Assessment of in vitro and in vivo antimicrobial activity of an isonitrosomalononitrile silver(I) salt

. The design and evaluation of antimicrobial activities of an isonitrosomalononitrile silver(I) salt was reported. This highly stable water-soluble silver salt shows Minimum Inhibitory Concentrations (MIC) values ranging from 0.15 to 5 µg/mL towards both sensitive and resistant Gram-positive and negative bacteria. Furthermore, this silver salt has been investigated for its ability to treat a S. aureus infected Galleria mellonella larvae animal model with promising results. Thus, our results demonstrated that 80% of the treated larvae survived after 24h with respect to 10% of the untreated ones, respectively.


Introduction
Extensive misuse of antibiotics worldwide lead to the emergence of antimicrobial resistance as a major threat to healthcare.[1] Thus, numerous scientists reported the end of the antibiotic age [2][3][4] and the emergence of multidrug-resistant bacteria (superbugs) such as vancomycinresistant Enterococcus (VRE), Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (MRPA).[5][6] Furthermore, the development of novel resistance should therefore be a prime activity of research in industry.[7] In this context, silver-based drugs [8][9][10][11][12][13] constitute an interesting area of research since one of their possible mechanism of action in vivo could be denaturing the deoxyribonucleic acid (DNA) molecule by disrupting the hydrogen bonding between the two anti-parallel strands.[14] Indeed, the antimicrobial activity of silver by limiting the growth of bacteria and biofilms [15] is well established [16] and its use applied into many medical products (e.g.catheters, plasters).Thus, it has been suggested that such a class of derivatives such as carbene-silver acetates could be of interest to become drug candidates.[17][18] Furthermore, the development of more lipophilic benzimidazole-derived carbene-silver complexes presenting potent activities against numerous bacteria were recently reported.[19][20] This paper describes the synthesis and biological antimicrobial evaluation of a new isonitrosomalononitrile silver salt against both Gram-positive and negative bacteria and demonstrates its in vivo efficiency against a S. aureus infected G. mellonella larvae animal model.

Results and discussion
The preparation of the isonitrosomalononitrile silver(I) salt 4 was easily achieved in a two steps synthesis as outlined in Scheme 1.A solution of malononitrile 1 in acetic acid reacted with an aqueous solution of sodium nitrite to lead to the formation of the expected isonitrosomalononitrile 2. This latter can subsequently react with an aqueous solution of silver nitrate to afford the expected isonitrosomalononitrile silver(I) salt 4 as a stable yellow solid in 91% yield ( 13 C NMR (63 MHz, CD 3 OD) δ (ppm): 118.44, 112.50, 107.39).In parallel, we prepared the corresponding isonitrosomalononitrile potassium salt 3 parent derivative to compare the influence of the nature of the metal used on the potent antimicrobial activities encountered.In this case, the isonitrosomalononitrile potassium salt was prepared by mixing as previously a solution of sodium nitrite solution in acetic acid in the presence of malononitrile 1 for 1h then quenched by adding 2N hydrochloric acid solution.After extraction, the oily residue can be purified by flash chromatography to afford isonitrosomalononitrile 2 in 83% yield or treated by a potassium hydroxide solution to lead to the expected isonitrosomalononitrile potassium salt 3 as yellow powder in 86% yield.This powder can be crystalized by slow cooling of an oversaturated water solution heated at 50°C.This afforded large crystals suitable for X-ray structure analysis.

Scheme 1.
A suitable yellow single crystal of 3 was carefully selected under a polarizing microscope and deposited on a single loop for single crystal X-ray diffraction analysis (Figure 1).The data were collected on a Bruker X8 APEX2 diffractometer using an Incoatec Ag-Kα Microfocus source (λ= 0.56086 Å) at room temperature.The diffraction intensities were collected until 2θ= 36.36°.This value is related to the absence of reflections at high angles which is linked to the crystallinity of the crystals.The intensities were extracted from the frames using SAINT V7 53a (SAINT: Area-Detector Integration Software.Madison: Siemens Industrial Automation, Inc., 1996).A monoclinic unit cell a= 8.7459(4), b= 8.8229(3), c= 7.1182(3) and β= 106.23 (1) was deduced from all data set.Intensities were semi-empirically corrected from absorption effects using SADABS (SADABS: Area-Detector Absorption Correction.Madison: Siemens Industrial Automation, Inc., 1995).The observed systematic extinction conditions led to use the space group P2 1 /c.The crystal structure was determined by using the software SUPERFLIP [21] and further refined by full-matrix least-squares procedures using JANA2006 software.[22] At the end of the refinement, the final R value was 0.0253, obtained for 759 reflections (I>3σ(I)).All experimental conditions as well as structure refinement details are given in Table 1.

Crystal system Monoclinic
Space In the obtain structure, there is one independent crystallographic site for potassium and oxygen and three for both carbon and nitrogen.The atomic coordinates, isotropic and anisotropic displacement parameters are listed in Table 2. Table 2. Atomic positions, isotropic and anisotropic displacement parameters (Å 3 ) for 3.

Atom
The lengthening of bond lengths is of 1.136( 4 3.

Atoms Bond distance (Å) Atoms
Bond angle (°)   Now, despite all our attempts, we were unable to obtain crystals corresponding to the powder of isonitrosomalononitrile silver salt 4 suitable for X-ray analysis.The X-ray powder diffraction pattern is also different from the one of isonitrosomalononitrile potassium salt 3 which implies that the isonitrosomalononitrile silver salt 4 is differently arranged, in strong relation with the rather big different size of the respective ionic radii.All the synthesized water-soluble compounds were then screened for their potent antimicrobial activity against sensitive or resistant Gram-positive and negative bacteria strains as listed in Table 4. First, we must notice that isonitrosomalononitrile 2 and isonitrosomalononitrile potassium salt 3 do not possess any activity even at high concentrations against the tested bacterial strains.

Strains
Nevertheless, isonitrosomalononitrile silver salt 4 was found to be active against all the microorganisms tested with Minimum Inhibitory Concentrations (MICs) varying from 0.625 to 2.5 µg/mL whatever the considered bacterial strains.Thus, 4 is active in the same range against Gram-positive and negative bacteria and in no case, we have noticed a lack of activity.
Thus, the great difference in terms of antimicrobial efficiency between the different tested compounds 1-4 seems to be exclusively related to the presence of a silver atom in the case of  5).
Moreover, IC 50 's in the same range than those obtained for well-known AgNO 3 were encountered opening the way for animal testings.Due to their ability to possess an immune system similar to mammals [24] cheap Greater WaxMoth, Galleria mellonella [25][26] were widely chosen to evaluate the in vivo efficacy and stability of new antimicrobial compounds and determine the virulence of microbial pathogens [27] in 24-48 h.[28][29] A preliminary study was performed by using Galleria mellonella larvae as an animal model to determine their tolerance to isonitrosomalononitrile silver salt 4 as well as its in vivo activity against a S. aureus infection.Thus, no toxicity up to a concentration of 25 µg/mL (12 µM) was encountered whereas larvae inoculated with a dose of 50 µg/mL showed a 90% reduction in viability after 24h.Finally, larvae inoculated with isonitrosomalononitrile silver salt 4 (3.125, 6.25, 12.5, 25 µg/mL) do not present any signs of acute toxicity such cuticular darkening (Figure 4A).To ensure to the preliminary in vivo activity of isonitrosomalononitrile silver salt 4, larvae were infected with S. aureus ATCC25923 (20 µL of a solution containing 10 7 bacterial cells) and subsequently administered with or without isonitrosomalononitrile silver salt 4 (6.25 µg/mL) 4h post infection.Our results demonstrated that 80% of the treated larvae survived after 24h with respect to 10% of the untreated ones, respectively (Figure 4B).These preliminary but promising data open the way to perform more detailed studies typically for a topical use towards S. aureus Skin infections.

Conclusion
The design and evaluation of antimicrobial activities of a water-soluble isonitrosomalononitrile silver(I) salt with MIC values ranging from 0.15 to 5 µg/mL against both sensitive and resistant Gram-positive and negative bacteria was investigated.We have also demonstrated its ability to be used for the treatment of a S. aureus infected Galleria mellonella larvae animal model with promising results opening the way for a potent therapeutic use.Studies are under progress to evaluate and improve the potentiality of such silver salt derivatives as well as determine more precisely the potent mechanism of action of this new class of antimicrobial agents.

Experimental section
All solvents were purchased from VWR and used without further purification.Reagents were used as commercially available. 13C NMR spectra were recorded in CDCl 3 on a Bruker AC 300 spectrometer working at 75 MHz.Tetramethylsilane was used as internal standard.All chemical shifts are given in ppm.

Synthesis of isonitrosomalononitrile 2
7g of sodium nitrite (101 mmol) were slowly added at 0°C to 4 g of malononitrile (65 mmol) dissolved in water (20 mL) and acetic acid (10 mL).Stirring was maintained at room temperature in the dark overnight then quenched by adding 50 mL of a 2N HCl solution.The solution was then extracted with diethyl ether and dried over Na 2 SO 4 .After filtration, the solvents are removed under vacuum to give an oily residue which was purified by flash chromatography on silica gel using petroleum ether/ethylacetate as eluent.The product 2 is obtained as a pale-yellow oil in 83% yield. 1 H NMR (300 MHz, CD 3 OD) δ (ppm): 8.89 (s, 1H). 13

Synthesis of isonitrosomalononitrile potassium salt 3
7g of sodium nitrite (101 mmol) were slowly added at 0°C to 4 g of malononitrile (65 mmol) dissolved in water (20 mL) and acetic acid (10 mL).Stirring was maintained at room temperature in the dark overnight then quenched by adding at 0°C 50 mL of a KOH (4g, 71 mmol) methanolic solution.The reaction mixture is stirred at 0°C for 1h then excess of diethyl ether was added to afford the potassium salt precipitation.The precipitate was collected by filtration, then washed with diethyl ether.The yellow solid was then dried at 80°C under vacuum for 4 hours affording the expected compound 3 in 86% yield.

Inoculation of Galleria mellonella larvae
Ten larvae of G. mellonella (purchased at Animalis center) were stored in the dark at 15°C.
Larvae of the same age and weighing around 0.2 g were inoculated with 20 µL of water containing 5 10 7 S. aureus cells through the last pro-leg using a Myjector U100 insulin syringe (Terumo Europe, Leuven, Belgium).

In vivo toxicity assay
Larvae were injected with 20 µL of isonitrosomalononitrile silver salt 4 solution (31.

Determination of minimal inhibitory concentrations
Antimicrobial activity of the compounds was studied by determination of minimal inhibitory concentrations (MIC) according to the NCCLS guidelines M7-A2 using the microbroth dilution methods.The bacteria strains were grown on trypticase soy agar (Becton Dickinson) at 37 °C for 24h.Inocula were prepared in TCE (tryptone 0.1%, NaCl 8%, wt/vol) by adjusting the turbidity at 623 nm to obtain 1-3 10 5 CFU/mL.
Antimicrobial activities of the compounds were determined by using a broth microdilution method performed in sterile 96-well microplates.All compounds were solubilized in water at a concentration of 10mM and were transferred to each microplate well in order to obtain a two-fold serial dilution in 100 µL of broth and 100 µL of inoculum containing 2-6 10 5 CFU of each bacterium were added to each well.Some wells were reserved for positive controls and inoculum viability.After 24 h incubation, MIC was defined for each agent from duplicate observations as the lowest concentration of compound allowing no visible growth.

Cytotoxicity assays
The -A first set of cell cultures was incubated in PBS containing 10% WST-1 for 30 min at 37°C, 5% CO 2 .Cell viability was evaluated by the assessment of WST-1 absorbance at 450 nm in a microplate spectrophotometer.
-A second set of cell cultures was placed into Neutral Red medium (50 μg/ml Neutral Red in complete medium) and incubated for 3 hours at 37°C, 5% CO 2 .Then the Neutral Red medium was removed and the distaining solution (50% ethanol, 1% acetic acid, 49% distilled water; 50 µL per well) was added into the wells.The plates were shaken for 15-20 min at room temperature in the dark.Cell viability was evaluated by the assessment of absorbance at 540 nm in a microplate spectrophotometer.
Results were expressed as percentages of cell viability about the control (culture mediumonly), which corresponded to 100% cell viability.Dose-response curves were calculated by non-linear regression analysis with TableCurve V2 software.The Inhibitory Concentration 50% (IC 50 ) was defined as the concentration of 4 that induced a 50% decrease of viable cells.

Table of contents
Assessment of in vitro and in vivo antimicrobial activity of an isonitrosomalononitrile silver(I) salt Marie Colmont and Jean Michel Brunel The design and evaluation of antimicrobial activities of an isonitrosomalononitrile silver(I) salt was reported.This water-soluble silver salt shows Minimum Inhibitory Concentrations (MIC) values ranging from 0.15 to 5 µg/mL against a large panel of bacteria and is efficient to treat a S. aureus infected Galleria mellonella larvae animal model.

Figure 1 .
Figure 1.Crystal structure of isonitrosomalononitrile potassium salt presented along a) c, b) b, and c) a-axis and d) molecular structure of 3 with presentation of atom labelling scheme.

Table 3 .
Main bond distances and length in crystal of 3The X-ray diffraction analysis of the synthesized powder of 3 confirmed the correlation with the determined crystal structure of isonitrosomalononitrile potassium salt 3 and the purity of the sample.A Pattern matching performed using the same space group and unit cell parameters shows the perfect matching between both (Figure2).

Figure 2 .
Figure 2. Pattern matching profile of powder X-ray diffraction pattern of isonitrosomalononitrile potassium salt 3 The crystals obtained were nevertheless studied by X-ray diffraction.Their analysis shows a monoclinic unit cell with parameters a= 17.315(4)Å, b= 3.598(1)Å, c= 17.316(4)Å and β= 110.34(1)°,S.G.P2 1 .The poor quality of the crystals only allowed to propose a hypothetical model of that unknown phase presented on figure 3 which confirmed the differences with the expected the isonitrosomalononitrile silver salt 4.

Figure 3 .
Figure 3. Hypothetical model of the structure of the synthesized crystals of 4.

derivative 4 .
Indeed, the absence or replacement of this atom by a potassium atom lead to a loss of activity of the considered compound.Otherwise, since the MICs encountered are very close whatever the considered Gram-negative or Gram-positive bacteria as well as sensitive or resistant it is reasonable to envision that 4 possess a common mechanism of action against these pathogens.Furthermore, similar MICs were observed when targeting MDR bacteria or sensitive ones such as P. aeruginosa and PA01 as well as S. aureus ATCC25923 and CF Marseille.All these considerations could indicate that 4 possesses a mechanism of action which circumvents the mechanisms of resistance involved by the bacteria towards conventional antibiotics.The cytotoxic activity of isonitrosomalononitrile silver(I) salt 4 was also assessed by investigating the cell viability.Thus, two different physiological mechanisms involving the ability of mitochondrial dehydrogenases to transform WST-1 to formazan as well as the lysosomal membranes capacity to concentrate Neutral Red.All the results indicated that isonitrosomalononitrile silver(I) salt 4 has a moderate cytotoxicity with an IC 50 around 35 µM with respect to requested concentration necessary against all the bacteria tested.On the other hand, no differences are observed in the IC 50 values regarding the nature of the cell line, or the vital dye considered suggesting a global impact on different intracellular targets (Table

Table 1 .
Crystal data and structure refinement details for 3.