lepidiline 9a also showed high cytotoxicity in the A549 cell line, but other non-oxidized lepidilines: 1e–1g and 1e[PF6] were less active on the same cell line. It should be noted that all oxidized lepidilines 8a–8d revealed better cytotoxic activity against the A549 cell line (CC50 = 5.070–7.500 µM) than against the HeLa cell line (CC50 = 5.500–20.000 µM). In the experiments performed on HepG2 cells, the CC50 values indicated that all the tested lepidilines, except for 9a (CC50 = 6.900 µM), were not toxic (CC50 > 90 µM). The most cytotoxic against the selected cancer cell lines, lepidilines 1e–1g, and 1e[PF6] were rather nontoxic against normal cell lines—MRC-5 (CC50 = 36.667–149.667 µM), NCNT clone 929 (CC50 = 30.000–148.333 µM), LLC-MK2 (CC50 = 53.500–150.333 µM), and Vero (CC50 = 22.667–56.333 µM), with the exception of 1g, which is toxic against Velo cells. Compound 9a, cytotoxic to all the tested cancer cell lines, is also toxic against MRC-5, LLC-MK2, and Vero (CC50 = 0.040–0.347 µM); it is less toxic to NCNT 929 (CC50 = 3.750 µM). In the experiments performed on normal cells, the CC50 values for almost all oxidized lepidilines 8a–8d were > 50 µM, which indicates a large safety margin for these compounds.

2.3. In Vitro Antiviral Activity

The search for compounds with antiviral activity among the natural compounds and their modified analogues is a rapidly developing direction in pharmaceutical chemistry.
Screening of the antiviral activity of compounds 1a, 1c, 1a[PF6], 1c[PF6], 9b[PF6], 9c[PF6], 2a, 2b, and 10a–10d against viruses: Human herpesvirus 1 (HSV-1), Human herpesvirus 5 (HCMV), Human adenovirus 5 (AdV5), Human parainfluenza virus type 3 (HPIV-3), and Encephalomyocarditis virus (EMCV) demonstrated that all the compounds revealed a lack of antiviral activity against all investigated viruses in the nontoxic concentrations (Tables S3 and S4, Supplementary Materials).
The antiviral activity of 1e–1g, 1e[PF6], 8a–8d, and 9a was also evaluated against the same viruses (HSV-1, HCMV, AdV5, HPIV-3, and EMCV). The antiviral activity results were shown as an IC50 parameter (50% inhibitory concentrations).
According to the results collected in Table 2, we can conclude that all investigated compounds revealed a lack of antiviral activity against viruses: HSV-1, HPIV-3, AdV5, EMCV, and HCMV in the nontoxic concentrations.

3. Conclusions

A series of fluorinated imidazolium (compounds of type 1 and 9) and their oxa analogues (compounds of type 8), considered as close structural analogues of naturally occurring imidazolium alkaloids, known as lepidilines A and C, was prepared, and their anticancer, as well as antiviral activity, was examined. The target products were prepared via a straightforward, three-step protocol starting with benzylamines functionalized either with the F atom or with CF3 (or OCF3) groups. The presented study demonstrated, once more, a high utility of 2-unsubstituted imidazole N-oxides as key intermediates for the synthesis of polyfunctionalized imidazole derivatives.
The obtained new imidazolium salts demonstrated various cytotoxicity levels towards the tested normal and cancer cell lines. Notably, the introduction of fluorinated benzyl substituents resulted, in some cases, in a remarkable increase of bioactivity. For example, fluorinated analogues of lepidilines A and C, i.e., compounds 1e–1g and 1e[PF6], 8a–8d, and 9a were the most active against the HeLa or A549 cell lines. Their cytotoxicity was significantly higher in comparison with natural lepidiline A against the HeLa cell line. Remarkably, the most cytotoxic compound 9a was also toxic against normal cell lines. In contrast, derivatives 1e–f and 1e[PF6], 8a–8d were found to be rather nontoxic in the normal cell lines. All investigated compounds revealed no antiviral activity against HSV-1, HPIV-3, AdV5, EMCV, and HCMV in the range of nontoxic concentrations. The presented results confirmed the importance of fluorinated substituents for tuning the biological activity of organic compounds [28,29], including some naturally occurring imidazolium salts, such as lepidilines A and C, and their 4,5-diphenyl analogues.
The results obtained in the testing of the antiviral properties of lepidilines A and C, as well as their fluorinated analogues, suggest that the earlier reported antiviral activity of Maca extract [9] results rather from the presence of other compounds, e.g., indole derivatives or complex isothiocyanates, which were also identified as its components [31,32,33].

4. Materials and Methods

4.1. General Synthetic Procedures

Commercial chemicals and solvents were used as received. If not stated otherwise, products were purified by filtration through a short silica gel plug (200–400 mesh) by using freshly distilled solvents as eluents or by recrystallization. Melting points were determined in capillaries with an Aldrich Melt-Temp II, and they are uncorrected. NMR spectra were taken with a Bruker AVIII spectrometer (1H NMR (600 MHz), 13C NMR (151 MHz), and 19F NMR (565 MHz); chemical shifts are relative to the residual undeuterated solvent peaks (CDCl3: 1H NMR δ = 7.26, 13C NMR δ = 77.16 [34]) or to the external standard (CFCl3: 19F NMR δ = 0.00). IR spectra were measured with an Agilent Cary 630 FTIR spectrometer neat. Mass spectra (ESI) were obtained with a Varian 500-MS LC Ion Trap. Elemental analyses were obtained with a Vario EL III (Elementar Analysensysteme GmbH) instrument. Starting α-hydroxyiminoketones 5a [35] and 5b [36] were prepared following the general literature protocols.

4.1.1. Synthesis of Imidazolium Chlorides 1e–g and 9a

To a deoxygenated solution of imidazole 7 (1.0 mmol) in MeCN (2.0 mL) was added benzyl halide (1.2 mmol), and the resulting mixture was MW-irradiated at 110 °C in a closed vessel until the starting imidazole was fully consumed (TLC monitoring, typically ca. 45 min). After the solvent was removed under reduced pressure, the crude product was washed with several portions of dry Et2O (5 × 5 mL), and the solid imidazolium chloride was recrystallized from a CH2Cl2/hexane mixture.
1-Benzyl-3-(3-fluorobenzyl)-4,5-dimethylimidazolium chloride (1e): 195 mg (59%), pale yellow crystals, m.p. 190–192 °C. 1H NMR (600 MHz, CDCl3): δ 2.05 (sbr, 6 H, 2 Me), 5.47, 5.59 (2 s, 2 H each, 2 CH2), 6.95–7.00, 7.10–7.12, 7.26–7.34 (3 m, 2 H, 1 H, 6 H), 11.18 (s, 1 H, C(2)H). 13C NMR (151 MHz, CDCl3): δ 8.87, 8.88, 50.4 (d, 4JC,F = 1.4 Hz, CH2), 51.2, 114.8 (d, 2JC,F = 22.4 Hz, CH), 116.0 (d, 2JC,F = 21.1 Hz, CH), 123.6 (d, 4JC,F = 2.9 Hz, CH), 127.2, 127.3, 127.8, 129.0, 129.4, 131.1 (d, 3JC,F = 8.2 Hz, CH), 133.1, 135.8 (d, 3JC,F = 7.2 Hz, i-C), 137.6(br)*, 163.0 (d, 1JC–F = 248.3 Hz, i-C); broadened signal due to partial H/D exchange at C(2). 19F NMR (565 MHz, CDCl3): δ −111.2 (mc, CF). ESI-MS (m/z): 295.4 (100, [M − Cl]+). HRMS (ESI-TOF) m/z: [M − Cl]+ calcd for C19H20FN2 295.1611, found 295.1612.
1-Benzyl-4,5-dimethyl-3-[3-(trifluoromethoxy)benzyl]imidazolium chloride (1f): 285 mg (72%), colorless crystals, m.p. 108–110 °C. 1H NMR (600 MHz, CDCl3): δ 2.05 (sbr, 6 H, 2 Me), 5.49, 5.65 (2 s, 2 H each, 2 CH2), 7.07 (mc, 1 H), 7.13–7.16, 7.24–7.41 (2 m, 1 H, 7 H), 10.94 (s, 1 H, C(2)H). 13C NMR (151 MHz, CDCl3): δ 8.8, 8.9, 50.3, 51.2, 120.2, 120.4 (q, 1JC–F = 258.0 Hz, OCF3), 121.2, 126.4, 127.2, 127.4, 127.7, 129.0, 129.4, 131.1, 133.1, 135.9, 137.6(br), 149.6. 19F NMR (565 MHz, CDCl3): δ −57.8 (s, OCF3). IR (neat): ν 1554, 1260, 1208, 1171, 758, 701 cm−1. ESI-MS (m/z): 361.4 (100, [M – Cl]+). HRMS (ESI-TOF) m/z: [M − Cl]+ calcd for C20H20F3N2O 361.1528, found 361.1531.
1-Benzyl-4,5-dimethyl-3-[3-(trifluoromethyl)benzyl]imidazolium chloride (1g): 171 mg (45%), colorless crystals, m.p. 120–122 °C. 1H NMR (600 MHz, CDCl3): δ 2.05 (sbr, 6 H, 2 Me), 5.49, 5.71 (2 s, 2 H each, 2 CH2), 7.25–7.34 (m, 5 H), 7.45 (mc, 1 H), 7.47–7.50, 7.53–7.55, 7.63–7.65 (3 m, 1 H each), 10.96 (s, 1 H, C(2)H). 13C NMR (151 MHz, CDCl3): δ 8.87, 8.88, 50.4, 51.2, 123.7 (q, 1JC–F = 272.5 Hz, CF3), 124.2 (q, 3JCF = 3.6 Hz, CH), 125.8 (q, 3JC,F = 3.6 Hz, CH), 127.1, 127.4, 127.7, 129.0, 129.4, 130.2, 131.5 (q, 2JC,F = 32.6 Hz, i-C), 131.6, 133.1, 134.7, 137.5(br). 19F NMR (565 MHz, CDCl3): δ −62.6 (s, CF3). IR (neat): ν 1551, 1454, 1327, 1163, 1115, 1074, 701 cm−1. ESI-MS (m/z): 345.4 (100, [M−Cl]+). HRMS (ESI-TOF) m/z: [M − Cl]+ calcd for C20H20F3N2 345.1579, found 345.1577.
1-Benzyl-4,5-diphenyl-3-[3-(trifluoromethyl)benzyl]imidazolium bromide (9a): 488 mg (89%), colorless crystals, m.p. 216–217 °C. 1H NMR (600 MHz, CDCl3): δ 5.47, 5.68 (2 s, 2 H each, 2 CH2), 6.82 (mc, 1 H), 7.03–7.11, 7.20–7.31, 7.37–7.47, 7.77–7.80 (4 m, 6 H, 7 H, 4 H, 1 H), 11.38 (s, 1 H, C(2)H). 13C NMR (151 MHz, CDCl3): δ 51.0, 51.7, 123.5 (q, 1JC–F = 272.5 Hz, CF3), 124.5, 124.6, 125.3 (q, 3JC,F = 3.7 Hz, CH), 125.8 (q, 3JC,F = 3.7 Hz, CH), 128.5, 129.1(br), 129.16, 129.21, 129.3, 130.1, 130.6, 130.7, 130.75, 130.81, 130.9 (q, 2JC−F = 32.6 Hz, i-C), 132.2, 132.5, 132.7(br), 133.2, 134.3, 137.8(br). 19F NMR (565 MHz, CDCl3): δ −62.8 (s, CF3). IR (neat): ν 1554, 1450, 1327, 1170, 1122, 1074, 697 cm−1. ESI-MS (m/z): 469.6 (100, [M – Br]+). Anal. calcd for C30H24BrF3N2 (548.1): C 65.58, H 4.40, N 5.10; found: C 65.36, H 4.40, N 4.90.

4.1.2. Synthesis of Imidazolium Hexafluorophosphate 1e[PF6]

To a solution of imidazolium chloride 1e (89 mg, 0.27 mmol) in EtOH (2.0 mL) was added dropwise a solution of NH4PF6 (47 mg, 0.29 mmol) in H2O (1.0 mL), and the mixture was stirred for 30 min. The precipitate of the crude product was isolated, washed with dry Et2O (4 × 4 mL), and recrystallized from a CH2Cl2/i-Pr2O mixture by slow evaporation of the solvents at room temperature.
1-Benzyl-3-(3-fluorobenzyl)-4,5-dimethylimidazolium hexafluorophosphate (1e[PF6]): 53 mg (45%), colorless crystals, m.p. 110–112 °C. 1H NMR (600 MHz, CDCl3): δ 2.04, 2.05 (2 s, 3 H each, 2 Me), 5.21, 5.22 (2 s, 2 H each, 2 CH2), 6.89–6.91, 6.96–7.00, 7.19–7.21, 7.28–7.35 (4 m, 1 H, 2 H, 2 H, 4 H), 8.63 (s, 1 H, C(2)H). 13C NMR (151 MHz, CDCl3): δ 8.57, 8.63, 50.5 (d, 4JC,F = 2.1 Hz, CH2), 51.3, 114.8 (d, 2JC,F = 22.6 Hz, CH), 116.2 (d, 2JC,F = 20.9 Hz, CH), 123.5 (d, 4JC,F = 3.1 Hz, CH), 127.8, 128.1, 128.2, 129.2, 129.6, 131.4 (d, 3JC,F = 8.3 Hz, CH), 132.6, 134.7, 135.2 (d, 3JC,F = 7.4 Hz, i-C), 163.1 (d, 1JC–F = 248.1 Hz, i-C). 19F NMR (565 MHz, CDCl3): δ −72.0 (d, 1JP–F = 712.8 Hz, PF6), −111.1 (mc, CF). IR (neat): ν 1586, 1450, 1353, 1252, 1208, 824, 738 cm−1. ESI-MS (m/z): 295.4 (100, [M − PF6]+).

4.1.3. Synthesis of Alkoxy-Imidazolium Bromides 8

To a solution of corresponding imidazole N-oxide 6 (1.0 mmol) in CHCl3 (3.0 mL) was added excess alkyl bromide (1.1 mmol), and the resulting mixture was stirred at room temperature until the starting N-oxide was fully consumed (TLC monitoring, SiO2 EtOAc/MeOH 95:5). After the solvent was removed under reduced pressure, the resulting crude product was triturated with Et2O (4 × 10 mL) and dried under vacuum. Crude products were recrystallized from diisopropyl ether/dichloromethane mixtures by slow evaporation of the solvents at room temperature.
3-Benzyloxy-1-(3-fluorobenzyl)-4,5-dimethylimidazolium bromide (8a): 246 mg (63%), beige solid, m.p. 124–126 °C. 1H NMR (600 MHz, CDCl3): δ 1.94, 2.06 (2 s, 3 H each, 2 Me), 5.56, 5.65 (2 s, 2 H each, 2 CH2), 6.91–6.94, 7.00–7.04, 7.13–7.15, 7.31–7.35, 7.37–7.44, 7.49–7.51 (6 m, 1 H, 1 H, 1 H, 1 H, 3 H, 2 H), 11.08 (s, 1 H, C(2)H). 13C NMR (151 MHz, CDCl3): δ 7.3, 9.1, 50.6 (d, 4JC,F = 2.0 Hz, CH2), 84.0, 114.9 (d, 2JC,F = 22.5 Hz, CH), 116.1 (d, 2JC,F = 20.9 Hz, CH), 123.9 (d, 4JC,F = 3.0 Hz, CH), 124.4, 125.1, 129.2, 130.6, 130.7, 131.1 (d, 3JC,F = 8.3 Hz, CH), 131.5, 132.6(br), 135.5 (d, 3JC,F = 7.3 Hz, i-C), 163.0 (d, 1JC–F = 248.4 Hz, i-C). 19F NMR (565 MHz, CDCl3): δ −111.0 (mc, CF). IR (neat): ν 1592, 1454, 1245, 1141, 947, 917 cm−1. ESI-MS (m/z): 311.4 (100, [M – Br]+). Anal. calcd for C19H20BrFN2O (390.1): C 58.32, H 5.15, N 7.16; found: C 58.21, H 5.14, N 6.87.
3-Benzyloxy-4,5-dimethyl-1-[3-(trifluoromethoxy)benzyl]imidazolium bromide (8b): 410 mg (90%), pale yellow solid, m.p. 108–110 °C. 1H NMR (600 MHz, CDCl3): δ 1.92, 2.06 (2 s, 3 H each, 2 Me), 5.53, 5.70 (2 s, 2 H each, 2 CH2), 7.04 (mc, 1 H), 7.16–7.19, 7.32–7.42, 7.46–7.48 (3 m, 1 H, 5 H, 2 H), 11.07 (s, 1 H, C(2)H). 13C NMR (151 MHz, CDCl3): δ 7.3, 9.1, 50.6, 84.1, 120.3 (q, 1JC–F = 258.0 Hz, OCF3), 120.4, 121.2, 124.4, 125.2, 126.7, 129.2, 130.5, 130.7, 131.0, 131.5, 132.6(br), 135.4, 149.6 (q, 3JC,F = 1.6 Hz, i-C). 19F NMR (565 MHz, CDCl3): δ −57.8 (s, OCF3). IR (neat): ν 1446, 1238, 1215, 1148, 910, 707 cm−1. ESI-MS (m/z): 377.5 (100, [M – Br]+). Anal. calcd for C20H20BrF3N2O2 (456.1): C 52.53, H 4.41, N 6.13; found: C 52.39, H 4.42, N 5.85.
3-Benzyloxy-4,5-dimethyl-1-(2-(trifluoromethyl)benzyl)imidazolium bromide (8c): 330 mg (75%), colorless solid, m.p. 123–125 °C. 1H NMR (600 MHz, CDCl3): δ 1.95, 2.04 (2 s, 3 H each, 2 Me), 5.65, 5.69 (2 s, 2 H each, 2 CH2), 7.38–7.43, 7.50–7.58, 7.61–7.64, 7.71–7.73 (4 m, 3 H, 4 H, 1 H, 1 H), 10.42 (s, 1 H, C(2)H). 13C NMR (151 MHz, CDCl3): δ 7.3, 8.7, 47.8* (q, JC,F = 2.8 Hz, CH2), 84.2, 124.0 (q, 1JC–F = 273.8 Hz, CF3), 124.2, 125.2, 126.8 (q, 3JC,F = 5.6 Hz, CH), 128.2 (q, 2JC,F = 30.9 Hz, i-C), 129.2, 129.6, 130.5, 130.6(br), 130.87, 130.92, 131.9, 133.2(br), 133.4; *through-space C–F coupling. 19F NMR (565 MHz, CDCl3): δ −59.7 (s, CF3). IR (neat): ν 1446, 1312, 1170, 1103, 1040, 951 cm−1. ESI-MS (m/z): 361.7 (100, [M – Br]+). HRMS (ESI-TOF) m/z: [M – Br]+ calcd for C20H20F3N2O 361.1528, found 361.1529.
3-Benzyloxy-4,5-dimethyl-1-[4-(trifluoromethyl)benzyl]imidazolium bromide (8d): 229 mg (52%), colorless solid, m.p. 180–182 °C (decomp.). 1H NMR (600 MHz, CDCl3): δ 1.94, 2.07 (2 s, 3 H each, 2 Me), 5.55, 5.76 (2 s, 2 H each, 2 CH2), 7.37–7.44, 7.47–7.50, 7.58–7.60 (3 m, 3 H, 4 H, 2 H), 11.10 (s, 1 H, C(2)H). 13C NMR (151 MHz, CDCl3): δ 7.3, 9.1, 50.7, 84.1, 123.8 (q, 1JC–F = 272.3 Hz, CF3), 124.4, 125.2, 126.3 (q, 3JC,F = 3.7 Hz, 2 CH), 128.7, 129.3, 130.6, 130.7, 131.3 (q, 2JC,F = 32.7 Hz, i-C), 131.5, 132.8(br), 137.1. 19F NMR (565 MHz, CDCl3): δ −62.8 (s, CF3). IR (neat): ν 1420, 1320, 1141, 1111, 1066, 910, 772 cm−1. ESI-MS (m/z): 361.4 (100, [M – Br]+). Anal. calcd for C20H20BrF3N2O (440.1): C 54.44, H 4.57, N 6.35; found: C 54.32, H 4.70, N 6.44.
3-Benzyloxy-4,5-diphenyl-1-[3-(trifluoromethyl)benzyl]imidazolium bromide (8e): 147 mg (26%), colorless solid, m.p. 107–110 °C. 1H NMR (600 MHz, CDCl3): δ 5.38, 5.70 (2 s, 2 H each, 2 CH2), 6.85 (mc, 1 H), 7.12–7.23, 7.28–7.32, 7.38–7.46, 7.50–7.53, 7.72–7.74 (5 m, 8 H, 3 H, 4 H, 2 H, 1 H), 11.44 (s, 1 H, C(2)H). 13C NMR (151 MHz, CDCl3): δ 51.0, 84.6, 123.5 (q, 1JC–F = 272.4 Hz, CF3), 122.9, 124.3, 125.5 (q, 3JC,F = 3.6 Hz, CH), 125.9 (q, 3JC,F = 3.5 Hz, CH), 128.8, 128.87, 128.94, 129.2, 129.6, 129.7, 130.0, 130.2, 130.4, 130.7, 130.9, 131.0 (q, 2JC,F = 32.6 Hz, i-C), 131.1, 131.2, 132.0 (br), 134.1(br), 134.2. 19F NMR (565 MHz, CDCl3): δ −62.7 (s, CF3). IR (neat): ν 1539, 1457, 1316, 1159, 1122, 1077, 887, 757 cm−1. ESI-MS (m/z): 485.6 (100, [M – Br]+). Anal. calcd for C30H24BrF3N2O (564.1): C 63.73, H 4.28, N 4.95; found: C 63.57, H 4.16, N 4.69.
3-[(3-Methoxy)benzyloxy]-4,5-diphenyl-1-[3-(trifluoromethyl)benzyl]imidazolium bromide (8f): 250 mg (42%), colorless solid, m.p. 132–134 °C. 1H NMR (600 MHz, CDCl3): δ 3.71 (s, 3 H, Ome), 5.37, 5.71 (2 s, 2 H each, 2 CH2), 6.75–6.79, 6.85–6.87, 7.10–7.16, 7.29–7.32, 7.38–7.48, 7.51–7.54, 7.75–7.78 (7 m, 2 H, 2 H, 5 H, 2 H, 4 H, 2 H, 1 H), 11.52 (s, 1 H, C(2)H). 13C NMR (151 MHz, CDCl3): δ 50.9, 55.5, 84.7, 115.2, 116.9, 122.9, 123.0, 123.6 (q, 1JC–F = 272.5 Hz, CF3), 124.3, 125.6 (q, 3JC,F = 3.5 Hz, CH), 126.0 (q, 3JC,F = 3.6 Hz, CH), 128.8, 129.0, 129.66, 129.72, 129.9, 130.1, 130.4, 131.06 (q, 2JC,F = 32.5 Hz, i-C), 131.08, 131.3, 132.2, 132.9(br), 134.1(br), 134.2, 159.9. 19F NMR (565 MHz, CDCl3): δ −62.8 (s, CF3). IR (neat): ν 1599, 1491, 1446, 1327, 1267, 1167, 1115, 1074, 928 cm−1. ESI-MS (m/z): 515.6 (100, [M – Br]+). Anal. calcd for C31H26BrF3N2O2 (594.1): C 62.53, H 4.40, N 4.70; found: C 62.55, H 4.31, N 4.52.
3-[(3,5-Dimethyl)benzyloxy]-4,5-diphenyl-1-[3-(trifluoromethyl)benzyl]imidazolium bromide (8g): 320 mg (54%), colorless solid, m.p. 156–158 °C. 1H NMR (600 MHz, CDCl3): δ 2.18 (s, 6 H, 2 Me), 5.29, 5.73 (2 s, 2 H each, 2 CH2), 6.73, 6.84, 6.93 (3 mc, 2 H, 1 H, 1 H), 7.09–7.15, 7.28–7.31, 7.38–7.49, 7.52–7.55, 7.81–7.83 (5 m, 4 H, 2 H, 4 H, 2 H, 1 H), 11.55 (s, 1 H, C(2)H). 13C NMR (151 MHz, CDCl3): δ 21.1, 50.9, 85.1, 122.9, 123.6 (q, 1JC–F = 272.5 Hz, CF3), 124.4, 125.7 (q, 3JC,F = 3.8 Hz, CH), 126.0 (q, 3JC,F = 3.4 Hz, CH), 128.4, 128.7, 128.9, 129.4, 129.68, 129.72, 130.1, 130.3, 130.7, 131.0 (q, 2JC,F = 32.6 Hz, i-C), 131.1, 131.3, 132.0, 133.0, 134.1(br), 134.3, 138.5. 19F NMR (565 MHz, CDCl3): δ −62.8 (s, CF3). IR (neat): ν 1543, 1446, 1327, 1167, 1118, 1074, 760 cm−1. ESI-MS (m/z): 513.5 (100, [M – Br]+). Anal. calcd for C32H28BrF3N2O (592.1): C 64.76, H 4.76, N 4.72; found: C 64.69, H 4.54, N 4.39.

4.2. In Vitro Cytotoxicity and Antiviral Activity

4.2.1. Cytotoxicity Screening of Compounds at a Concentration of 10 µM

Cytotoxic properties of compounds 1a, 1c, 1a[PF6], 1c[PF6], 9b[PF6], 9c[PF6], 10a–10d, 2a, and 2b were assessed on Vero (CCL-81, Cercopithecus aethiops normal kidney cells), LLC-MK2 (CCL-7, Macaca mulatta normal kidney cells), MRC-5 (CCL-171, Human lung normal fibroblasts), NCTC clone 929 (CCL-1, Mus musculus normal subcutaneous connective tissue cells), and HeLa (CCL-2, Human cervix adenocarcinoma cells) cell lines. Cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).
All tested compounds were dissolved in DMSO (dimethyl sulfoxide, Sigma-Aldrich, Darmstadt, Germany) and then suspended in Minimum Essential Medium (MEM; Sigma-Aldrich, Darmstadt, Germany) supplemented with 2% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, Darmstadt, Germany), 2 mM L-glutamine (Sigma-Aldrich, Darmstadt, Germany), and 100 units/mL penicillin G with 100 mg/mL streptomycin (Sigma-Aldrich, Darmstadt, Germany).
Cells were propagated in Minimum Essential Medium (MEM; Sigma-Aldrich, Darmstadt, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, Darmstadt, Germany) and 100 units/mL penicillin G with 100 mg/mL streptomycin (Sigma-Aldrich, Darmstadt, Germany). Upon reaching 80–90% confluency, cells were harvested with 0.25% trypsin in 1 mM EDTA (Life Technologies, Warsaw, Poland) and seeded into 96-well microplates at 2 × 105 cells/mL. After overnight incubation at 37 °C in a humidified atmosphere containing 5% CO2, the culture medium was replaced with a 100 μL freshly prepared solution of tested compounds diluted with a maintenance medium supplemented with 2% FBS to obtain compound concentrations of 10 µM. The final concentration of DMSO in the medium was 0.1%. All experiments were carried out in triplicate. Compounds treated and untreated cells (control group) were incubated at 37 °C for 48 h in a humidified atmosphere containing 5% CO2.
After incubation with drugs, the cells were treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye solution (MTT, Sigma-Aldrich, Darmstadt, Germany) (25 μL, 5 mg/mL) for 2 h and lysed with solvent solution (100 μL) containing: DMF (Sigma-Aldrich, Darmstadt, Germany) (45 mL), SDS (Sigma-Aldrich, Darmstadt, Germany) (13.5 g), and distilled water (55 mL). After overnight incubation at 37 °C, optical density at 550 nm and a reference wavelength of 670 nm were measured on a microplate spectrophotometer, Varioskan Lux (Thermo Fisher Scientific, Waltham, MA, USA). Compounds demonstrating cell viability ≥50% determined in both cytotoxicity as well as antiviral screening were selected for further studies.

4.2.2. Cytotoxicity Assay in the Range of 0.1–1000 µM on Normal and Cancer Cell Lines

Cytotoxic properties of compounds 1e–1g, 1e[PF6], 8a–8d, and 9a were assessed on seven cell lines, including four normal cell lines: Vero (CCL-81, Cercopithecus aethiops normal kidney cells), LLC-MK2 (CCL-7, Macaca mulatta normal kidney cells), MRC-5 (CCL-171, Human lung normal fibroblasts), and NCTC clone 929 (CCL-1, Mus musculus normal subcutaneous connective tissue cells) and three cancer cell lines: A549 (CCL-185, Human lung carcinoma cells), HeLa (CCL-2, Human cervix adenocarcinoma cells), and HepG2 (HB-8065, Human hepatocellular carcinoma). Cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).
All tested compounds were dissolved in DMSO (dimethyl sulfoxide, Sigma-Aldrich, Darmstadt, Germany) and then suspended in Minimum Essential Medium (MEM; Sigma-Aldrich, Darmstadt, Germany) supplemented with 2% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, Darmstadt, Germany), 2 mM L-glutamine (Sigma-Aldrich, Darmstadt, Germany), and 100 units/mL penicillin G with 100 mg/mL streptomycin (Sigma-Aldrich, Darmstadt, Germany).
Investigated cells were propagated in Minimum Essential Medium (MEM; Sigma-Aldrich, Darmstadt, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, Darmstadt, Germany) and 100 units/mL penicillin G with 100 mg/mL streptomycin (Sigma-Aldrich, Darmstadt, Germany). After reaching 80–90% confluency, cells were harvested with trypsin (Life Technologies, Warsaw, Poland) and seeded into 96-well microplates at 2 × 105 cells/mL. After overnight incubation at 37 °C in a humidified atmosphere containing 5% CO2, the culture medium was replaced with a 100 μL freshly prepared solution of tested compounds diluted with a maintenance medium supplemented with 2% FBS to obtain compound concentrations in the range from 0.1 to 1000 µM. The final concentration of DMSO in the medium was 0.1%. All experiments were carried out in triplicate. Cells exposed to investigated compounds and unexposed cells (control group) were incubated at 37 °C for 48 h in a humidified atmosphere containing 5% CO2 [37,38]. The cytotoxicity was evaluated by the MTT assay.
After the incubation, cells were treated for 2 h with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye solution (MTT, Sigma-Aldrich, Darmstadt, Germany) and lysed with solvent solution. After overnight incubation at 37 °C optical density at 550 nm, and a reference wavelength of 670 nm was measured on a microplate spectrophotometer, Varioskan Lux (Thermo Fisher Scientific, Waltham, MA, USA). The cytotoxic concentration (CC50) was defined as the concentration required to reduce cell viability by 50% compared to untreated controls and was calculated by linear regression analysis of the dose–response curves obtained from the data.

4.2.3. Antiviral Screening of Compounds at a Concentration of 10 µM

Antiviral properties of compounds 1a, 1c, 1a[PF6], 1c[PF6], 9b[PF6], 9c[PF6], 10a–10d, 2a, and 2b were assessed against against five viruses: Human herpesvirus 1 (HSV-1, VR-539), Human parainfluenza virus type 3 (HPIV-3, VR-93), Human adenovirus 5 (AdV5, VR-5), Human herpesvirus 5 (HCMV, VR-977), and Encephalomyocarditis virus (EMCV, VR-1479). Viruses were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).
All tested compounds were dissolved in DMSO (dimethyl sulfoxide, Sigma-Aldrich, Darmstadt, Germany) and then suspended in Minimum Essential Medium (MEM; Sigma-Aldrich, Darmstadt, Germany) supplemented with 2% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, Darmstadt, Germany), 2 mM L-glutamine (Sigma-Aldrich, Darmstadt, Germany), and 100 units/mL penicillin G with 100 mg/mL streptomycin (Sigma-Aldrich, Darmstadt, Germany).
Vero, LLC-MK2, NCTC clone 929, MRC-5, and HeLa cells were propagated in Minimum Essential Medium (MEM; Sigma-Aldrich, Darmstadt, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, Darmstadt, Germany) and 100 units/mL penicillin G with 100 mg/mL streptomycin (Sigma-Aldrich, Darmstadt, Germany). Upon reaching 80–90% confluency, cells were harvested with 0.25% trypsin in 1 mM EDTA (Life Technologies, Warsaw, Poland) and seeded onto 96-well microplates at 2 × 105 cells/mL. After overnight incubation of cells at 37 °C in a humidified atmosphere containing 5% CO2, the culture medium was removed, and cells were inoculated with the respective virus solution in MEM supplemented with 2% FBS and antibiotics (HSV-1 MOI 0.005, 1000 virions/mL; HPIV-3 MOI 0.01, 2000 virions/mL; AdV5 MOI 0.005, 1000 virions/mL, EMCV MOI 0.005, 1000 virions/mL, HCMV 20 PFU (plaque forming units) per well). After a 1-h (HSV-1, HPIV-3, AdV5, and EMCV) or 2-h adsorption period (HCMV), the residual virus was removed, and the infected cells were further incubated with a 100 μL freshly prepared solution of tested compounds diluted with a maintenance medium supplemented with 2% FBS to obtain compound concentrations of 10 µM. The final concentration of DMSO in the medium was 0.1%. All experiments were carried out in triplicate. The cells monolayers were incubated with the compounds at 37 °C in a humidified atmosphere containing 5% CO2 until the typical cytopathic effect (CPE) was visible. Viral infection was evaluated by the MTT assay or plaque reduction assay (HCMV). After incubation with drugs, the cells were treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye solution (MTT, Sigma-Aldrich, Darmstadt, Germany) (25 μL, 5 mg/mL) for 2 h and lysed with solvent solution (100 μL) containing: DMF (Sigma-Aldrich, Darmstadt, Germany) (45 mL), SDS (Sigma-Aldrich, Darmstadt, Germany) (13.5 g), and distilled water (55 mL). After overnight incubation at 37 °C, the optical density at 550 nm and a reference wavelength of 670 nm were measured on a microplate spectrophotometer, Varioskan Lux (Thermo Fisher Scientific, Waltham, MA, USA). The number of HCMV plaques was counted under an inverted microscope Olympus IX73 (Olympus, Tokyo, Japan). Compounds demonstrating cell viability ≥50% determined with both cytotoxicity as well as antiviral screening, were selected for further studies.

4.2.4. Antiviral Activity Assay in the Range of 0.1–1000 µM

Antiviral properties of compounds 1e–1g, 1e[PF6], 8a–8d, and 9a were assessed against five viruses: Human herpesvirus 1 (HSV-1, VR-539), Human parainfluenza virus type 3 (HPIV-3, VR-93), Human adenovirus 5 (AdV5, VR-5), Human herpesvirus 5 (HCMV, VR-977), and Encephalomyocarditis virus (EMCV, VR-1479). Viruses were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).
All tested compounds were dissolved in DMSO (dimethyl sulfoxide, Sigma-Aldrich, Darmstadt, Germany) and then suspended in Minimum Essential Medium (MEM; Sigma-Aldrich, Darmstadt, Germany) supplemented with 2% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, Darmstadt, Germany), 2 mM L-glutamine (Sigma-Aldrich, Darmstadt, Germany), and 100 units/mL penicillin G with 100 mg/mL streptomycin (Sigma-Aldrich, Darmstadt, Germany).
Investigated cells were propagated in Minimum Essential Medium (MEM; Sigma-Aldrich, Darmstadt, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, Darmstadt, Germany) and 100 units/mL penicillin G with 100 mg/mL streptomycin (Sigma-Aldrich, Darmstadt, Germany). After reaching 80–90% confluency, cells were harvested with trypsin (Life Technologies, Warsaw, Poland) and seeded into 96-well microplates at 2 × 105 cells/mL after overnight incubation of cells at 37 °C in a humidified atmosphere containing 5% CO2.
The culture medium was removed from confluent cells grown in 96-well microplates, and the cells were inoculated with virus solutions in MEM supplemented with 2% FBS and antibiotics (HSV-1 MOI 0.005, 1000 virions/mL; HPIV-3 MOI 0.01, 2000 virions/mL; AdV5 MOI 0.005, 1000 virions/mL; EMCV 0.005, 1000 virions/mL; HCMV 20 PFU/well (plaque-forming units per well). After a 1-h (HSV-1, HPIV-3, AdV5, and EMCV) or 2-h adsorption period (HCMV), residual viral particles were removed, and infected cells were further incubated with MEM supplemented with 2% FBS containing compound concentrations in the range from 0.1 to 1000 µM [38]. All experiments were carried out in triplicate. The cell monolayers were incubated with the investigated compounds at 37 °C in a humidified atmosphere containing 5% CO2 until a cytopathic effect (CPE) was visible. Viral infection was evaluated by the MTT assay (as described previously) or plaque reduction assay. HCMV plaques were counted under an inverted microscope, Olympus IX73 (Olympus, Tokyo, Japan). Antiviral activity was expressed as the concentration required to reduce the number of viral plaques to 50% of the control (virus-infected but untreated).

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/molecules27113524/s1: Synthetic protocols for the preparation of imidazole N-oxides 6, imidazoles 7, and copies of 1H and 13C NMR spectra of all the new imidazole N-oxides 6 and imidazolium salts of types 1, 8, and 9. Results of the initial cytotoxicity and antiviral screening of the selected lepidilines and their known analogues, i.e., 1a, 1c, 1a[PF6], 1c[PF6], 2a, 2b, 9b[PF6], 9c[PF6], and 10a–10d).

Author Contributions

Conceptualization, G.M. and M.J.; methodology and organic synthesis, G.M. and M.J.; biological assays, M.D.-B. and A.B.O.; investigation and synthesis, M.K. and M.C.; biological assays, M.D.-B. and A.B.O.; writing—original draft preparation, G.M. and M.J.; biological part, M.D.-B. and A.B.O.; writing—review and editing, G.M., M.J. and A.B.O.; and supervision, G.M. All authors have read and agreed to the published version of the manuscript.

Funding

The authors thank the National Science Center (NCN, Cracow, Poland) for financial support in the framework of the Beethoven-2 grant (#2016/23/G/ST5/04115/1) (G.M., M.K., M.C., and M.J.). The biological part was financed by POL-OPENSCREEN (DIR/WK/2018/06) and the Statutory Found of IBM PAS (M.D.-B. and A.B.O.).

Data Availability Statement

All data are available in this publication.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Leon, J. The “Maca” (Lepidium meyenii), a little known food plant of Peru. Econ. Bot. 1964, 18, 122–127. [Google Scholar] [CrossRef]
  2. Dini, A.; Migliuolo, G.; Rastrelli, L.; Saturnino, P.; Schettino, O. Chemical composition of Lepidium meyenii. Food Chem. 1994, 49, 347–349. [Google Scholar] [CrossRef]
  3. Beharry, S.; Heinrich, M. Is the hype around the reproductive health claims of Maca (Lepidium meyenii Walp) justified? J. Ethnopharmacol. 2018, 211, 126–170. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Cheng, C.; Shen, F.; Ding, G.; Liu, A.; Chu, S.; Ma, Y.; Hou, X.; Hao, E.; Wang, X.; Hou, Y.; et al. Lepidiline A improves the balance of endogenous sex hormones and increases fecundity by targeting HSD17B1. Mol. Nutr. Food Res. 2020, 64, 1900706. [Google Scholar] [CrossRef] [PubMed]
  5. Cui, B.; Zheng, B.L.; He, K.; Zheng, Q.Y. Imidazole Alkaloids from Lepidium meyenii. J. Nat. Prod. 2003, 66, 1101–1103. [Google Scholar] [CrossRef] [PubMed]
  6. Jin, W.; Chen, X.; Dai, P.; Yu, L. Lepidiline C and D: Two new imidazole alkaloids from Lepidium meyenii Walpers (Brassicaceae) roots. Phytochem. Lett. 2016, 17, 158–161. [Google Scholar] [CrossRef]
  7. Mlostoń, G.; Kowalczyk, M.; Celeda, M.; Gach-Janczak, K.; Janecka, A.; Jasiński, M. Synthesis and cytotoxic activity of lepidilines A-D: Comparison with some 4,5-diphenyl analogues and related imidazole-2-thiones. J. Nat. Prod. 2021, 84, 3071–3079. [Google Scholar] [CrossRef]
  8. Mlostoń, G.; Celeda, M.; Poper, W.; Kowalczyk, M.; Gach-Janczak, K.; Janecka, A.; Jasiński, M. Synthesis, selected transformations, and biological activity of alkoxy analogues of lepidilines A and C. Materials 2020, 13, 4190. [Google Scholar] [CrossRef]
  9. del Valle Mendoza, J.; Pumarola, T.; Gonzales, L.A.; del Valle, L.J. Antiviral activity of maca (Lepidium meyenii) against human influenza virus. Asian Pac. J. Trop. Med. 2014, 7, S415–S420. [Google Scholar] [CrossRef] [Green Version]
  10. Sharma, D.; Narasimhan, B.; Kumar, P.; Judge, V.; Narang, R.; De Clerq, E.; Balzarini, J. Synthesis, antimicrobial and antiviral evaluation of substituted imidazole derivatives. Eur. J. Med. Chem. 2009, 44, 2347–2353. [Google Scholar] [CrossRef]
  11. Zhang, L.; Peng, X.-M.; Damu, G.L.V.; Geng, R.-X.; Zhou, C.-H. Comprehensive review in current developments of imidazole-based medicinal chemistry. Med. Res. Rev. 2014, 34, 340–437. [Google Scholar] [CrossRef] [PubMed]
  12. Seck, I.; Nguemo, F. Triazole, imidazole, and thiazole-based compounds as potential agents against coronavirus. Results Chem. 2021, 3, 100132. [Google Scholar] [CrossRef] [PubMed]
  13. Denel-Bobrowska, M.; Olejniczak, A.B. Non-nucleoside structured compounds with antiviral activity—Past 10 years (2010–2020). Eur. J. Med. Chem. 2022, 231, 114136. [Google Scholar] [CrossRef]
  14. Purser, S.; Moore, P.R.; Swallow, S.; Gouverneur, V. Fluorine in medicinal chemistry. Chem. Soc. Rev. 2008, 37, 320–330. [Google Scholar] [CrossRef]
  15. Gillis, E.P.; Eastman, K.J.; Hill, M.D.; Donnelly, D.J.; Meanwell, N.A. Applications of fluorine in medicinal chemistry. J. Med. Chem. 2015, 58, 8315–8359. [Google Scholar] [CrossRef] [PubMed]
  16. Zhou, Y.; Wang, J.; Gu, Z.; Wang, S.; Zhu, W.; Acena, J.L.; Soloshonok, V.A.; Izawa, K.; Liu, H. Next generation of fluorine-containing pharmaceuticals, compounds currently in phase II-III clinical trials of major pharmaceutical companies: New structural trends and therapeutic areas. Chem. Rev. 2016, 116, 422–518. [Google Scholar] [CrossRef]
  17. Arduengo, A.J., III; Harlow, R.L.; Kline, M. A stable crystalline carbene. J. Am. Chem. Soc. 1991, 113, 361–363. [Google Scholar] [CrossRef]
  18. Arduengo, A.J., III. Looking for stable carbenes:  The difficulty in starting anew. Acc. Chem. Res. 1999, 32, 913–921. [Google Scholar] [CrossRef]
  19. Hopkinson, M.N.; Richter, C.; Schedler, M.; Glorius, F. An overview of N-heterocyclic carbenes. Nature 2014, 510, 485–496. [Google Scholar] [CrossRef]
  20. Huynh, H.V. Electronic properties of N-heterocyclic carbenes and their experimental determination. Chem. Rev. 2018, 118, 9457–9492. [Google Scholar] [CrossRef]
  21. Hough, W.L.; Smiglak, M.; Rodriguez, H.; Swatloski, R.P.; Spear, S.K.; Daly, D.T.; Pernak, J.; Grisel, J.E.; Carliss, R.D.; Soutullo, M.D.; et al. The third evolution of ionic liquids: Active pharmaceutical ingredients. New J. Chem. 2007, 31, 1429–1436. [Google Scholar] [CrossRef]
  22. Riduan, S.N.; Zhang, Y. Imidazolium salts and their polymeric materials for biological applications. Chem. Soc. Rev. 2013, 42, 9055–9070. [Google Scholar] [CrossRef] [PubMed]
  23. Mlostoń, G.; Jasiński, M.; Wróblewska, A.; Heimgartner, H. Recent progress in the chemistry of 2-unsubstituted 1H-imidazole 3-oxides. Curr. Org. Chem. 2016, 20, 1359–1369. [Google Scholar] [CrossRef] [Green Version]
  24. Mlostoń, G.; Celeda, M.; Urbaniak, K.; Jasiński, M.; Bakhonsky, V.; Schreiner, P.R.; Heimgartner, H. Synthesis and selected transformations of 2-unsubstituted 1-(adamantyloxy)imidazole 3-oxides: Straightforward access to non-symmetric 1,3-dialkoxyimidazolium salts. Beilstein J. Org. Chem. 2019, 15, 497–505. [Google Scholar] [CrossRef] [Green Version]
  25. Tao, X.-L.; Lei, M.; Wang, Y.-G. Unexpected microwave reaction of 1,3-disubstituted imidazolium slats: A novel synthesis of 1,3-disubstituted imidazole-2-thiones. Synth. Commun. 2007, 37, 399–408. [Google Scholar] [CrossRef]
  26. Laus, G.; Kahlenberg, V.; Wurst, K.; Müller, T.; Kopacka, H.; Schottenberger, H. Synthesis and crystal structures of new 1,3-disubstituted imidazoline-2-thiones. Z. Nat. B 2013, 68, 1239–1252. [Google Scholar] [CrossRef] [Green Version]
  27. López-Lázaro, M. How many times should we screen a chemical library to discover an anticancer drug? Drug Discov. Today 2015, 20, 167–169. [Google Scholar] [CrossRef]
  28. Swallow, S. Fluorine in medicinal chemistry. Prog. Med. Chem. 2015, 54, 65–133. [Google Scholar] [CrossRef]
  29. Isanbor, C.; O’Hagan, D. Fluorine in medicinal chemistry: A review of anti-cancer agents. J. Fluor. Chem. 2006, 127, 303–319. [Google Scholar] [CrossRef]
  30. Cavaliere, A.; Probst, K.C.; Westwell, A.D.; Slusarczyk, M. Fluorinated nucleosides as an important class of anticancer and antiviral agents. Future Med. Chem. 2017, 9, 1809–1833. [Google Scholar] [CrossRef]
  31. Clément, C.; Diaz Grados, D.A.; Avula, B.; Khan, I.A.; Mayer, A.C.; Ponce Aguirre, D.D.; Manrique, I.; Kreuzer, M. Influence of colour type and previous cultivation on secondary metabolites in hypocotyls and leaves of maca (Lepidium meyenii Walpers). J. Sci. Food Agric. 2010, 90, 861–869. [Google Scholar] [CrossRef] [PubMed]
  32. Corazza, O.; Martinotti, G.; Santacroce, R.; Chillemi, E.; Di Giannantonio, M.; Schifano, F.; Cellek, S. Sexual enhancement products for sale online: Raising awareness of the psychoactive effects of Yohimbine, Maca, Horny Goat Weed, and Ginkgo biloba. BioMed Res. Int. 2014, article ID 841798. [Google Scholar] [CrossRef] [Green Version]
  33. Zhao, J.; Muhammad, I.; Dunbar, D.C.; Mustafa, J.; Khan, I.A. New alkamides from Maca (Lepidium meyenii). J. Agric. Food Chem. 2005, 53, 690–693. [Google Scholar] [CrossRef] [PubMed]
  34. Fulmer, G.R.; Miller, A.J.M.; Sherden, N.H.; Gottlieb, H.E.; Nudelman, A.; Stoltz, B.M.; Bercaw, J.E.; Goldberg, K.I. NMR chemical shifts of trace impurities: Common laboratory solvents, organics, and gases in deuterated solvents relevant to the organometallic chemist. Organometallics 2010, 29, 2176–2179. [Google Scholar] [CrossRef] [Green Version]
  35. Diels, O.; Jost, H. Ueber die Darstellung des Diacetyls und ein Polymerisationsproduct desselben. Ber. Dtsch. Chem. Ges. 1902, 35, 3290–3299. [Google Scholar] [CrossRef] [Green Version]
  36. Watson, T.; Taylor, J.; Marks, M.S. CCXCVIII—The configurations of the benzilmonoximes. J. Chem. Soc. 1930, 2302–2307. [Google Scholar] [CrossRef]
  37. Leśnikowski, Z.J.; Paradowska, E.; Olejniczak, A.B.; Studzińska, M.; Seekamp, P.; Schüssler, U.; Gabel, D.; Schinazi, R.F.; Plešek, J. Towards new boron carriers for boron neutron capture therapy: Metallacarboranes and their nucleoside conjugates. Bioorganic Med. Chem. 2005, 13, 4168–4175. [Google Scholar] [CrossRef] [PubMed]
  38. Olejniczak, A.B.; Adamska, A.M.; Paradowska, E.; Studzinska, M.; Suski, P.; Leśnikowski, Z.J. Modification of selected anti-HCMV drugs with lipophilic boron cluster modulator. Acta Pol. Pharm. Drug Res. 2013, 70, 489–504. [Google Scholar]
Molecules 27 03524 g001 550
Figure 1. Structures of naturally occurring alkaloids known as lepidilines (A–D).
Figure 1. Structures of naturally occurring alkaloids known as lepidilines (A–D).
Molecules 27 03524 g001
Molecules 27 03524 g002 550
Figure 2. The structure of N-adamantanyl-functionalized ‘Arduengo salt’ 2a and its bis-oxidized analogue, 2b.
Figure 2. The structure of N-adamantanyl-functionalized ‘Arduengo salt’ 2a and its bis-oxidized analogue, 2b.
Molecules 27 03524 g002
Molecules 27 03524 sch001 550
Scheme 1. Synthesis of fluorinated lepidiline analogues 1e–1g using 2-unsubstituted imidazole N-oxides 6a–6c as the key intermediates.
Scheme 1. Synthesis of fluorinated lepidiline analogues 1e–1g using 2-unsubstituted imidazole N-oxides 6a–6c as the key intermediates.
Molecules 27 03524 sch001
Molecules 27 03524 sch002 550
Scheme 2. O-Benzylation reactions of imidazole N-oxides 6a, b, d, and e with benzyl bromide leading to alkoxyimidazolium salts 8a–8d.
Scheme 2. O-Benzylation reactions of imidazole N-oxides 6a, b, d, and e with benzyl bromide leading to alkoxyimidazolium salts 8a–8d.
Molecules 27 03524 sch002
Molecules 27 03524 sch003 550
Scheme 3. Synthesis of 4,5-diphenyl-functionalized fluorinated alkoxyimidazolium and imidazolium salts 8e–8g and 9a, respectively.
Scheme 3. Synthesis of 4,5-diphenyl-functionalized fluorinated alkoxyimidazolium and imidazolium salts 8e–8g and 9a, respectively.
Molecules 27 03524 sch003
Molecules 27 03524 g003 550
Figure 3. Ion exchange in selected lepidiline-derived imidazolium salts of types 1 and 9 and the structures of imidazole-2-thiones 10 used in this study.
Figure 3. Ion exchange in selected lepidiline-derived imidazolium salts of types 1 and 9 and the structures of imidazole-2-thiones 10 used in this study.
Molecules 27 03524 g003
Table
Table 1. Cytotoxic effect of compounds 1e–1g, 1e[PF6], 8a–8d, and 9a on the normal and cancer cell lines.
Table 1. Cytotoxic effect of compounds 1e–1g, 1e[PF6], 8a–8d, and 9a on the normal and cancer cell lines.
CompoundCC50 [µM]
Normal Cell LinesCancer Cell Lines
VeroLLC-MK2NCTC Clone 929MRC-5HeLaA549HepG2
1e22.667 ± 1.52853.500 ± 2.17979.167 ± 1.44352.667 ± 0.5770.039 ± 0.00137.667 ± 3.786423.333 ± 7.638
1e[PF6]56.333 ± 3.215150.333 ± 2.517148.333 ± 11.547149.667 ± 4.1670.080 ± 0.00138.000 ± 3.464316.667 ± 5.774
1f38.000 ± 4.00059.000 ± 1.00048.333 ± 2.88736.667 ± 2.3090.039 ± 0.0011.600 ± 0.173152.000 ± 2.000
1g0.107 ± 0.01266.167 ± 2.25530.000 ± 2.00054.333 ± 1.1550.053 ± 0.00610.333 ± 1.041130.000 ± 5.000
8a250.000 ± 10.000326.677 ± 7.640256.611 ± 10.410206.170 ± 8.61120.000 ± 3.4607.500 ± 0.500361.667 ± 2.890
8b143.333 ± 2.890256.667 ± 6.77090.667 ± 4.16024.333 ± 2.0807.333 ± 0.7605.500 ± 0.500162.667 ± 6.430
8c53.667 ± 1.53355.000 ± 1.00028.500 ± 0.50045.667 ± 3.79016.000 ± 1.0007.370 ± 0.32095.000 ± 1.000
8d229.333 ± 9.0207.500 ± 1.000283.333 ± 10.41059.667 ± 2.5205.500 ± 0.5005.070 ± 0.120321.670 ± 5.770
9a0.347 ± 0.0060.040 ± 0.0023.750 ± 0.0870.090 ± 0.0100.019 ± 0.0010.035 ± 0.0056.900 ± 0.361
Table
Table 2. Antiviral activity of compounds 1e–1g, 1e[PF6], 8a–8d, and 9a.
Table 2. Antiviral activity of compounds 1e–1g, 1e[PF6], 8a–8d, and 9a.
CompoundIC50 [µM]
HSV-1HPIV-3EMCVHCMVAdV5
1e>22.667>53.500>79.167>52.667>0.039
1e[PF6]>56.333>150.333>148.333>149.667>0.080
1f>38.000>59.000>48.333>36.667>0.039
1g>0.107>66.167>30.000>54.333>0.053
8a>250.000>326.667>256.611>206.170>20.000
8b>143.333>256.667>90.667>24.333>7.333
8c>53.667>55.000>28.500>45.667>16.000
8d>229.333>7.500>283.333>59.667>5.500
9a>0.347>0.040>3.750>0.090>0.019
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Mlostoń, G.; Kowalczyk, M.; Celeda, M.; Jasiński, M.; Denel-Bobrowska, M.; Olejniczak, A.B. Fluorinated Analogues of Lepidilines A and C: Synthesis and Screening of Their Anticancer and Antiviral Activity. Molecules 2022, 27, 3524. https://doi.org/10.3390/molecules27113524

AMA Style

Mlostoń G, Kowalczyk M, Celeda M, Jasiński M, Denel-Bobrowska M, Olejniczak AB. Fluorinated Analogues of Lepidilines A and C: Synthesis and Screening of Their Anticancer and Antiviral Activity. Molecules. 2022; 27(11):3524. https://doi.org/10.3390/molecules27113524

Chicago/Turabian Style

Mlostoń, Grzegorz, Mateusz Kowalczyk, Małgorzata Celeda, Marcin Jasiński, Marta Denel-Bobrowska, and Agnieszka B. Olejniczak. 2022. "Fluorinated Analogues of Lepidilines A and C: Synthesis and Screening of Their Anticancer and Antiviral Activity" Molecules 27, no. 11: 3524. https://doi.org/10.3390/molecules27113524

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