Cu(I)–N-heterocyclic carbene complexes bearing a naphthyl substituent: synthesis, characterization, and antimicrobial activities

Abstract A series of 1-(alkyl)-3-(1-naphthyl)imidazolinium bromides (2a–d) as N-heterocyclic carbene (NHC) precursors were synthesized from 3-(1-naphthyl)-1H-imidazolinium chloride (1). By reacting these NHC precursors with fresh CuCl, Cu(I)–NHC complexes (3a–d) were synthesized. The structures of the compounds were elucidated using 1H, 13C-NMR, FT-IR, ESI-MS, X-ray diffraction, scanning electron microscopy with energy-dispersive X-ray spectroscopy analysis, and elemental analysis techniques. The thermal properties of selected complexes were determined. Salts (1, 2a–d) and Cu(I)–NHC complexes (3a–d) were investigated against test microorganisms to determine their antimicrobial potency. The imidazolinium salts 2b and 2d displayed remarkable antibacterial efficacy, particularly against gram-positive bacteria; however, the corresponding Cu(I)–NHC derivatives (3a–d) did not exhibit a similar level of potency probably due to limited solubility. Overall, it can be concluded that incorporation of N-substituent species with O-heteroatoms, instead of benzyl, allyl, or hexyl groups on NHC, can enhance the activity against microbial derivatives. Graphical Abstract


Introduction
Activities of pharmacologically important metal-based drug molecules have become remarkable due to their antimicrobial and anticancer properties [1, 2].There is an urgent need for new generation antimicrobial drugs with the increase of multi-drug resistant bacteria [3].Metal-based drugs for the control of various infectious diseases are a new group of antimicrobial agents.Structural tunability achievable with metal ions and ligands enables creation of active metal-drugs involved in specific interactions with proteins, nucleic acids, antibiotics, and many other biomolecules [4].Copper is one of the most interesting biometals for new metalbased drugs with strong potential in therapeutic applications [5].It is known to interact with biomolecules such as albumin, ceruloplasmin as an important trace metal [5(a), 6].
Phenylboronic acids are one of the most effective substrates as a cross-linking agent in the formation of new products with the formation of C-X (X ¼ C, N, and heteroatom) bonds [35].One of the new approaches can be to obtain valuable phenol derivatives as intermediates in the preparation of pharmaceutical and therapeutic molecules by the production of C-O bond from aryl boronic acids and solvent through a CEL-like mechanism [20,36] accompanied by Cu-NHC catalyst [37].Cu(I)-NHC catalysts can be prepared with several synthetic procedures [26,[38][39][40][41]: the synthesis of Cu(I)-NHC by reaction of azoli(ni)um salt with copper(I) halide salt in the presence of a suitable base (route 1), the reaction of Ag 2 O with azoli(ni)um salt to yield Ag(I)-NHC, and then transmetalation with copper to form Cu(I)-NHC complexes (route 2) and treatment of azoli(ni)um salts with Cu 2 O to yield the Cu(I)-NHC complexes (route 3).
In the present work, synthesis and characterization of copper(I)-NHC complexes bearing naphthyl substituent were investigated, then studied for efficiencies against test microorganisms to determine their antimicrobial potency.

General considerations
Reactions sensitive to humidity and oxygen were performed using dried-degassed solvent in a dry argon atmosphere, Schlenk balloon, and vacuum line techniques.Before the experiment, the glassware was heated under vacuum and filled with dry argon.The reagents, solvents, and materials used were purchased from commercial suppliers.The solvents used (analytical grade) were dried by standard methods and distilled under an argon atmosphere.NMR spectra were recorded at 297 K on a JEOL ECZ500R (11.75 T) NMR instrument at 500 MHz ( 1 H) and 125 MHz ( 13 C) NMR and a Varian Mercury AS 400 NMR instrument at 400 MHz ( 1 H) and 100.56 MHz ( 13 C).Chemical shifts (d) are quoted as parts per million (ppm) and the coupling constants J are reported in Hz.FT-IR spectra were recorded using a Perkin Elmer Spectrum Two Model.Melting points were found using an Electrothermal 9100.The reactions were monitored by TLC with silica gel plates 60 F254.The TLC image was observed with UV light.Elemental data were obtained on a LECO, CHNS-932 elemental analyzer.Analysis results are given in the Supporting Information.The TGA of the compounds were obtained by Perkin Elmer STA 8000, carried out in flowing nitrogen atmosphere from 25 to 1000 � C in a platinum crucible.The heating rate was 10 � C min À 1 and sample masses were 1 mg.Thermoscientific TSQ quantis LC-MS/MS system was used in the direct infusion ESI-MS/MS method.Pump speed is 20 lL min À 1 .Initial source device setting are as follows: spray voltage: 3500 V, ion transfer tube temperature: 275 � C, vaporizer temperature: 75 � C, sheath gas (arb): 15, aux gas (arb): 5.The samples were diluted to 1 ppm and dissolved in dichloromethane.The surface morphology of the complexes was examined via the use of scanning electron microscopy (SEM, EVOLS10, Zeiss) equipped with energy-dispersive X-ray spectroscopy (EDX) detectors.Intensity data of the compounds were collected with a STOE IPDS II diffractometer at room temperature using graphite monochromated Mo Ka radiation by applying the x-scan method.

Synthesis of 3-(1-naphthyl)-1H-imidazolinium chloride (1)
This compound was synthesized from N-(1-naphthyl)ethylenediamine dihydrochloride (4.0 g, 15.43 mmol) and CH(OEt) 3 (40.0mL) according to the method known in the literature [42].The mixture was stirred for 5 h in an oil bath at 120 � C under argon.In the meantime, EtOH, which was formed as a by-product, was removed from the medium by distillation.It was then cooled to r.

General procedure for the synthesis of copper(I)-NHC complexes
The copper(I)-NHC complexes (3a-d) were prepared from the related 1-(alkyl)-3-(1naphthyl)-imidazolinium chloride (2) derivative (1.0 mmol), fresh copper chloride (1.0 mmol, 0.099 g), and K 2 CO 3 (2.0 mmol, 0.276 g) in dry acetone (10.0 mL) using standard Schlenk techniques according to the method known in the literature [26].The reaction mixture was refluxed for 24 h, and then the acetone was evaporated in vacuum.The residue was washed with water and dried.The product was allowed to crystallize from the CHCl 3 /Et 2 O system affording a light orange compound.

X-ray crystallography
X-ray diffraction data of 2b were collected on a STOE IPDS II diffractometer at room temperature using graphite-monochromated Mo Ka radiation by applying the x-scan method.Data collection and cell refinement were carried out using X-AREA [43] while data reduction was applied using X-RED32 [43].The structure was solved by a dualspace algorithm using SHELXT-2018 [44] and refined by full-matrix least-squares calculations on F 2 using SHELXL-2019 [45].All H atoms were located in difference maps and then treated as riding atoms, fixing the bond lengths at 0.93 and 0.97 Å for aromatic CH and CH 2 atoms, respectively.The displacement parameters of the H atoms were fixed at U iso (H) ¼ 1.2 U eq .Crystal data, data collection, and structure refinement details are collected in Table 1.Molecular graphics were created by using OLEX2 [46].

In vitro antimicrobial activity assays
To evaluate the antimicrobial activities of the synthesized compounds, two well-established assays were employed: agar-plate disc diffusion and broth microdilution.For these assays, a set of nine indicator strains, obtained from the Ankara Refik Saydam Hıfzısıhha Institute in T€ urkiye, were used: Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27853, Candida albicans ATCC 10231, and Aspergillus niger ATCC 16404.The results were evaluated according to the guidelines set by the Clinical and Laboratory Standards Institute (CLSI) [47][48][49][50].

Agar-plate disc diffusion assay
The antimicrobial activity was qualitatively measured using the agar-plate disc diffusion assay.To perform the assay, fresh solid cultures of indicator strains were grown on Mueller-Hinton agar (MHA; Oxoid) for bacteria and Sabouraud dextrose agar (SDA; Oxoid) for fungi.These cultures were then dissolved in NaCl (0.85%) to achieve an OD 625 value corresponding to 0.5 McFarland (approximately 1-4 � 10 8 cfu mL À 1 ).Then, 100 mL of the diluted cultures were spread on MHA and SDA plates.Sterile filter paper discs were loaded with the compounds (100 lg) and DMSO (used as a blank control) in a volume of 20 lL.Standard antibiotics were also included in the assay (amikacin and amphotericin B in 100 mg quantity).The plates were initially incubated at 4 � C for 2 h to allow for diffusion, followed by further incubation at 37 � C for 16 h (for bacteria) or at 26 � C for 48 h (for fungi).Each experiment was conducted in duplicate.The mean values of the inhibition zone diameter (mm ± SD) were calculated.

Broth microdilution assay
The minimal inhibitory concentration (MIC) values of each compound against the indicator strains were determined using a broth microdilution test.To prepare the samples, a double dilution series ranging from 512 to 0.125 mg mL À 1 was created by adding 100 mL of each compound and standard antibiotics to the wells of round-bottomed microtiter plates containing 100 mL of cation-adjusted Mueller-Hinton broth (CA-MHB; Oxoid).The final components added to the wells included ten-fold diluted cultures with an OD 625 value equivalent to a 0.5 McFarland solution, which corresponded to approximately 1-4 � 10 8 cfu mL À 1 .Incubation conditions were set at 37 � C for 16-20 h for bacteria and 26 � C for 48 h for fungi.Each assay included three technical replicates.The MIC value for each bacterial strain was determined as the lowest concentration that did not result in turbidity.For assessing antifungal activity, the MIC 50 value (the concentration that inhibited �50% of growth compared to the control) was determined.To ensure accuracy, control reactions were included in the assay.The controls were as follows: CA-MHB without the compound but with the inoculum, and CA-MHB with the compound but without the inoculum.Furthermore, to determine the minimal bactericidal concentration (MBC; the minimum concentration of the compound required to kill the microorganism) and minimal fungicidal concentration (MFC), 2 mL culture samples from each well were inoculated onto agar plates without any compounds.These plates were then incubated under the specified growth conditions as mentioned in the text.

Synthesis and characterizations of imidazolinium salts and Cu(I)-NHC complexes
NHC ligands have become one of the important tools in organic chemistry with many commercial applications.In modern catalysis, Cu-NHC complexes (remarkable Cu(IPr)Cl) are used in important transformations such as cyclopropanation of terminal alkenes, carbene transfer reactions, conjugate reduction of a,b-unsaturated ketones and esters, and hydrosilylation of ketones in the development process of air-and moisture-stable catalysts [51].Synthesis of copper(I)-NHC complexes was accomplished by first synthesizing azolinium salts (2a-d) as NHC precursor in a two-step procedure, as shown in Figure 1.All azolinium salts were prepared similarly to the published method [42].The two-step methodology began by reacting N-(1-naphthyl)ethylenediamine dihydrochloride with triethyl orthoformate and the solution was heated at 120 � C for 5 h, observing ethanol as a by-product in the distillation apparatus, a ringclosing product 1 was prepared with 81% yield.The asymmetric 1-(alkyl)-3-(1- naphthyl)imidazolinium salts (2a-d) were synthesized from deprotonation of 3-(1naphthyl)-1H-imidazolinium chloride (1) with NaHCO 3 followed by reaction with alkyl bromides.
The naphthyl-substituted imidazolinium salts, 2a-d, were stable to air and moisture (both in solid and in solution).The structural characterizations of naphthyl-substituted imidazolinium salts were elucidated by 1 H, 13 C-NMR spectroscopy, FT-IR, and X-ray diffraction techniques.The structures of 2a-d can be easily demonstrated by 1 H NMR spectroscopic data.The typical carbonic protons (C 2 -H) of 2a-d were at d ¼ 9.63, 9.14, 9.36, and 9.09 ppm as sharp singlets, while carbon atom signals were at d ¼ 158.3, 159.85, 158.56, 158.3 ppm for the C 2 -H, respectively (see Figures S2-S5).
The coordination of imidazolinium salts with copper(I) can be monitored in the absence of pro-carbenic protons (see Figures S6-S9), demonstrating the formation of Cu(I)-NHC complexes (Figure 2).Since concentrated solutions of 3a-d could not be prepared with deuterated solvents, 13 C-NMR could not be taken to see the carbene peak.Therefore, different methods have been used to support structure characterization, such as elemental analysis, SEM with EDX analysis, mass analysis, and thermal analysis.
FT-IR spectra of imidazolinium salts (1, 2a-d) and Cu(I)-NHC complexes (3a-d) were recorded (Figures S10 and S11).The vibrational modes involving C ¼ N bonds in 1 and 2a-d salts were at 1631, 1638, 1660, 1652, and 1645 cm À 1 , respectively.A significant shift in this area was observed upon coordination of Cu(I) of this tentative C ¼ N vibrational mode to 1669 cm À 1 for 3a, 1662 cm À 1 for 3b, 1663 cm À 1 for 3c, and 1667 cm À 1 for 3d consistent with the coordination of Cu to the NHC group.Thermal gravimetric analysis measurements of selected Cu(I)-NHC complexes (3c and 3d) showed weight loss occurred in three steps (see Figures S17 and S18).The TGA curves of these complexes show decomposition temperatures above 110 � C. The first mass loss at 110-240 � C is attributed to the alkyl group fragments attached to the nitrogen atom of 3c (or 3d) (�15%).In the second step, the bromide is lost at 240-720 � C (�28%).In the third step, decomposition of the imidazole group occurs at 720-860 � C (�16%).The complex exhibits a residue above 860 � C, corresponding to copper nitride.After this decomposition step, the mass loss is around 57% up to 1000 � C. The residue with the mass 43% may be assigned to naphthyl ring, imidazole, and copper.The obtained results indicate that the copper(I) complexes have high thermal stability.
The ESI-MS chromatograms of 3a, 3b, and 3c are given in the Supporting Information.In the mass spectrum of 3a the peaks were observed in high abundance at 484.56 and 421.17, respectively (see Figures S12-S14).The surface morphology and determination of the elemental compositions of the selected copper(I)-NHC complexes (3b and 3d) were carried out using SEM with EDX analysis.Morphological structure images at different magnifications and EDX spectra are given in Figures S15  and S16.The EDX measurements of 3b and 3d confirmed that the structure on the surface contains C, N, O, Br, and Cu.
Molecular structure of 2b with the atom-numbering scheme is depicted in Figure 3, while important bond lengths and angles are listed in Table 2.
In the molecular structure of 2b, no intramolecular interactions are observed.In the crystal structure, the cation is linked to the bromide anion via a C1-H1� � �Br1 interaction while atom C12 at (x, y, z) acts as a hydrogen-bond donor to bromide anion at (x, À y þ 3/2, z À 1/2).Together, these interactions form a form a C 1 2 8 ð Þ [53] molecular chain along the c axis (Figure 4).The detailed geometry of the intermolecular interactions is given in Table 3.

Antimicrobial activity of the compounds against the indicator strains
The agar disc diffusion data clearly demonstrated the efficacy of the tested compounds against gram-positive bacteria.Among them, 2b exhibited the highest inhibitory effect, as evidenced by the largest inhibition zone measuring 17 mm when tested against S. aureus ATCC 25923.This result indicates that this particular strain was highly sensitive to 2b, as indicated in Table 1.Furthermore, 2c and 2d also displayed significant effectiveness against the growth of B. subtilis ATCC 6633 and S. aureus ATCC 25923.The inhibition zones observed around the discs for these compounds were 13.5 and 13 mm, respectively (Table 4).
In line with the qualitative data, the MIC values for 2b and 2d were determined to be 64 mg mL À 1 against B. subtilis ATCC 6633 and S. aureus ATCC 25923, while the MIC value for E. faecalis ATCC 29212 was 128 mg mL À 1 .Conversely, the remaining complexes exhibited lower antibacterial activity against the indicator strains, with MIC values exceeding 512 mg mL À 1 (Table 5).Interestingly, the MBC values for 2b and 2d were equal to their respective MIC values against B. subtilis ATCC 6633 and S. aureus ATCC 25923.In addition, the MBC value of 2d against E. faecalis ATCC 29212 was 128 mg mL À 1 , the value the same as its MIC whereas the MBC of 2b against E. faecalis ATCC 29212 was 256 mg mL À 1 .It is noteworthy that the MIC and MBC values of 3a-d were higher than 512 mg mL À 1 against all the tested strains (Tables 5 and 6).
In summary, imidazolinium salts, especially 2b and 2d, demonstrated the most significant inhibitory activity against B. subtilis ATCC 6633 and S. aureus ATCC 25923.Additionally, both compounds displayed effectiveness against E. faecalis ATCC 29212.In contrast, none of the Cu(I)-NHC complexes demonstrated a comparable level of potency to that exhibited by the imidazolinium salts.In fact, considering the significant potency and efficacy displayed by the NHC-metal complexes in both bacterial and cancer cell studies, they emerge as potential candidates for further development as therapeutic agents [54,55].For instance, Touj et al. [26] reported both antimicrobial (against E. coli and S. aureus) and anticancer efficacies of (NHC)Cu(I) complexes.N-Substituent species containing O-heteroatom on NHC have been shown to improve the activity with cytotoxic activity against microbial derivatives [54].In addition, Patil et al. [55] showed that NHC-silver complexes have potent activity against E. coli and S. aureus.On the other hand, the lower antimicrobial activity of our Cu(I)-NHC complexes against the indicator strains might be due to their less solubility even in DMSO compared to imidazolinium counterparts.
Staphylococcus aureus serves a dual role as a commensal bacterium, commonly found among approximately 30% of the human population as part of the normal microbiota [56], and as a prominent causative agent of various bacterial infections.These infections encompass a range of conditions, including bacteremia, pleuropulmonary infections, infective endocarditis, osteoarticular diseases, skin and soft tissue     infections, as well as device-related infections [57].Moreover, S. aureus possesses a substantial threat to human health due to its ability to develop resistance to antibiotics, thereby impeding effective treatment strategies.It also produces certain molecules that confer protection against neutrophil-mediated killing, thus enabling it to overcome the host's defense system [58].Enterococcus faecalis is an opportunistic pathogen exhibiting resistance to several antibiotics.Furthermore, it possesses the capability to transfer antibiotic resistance to other pathogenic organisms, thereby exacerbating the challenge of antimicrobial resistance in healthcare settings [59].In turn, B. subtilis is rarely related with infections such as bacteremia/septicemia, endocarditis, meningitis, food poisoning and infections of respiratory, urinary tract, and gastrointestinal tract [60].
In light of these challenges, 2b and 2d under investigation hold potential for advancement of research studies aimed at addressing S. aureus, E. faecalis, and B. subtilis-related infections.Further exploration of the efficacy of these compounds in combating gram-positive bacterial infections is warranted.Future research will focus on indepth investigations to elucidate their mechanisms of action, pharmacokinetic profiles, and safety profiles, which are essential aspects in the development of effective drugs for bacterial infections and cancer diseases.

Conclusion
A series of imidazolinium salts (2a-d) containing naphthyl substituent and their Cu(I)-NHC complexes (3a-d) were synthesized.The structure characterizations were elucidated using 1 H, 13 C-NMR, FT-IR, ESI-MS, X-ray diffraction, SEM with EDX analysis, and elemental analysis techniques.The thermal degradation curves of 3c and 3d were investigated.The biological activities of all compounds against some bacterial species were investigated.The 2b and 2d imidazolinium salts were the most potent antimicrobials against bacterial strains.This result indicates that the N-substituent species containing O-heteroatom rather than benzyl, allyl, hexyl on NHC can improve the activity against microbial derivatives.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
Financial support from TUBITAK-2209 is gratefully acknowledged.

Figure 3 .
Figure 3.A view of the asymmetric unit of 2b showing the atom-labeling scheme.Displacement ellipsoids are drawn at the 20% probability level, and dotted lines display the H-bonding interaction.

Figure 4 .
Figure 4. Part of the crystal structure of 2b showing the intermolecular interactions represented by dotted lines.For clarity, H atoms not involved in the H-bonding have been omitted.

a
Response of indicator strains after exposure after exposure to the given complexes.S: sensitive; I: intermediary; R: resistant.

Table 1 .
Crystal data and structure refinement parameters for 2b.

Table 4 .
Agar disc diffusion results of the compounds against the indicator strains.

Table 5 .
MIC results of the compounds against the indicator strains.

Table 6 .
MBC/MFC values of the compounds against the indicator strains.