Synthesis and Characterization of Camphorimine Au(I) Complexes with a Remarkably High Antibacterial Activity towards B. contaminans and P. aeruginosa

Fourteen new camphorimine Au(I) complexes were synthesized and characterized by spectroscopic (NMR, FTIR) and elemental analysis. The structural arrangement of three selected examples were computed by Density Functional Theory (DFT) showing that the complexes essentially keep the {AuI-CN} unit. The Minimum Inhibition Concentrations (MIC) were assessed for all complexes showing that they are active towards the Gram-negative strains E. coli ATCC25922, P. aeruginosa 477, and B. contaminans IST408 and the Gram-positive strain S. aureus Newman. The complexes display very high activity towards P. aeruginosa 477 and B. contaminans IST408 with selectivity towards B. contaminans. An inverse correlation between the MIC values and the gold content was found for B. contaminans and P. aeruginosa. However, plots of MIC values and Au content for P. aeruginosa 477 and B. contaminans IST408 follow distinct trends. No clear relationship could be established between the MIC values and the redox potentials of the complexes measured by cyclic voltammetry. The MIC values are essentially independent of the redox potentials either cathodic or anodic. The complexes K3[{Au(CN)2}3(A4L)] (8, Y = m-OHC6H4) and K3[{Au(CN)2}3(B2L)]·3H2O (14, Z = p-C6H4) display the lower MIC values for the two strains. In normal fibroblast cells, the IC50 values for the complexes are ca. one order of magnitude lower than their MIC values, although higher than that of the precursor KAu(CN)2.


Introduction
The medicinal properties of camphor have been recognized since ancient times, having a long history of traditional applications. Pharmacological uses of camphor include liniments and balms for relief of muscular pain, inhalants for nasal decongestion, antitussives, and expectorants. The activity of camphor on nasal decongesting was attributed to the stimulation of cold receptors in the nose [1][2][3]. Such behavior triggered our interest in the ability of camphor derivatives, in particular camphor derived complexes, to interact with other biological targets and also to evaluate their antibacterial properties. With that purpose, we prepared silver and copper complexes and investigated their antimicrobial Since the camphor ligands are neutral, no such type of interaction is feasible. However, adducts with a variety of metal to ligand ratios [{KAu(CN) 2 } n (L)] (n = 1, 3, 5, 7) were reproductively obtained. A tentative to rationalize such a reactivity trend is based on the interaction of the potassium ion with several nitrogen atoms of the neighbor molecules as found for the precursor potassium gold dicyanide by X-ray diffraction analysis. Potassium gold dicyanide displays a polynuclear tridimensional structure formed by alternating linear anionic (Au(CN) 2 − ) and cationic (K + ) layers, with each potassium ion interacting with several nitrogen atom of distinct Au(CN) 2 units [21]. By reaction of the camphor derivatives ( A L, B L or C L) with potassium gold dicyanide (KAu(CN)2), two main types of complexes were obtained: those that kept the KAu(CN)2 unit and those that lost the KCN moiety to afford the Au(CN) unit center (Scheme 1).
The anionic [Au(CN)2] − unit is known to form coordination polymers acting as a spacer between gold and cationic Cu(II), Zn(II), Ni(II), Co(II), Sn(II) metal centers [18][19][20]. Since the camphor ligands are neutral, no such type of interaction is feasible. However, adducts with a variety of metal to ligand ratios [{KAu(CN)2}n(L)] (n = 1, 3, 5,7) were reproductively obtained. A tentative to rationalize such a reactivity trend is based on the interaction of the potassium ion with several nitrogen atoms of the neighbor molecules as found for the precursor potassium gold dicyanide by X-ray diffraction analysis. Potassium gold dicyanide displays a polynuclear tridimensional structure formed by alternating linear anionic (Au(CN)2 − ) and cationic (K + ) layers, with each potassium ion interacting with several nitrogen atom of distinct Au(CN)2 units [21].
Although the camphorimine gold adducts with nuclearity higher than three are intriguing, they will be considered as dopped gold potassium dicyanide species and, therefore, they are not further discussed or their biological properties studied.
Depending As far as we know, formation of {Au(CN)} from potassium gold dicyanide was not reported before.
All complexes were formulated based on analytical and spectroscopic properties (See Experimental). In order to elucidate the structural arrangement and geometry of the complexes, computational calculations by DFT were undertaken, since no suitable crystals could be obtained to perform single crystal X-ray diffraction analysis.

Computational Calculations
DFT calculations were carried out using GAMESS-US [22] version R3 with a B3LYP functional, using a SBKJC basis set. Although the camphorimine gold adducts with nuclearity higher than three are intriguing, they will be considered as dopped gold potassium dicyanide species and, therefore, they are not further discussed or their biological properties studied.
Depending . No such type of complexes was obtained for ligand C L. As far as we know, formation of {Au(CN)} from potassium gold dicyanide was not reported before.
All complexes were formulated based on analytical and spectroscopic properties (See Experimental). In order to elucidate the structural arrangement and geometry of the complexes, computational calculations by DFT were undertaken, since no suitable crystals could be obtained to perform single crystal X-ray diffraction analysis.
Structures with one and two cyanide groups per gold atom were attempted. However, all the essayed structures with the fragment {Au(CN) 2 } underwent dissociative pathways releasing the ligand and regenerating the [Au(CN) 2 ] − unit. All the structures converged to [Au(CN)L] in a linear arrangement (see complex 13, Figure 1). In some cases, the released KCN can co-crystallize (1, Figure 1) or a second unit of AuCN is incorporated (complex 6 in Figure 1). The second gold cyanide unit binds to the {Au(CN)L} fragment through a week Au-Au bond (bond order 0.178) as well as a week Au-O bond (bond order 0.234). Although stronger, the Au-N bond is very labile with a bond order of 0.324. the released KCN can co-crystallize (1, Figure 1) or a second unit of AuCN is incorporated (complex 6 in Figure 1). The second gold cyanide unit binds to the {Au(CN)L} fragment through a week Au-Au bond (bond order 0.178) as well as a week Au-O bond (bond order 0.234). Although stronger, the Au-N bond is very labile with a bond order of 0.324.

Antibacterial Activity
The antibacterial properties of the camphorimine Au(I) complexes were assessed experimentally through the determination of the Minimum Inhibitory Concentration (MIC) against the Gram-positive strain S. aureus Newman and the Gram-negative strains E. coli ATCC25922, P. aeruginosa 477, and B. contaminans IST408. Experimental results show that all the complexes are active towards the bacterial strains under evaluation. As a general trend, complexes perform better for P. aeruginosa 477 and B. contaminans IST408 than for E. coli ATCC25922 or S. aureus Newman, although complexes 1, 2, and 10 display a reasonable activity against all the strains under study (Table 1).

Antibacterial Activity
The antibacterial properties of the camphorimine Au(I) complexes were assessed experimentally through the determination of the Minimum Inhibitory Concentration (MIC) against the Gram-positive strain S. aureus Newman and the Gram-negative strains E. coli ATCC25922, P. aeruginosa 477, and B. contaminans IST408. Experimental results show that all the complexes are active towards the bacterial strains under evaluation. As a general trend, complexes perform better for P. aeruginosa 477 and B. contaminans IST408 than for E. coli ATCC25922 or S. aureus Newman, although complexes 1, 2, and 10 display a reasonable activity against all the strains under study (Table 1).  As previously reported, the ligands are not active against the upper mentioned strains [9].
In order to try to correlate the characteristics (metal content, ionic/neutral character, nuclearity) of the complexes with their antibacterial activity, the MIC values obtained for P. aeruginosa 477 and B. contaminans IST408 (Table 1) were graphically depicted versus the metal content of each complex (Figures 2 and 3).
As previously reported, the ligands are not active against the upper mentioned strains [9].
In order to try to correlate the characteristics (metal content, ionic/neutral character, nuclearity) of the complexes with their antibacterial activity, the MIC values obtained for P. aeruginosa 477 and B. contaminans IST408 (Table 1) were graphically depicted versus the metal content of each complex (Figures 2 and 3).  Table 1).  Table 1).  Table 1).
As previously reported, the ligands are not active against the upper mentioned strains [9].
In order to try to correlate the characteristics (metal content, ionic/neutral character, nuclearity) of the complexes with their antibacterial activity, the MIC values obtained for P. aeruginosa 477 and B. contaminans IST408 (Table 1) were graphically depicted versus the metal content of each complex (Figures 2 and 3).  Table 1).  Table 1).  Table 1). Figures 2 and 3 clearly evidence a direct relationship between the antibacterial activities of the complexes and their gold content for the B. contaminans and P. aeruginosa strains, i.e., the higher the gold content, the higher the activity, the lower the MIC values. However, the correlation pattern is considerably different for the two strains. That is not the case for P. aeruginosa for which complex 3 displays one of the lowest MIC values (Figure 2). Such trend evidence that different mechanisms of action operate for the two bacterial species. In fact, the plots defined for P. aeruginosa ( Figure 2) stay away from the point defined by the gold cyanide precursor. In this case, the gold cyanide content remains relevant but structural effects due to the gold cyanide unit {Au(CN) 2 − } seem less important than for B. contaminans. In the case of complexes with bi-camphor ligands, symmetry conceivably play a role in the activity, since the para spacer at complex 14 considerably enhances the antibacterial activity compared to the meta spacer at complex 13 ( Figure 2). The high symmetry of the para bicamphor ligand [23] rends almost equally accessible four binding atoms (N, O) to interaction with bacteria receptors.

Plots in
The effect of the nuclearity, number, and eleFctronic characteristics of the ligands at the antibacterial properties of the complexes does not show a trend. However, it is evident that the ligands fine-tune the antibacterial activity, according to the slope of the lines (Figures 2 and 3).
The distinct trend for the activity/gold content observed for the two bacterial strains (B. contaminans and P. aeruginosa) cannot be attributed to the cell wall structural arrangement, since both species are Gram-negative, with a complex cell wall composed of an inner and an outer membrane.

Toxicity
The assessment of the cytotoxicity of new complexes is an essential step in the investigation of their application as prospective drugs. The first screenings are in vitro studies using normal cells such as human fibroblasts. The MTT assay was selected to assess cell viability by measuring the activity of a mitochondrial succinate dehydrogenase as the end-point of cytotoxicity [24,25]. Results obtained with the gold camphorimine complexes indicated that in general they have high cytotoxicity presenting IC 50 values in the range 0.15-0.40 µg/mL. Such cytotoxic activity is attributed to the contribution of the core of the complexes based on gold cyanide. From the results obtained, KAu(CN) 2 displayed an IC 50 value (0.06 µg/mL) well below (one order of magnitude) than that of the camphorimine gold complexes.

Redox Properties
Electron transfer is often involved in biological processes, so the study of the electrochemical properties of the camphorimine gold complexes could allow some insight into their redox properties. The study was undertaken by cyclic voltammetry in acetonitrile using Bu 4 NBF 4 as electrolyte (see Experimental for details). The data obtained is depicted in Table 1. As a general trend, the complexes display irreversible anodic waves at potentials lower than that of K[Au(CN) 2 ] (E ox p = 1.72 V, Figure 4a). The cathodic processes are also irreversible and fall within the potentials −1.65 and −1.85 V (Table 1) (Figures 4a and 5a) that fall in the range of values reported for the free ligands [5,26,27]. also irreversible and fall within the potentials −1.65 and −1.85 V (Table 1). No cathodic process or adsorption wave indicative of gold formation were observed for K[Au(CN)2].
The plots of the anodic and cathodic potentials versus the MIC values for P. aeruginosa 477and B. contaminans were depicted in Figures 4 and 5, respectively. A random distribution is observed in what refers to points defined by the MIC values and the reduction potentials (Figures 4a and 5a) that fall in the range of values reported for the free ligands [5,26,27].  The anodic processes accommodate within two ranges of potentials either for P. aeruginosa or B. contaminans (Figures 4b and 5b). The complexes with just one ligand (1,2,11,13) tend to fit into the range of the lower potentials, while those with two ligands (5,7,10)   also irreversible and fall within the potentials −1.65 and −1.85 V (Table 1). No cathodic process or adsorption wave indicative of gold formation were observed for K[Au(CN)2]. The plots of the anodic and cathodic potentials versus the MIC values for P. aeruginosa 477and B. contaminans were depicted in Figures 4 and 5, respectively. A random distribution is observed in what refers to points defined by the MIC values and the reduction potentials (Figures 4a and 5a) that fall in the range of values reported for the free ligands [5,26,27].  The anodic processes accommodate within two ranges of potentials either for P. aeruginosa or B. contaminans (Figures 4b and 5b). The complexes with just one ligand (1,2,11,13) tend to fit into the range of the lower potentials, while those with two ligands (5,7,10) fit the potential values that include the KAu(CN)2 precursor. The electron releasing/withdrawing properties of the camphorimine ligands (L = OC10H14NY) may drive the redox and structure of the complexes, since electron donor ligands (Y = C6H4NH2, C6H4N; The anodic processes accommodate within two ranges of potentials either for P. aeruginosa or B. contaminans (Figures 4b and 5b). The complexes with just one ligand (1,2,11,13) tend to fit into the range of the lower potentials, while those with two ligands (5,7,10) fit the potential values that include the KAu(CN) 2 precursor. The electron releasing/withdrawing properties of the camphorimine ligands (L = OC 10 H 14 NY) may drive the redox and structure of the complexes, since electron donor ligands (Y = C 6 H 4 NH 2 , C 6 H 4 N; Z = m-C 6 H 4 , Scheme 1) tend to produce mono ligand-complexes that oxidize at the lower range of potentials.

Materials and Methods
The complexes were synthesized under nitrogen using Schlenk and vacuum techniques. Camphor ligands (OC 10 H 14 NY: Y = NH 2 , C 6 H 5 , C 6 H 4 NH 2 -4, C 6 H 4 CH 3 -4, C 6 H 4 OH-3, NC 10 H 14 NC 6 H 4 ) were prepared according to reported procedures [27]. Gold potassium dicyanide, camphor, the amines, and hydrazine were purchased from Sigma Aldrich. Acetonitrile (PA grade) was purchased from Carlo Erba, purified by conventional techniques [28] and distilled before use. The FTIR spectra were obtained from KBr pellets using a JASCO FT/IR 4100 spectrometer. The NMR spectra ( 1 H, 13 C, DEPT, HSQC, and HMBC) were obtained from CD 3 CN, DMSO, or CDCl 3 solutions using a Bruker Avance II+ (300 or 400 MHz) spectrometers. The NMR chemical shifts are referred to TMS (δ = 0 ppm). The redox properties were studied by cyclic voltammetry using a three compartments cell equipped with a Pt wire electrode and interfaced with a VoltaLab PST050 equipment. The cyclic voltammograms were obtained using solutions of NBu 4 1 2 h. Then, acetonitrile (7 mL) was added, and the mixture stirred for 3 days. Upon solvent evaporation, a yellow precipitate was obtained that was washed with ether and dried under vacuum. Yield 57 mg, 73%.  13 13

Computational Calculations
The optimization of the structures and the molecular geometry of the complexes were carried out by DFT calculations using GAMESS-US [22] version R3, with a B3LYP functional, using a SBKJC basis set [29]. The structures were confirmed stationary points by Hessians with non-negative eigen values and six near zero rotational and translational frequencies.

Bacterial Strains
The bacterial strains E. coli ATCC25922, P. aeruginosa 477, B. contaminans IST408, and S. aureus Newman were used in the present work and were kept at −80 • C in 20% glycerol. When in use, bacterial strains were maintained in Luria Broth (LB) solid media (Sigma).

Minimal Inhibitory Concentration Assessment
The Au(I) complexes minimal inhibitory concentration (MIC) towards the abovementioned bacterial strains was determined by microdilution methodologies using Mueller-Hinton broth (MH), as previously described [30]. Positive (no compound) and negative controls (no bacterial inoculum) were included in each microdilution experiment.

Toxicity Assessment in Normal Cells
The in vitro cytotoxicity of the gold complexes was evaluated in adult human dermal fibroblasts (HDF) (Sigma-Aldrich) using the MTT assay as previously described [29]. Cells were cultured in Fibroblast Growth Medium (Sigma-Aldrich) following the instructions of the supplier for these cells. Cells laid in 96 well plates were incubated with serial dilutions of the compounds for 24 h. Each compound's dilution was performed in four wells. For each assay controls (cells without test compound) were done. At least two independent experiments were performed for each cytotoxicity analysis. The cytotoxicity of each compound was expressed by the IC 50 , the concentration of compound causing 50% decrease of cellular viability.

Conclusions
Camphorimine gold(I) cyanide complexes display relevant antibacterial activity towards Gram-negative E. coli ATCC25922 and Gram-positive S. aureus Newman bacterial strains and a remarkably high antibacterial activity towards the Gram-negative strains P. aeruginosa 477 and B. contaminans IST408. Depending on the characteristics of the camphorimine ligands, the gold content and the structural arrangement, the complexes reach rather low MIC values (2.4 µg/mL for B. contaminans IST408 (14) or 3.9 µg/mL for P. aeruginosa 477 (8) An insight into the relationship between the MIC values and the gold content shows that an inverse correlation exists, i.e., the higher the Au content, the lower the MIC values. The plots of the MIC values versus Au (%) reveal that the performance of the complexes towards the two strains is distinct. In the case of B. contaminans, the antimicrobial activity correlates directly with that of the potassium gold cyanide precursor, while in the case of P. aeruginosa, it is essentially independent of it. Except for compound 14, complexes of general formula K[Au(CN) 2 L] (1,4,9) and K[Au(CN) 2 L 2 ] (10) display the highest activities against B. contaminans. These complexes have in common camphorimine ligands with amine ( A1 L, A5 L) and hydroxy ( A6 L) substituents, which are easily ionizable, a fact that is considered to improve their low MIC values supported by the observation that the highest MIC value was found for complex [Au(CN)L 2 ] (5) with low ionizable character.
In the case of P. aeruginosa, the plots show that there is no correlation between the values (MIC and % Au) for the Au(I) camphorimine complexes and the precursor KAu(CN) 2 , which sits aside from all lines. In this case, the MIC values for most of the complexes are lower than that of KAu(CN) 2 and there is no evidence for enhancement of the antibacterial activity enabled by protic or hydrogen bonding substituents at the camphorimine ligand. Such trend shows that the coordination of the camphorimine ligands have a positive effect on the antibacterial activity, independently of the substituent.
No correlation was found between the redox potentials and the MIC values, in agreement with structural rather than electronic aspects are driving the interaction of the complexes with bacteria cells.