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

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.


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, oxaanalogues 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 heteroorganic 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 metaposition 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).

Chemistry
The preparation of lepidilines 1a and 1c was performed following the general procedure described previously employing the respective 2-unsubstituted 4,5dimethylimidazole 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]. 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 CF 3 and OCF 3 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). 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 heteroorganic 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 metaposition 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).

Chemistry
The preparation of lepidilines 1a and 1c was performed following the general procedure described previously employing the respective 2-unsubstituted 4,5dimethylimidazole 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].

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 CF 3 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-4,5dimethylimidazoles 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.
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 Noxides 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-4,5dimethylimidazoles 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. Scheme 1. Synthesis of fluorinated lepidiline analogues 1e-1g using 2-unsubstituted imidazole Noxides 6a-6c as the key intermediates.
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 Oalkylation. 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).
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 CHCl 3 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 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 In extension of the study, collection of lepidiline analogues with a 4,5-dimethylsubstituted imidazolium core was supplemented by 4,5-diphenyl derivative functionalized with the m-CF 3 -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 1,4,5trisubstituted 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 extension of the study, collection of lepidiline analogues with a 4,5-dimethylsubstituted 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. Scheme 3. Synthesis of 4,5-diphenyl-functionalized fluorinated alkoxyimidazolium and imidazolium salts 8e-8g and 9a, respectively.
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 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[PF 6 ] and 9[PF 6 ] by counterion exchange via treatment with ammonium hexafluorophos- phate 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).  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).

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

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[PF 6  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[PF 6 ], 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 CC 50 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[PF 6 ], and 9a were found to be the most cytotoxic against the HeLa cell line, with CC 50 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[PF 6 ]) were less active (CC 50 = 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 OCF 3 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 PF 6 − (compound 1e[PF 6 ]) 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[PF 6 ], and 8a-8d) revealed that oxidized analogues 8a-8d were much less active against HeLa cells, with CC 50 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[PF 6 ].
Lepidiline 9a also showed high cytotoxicity in the A549 cell line, but other nonoxidized lepidilines: 1e-1g and 1e[PF 6 ] 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 (CC 50 = 5.070-7.500 µM) than against the HeLa cell line (CC 50 = 5.500-20.000 µM).

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.
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.

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 CF 3 (or OCF 3 ) 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[PF 6 ], 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[PF 6 ], 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].

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 ( 1 H NMR (600 MHz), 13 C NMR (151 MHz), and 19 F NMR (565 MHz); chemical shifts are relative to the residual undeuterated solvent peaks (CDCl 3 : 1 H NMR δ = 7.26, 13 C NMR δ = 77.16 [34]) or to the external standard (CFCl 3 : 19 F 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.

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 Et 2 O (5 × 5 mL), and the solid imidazolium chloride was recrystallized from a CH 2 Cl 2 /hexane mixture.
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 × 10 5 cells/mL. After overnight incubation at 37 • C in a humidified atmosphere containing 5% CO 2 , 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% CO 2 .