A Novel Ag-N-heterocyclic Carbene Complex Bearing the Hydroxyethyl Ligand: Synthesis, Characterization, Crystal and Spectral Structures and Bioactivity Properties

: In this study, a novel silver N-heterocyclic carbene (Ag-NHC) complex bearing hydroxyethyl substituent has been synthesized from the hydroxyethyl-substituted benzimidazolium salt and silver oxide by using in-situ deprotonation method. A structure of the Ag-NHC complex was characterized by using UV-Vis, FTIR, 1 H-NMR and 13 C-NMR spectroscopies and elemental analysis techniques. Also, the crystal structure of the novel complex was determined by single-crystal X-ray di ﬀ raction method. In this paper, compound 1 showed excellent inhibitory e ﬀ ects against some metabolic enzymes. This complex had Ki of 1.14 0.26 µ M against human carbonic anhydrase I (hCA I), 1.88 ± 0.20 µ M against human carbonic anhydrase II (hCA I), and 10.75 ± 2.47 µ M against α -glycosidase, respectively. On the other hand, the Ki value was found as 25.32 ± 3.76 µ M against acetylcholinesterase (AChE) and 41.31 ± 7.42 µ M against butyrylcholinesterase (BChE), respectively. These results showed that the complex had drug potency against some diseases related to using metabolic enzymes.


Introduction
Medical applications of the silver metal were discovered a long time ago [1]. The most commonly used of silver compounds are silver nitrate and silver sulfonamides [2]. Today, most organisms have been known to develop antimicrobial resistance to drugs. Therefore, researchers have tried to develop novel, stronger and multimodal alternatives that have the least antibiotic effects on the human body [3]. At the clinical stage, silver has been shown to exhibit broad activity against antibiotics including those resistant to deadly microbes [4]. In recent studies, silver complexes exhibit less toxicity to the human body than other metal complexes, which have made them desirable antimicrobial substances [5].
The organic ligands, which coordinated to the silver metal center, make important contributions to biological activity. Among them, N-heterocyclic carbenes (NHCs) are one of the most important organic Also, FTIR spectra assay were kept in the range 400-4000 cm −1 on Perkin Elmer Spectrum 100 FTIR spectrometer (Waltham, MA, USA). The UV spectrum was measured with Shimadzu UV-1601 instrument (Duisburg, Germany). The 1 H and 13 C NMR spectra were recorded using either a Bruker 400 Merkur spectrometer (Billerica, MA, USA) operating at 1 H NMR (400 MHz) and 13 C NMR (100 MHz) in DMSO-d 6 with tetramethylsilane as an internal reference by Inonu University Catalysis Research and Application Center in Malatya, Turkey. Elemental analyses were performed by Inonu University Scientific and Technology Centre (Malatya, Turkey) on LECO, CNHS932 Elemental Analyzers (Haan, Germany).

Biochemical Studies
The inhibiting effects of the Ag-NHC complex 1 on both carbonic anhydrase isoenzymes (hCA I and II) are described by Verpoorte et al. [32] and detailed in previous studies [33,34] and recorded spectrophotometrically using p-nitrophenylacetate substrate (PNA) at 348 nm [35,36]. Indeed, the BChE and AChE inhibitory effects of Ag-NHC complex 1 were performed according to the Ellman's assay [37] and spectrophotometrically recorded at 412 nm [38]. Butyrylthiocholine iodide and acetylthiocholine iodide was substrates for both enzymatic reaction. On the other hand, 5,5 -dithio-bis (2-nitro-benzoic) acid molecule was used to measure AChE and BChE activities, respectively [39]. Additionally, the α-glycosidase inhibitory effect of Ag-NHC complex 1 was carried out using the p-nitrophenyl-D-glycopyranoside molecule (p-NPG) as the substrate according to the method of Tao et al. [40] Samples of this work were prepared by dissolving as mg/mL. This assay was performed according to previous studies [41,42].

X-ray Crystallography
X-ray single crystal diffraction data for Ag-NHC complex 1 was collected at room temperature on a Rigaku-Oxford Xcalibur diffractometer (Oxford, UK) with an EOS-CCD detector using graphite-monochromatic MoKα radiation (λ = 0.71073 Å) with CrysAlisPro software (Oxford, UK) [43]. Data reduction and analytical absorption correction were performed by CrysAlisPro program [44]. Utilizing Olex2 [45], structure was solved using Intrinsic Phasing method with SHELXT [46] and refined by full-matrix least squares on F2 in SHELXL [47]. Anisotropic thermal parameters were applied to all non-hydrogen atoms. All hydrogen atoms were placed using standard geometric models and with their thermal parameters riding on those of their parent atoms. Some positional disorders were observed for hydroxyethyl groups in the structure, and to ensure satisfactory refinement of these disordered hydroxyethyl groups, constraint and restraint instructions such as EADP, DFIX, and DELU were applied. A summary of crystal data, experimental details, and refinement results for the Ag-NHC complex 1 is given in Table 1. Crystallographic data as cif file for the structure reported in this paper

Synthesis
The Ag-NHC complex 1 bearing hydroxyethyl-liganded have illustrated in Scheme 1. The complex was synthesized from the hydroxyethyl-substituted benzimidazolium salt [28] and silver oxide via in-situ deprotonation method. The reaction mixture was stirred in dark during 48 h. at room temperature. The Ag-NHC complex 1 was obtained as a white solid at 80% yield. The novel stable complex was well soluble in halogenated solvents such as dichloromethane and chloroform. Also, the Ag-NHC complex 1 was well soluble in polar solvents such as dimethylsulfoxide and dimethylformamide. But, this complex was less soluble in polar solvents such as water and ethanol.

Synthesis
The Ag-NHC complex 1 bearing hydroxyethyl-liganded have illustrated in Scheme 1. The complex was synthesized from the hydroxyethyl-substituted benzimidazolium salt [28] and silver oxide via in-situ deprotonation method. The reaction mixture was stirred in dark during 48 h. at room temperature. The Ag-NHC complex 1 was obtained as a white solid at 80% yield. The novel stable complex was well soluble in halogenated solvents such as dichloromethane and chloroform. Also, the Ag-NHC complex 1 was well soluble in polar solvents such as dimethylsulfoxide and dimethylformamide. But, this complex was less soluble in polar solvents such as water and ethanol. Scheme 1. Synthesis of the hydroxyethyl-substituted Ag-NHC complex 1.

NMR Study
The structure of the Ag-NHC complex 1 was characterized by 1 H NMR and 13 C NMR spectroscopic methods. When the 1 H NMR data was examined, the characteristic proton peak that observed at Crystals 2020, 10, 171 5 of 15 10.42 ppm for the starting benzimidazolium salt [28] was not observed in the novel Ag-NHC complex. The -CH 3 and -CH 2 proton peaks belonging to the ethyl group (-NCH 2 CH 3 ) have been observed as triplets at 1.48 ppm (J = 7.2 Hz) and as multiplets at 4.55 ppm, respectively. The two -CH 2 proton peaks belonging to the hydroxyethyl group (-NCH 2 CH 2 OH) have been observed as triplet at 3.87 ppm (J = 5.2 Hz) and as multiplets at 4.58 ppm respectively. The broad (wide) singlet peak was observed at 5.12 ppm for hydroxy proton in the 1 H NMR spectrum. The aromatic proton peaks of benzimidazole have observed as multiplited between 7.42 and 7.83 ppm. When the 13 C NMR data was examined, the characteristic carbon peak that observed at 141.7 ppm for the 2-CH of the starting benzimidazolium salt [28] was not observed in the novel Ag-NHC complex. Furthermore, the characteristic Ag-C carbene resonance for the novel Ag-NHC complex 1 was observed in the 13 C NMR spectra appeared highly downfield at 190.0 ppm. The -CH 2 aliphatic carbon atom bonding to -OH on the hydroxyethyl group has been observed at 60.9 ppm. The -CH 2 aliphatic carbon atom bonding to the nitrogen on the hydroxyethyl group (-NCH 2 CH 2 OH) has been observed at 51.7 ppm. The -CH 3 and -CH 2 aliphatic carbon peaks belonging to the ethyl group (-NCH 2 CH 3 ) have been observed at 16.5 and 44.2 ppm respectively. The aromatic carbon peaks of benzimidazole observed between 112.2 and 134.4 ppm. All 1 H NMR data ( Figure 1) and 13 C NMR data ( Figure 2) for the novel Ag-NHC complex 1 have compatible with the literature [6,7]. The structure of the Ag-NHC complex 1 was characterized by 1 H NMR and 13 C NMR spectroscopic methods. When the 1 H NMR data was examined, the characteristic proton peak that observed at 10.42 ppm for the starting benzimidazolium salt [28] was not observed in the novel Ag-NHC complex. The -CH3 and -CH2 proton peaks belonging to the ethyl group (-NCH2CH3) have been observed as triplets at 1.48 ppm (J = 7.2 Hz) and as multiplets at 4.55 ppm, respectively. The two -CH2 proton peaks belonging to the hydroxyethyl group (-NCH2CH2OH) have been observed as triplet at 3.87 ppm (J = 5.2 Hz) and as multiplets at 4.58 ppm respectively. The broad (wide) singlet peak was observed at 5.12 ppm for hydroxy proton in the 1 H NMR spectrum. The aromatic proton peaks of benzimidazole have observed as multiplited between 7.42 and 7.83 ppm. When the 13 C NMR data was examined, the characteristic carbon peak that observed at 141.7 ppm for the 2-CH of the starting benzimidazolium salt [28] was not observed in the novel Ag-NHC complex. Furthermore, the characteristic Ag-Ccarbene resonance for the novel Ag-NHC complex 1 was observed in the 13 C NMR spectra appeared highly downfield at 190.0 ppm. The -CH2 aliphatic carbon atom bonding to -OH on the hydroxyethyl group has been observed at 60.9 ppm. The -CH2 aliphatic carbon atom bonding to the nitrogen on the hydroxyethyl group (-NCH2CH2OH) has been observed at 51.7 ppm. The -CH3 and -CH2 aliphatic carbon peaks belonging to the ethyl group (-NCH2CH3) have been observed at 16.5 and 44.2 ppm respectively. The aromatic carbon peaks of benzimidazole observed between 112.2 and 134.4 ppm. All 1 H NMR data ( Figure 1) and 13 C NMR data ( Figure 2) for the novel Ag-NHC complex 1 have compatible with the literature [6,7].

FTIR Study
Herein, the FTIR spectroscopy has been used to describe the functional groups available in the complex. The FTIR spectrum of the novel Ag-NHC complex 1 has illustrated in Figure 3. It has been recorded for the wavenumbers region between 4000 and 450 cm −1 . When the investigation of the FTIR spectrum, the symmetrical C-H stretching frequency of the benzene rings observed intense at 3119 and 3155 cm −1 . The symmetrical C-H stretching frequency of the -CH 2 -and -CH 3 [48,49]. According to the literature, Ag-C stress vibrations are expected in the frequency region of 400-155 cm −1 [50].

FTIR Study
Herein, the FTIR spectroscopy has been used to describe the functional groups available in the complex. The FTIR spectrum of the novel Ag-NHC complex 1 has illustrated in Figure 3. It has been recorded for the wavenumbers region between 4000 and 450 cm −1 . When the investigation of the FTIR spectrum, the symmetrical C-H stretching frequency of the benzene rings observed intense at 3119 and 3155 cm −1 . The symmetrical C-H stretching frequency of the -CH2-and -CH3 groups in the ethyl and the -CH2groups in the hydroxyethyl becomes intense at 2869, 2934 and 2975 cm −1 . The symmetrical C=C-C stretching frequency of the benzene rings become intense at 1555 cm −1 . The symmetrical conjugated C=C bond stretching frequency of the benzene rings becomes intense at 1344 cm −1 . The band at 1039 cm −1 and 1054 cm −1 corresponds to the primary alcohol (hydroxyethyl group) C-O stretching mode. The band at 1288 cm −1 corresponds to the primary alcohol (hydroxyethyl group) O-H in-plane bending vibration. The symmetrical for Ccarbene-N stretching frequency in the benzimidazole group observed intense at 1397 cm −1 [48,49]. According to the literature, Ag-C stress vibrations are expected in the frequency region of 400-155 cm −1 [50].

UV-Vis Study
The UV-Vis spectra of the novel Ag-NHC complex of dissolved in chloroform at 25 °C showed up four absorption bands at 220, 250, 270 and 320 nm, respectively. The UV-Vis spectra of the novel Ag-NHC complex 1 were recorded in (CHCl3) solutions at a concentration of 15 or 10 µM and were depicted in Figure 4 with a range 200～400 nm. The novel Ag-NHC complex 1 showed a new absorption peak at 320 related to MLCT (metal-ligand charge transfer) (Ag + to NHC ligand) ( Figure  4) [51]. This peak is known as the wide range bands, both π → π *, n → π * and d-d transitions of (C = N) and charge-transfer transition arising from π electron interactions between metal and ligand that involves either a metal-to-ligand or ligand-to-metal electron transfer [52]. The absorption bands

UV-Vis Study
The UV-Vis spectra of the novel Ag-NHC complex of dissolved in chloroform at 25 • C showed up four absorption bands at 220, 250, 270 and 320 nm, respectively. The UV-Vis spectra of the novel Ag-NHC complex 1 were recorded in (CHCl 3 ) solutions at a concentration of 15 or 10 µM and were Crystals 2020, 10, 171 7 of 15 depicted in Figure 4 with a range 200~400 nm. The novel Ag-NHC complex 1 showed a new absorption peak at 320 related to MLCT (metal-ligand charge transfer) (Ag + to NHC ligand) (Figure 4) [51]. This peak is known as the wide range bands, both π → π *, n → π * and d-d transitions of (C = N) and charge-transfer transition arising from π electron interactions between metal and ligand that involves either a metal-to-ligand or ligand-to-metal electron transfer [52]. The absorption bands below 220~270 nm in CHCl 3 are practically identical and can be attributed to π → π* transitions in the benzene and benzimidazole ring [51,53].

UV-Vis Study
The UV-Vis spectra of the novel Ag-NHC complex of dissolved in chloroform at 25 °C showed up four absorption bands at 220, 250, 270 and 320 nm, respectively. The UV-Vis spectra of the novel Ag-NHC complex 1 were recorded in (CHCl3) solutions at a concentration of 15 or 10 µM and were depicted in Figure 4 with a range 200～400 nm. The novel Ag-NHC complex 1 showed a new absorption peak at 320 related to MLCT (metal-ligand charge transfer) (Ag + to NHC ligand) ( Figure  4) [51]. This peak is known as the wide range bands, both π → π *, n → π * and d-d transitions of (C = N) and charge-transfer transition arising from π electron interactions between metal and ligand that involves either a metal-to-ligand or ligand-to-metal electron transfer [52]. The absorption bands below 220～270 nm in CHCl3 are practically identical and can be attributed to π → π* transitions in the benzene and benzimidazole ring [51,53].
In the crystal packing of the complex 1, cation molecules linked to each other via bromide anions with O1-H1···Br2i [H1···Br2 = 2.58 Å, O1-Br2 hydrogen bonding interactions forming an infinite chain along the b-and c-axis. The crystal structure is also stabilized by C-H···Br and C-H···O type intra-and intermolecular weak interactions. As can be seen in Figure 6, the Ag2 atoms settle in each corner of the unit cell and one is in the middle of the unit cell, while the bromide anions are between these corners Ag2 atoms. The crystal structure is also stabilized by C-H•••Br and C-H•••O type intra-and intermolecular weak interactions. As can be seen in Figure 6, the Ag2 atoms settle in each corner of the unit cell and one is in the middle of the unit cell, while the bromide anions are between these corners Ag2 atoms.

Enzyme Inhibition Studies
Inhibitors of metabolic enzymes can constitute new therapeutics against cancer or may have potential as antibacterial and antifungal drugs [56,57]. Recently, the inhibition of human CAs by sulfonamide compounds has been recorded to inhibit the growth of pathogenic microorganisms. Selective inhibition of CA II constitutes a viable approach to fight against the disturbances caused by the harmful effects of CA II enzyme [58][59][60]. When testing the results, the following inhibition activity relevance could be considered and summarized: Figure 6. Representation of the packing diagram for the Ag-NHC complex 1. The Ag2 atoms settle in each corner of the unit cell and one is in the middle, while the bromide anions are between these corner silver atoms. The Ag, Br and O atoms are shown as balls, while the other atoms are shown in a wireframe style. For the sake of clarity, hydrogen atoms that do not to play role in the bonding are omitted.

Enzyme Inhibition Studies
Inhibitors of metabolic enzymes can constitute new therapeutics against cancer or may have potential as antibacterial and antifungal drugs [56,57]. Recently, the inhibition of human CAs by sulfonamide compounds has been recorded to inhibit the growth of pathogenic microorganisms. Selective inhibition of CA II constitutes a viable approach to fight against the disturbances caused by the harmful effects of CA II enzyme [58][59][60]. When testing the results, the following inhibition activity relevance could be considered and summarized: 1.