Synthesis, Structural Analysis, and Biological Activities of Some Imidazolium Salts

Four newly synthesized imidazolium salts were characterized by nuclear magnetic resonance, vibrational spectra, and mass spectra. Then, the density functional theory calculations were performed to obtain the molecular configurations on which the theoretical nuclear magnetic resonance and infrared spectra were consequently obtained. The comparison of calculated spectra with the experimental spectra for each molecule leads to the conclusion that the theoretical results can be assumed to be a good approach to their molecular configurations. The in vitro biological activities of the salts on the selected bacteria and cancer cell lines were determined by using the broth dilution method according to Clinical and Laboratory Standards Institute guidelines. The 1,3-bis(2-hydroxyethyl) imidazolidinium bromide and 3-(2-ethoxy-2-oxoethly)-1-(3-aminopropyl)-1H-imidazol-3-ium bromide showed efficiency on Bacillus cereus ATCC 11778. The 3-bis(2-carboxyethyl)-4-methyl-1-H-imidazol-3-ium bromide was effective on HeLa while a similar effect was observed on Hep G2 with 3-(2-carboxyethyl)-1-(3-aminopropyl)-1H-imidazol-3-ium bromide.


Introduction
Imidazole rings are building blocks in amino acids [1] together with the fact that their membership in the development of new antifungal drugs [2,3] and antibiotics [4,5] are crucial. Its derivatives are widely used in other medicinal applications [6]. e pregnane derivatives with imidazole moiety and triazole moiety, for example, were tested on the prostate, breast, and lung cancer cell lines, and dose-effective proliferation of the cells was determined [7]. Similarly, the novel hybrid compounds of imidazole scaffold-based 2-benzylbenzofloran have been prepared and used in cytotoxic activity studies on various cancer cell lines [8].
As a five-membered aromatic ring containing two nonadjacent nitrogen atoms [9], imidazole is also subjected to various computational chemistry research beyond its biological applications. Its ability to capture CO 2 was determined in the investigations of the greenhouse effect compensation in the framework of van der Waals bonded host-guest relation [10]. e hydroxyl conductivity in polymembranes based on imidazole salts was simulated using radial distribution functions and found that the imidazole groups provide better conductivity than that of water and methanol [11]. Moreover, the specific imidazole derivatives exhibited good cross section values for two-photon absorption [12]. e detoxification of phosphotriesters by imidazole rings was clarified comparing the same effects with methylimidazoles depending on the methyl positioning [13]. N-Heterocyclic carbenes (NHCs) are the imidazolebased carbene groups which are isolated and crystallized by the deprotonation of imidazole salts [14]. Also, the imidazole salts naturally transform into NHCs over metal complex building reactions, as exampled on the synthesis and characterization of the silver-NHC complexes [15] and the iron-imidazole salts [16]. e constitution and functions of imidazole ligands in organometallic chemistry and inorganic chemistry have been widely studied, and these particular researches have been evaluated as a scientific competition field due to its importance in the related industry [17]. Also, they are set as alternative to usual ligands in the carbon-carbon coupling reactions of the pharmaceutical reagents [18]. e synthesis and spectroscopic characterizations of four new NHC ligands, namely, 1,3-bis (2-hydroxyethyl) imidazolidinium bromide (L A ),

Instrumentation and Methods.
e 1 H and 13 C NMR spectra of the compounds in deuterium oxide (D 2 O) were recorded on Varian 300 MHz and Varian 75.5 MHz, respectively. e IR spectra by KBr pellets were recorded in the range 450-4000 cm −1 by a PerkinElmer BXII spectrometer. e mass spectra were acquired by the electron impact technique using a ermo Finnegan Trace DSQ GC/MS. Elemental analyses for C, H, and N were realized on the dried samples using a PerkinElmer 2400 CHN analyzer. e absorbance measurements in determining the biological activities of the material were carried out in ermo Scientific Multiskan Go multiplate spectrophotometer.

1,3-Bis(2-hydroxyethyl) Imidazolidinium Bromide, L A .
Imidazole (10 mmol, 0.68 g) was dissolved in tetrahydrofuran (THF), and bromoethanol (22 mmol, 2.75 g) was added as the mixture was stirred for 20 hours. e completion of the reaction was monitored by thin-layer chromatography (TLC) in ethyl acetate/hexane (1 : 5) analyses, and the solid residue was filtered out with a sintered glass funnel. e solvent in the filtrate was evaporated using a rotary evaporator, and the product was dried in a vacuum desiccator. After that, the product was purified by column chromatography (ethyl acetate/hexane, 1 : 5). e best yield was obtained when the reaction was carried out at room temperature with a 1 : 2 mole ratio of the reagents. 1.54 g of the final product was obtained with 65% yield. It was in yellowish liquid form. e elemental analyses result for L A with the chemical formula C 7 H 13 BrN 2 O 2 are C, 35.46%; H, 5.53%; and N, 11.82%; found: C, 35.35%; H, 5.43%; and N, 11.73%. e mass spectroscopy reads (m/z) 158.22 (M + H) + which is consistent with the expected molecular weight.
e procedure applied in the synthesis of L A was also used for the synthesis of L C by replacing imidazole and bromoethanol with 4-methylimidazole (10 mmol, 0.82 g) and 3-bromopropionic acid (22 mmol, 3.366 g), respectively. After that, the product was purified by column chromatography (ethyl acetate/hexane, 1 : 5

3-(2-Carboxyethyl
e procedure applied in the synthesis of L B was also used for the synthesis of L D by replacing ethyl bromoacetate with 3-bromopropanoic acid (11 mmol, 1.683 g), respectively. e product was purified by column chromatography (ethyl acetate/hexane, 1 : 5). It was pale brown oily liquid. 1 Table 1: Some selected geometrical parameters of the common properties of the investigated molecules. e data of the molecule showed by L in the last column reflect the X-ray diffraction measurements and calculated values on 1,3-bis(acetamide)imidazol-3ium bromide as given in [22]. e numbers in the paranthesis indicates the experimental error margins on the specific measurements. e labeled and numbered atoms in the first column are presented for each molecule in Scheme 1. bacteria or the yeast culture, and 20 μl of the chemical compound solution. All microplates were incubated at 37°C for 24 hr. e absorbance was measured at 600 nm.

Cytotoxicity.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to observe the cytotoxicity of synthesized compounds on human cervical cancer cell line (HeLa), human liver cancer cell line (Hep G2), and healthy mouse embryonic fibroblast cell line (MEF). All cell lines were provided with American Type Culture Collection (ATCC, Manassas, VA, USA). A certain population of the cells were incubated in Dulbecco's modified Eagle's Medium (DMEM) provided with Life Technologies GIBCO, Grand Island, NY, USA involving 1% penicillin streptomycin and 1% L-glutamine and HAMS F12 (1 : 1) broth medium at 37°C under 5% CO 2 . e cells were planted in 96-multiwell plates with approximately equal numbers of 10 5 , and they were allowed further incubation for 24 hr. e seven different doses of each compound were applied to the cancer cell lines and the MEF cells. e applied doses were 400 μm, 200 μm, 100 μm, 50 μm, 25 μm, 12.5 μm, and 6.25 μm, and the dose application time was 24 hr. e surviving control for each cell was carried out against the cells not exposed to any dose. en, 20 μl/plate of 5 mg/ml MTTsolution was added into the each well and left to further incubation between 2 and 4 hr. e excess MTT solutions were removed from the wells, and 200 μl of ultra-pure DMSO was added. e set was left in dark for 5 min before measuring the color intensities on a 492 nm spectrophotometer.

Computational Modeling.
All calculations were carried out in the framework of Gaussian 09 package. e molecules were optimized in their ground state using Becke3-Lee-Yang-Parr (B3LYP) exchange correlation functional method and 6-311G + (2d, p) basis set within density functional theory (DFT). en, the NMR spectra and the IR spectra were calculated on the optimized geometries using the same method and the same basis set. e gaugeindependent atomic orbital (GIAO) method was adopted to acquire theoretical 1 H and 13 C NMR shifts, which were   converted to that of tetramethylsilane scale. e IR spectra were scaled by the factor 0.9613 due to the theoretical miscalculations [19][20][21]. e vibrational modes were assigned by observing the animation property of the frequency calculations provided with Gaussian 09 package.

Result and Discussion
NMR and IR spectra are reliable methods to elucidate an organic material or some of metal complexes. ey can also be used to verify calculated molecular structure by comparing calculated spectra with corresponding experimental spectra and thus determining structural parameters and complete description of chemicals investigated.

Molecular Structures.
e NMR measurements were taken in dilute D 2 O solution, while the IR spectra were recorded in the solid KBr pellet. erefore, NMR and infrared spectra may reflect different molecular structures as the molecule surrounded by D 2 O molecules in NMR spectra while the intermolecular interactions exist in the IR spectra, especially on -CH and -OH bonds [22]. On the theoretical calculation side, the crystalline phase calculations were excluded due to the single crystal form of the molecules obtained for X-ray analysis. e gas phase calculations were adopted considering differences with other phases with 2% maximum error margin which were especially on the -CH and -OH bond lengths. e process of the theoretical modeling of the molecules has initially been realized by using the potential energy surface scanning method for the selected dihedral angles of each molecule, thus obtaining the lowest energy conformers in the gas phase, and the resulting optimized conformers are given in Scheme 1. e imidazole rings except that of L C with its CH 3 attachment and the CH 2 groups directly bonded to the nitrogen of the rings are common in the all molecules.  Table 1. Although the molecules differ from each other by their moieties bonded to the imidazole rings, the bond lengths, the bond angles, and the dihedral angles belong to the ring, and its immediate vicinity are in good agreement with each other and with the corresponding X-ray diffractometer (XRD) results of previously investigated similar molecule 1,3-bis(acetamide) imidazol-3-ium bromide which was crystallized successfully [22]. e dihedral angles chosen on the imidazole ring are either about 0°or 180°, implying the aromatic structure as expected. e C-N bond lengths which bind the rings and the aliphatic moieties, that is, C 4 -N 2 and N 1 -C 5 are equal in L A reflecting the symmetric structure of the molecule. However, these are different in L B , L C , and L D because of the different moieties on the both side of their aromatic rings. Although the symmetry of L C was broken by the C-CH 3 group instead of C-H in the ring, no drastic changes were observed in the concerning ring parameters. It can be inferred that the common geometrical parameters are consistent with each other with a priori theoretical confirmations before NMR and IR spectra clarifications of the calculated molecular structures.

Nuclear Magnetic Resonance Spectra.
e experimental and theoretical chemical shifts of 1 H NMR and 13 C NMR spectra of L A , L B , L C , and L D molecules are given in Table 2.
e calculations were carried out in the gas phase considering their optimized geometries given in Scheme 1. e proton signals for -NH 2 and -OH in all were absent in the experimental 1 H NMR spectra because the solvent was D 2 O concerning the solubility of the specimens which exchanged NH 2 and OH protons with deuterium. Although their detailed analysis left to the IR spectra in the following section, the calculated shifts δ 1.73 ppm of OH in L A and δ 0.5 ppm and δ 0.7 ppm of NH 2 in L B and L D , respectively, are in the typical chemical shift range of R-NH 2 and R-OH groups. e imidazole proton (NCHN, H 1 ) shifts of L A , L C , and L D with 9.06 ppm, 8.84 ppm, and 9.11 ppm, respectively, are noticeably bigger than that of L B with 7.79 ppm. at can be due to the intramolecular interactions of the NCHN

3bis(2-hydroxyethyl) imidazolidinium bromide, (b) 3-(2-ethoxy-2-oxoethly)-1-(3-aminopropyl)-1H-imidazol-3-ium bromide, (c) 1,3-bis(2-carboxyethyl)-4-methyl-1H-imidazol-3-ium bromide, and (d) 3-(2-carboxyethyl)-1-(3-aminopropyl)-1H-imidazol-3-ium bromide.
hydrogen with the close oxygen atoms since the oxygen reduces the electron density on the hydrogen depending on the distance between them, thus causing higher NMR shifts. Indeed, the H 1 -O 1A in L A , the H 1 -O 1C in L C , and the H 1 -O 1D in L D are 2.61Å, 2.42Å, and 2.41Å, respectively. ese distances which were measured from the theoretical models of the molecules are quite longer than that 2.85Å distance of the H 1 -O 1B measured in L B which results in small shifts for the H 1 of L B . is is supported by the fact that the other imidazole proton shifts of H 2 and H 3 are all in close values about 7.7 ppm for all molecules as no distinct intermolecular interactions possible on H 2 and H 3 in any of them. In addition, the NMR signals of CH 2 protons in the aliphatic chains of all molecules and the smallest proton shifts of CH 3 protons in L B and L C molecules are theoretically and experimentally in agreement with the expected NMR results.
ese NMR data are in accordance with the previous study on a similar imidazole salt [23,24] in which the protons belong to the aliphatic chain found in 4.15 ppm-2.08 ppm, the carbons belong to the imidazole ring, and the aliphatic chain is found in 139.2 ppm-123 ppm and 62.5 ppm-33.9 ppm as in this study. e eight R-squared tests using the data in Table 2 provide least 99.8% agreement between the experimental and the theoretical 1 H and 13 C NMR for L A , L B , L C , and L D . e theoretical results for NMR are in very good agreement with the experimental results as well as the observation of the expected specific values. us, one can infer that the calculated atomic configurations of the all molecules are good estimations except the exclusion of the interchangeable hydrogens. Figure 1, the experimental infrared spectra of L A , L B , L C , and L D in the 450-4000 cm −1 region are given against their IR spectra calculations. e detailed account of the IR spectra including the in-plane vibrations of the stretching, scissoring, and rocking and the out-of-plane vibrations of the wagging and twisting for each molecule is presented for L A , L B , L C , and L D in Tables 3-6, respectively. Table 3: e experimental and theoretical vibrational wave numbers for the infrared spectra of 1,3-bis (2-hydroxyethyl) imidazolidinium bromide (L A ) with its symbolled and numbered atoms in Figure 1 Figure 1 ], stretching; δ, in-plane bending; c, out-of-plane bending; s, symmetric; as, asymmetric; sc, scissoring; r, rocking; t, twisting; w, wagging.

experimental and theoretical vibrational wave numbers for the infrared spectra of 3-(2-ethoxy-2-oxoethly)-1-(3-aminopropyl)-1H-imidazol-3-ium bromide (L B ) with its symbolled and numbered atoms in
Bioinorganic Chemistry and Applications 7 in agreement with the previously observed ν(C�O) stretching [25] and theoretically calculated values as reflected in Table 3. e imidazole ν(C�N) stretching of L A appears as individual signals while they are coupled under broad peaks with L B , L C , and L D molecules as seen about 1650 cm −1 while the frequencies for ν(C�N) stretching are in agreements with the corresponding data as given in [26]. e aliphatic ν(C-N) stretching in 1069-1235 cm −1 interval and the aliphatic ν(C-C) stretching in 917-1099 cm −1 are consistent with the previous corresponding measurements [27].
Beyond the consistency of the common imidazole properties of the molecules, we also give unique infrared signals of molecules in Table 3. C-O stretching belong to the H 2 C-OH group in L A , in-plane δ vibrations of CH 3 at the end of the aliphatic chain of L B , and at the attachment of the imidazole ring of L C are such unique vibrations. e calculated and observed wave numbers of these vibrations are consistent with the concerning previous studies [28,29]. e molecules in consideration analyzed by infrared spectrum because of the lack of exchangeable protons of -OH and -NH 2 in the NMR spectra of the chemicals and the existence of these groups is proved in the molecules. Also, the IR spectra of the molecules reverifies the theoretically obtained configurations of them as they were first verified by the comparison the theoretical NMR spectra with that of the experimental NMR spectra.

Antibacterial and Cytotoxic Activities.
e antibacterial tests of the synthesized four molecules were run on the six Table 5: e experimental and theoretical vibrational wave numbers for the infrared spectra of 1,3-bis(2-carboxyethyl)-4-methyl-1Himidazol-3-ium bromide (L C ) with its symbolled and numbered atoms in Figure 1(c). yeast as much as the antibiotics. L C and L D show no noticeable inhibitory effects on the target bacteria, and thus, their absorbance values as function of their concentration were not given for the sake of brevity. Figure 3(a) shows the cytotoxic activity of L C on HeLa and Hep G2 cell lines against healthy MEF cell lines, as does Figure 3(b) for the cytotoxic activity of L D . e cytotoxic activities of L A and L B are not exhibited as they showed lesser cytotoxic activities in comparison with L C and L D . e percentage cell viability of HeLa and Hep G2 cell lines was significantly reduced by L C and L D at the end of 24 hr application of the doses. In addition, L C did not harm the healthy MEF cell lines for any dose so that its half inhibitory concentrations (IC 50 ) cannot be calculated. Meantime, L D showed some activity on the MEF cells with the high doses together with the fact that the activity was not as strong as it did on the cancer cell lines. e antibacterial and cytotoxic effectiveness of the synthesized molecules are summarized in Table 7 by presenting their IC 50 concentrations in the units of µM on the bacteria together with the IC 50 values of the antibiotics and on the cancer cell lines. e lack of the IC 50 values of L C and L D for antibacterial activity in Table 7 indicates that the IC 50 values of them cannot be calculated due to their very weak effect on the bacteria sample within the dose range considered. L A and L B were, respectively, thrice and twice more effective on Bacillus cereus than the antibiotic (gentamicin) as the L A equals the antibiotic (amphotericin-b) effect on Candida albicans.   e cell viability assay of the chemicals exhibits no harmful effect on the healthy MEF cell lines as their IC 50 values cannot be calculated within the dose range considered. e L C inhibition on HeLa and the L D inhibition on Hep G2 are distinctive when they are compared with the inhibition of the other imidazole-based chemicals on different cancer cell lines in the concerning studies. e IC 50 values of the L C on HeLa and the L D on Hep G2 were 81 µM and 57 µM, respectively. e similar imidazole compounds which have alkyl moieties were tested on the cancer cells different from the cells used in this study [8]. e IC 50 values of 1-(benzofuran-2-yl (phenyl)methyl)-3-allyl-2-ethyl-1H-imidazol-3-ium bromide and 1-(benzofuran-2-yl(phenyl)methyl)-3-butyl-2-ethyl-1Himidazol-3-ium iodide on leukemia (HL-60), lung carcinoma (A549), colon carcinoma (SW480), breast carcinoma (MCF-7), and myeloid liver carcinoma (SMMC-7721) cancer lines have been detected over 40 µM. Additionally, the IC 50 activity of 3β-hydroxy-21-(1H-imidazol-1-yl)pregna-5,16dien-20-one on prostate cancer (PC-3), breast cancer (MCF7), and lung cancer (SK-LU-1) were 20 µM, 19 µM, and 18 µM, respectively [7]. ese results are quantitatively better than 81 µM and 57 µM on Hep G2 and HeLa. However, this is compensated by the fact that the L C and L D have not any harmful effect on the healthy MEF cell lines.

Conclusions
Novel imidazole salts, or N-heterocyclic carbene ligands, namely, 1,3-bis(2-hydroxyethyl) imidazolidinium bromide L A , 3-(2-ethoxy-2-oxoethly)-1-(3-aminopropyl)-1H-imidazol-3-ium bromide L B , 1,3-bis(2-carboxyethyl)-4-methyl-1H-imidazol-3ium bromide L C , and 3-(2-carboxyethyl)-1-(3-aminopropyl)-1H-imidazol-3-ium bromide L D were synthesized, and they were preliminary confirmed by GC-MS and elemental analysis methods. eir molecular structures were theoretically determined, and they were confirmed by comparing calculated 1 H, 13 C NMR, and IR spectra with those of experimentally observed data. Also, the calculated structures were verified by the XRD results on a similar imidazole salt [22]. e antimicrobial and cytotoxic activities of the synthesized ligands on some specific bacteria and cancer cell lines were measured using spectrophotometric methods. It is seen that L A showed better inhibition than the selected antibiotic on Bacillus cereus ATCC 11778 while it is effective on the selected bacteria and the yeast together with L B . On the cytotoxicity evaluation, L C showed considerable inhibition effect on HeLa, as does L D on Hep G2. Although their IC 50 doses are quite high in comparison with the similar chemicals in the literature, the cytotoxicity of L C and L D is affirmed by not causing harmful effect on the healthy MEF cells as much as they do on the cancel cell lines.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.