Antibacterial Thiamine inspired silver (I) and gold (I) N-heterocyclic carbene compounds

Abstract

Three new coinage metal carbene complexes of silver and gold were synthesized from a thiamine inspired proligand. The compounds were characterized by HRMS, NMR spectroscopy (¹H, ¹⁹F, ³¹P and ¹³C), FT-IR and elemental analysis. The coordination environment around the metal centers was correlated to the diffusion coefficients obtained from DOSY-NMR experiments and was in agreement with the nuclearity observed in the solid-state by single crystal X-ray crystallography. The silver and gold carbene compounds were subjected to MIC studies against a panel of pathogenic bacteria, including multidrug resistant strains, with the gold carbene derivative showing the most potent antimicrobial activity against Gram-positive methicillin resistant Staphylococcus aureus (MRSA).

Graphical Abstract

Bioinspired gold carbene compound effective antibacterial against S. aureus

1. Introduction

The antibacterial properties of certain metal salts and alloys have been known since antiquity due to the biocidal oligodynamic effect.[1,2] For example, silver salts have been a recurring treatment for burns,[3] now still in use in the form of silver sulfadiazine (Silvadene) ointments.[4,5] Owing to the expansion of multidrug resistant bacterial strains, d-block transition metal containing drugs are becoming the target of new research as potential drug candidates, mainly for antitumoral,[6] antifungal,[7] antibacterial[8,9] or antiparasitic purposes.[10,11] A recent study found that compounds containing transition metal atoms exhibit a higher antimicrobial propensity compared to strictly organic molecules.[12] Transition metals, enabled by their unique reactivity and coordination chemistry, allow for multiple mechanisms of action[13,14] as well as the circumvention of certain resistance mechanisms by accessing novel chemical targets.[15]

When the surface of the skin is damaged, the nutrient-rich nature of the subepidermal environment is exposed, providing a hospitable and rich habitat for microorganisms to thrive. For this reason, wounds are highly susceptible to infection, particularly by pathogens already on the skin near the site of the wound.[16,17] Three of the bacterial species that are commonly associated with wound infections (Staphyolococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa) belong to the so called ESKAPE pathogens: nosocomial, virulent, multi-drug resistant bacteria for which new, effective treatments are critically needed.[18] Scarcity of effective therapeutics has made treatment of bacterial infections difficult to manage and has put the antibiotic crisis among the most important threats to global healthcare systems.[19,20] Treatment of drug-resistant bacteria with compounds exhibiting novel mechanisms of action is more important than ever.

Transition metal complexes composed of a coinage metal (Cu, Ag, Au) and an N-heterocylic carbene ligand (NHC) have gained interest as antimicrobial metallodrugs.[8,20–29] The employment of coinage metal NHC complexes is ideal owing to the well-established high yield metalation protocols[31–36] and opportunities for transmetalation.[37] The vast chemical space to modify the easily accessible supporting imidazolium and related N-heterocyclic ligand scaffolds[38–40] enables enhanced antimicrobial activity.[41] Additionally, it can impart target specificity[42] for use in theranostics,[43] making these complexes a tremendous pool of potential drug candidates.

In our research groups we have recently turned our interest to exploring bioinspired ligands based on the Vitamin B1 (Thiamine) as a potential scaffold for the controlled release of antibacterial transition metal ions (Scheme 1). Thiamine features a thiazole based carbene that catalyzes the benzoin condensation reaction in human cells.[44] The presence of this persistent carbene allows for a metal binding site and the development of novel transition metal-thiamine inspired compounds with potential antibacterial properties. Herein we report the synthesis and characterization of our first-generation thiamine inspired silver and gold compounds and their antimicrobial activity against antibiotic-resistant bacteria.

Scheme 1.

Vitamin B1 and proligand 1. Below preparation of proligand 1 and its solid-state structure.

2. Results and Discussion

2.1. Synthesis of metal complexes

Proligand 1 was prepared by reacting 1,4,5-trimethylimidazol[45] and 2-trifluoromethylbenzylbromide in refluxing acetonitrile for 12 hours. The product was purified by precipitation of the crude reaction mixture with cold diethyl ether followed by recrystallization from the minimal amount of acetone yielding 1 in 81 % yield. To allow for activity comparison between silver and gold analogues the imidazol-2-ylidene fragment was favored vs. the thiazol-2-ylidene which is known to yield air sensitive compounds of the lighter coinage metals. No silver thiazol-2-ylidene complexes have been reported on the Cambridge Crystallographic Data Centre. However, there are some examples of copper thiazol-2-ylidene[46–49] and gold thiazol-2-ylidene species [50–54]. Additionally, a-CF₃ group was substituted in the benzylic part of the proligand to and enhance lypophilicity[58,59] and have an additional biologically exogenous spectroscopic handle for future experiments on the biological fate of these species [55–57]. Detailed procedures regarding synthetic protocols can be found in the supporting information. Proligand 1 showed a downfield imidazolium ¹H-NMR signal at δ 9.04 ppm in d⁶-DMSO. In the solid-state, compound 1 crystallizes with 4 molecules of water per unit-cell that hydrogen bond with the bromide anions (Scheme 1).

The silver imidazol-2-ylidene complex 2 was prepared by reacting proligand 1 with Ag₂O in dichloromethane solution in the absence of light. This method has proven to be an efficient route to generate silver carbene complexes before and is compatible with the cocrystallized water molecules of the proligand.[31] Metalation was followed by the disappearance of imidazolium proton resonance in the ¹H-NMR spectrum and the compound was isolated by precipitation of the filtered reaction mixture with pentane. Upon metal binding the imidazol-2-ylidene ¹³C-NMR signal of 2 appeared at δ 179.2 ppm. Complex 4 was prepared by transmetalation of 2 using chloro(dimethyl sulfide)gold (I) producing an AgBr precipitate and dimethylsulfide as byproducts (Scheme 2).

Scheme 2.

Metalation of 1 using Ag₂O to yield compound 2 and 3. Compound 4 was prepared by transmetalation of 2.

The NMR data confirmed metalation but was not conclusive to confidently assign the structure of the metal compounds. No desymmetrization of the ¹⁹F-NMR-CF₃ signal was observed either that could point to the nuclearity of the compounds. In solution, the complexes were studied by HRMS (ESI) to investigate the coordination environment around the metal center. Compound 2 is a cationic biscarbene complex (SI Fig. 18), the counter ion being [AgBr₂]⁻ as supported by the elemental analysis results. Compound 4 is a neutral complex, where the gold atom is bound to a single carbene proligand and a chlorine atom (SI Fig. 20).

To impart additional stability to the cationic silver biscarbene 2 the [AgBr₂]⁻ counter anion was substituted for a non-coordinating hexafluorophosphate [PF₆]⁻ anion. Biscarbene 3 can be prepared from 2 and KPF₆ in dichloromethane causing immediate precipitation of AgBr and KBr or more conveniently in one step directly from 1, Ag₂O and KPF₆. Incidentally, the substitution of the anion later allowed us to compare the bactericidal activity of the silver biscarbene complexes without interference from the silver provided by the [AgBr₂]⁻ anion in the silver carbene complex 2.

It is known that the nuclearity of silver carbenes can be related to their biocidal potency.[60] To gain a further understanding of the solution dynamics and nuclearity of these compounds, the experimental diffusion coefficients (D) were obtained by DOSY-NMR and support the assignment of nuclearity by mass spectrometry, with a lower diffusion coefficient observed for the larger biscarbene 3 molecule (D = 2.21 cm²/s) vs. a higher coefficient observed for the smaller monocarbene 4 (D = 2.77 cm²/s) which is comparable in size to the the free proligand 1 with a diffusion coefficient of (D = 2.65 cm²/s).

The solid-state structures obtained by single crystal X-ray diffraction studies support the behaviour observed in solution. Colourless crystals of 3 were grown from a dichloromethane solution layered with pentane and cooled to −25 °C. Complex 3 crystallizes in the P-1 space group (Figure 1) with 4 independent molecules per unit cell and is a linear cationic biscarbene with an Ag-C_carbene bond lengths of 2.091(2) Å and 2.094(2) Å. The molecular structure reveals that in the solid-state the benzylic residues are staggered by the interaction of the trifluorobenzyl rings [approximately 4.0 Å between the phenyl ring centroids] whereas in solution the carbenes can freely rotate anchored through the silver atom. The imidazol-2-ylidene rings are locked as well through the methylene carbon which causes them to twist with a 44° angle between the planes defined by the NHC rings. The linear C-Ag-C coordination axis is slightly bent (∠C6-Ag1-C20: 8.5°) presumably influenced by the trifluoromethylphenyl stacking acting through the methylene linker into the NHC heterocycle.

Figure 1.

Solid-state structure of 3. Hydrogen atoms and [PF₆]⁻ anion removed for clarity. Distances are given in Angstroms (Å).

Single crystals of compound 4 were grown as brittle colourless plates by slow vapor diffusion of pentane into a concentrated solution of dichloromethane cooled to −25 °C. The gold carbene crystallizes in the P2₁/c space group with 4 independent molecules per unit cell. The linear structure is shown in Figure 2. The Au-C_carbene distance retracts to 1.946(7) Å, while the Au-Cl distance is elongated to 2.3353(13) Å.

Figure 2.

Solid-state structure of 4. Hydrogen atoms removed for clarity. Distances are given in Angstroms (Å).

2.2. Antimicrobial activity studies

Minimum inhibitory concentration (MIC) values were obtained for the compounds against Gram-positive and Gram-negative species that are commonly associated with wound infections, including antibiotic resistant strains (Table 1). Solutions of the compounds were sterilized and filtered through 0.22 micron filters to eliminate the potential effect of metal nanoparticles on the bacterial growth.[61] Proligand 1 did not exhibit antimicrobial activity against any of the selected strains. Silver biscarbenes 2 and 3, which are coordinatively saturated NHC metal complexes both in the solid-state and solution, similarly were not effective in killing bacteria even with [PF₆]- as counterion, pointing to a poor release of silver resulting in low availability of antibacterial metal ions. On the other hand, gold carbene 4 showed the most potent activity against methicillin-resistant strains of the Gram-positive bacterium S. aureus while displaying less activity against the Gram-negative A. baumannii and P. aeruginosa that have an outer membrane, which might prevent the compound’s uptake. This Gram-positive preference is known for other gold antibacterials and is modulated by the lipophilicity [62–64]. The lipophilicity of our compounds was estimated by RP-HPLC using the retention time values on a C₁₈ column. Compound 4 is more lipophilic (t_r = 11 min) compared to the parent proligand (t_r = 8 min). MIC values for the MRSA strains in this study show gold carbene 4 to be more efficacious than the known silver topical antimicrobial silver sulfadiazine (6) and are as effective as AgNO₃ (5). The gold carbene compound also outperforms the common gold precursor chloro(dimethylsulphide)gold(I) (7), likely due to the robust stability provided by the stronger Au-C_carbene bond compared to the Au-SMe₂ bond that is more easily exchanged.

Table 1.

Minimum inhibitory concentrations [MIC, μg/mL (μM)] for antibiotic-resistant bacteria and bacterial species commonly associated with wound infections.

Compound	1	2	3	4	5	6	7
					AgNO3
S. aureus MU50a,d	>256 (>733)	>256 (>281)	>256 (>324)	8 (16)	16 (94)	128 (358)	64 (217)
S. aureus USA100 635a,d	>256 (>733)	>256 (>281)	>256 (>324)	16 (32)	16 (94)	128 (358)	64 (217)
S. aureus USA300 AH1263a,d	>256 (>733)	>256 (>281)	>256 (>324)	16 (32)	16 (94)	128 (358)	64 (217)
A. baumannii AYEb,c	>256 (>733)	>256 (>281)	>256 (>324)	32 (64)	4 (24)	64 (179)	64 (217)
A. baumannii 5075b,c	>256 (>733)	>256 (>281)	>256 (>324)	32 (64)	4 (24)	64 (179)	64 (217)
P. aeruginosa PAO1b	>256 (>733)	>256 (>281)	256 (324)	256 (511)	2 (12)	64 (179)	64 (217)
P. aeruginosa PA14b	>256 (>733)	>256 (>281)	256 (324)	>256 (>511)	2 (12)	32 (90)	64 (217)

^aDenotes Gram-positive bacterium.

^bDenotes Gram-negative bacterium.

^cDenotes multi-drug resistant strain.

^dMethicillin-resistant.

Conclusion

In summary, new bioinspired silver and gold imidazol-2-ylidene complexes were synthetized and characterized. The silver bis(carbene) complexes were not successful in inhibiting bacterial growth. However, the gold carbene complex showed promising activity against Gram-positive bacteria and outperformed the widely prescribed silver sulfadiazine antimicrobial. Our future steps follow this path and are geared toward synthetizing more potent, broad spectrum and more biomimetically relevant gold thiazol-2-ylidene compounds to test against these multidrug-resistant pathogens.

4. Experimental Methods

4.1. General methods

NMR (¹H, ¹³C{¹H}, ¹⁹F, ³¹P) data was acquired on a Bruker AVANCE III 500 Cryoprobe spectrometer and processed using MestReNova software. ¹⁹F-NMR was referenced to internal TMS signal (δ = 0 ppm)[65] using the absolute referencing function in the Mestrenova software package and peak assignment was aided by 2D-NMR experiments as needed. FT-IR spectra were obtained in a Nicolet iS50 FT-IR spectrometer using attenuated total reflectance (ATR). Mass spectrometry data was provided by the UT Austin Mass Spectrometry Facility. X-Ray Crystallography was performed in the UT Austin – X-Ray Diffraction Laboratory. CCDC 1963913–1963915 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif. or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre. Elemental analysis was performed by Midwest Microlab Inc. Indianapolis, IN. Chemicals and solvents were purchased from commercial vendors and used as received. 1,4,5-trimethylimidazol was prepared as previously reported.[45]

4.2. MIC assay

Minimum inhibitory concentrations were determined using the broth microdilution method outlined by Hancock et. al.[66] Stock compounds were prepared at 10 mg/mL in sterile DMSO. From these stocks, 1 mg/mL solutions were prepared in Mueller-Hinton broth and filtered through a 0.22 μm filter. Dilutions of each compound, ranging from 1 μg/mL to 256 μg/mL (final concentrations) were prepared in Mueller-Hinton broth using the 1 mg/mL stocks. Compound dilutions were added to wells of a 96-well plate. Overnight bacterial cultures were diluted to ~10⁶ CFU/mL in Mueller-Hinton broth and were added to wells in equal volume to that of compound dilutions, for a total volume of 100 μl. Plates were incubated for 24 hours at 37° C. The following day, plates were evaluated by eye to determine the MIC value. The concentration value corresponding to the first row that no longer had visible bacterial growth, as evaluated by turbidity in the wells, was recorded as the MIC. The reported values are representative of at least biological duplicates, each made up of technical triplicates.

4.3. Synthesis of 3-((2-trifluoromethyl)benzyl)-1,4,5-trimethylimidazolium bromide (1)

2-trifluoromethylbenzyl bromide (8.37 mmol, 2.00 g) and 1,4,5-trimethylimidazol (8.37 mmol, 0.92 g) were suspended in acetonitrile (8 mL). The mixture was refluxed 12 h. Upon cooling the yellow solution was poured into a beaker containing cold diethyl ether (30 mL). Upon addition an off-white solid precipitated, which was collected by vacuum filtration and washed with additional diethyl ether (3 × 10 mL). The solid was recrystallized from acetone at −25 °C. Title compound (1) was obtained as a white crystalline solid in 81% yield (2.37 g). ¹H NMR (500 MHz, DMSO-d₆) δ 9.04 (s, 1H, NHC-H), 7.88 (d, J = 7.7 Hz, 1H, Aryl-H), 7.72 (t, J = 7.6 Hz, 1H, Aryl-H), 7.64 (t, J = 7.6 Hz, 1H, Aryl-H), 7.09 (d, J = 7.8 Hz, 1H, Aryl-H), 5.60 (s, 2H, -CH₂-), 3.79 (s, 3H, N-CH₃), 2.27 (s, 3H, -CH₃), 2.09 (s, 3H, -CH₃). ¹⁹F NMR (470 MHz, DMSO-d₆) δ −59.38 (Aryl-CF₃). ¹³C NMR (126 MHz, DMSO-d₆) 135.4 (NHC-CH), 133.6 (Aryl-CH), 132.1 (m, C_q), 129.1 (Aryl-CH), 128.7 (Aryl-CH), 127.9 (C_q), 126.6 (q, J = 5 Hz, HC-C-CF₃), 126.3 (C_q), 126.3 (q, J = 30 Hz, C-CF₃), 124.1 (q, J = 275 Hz, -CF₃) 46.4 (m, -CH₂-), 33.58 (N-CH₃), 7.86 (-CH₃), 7.80 (-CH₃). HRMS (ESI): 269.1258 m/z [Calculated: 269.1260 m/z, C₁₄H₁₆F₃N₂⁺]. Melting Point 174–175 °C. Elemental Analysis Anal. Calc. for C₁₄H₁₆N₂F₃Br. ½ H₂O: C, 46.94; H, 4.78; N, 7.82. Found: C, 47.00; H, 4.84; N, 7.80.

4.4. Synthesis of 2 [C₂₈H₃₀N₄F₆Br₂Ag₂]

To a solution of proligand 1 (0.57 mmol, 200 mg) in dichloromethane (15 mL) Ag₂O (0.29 mmol, 66 mg) was added. The mixture was stirred in the absence of light for 12 hours. The solution was filtered through celite and the solvent removed under reduced pressure until approximately 1 mL remained and it was poured into a stirring vial containing pentane (10 mL). After stirring for a few minutes, a white solid precipitated which was identified as the title compound. (156 mg, 60 %). ¹H NMR (500 MHz, DMSO-d₆) δ 7.77 (d, J = 7.7 Hz, 2H, Aryl-H), 7.58 (t, J = 7.6 Hz, 2H, Aryl-H), 7.50 (t, J = 7.6 Hz, 2H, Aryl-H), 6.67 (d, J = 7.8 Hz, 2H, Aryl-H), 5.44 (s, 4H, -CH₂-), 3.72 (s, 6H, N-CH₃), 2.19 (s, 6H, -CH₃), 1.93 (s, 6H, -CH₃). ¹⁹F NMR (470 MHz, DMSO-d₆) δ −59.66 (Aryl-CF₃). ¹³C NMR (126 MHz, DMSO-d₆) δ 179.2 (NHC-C-Ag), 135.1 (C_q), 133.1 (Aryl-CH), 128.1 (Aryl-CH), 126.8 (C_q), 126.7 (Aryl-CH), 126.2 (q, J = 5.5 Hz,, HC-C-CF₃), 125.5 (q, J = 30.5 Hz, C-CF₃), 125.4 (C_q), 123.1 (q, J = 275 Hz, -CF₃), 48.2 (m, -CH_2-), 36.1 (N-CH₃), 8.59 (-CH₃), 8.29 (-CH₃). HRMS (ESI): 643.1420 m/z [Calculated: 643.1423 m/z, C₂₈H₃₀AgF₆N₄⁺] Melting Point 158–159 °C Elemental Analysis Anal. Calc. for C₂₈H₃₀N₄F₆Br₂Ag₂: C, 36.87; H, 3.32; N, 6.14. Found: C, 36.89; H, 3.48; N, 6.02.

4.5. Synthesis of 3 [C₂₈H₃₀N₄F₁₂AgP]

Proligand 1 (0.29 mmol, 100 mg), Ag₂O (0.15 mmol, 34 mg) and KPF₆ (0.15 mmol, 27 mg) were combined in a vial and suspended in dichloromethane (5 mL). The suspension was stirred for 12 hours in the absence of light. The brown suspension was filtered through a 200 μm PTFE filter the resulting clear solution was concentrated to approximately 0.5 mL. Addition of pentane (10 mL) caused the title compound to precipitate as a white powdery solid which was washed with additional pentane (2 × 1 mL). Yield (104 mg, 91 %). Alternatively, this compound can be prepared by salt metathesis of 1 using KPF₆. ¹H NMR (500 MHz, DMSO-d₆) δ 7.72 (dd, J = 7.8, 1.4 Hz, 2H, Aryl-H), 7.53 (td, J = 7.7, 1.4 Hz, 2H), 7.47 (t, J = 7.6 Hz, 2H, Aryl-H), 6.63 (d, J = 7.7 Hz, 2H, Aryl-H), 5.39 (s, 4H, -CH₂-), 3.69 (s, 6H, N-CH₃), 2.18 (s, 6H, -CH₃), 1.93 (s, 6H, -CH₃).¹⁹ F NMR (471 MHz, DMSO-d₆) δ −59.76 (Aryl-CF), −69.92 (PF ⁻₆), −71.43 (PF₆⁻) ¹³C NMR (126 MHz, DMSO-d₆) δ 179.2 (NHC-C-Ag), 135.0 (C_q), 133.0 (Aryl-CH), 128.0 (Aryl-CH), 126.8 (C_q), 126.6 (Aryl-CH), 126.0 (q, J = 5.7 Hz, HC-C-CF₃), 125.5 (q, J = 30.4 Hz, C-CF₃), 125.4 (C_q), 124.0 (q, J = 275 Hz, -CF₃), 48.2 (m, -CH_2-) 36.0 (N-CH₃), 8.56 (-CH₃), 8.26 (-CH₃). ³¹P NMR (202 MHz, DMSO-d₆) δ −144.74 (hept, J = 710 Hz). HRMS (ESI): 643.1418 m/z [Calculated: 643.1423 m/z, C₂₈H₃₀AgF₆N₄⁺] Melting Point 151–153 °C Elemental Analysis Anal. Calc. for C₂₈ H₃₀N₄F₁₂AgP CH₂Cl₂: C, 39.84; H, 3.69; N, 6.41. Found: C, 40.51; H, 3.70; N, 6.61.

4.5. Synthesis of 4 [C₁₄H₁₅N₂F₃AuCl]

Dimethyl sulphide gold chloride (0.22 mmol, 65 mg) was dissolved in dichloromethane (10 mL). Compound 2 (0.11 mmol, 100 mg) was added as a solid producing immediate precipitation of a grey solid (AgBr). After stirring for 2.5 hours the solution was filtered through celite and rotary evaporated to approximately 1 mL. The solution was poured into pentane (10 mL) and the title compound precipitates as a white powder recovered by vacuum filtration (76 mg, 69%). ¹H NMR (500 MHz, DMSO-d₆) δ 7.83 (d, J = 7.7 Hz, 1H, Aryl-H), 7.65 (t, J = 7.6 Hz, 1H, Aryl-H), 7.55 (t, J = 7.6 Hz, 1H, Aryl-H), 6.68 (d, J = 7.6 Hz, 1H, Aryl-H), 5.53 (s, 2H, -CH_2-), 3.76 (s, 3H, N-CH₃), 2.22 (s, 3H, -CH₃), 1.97 (s, 3H, -CH₃). ¹⁹F NMR (470 MHz, DMSO-d₆) δ −59.23 (Aryl -CF₃). ¹³C NMR (126 MHz, DMSO-d₆) δ 168.3 (NHC-C-Au), 134.4 (C_q), 133.2 (Aryl-CH), 128.2 (Aryl-CH), 126.7 (C_q), 126.3 (Aryl-CH), 126.2 (q, J = 5.7 Hz, HC-C-CF₃), 125.5 (q, J = 30.5 Hz, C-CF₃), 125.0 (C_q), 124.1 (q, J = 275 Hz, -CF₃), 47.8 (m, -CH₂-), 35.4 (N-CH₃), 8.63 (-CH₃), 8.28 (-CH₃). HRMS (ESI): 523.0433 m/z [Calculated: 523.0434 m/z, C₁₄ H₁₅AuClF₃N₂Na⁺]. Melting Point 231–232 °C. Elemental Analysis Anal. Calc. for C₁₄H₁₅N₂F₃AuCl: C, 33.58; H, 3.02; N, 5.59. Found: C, 33.27; H, 3.00; N, 5.36.