Synthesis, crystal structure and antimicrobial activities of two silver(I) complexes based on bis(pyrazole) and carboxylic acid co-ligands

Two dinuclear and centrosymmetric silver-containing complexes, namely [Ag2(3,5-dmpz)2(3,5-dmpz methanol)2(tbtc)] (1) and [Ag2(3,5-dmpz)2(3,5-dmpz methanol)2(dbtc)] (2) (3,5-dmpz: dimethylpyrazole; 3,5-dmpz methanol: 3,5-dimethylpyrazole methanol; H2tbtc: tetrabromoterephthalic acid; H2dbtc: 2,5-dibromoterephthalic acid), were prepared and characterized. X-ray crystallographic data of 1 and 2 revealed that trigonal environment of the Ag ions has a “compressed Y’s” geometry. The antimicrobial activity of 1 and 2 was tested against the Gram-positive, Gram-negative bacteria and fungus, displaying the better inhibiting activity than the individual AgNO3 and 3,5-dimethylpyrazole, especially for Candida albicans (IC50 2.5 ± 0.1 mg mL −1 and 2.8 ± 0.2 mg mL−1). The quantified IC50 values displayed that complexes 1 and 2 were more effective against fungus than the Gram-negative bacteria and Gram-positive bacteria.


Introduction
The heavy metal silver ion has strong cytotoxicity to microorganisms due to its inhibition to bacterial reproduction and even bactericidal effect through its interaction with nucleic acids and bases in DNA [1,2].In comparison with simple silver salts, the kinds of complexes of silver ions which represent unconventional forms of silver salts are provided with tunable properties of inhibiting microorganisms [3][4][5][6][7][8][9][10][11][12].Therefore, people are more focused on the antibacterial effect instead of various coordination patterns of Ag [13,14].Since the beginning of the twentieth century, silver compounds have been widely used in clinical disinfection and diminishing inflammation.It has been found that silver complexes of various ligands, such as carboxylic acids, amino acids, nitrogen-heterocyclic carbine, phosphorus and sulfur atoms as donor atoms, show selective inhibitory effects on diverse cancer cells [15][16][17][18][19].For example, the salicylic acid complex of silver, which is used as NSAIDS, can reduce the risk of colon cancer and prevent against lung, esophageal and gastric cancers [20][21][22].The antioxidant Ag complexes of carboxylic acids also can be used as fungicides in medicine, cosmetics and food due to their interaction with sulfur-containing proteins in the body to prevent cell reproduction [22,23].Imidazole-based drugs have an excellent inhibitory effect on anaerobic microorganisms.Some silver complexes containing imidazole and its derivative groups have been found to be used in the treatment of antimicrobial resistance [24][25][26].
Whether the silver complex has bacteriostatic effect, one of the key factors is the biological active groups of the ligands and combination mode between the silver ion center and the ligands.Our group has previously reported tetranuclear complex with AgN 2 O structure based on pyrazole derivatives, which is a promising candidate for bacteriostatic agent [27].In this paper, attempts have been made to prepare binuclear Ag complexes of the tripodal ligand TMPzA (Tris (3,5-dimethylpyrazolomethyl) amine) by the reaction of TMPzA, silver salt and carboxylate acids in 1:1:0.5 molar proportion.While it is similar to our previous study [28], the coordination compounds of the expected tripodal ligand fail to occur.In the complexes, the ligand TMPzA also shows different extents of decomposition and it cleaves into smaller building blocks: 3,5-dimethylpyrazole and 3,5-dimethylpyrazole methanol.The synthesized complexes were then tested against different representative Gram-negative, Grampositive bacteria and fungus.

Materials and techniques
All chemicals employed were commercially available and used as received without further purification.Bacterial strains were provided by the laboratory of Department of Environment and Safety Engineering, Taiyuan Institute of Technology and Taiyuan Center for Disease Control and Prevention.The ligand TMPzA was synthesized according to the procedure of W. L. DRIESSEN et al. [29].Infrared spectra were recorded on a Bruker VERTEX-70 spectrometer as KBr pellets in the frequency range 4000-400 cm −1 .The elemental analyses (C, H, N contents) were determined on a CE instruments EA 1110 analyzer.NMR spectra were recorded at the Analytical Testing Center, Tai Yuan Institute of Technology using a AVANCE III HD 400 MHz spectrometer.

Synthesis of complexes (1) and (2)
To a solution of 1 mmol AgNO 3 (170 mg, 1 mmol) in 20 mL CH 3 OH, a stoichiometric amount of the ligand TMPzA (341 mg, 1 mmol) and the bridged carboxylate (0.5 mmol) tetrabromoterephthalic acid for 1, 2,5-dibromoterephthalic acid for 2 were added.The mixture was stirred at room temperature for 1 h, and it was protected from light.Complexes 1 and 2 were obtained from the mother solution for several days.Yield (calculated on the basis of the Ag): 62% for 1 and 73% for 2.

X-ray data collection and structure refinement
Crystal data and details of the structure determinations are compiled in Table S1.All the single crystal data were collected at 298 K on a Bruker ApexII CCD diffractometer with Mo-Kα radiation (λ = 0.71073 Å).Absorption corrections were performed with the SADABS software packages.The structures were solved by using SHELXT 2014 and refined using SHELXL 2018 [30,31].All structures were solved by a combination of direct methods and difference Fourier syntheses and against F 2 by the full matrix least-squares technique.All non-hydrogen atoms were refined anisotropically.The hydrogen atoms on non-carbon atoms were located from difference Fourier maps, and the hydrogen atoms connecting on the carbon atoms were determined with theoretical calculation and refined isotropically.

Antimicrobial test
Complexes 1, 2 and 3,5-dimethylpyrazole were dissolved in DMSO, and AgNO 3 was dissolved in water to prepare the solutions with the concentration of 0.1 mol/L.The oxford cup agar diffusion method was used for antimicrobial test against each species.The solid culture medium after sterilized was poured while it was hot into the petri plates and coated uniformly to the slice thickness of 1-3 mm.Next, 200 μL volume of different solutions of complex 1, 2, AgNO 3 , 3,5-dimethylpyrazole and DMSO (as a control) were injected into the oxford cup to ensure the equivalency.
The incubation was carried out at 37 °C for 48 h, and the zones diameter (mm) of inhibition was measured and recorded using a millimeter scale after incubation.Every test was repeated three times to eliminate errors possibly that occurred.

IC 50 determination
The five different concentrations of complexes 1 and 2 including 0.1 mg mL −1 , 2.5 mg mL −1 , 5.0 mg mL −1 , 7.5 mg mL −1 and 10 mg mL −1 were prepared.Every tested agent was mixed with liquid nutrient medium and shakes followed by co-culturing for a period of time.Then, the absorbance of bacterial fluid at 600 nm was determined as the absorbance is proportional to concentration of microorganism.The bacteriostatic rate was calculated by the equa- , where OD 0 represents the absorbance of bacterial fluid without any tested agent.Finally, bacteriostatic rate plot was taken to concentration of tested sample and the value of IC 50 was obtained.

Synthesis of 1 and 2
As shown in scheme 1, the ligand TMPzA reacted with AgNO 3 and bridge ligands in 1:1:0.5 mol ratio in CH 3 OH at room temperature to form complex (1) and (2).In the process of coordination, the TMPzA cleaves into 3,5-dmpz and 3,5-dmpz methanol that coordinate to Ag with the N atoms of the pyrazole rings.Also, the O atoms of the carboxylic acids coordinate to two Ag atoms forming dinuclear complexes.
The complexes 1 and 2 possess the similar structure, and we take 1 as example.As shown in Fig. 1 [37].It is interesting that 1 and 2 are linked together through weak Ag…Br interactions to produce 1D chains as exhibited in Figs. 3 and 4. The distances of 3.241 Å and 3.422 Å between Br atom of one molecule and the Ag atom from another molecule of complexes 1 and 2, respectively, indicate the presence of weak noncovalent Ag…Br interactions [38,39].
IR spectra (Fig. S2) showed the carboxylate groups of tbtc and dbtc coordinated with two Ag atoms in a monodentate mode, bridging the two metal centers, which were consistent with the crystal structure analysis.The characteristic υ as bands of carboxylate groups (1649 and 1580 cm −1 for 1, 1621 and 1599 cm −1 for 2) and υ s band (1284 cm −1 for 1 and 1310 cm −1 for 2) with values of Δ = υ as (COO − ) − υ s (COO − ) Scheme 1 Schematic diagram of the reactions for the synthesis of the silver(I) complexes 1 and 2 = 360 cm −1 and 265 cm −1 for 1 and 311 cm −1 and 289 cm −1 for 2, which indicated the monodentate (Δ > 200 cm −1 ) coordination modes in tbtc and dbtc [40].

Antimicrobial activity
Antibacterial activity of complexes 1, 2 and 3,5-dimethylpyrazole, AgNO 3 as well as DMSO was assayed in vitro for their ability to inhibit the growth of Gram-positive bacteria (Bacillus subtilis (ATCC 9372), Staphylococcus aureus (ATCC 6538) and Micrococcus luteus (ATCC 7468)), Gramnegative bacteria (Escherichia coli (ATCC 25922)) and the fungus Candida albicans (ATCC 14053).The activities were evaluated by measuring the zones of diameter of inhibition.When the concentration of tested sample was 0.1 mg/mL, the results are illustrated as in Table 1.

Conclusions
In this paper, we have synthesized and characterized two dinuclear and centrosymmetric silver-containing complexes of 3,5-dimethyl pyrazole and 3,5-dmpz methanol resulting from the breakdown of TMPzA.In the complexes, two Ag centers were linked by the carboxylate bridges and formed trigonal environment.The coordination mode of N-Ag-N contained in AgN 2 O is infrequent among the masses of silver complexes.Interestingly, the Ag…Br-aryl interaction has been used to construct a metal-organic 1D chain structure.We also tried to synthesize compound using 3,5-dimethyl pyrazole and 3,5-dimethyl methanol instead of TMPzA, but we failed to obtain complexes 1 and 2. It could be attributed to the advantages of using TMPzA directly which can provide 3,5-dimethylpyrazole and 3,5-dimethylpyrazole methanol at the same time when it cleaved, so that Ag ions can quickly combine to them simultaneously.The antibacterial and antifungal activities of complexes 1 and 2 were also assessed.The IC50 values for the complexes indicated that they were more effective against fungus than the bacteria and the results also showed that they were moderate antibacterial agents compared with other similar silver complexes based on nitrogen-contained ligands.

Fig. 3 Fig. 4
Fig. 3 1D chain of complex 1 constructed via Ag…Br interactions indicated by dotted line, hydrogen atoms were omitted for clarity