Synthesis, Structure, Biological Activity, and Luminescence Properties of a “Butterfly”-Type Silver Cluster with 3-Benzyl-4-phenyl-1,2,4-triazol-5-thiol

A new silver(I) cluster [Ag8L4(Py)(Pype)]·4Py·11H2O (I) with 3-benzyl-4-phenyl-1,2,4-triazol-5-thiol (L) was synthesized via the direct reaction of AgNO3 and L in MeOH, followed by recrystallization from a pyridine–piperidine mixture. The compound I was isolated in a monocrystal form and its crystal structure was determined via single crystal X-ray diffraction. The complex forms a “butterfly” cluster with triazol-5-thioles. The purity of the silver complex and its stability in the solution was confirmed via NMR analysis. Excitation and emission of the free ligand and its silver complex were studied at room temperature for solid samples. The in vitro biological activity of the free ligand and its complex was studied in relation to the non-pathogenic Mycolicibacterium smegmatis strain. Complexation of the free ligand with silver increases the biological activity of the former by almost twenty times. For the newly obtained silver cluster, a bactericidal effect was established.

A well-studied family of biologically active ligands that can be used to create new metallodrugs includes the derivatives of 1,2,4-triazoles, which have been known for more than 100 years and the methods of their synthesis and chemical properties have been studied extensively [27].Their biological activity has been proven via many years of clinical use as drugs registered in the European Pharmacopoeia, for example, Alprazolam (sedative), Anastrozole (treatment for breast cancer), Brotizolam (sedative), Fluconazole (antifungal), Itraconazole (antifungal), Letrozole (treatment for breast cancer), Ribavirin (antiviral), Terconazole (antifungal), and Trapidi (antiaggregant) [28].Despite more than a century-long history of studying triazoles, they still attract the attention of researchers [29].When designing novel bioactive metal complexes, it is very important to know the mechanism of pharmacokinetics; in the case of 1,2,4-triazole derivatives, these data are already available [30][31][32], which can facilitate and reduce the cost of further testing of the corresponding metallodrugs.
To date, reliable data have been obtained on the antifungal, antioxidant, anticonvulsant, anti-inflammatory activity of metal complexes with 1,2,4-triazoles and their potential use for the treatment of tuberculosis, diabetes, and different types of cancer [33,34].It was shown that triazole-derived Schiff base ligands and their cobalt(II), nickel(II), copper(II), and zinc(II) complexes reveal antibacterial and antifungal activity, of which the study proved that the metal complexes showed better activity than the ligands after coordination [35,36].It was also shown that the addition of other donor atoms (oxygen, nitrogen, and sulfur) to triazole derivatives leads to the formation of biologically active complexes.For example, vanadyl(IV) complexes with mono-and di-substituted ONS donor triazoles reveal antibacterial, antifungal, and cytotoxic activity [37,38].
According to the previous studies, silver cation is a promising complex-forming ion in the design of metallodrugs [39][40][41][42].Silver-containing compounds are currently used for the therapy of skin diseases and burns: silver sulfadiazine [39], silver proteinate (Protargol) [43], and silver nitrate [44].Since silver has long been used in the treatment of various diseases, its pharmacokinetics, like 1,2,4-triazole derivatives, are also well studied [45,46].Having analyzed the Cambridge structural database, we found that only a few dozen structurally characterized silver complexes with 1,2,4-triazole derivatives are known, and to the best of our knowledge, the biological activity of these compounds have not been studied.
Bioactive luminescent metallodrugs are of great interest in the field of bioinorganic chemistry due to the potential applications of these compounds for the imaging of living cells [47].This approach can be used to track the distribution of luminescent substances within a cell and study their uptake by cells [48].Undoubtedly, the leaders in bioimaging among compounds used in modern medicine and biology are complexes based on lanthanides [49,50].However, currently more and more research is devoted to promising luminescent probes in living systems based on Ag(I) (with simultaneous antibacterial properties).It was found that luminescent properties of Au(I) and Ag(I) complexes with N-heterocyclic carbenes can be used to investigate biodistribution [51].
Thus, the goal of this study was to develop a method for the synthesis of a new silver complex with a 1,2,4-triazole sulfur-containing derivative-3-benzyl-4-phenyl-1,2,4triazol-5-thiol (L, Scheme 1)-and determine its molecular structure, confirm the purity, and analyze the luminescent properties of the free ligand and the complex.Also, the purpose of this study was to find out whether the free ligand and its silver complex exhibit biological activity against M. Smegmatis (a model non-virulent strain of M. tubercolisis) and how the coordination of the ligand affects these properties.This current work is a part of our ongoing research to find new anti-tuberculosis metallodrugs [22,[24][25][26]62].

Synthesis and Characterization
The reaction of L and AgNO3 in a methanol solution with further crystallization from the mixture of pyridine (Py) and piperidine (Pype) (Scheme 2) led to the formation of the Scheme 1. Keto-enol tautomerization of L.

Synthesis and Characterization
The reaction of L and AgNO 3 in a methanol solution with further crystallization from the mixture of pyridine (Py) and piperidine (Pype) (Scheme 2) led to the formation of the product [Ag 8 L 4 (Py)(Pype)]•4Py•11H 2 O (I) in a form of polycrystals, suitable for single-crystal X-ray diffraction analysis.A mixture of pyridine-piperidine was used as a solvent, since similar compounds were previously obtained by adding pyridine and triphenylphosphine to a white precipitate, which indicates the need for the presence of strong bases of a donor nature to convert the resulting compound into a soluble form [63].The reaction proceeds in air without any precautions about daylight.Decanted and twice washed with cold distilled water, the major product was isolated in a 90% yield.Scheme 1. Keto-enol tautomerization of L.

Synthesis and Characterization
The reaction of L and AgNO3 in a methanol solution with further crystallization from the mixture of pyridine (Py) and piperidine (Pype) (Scheme 2) led to the formation of the product [Ag8L4(Py)(Pype)]•4Py•11H2O (I) in a form of polycrystals, suitable for singlecrystal X-ray diffraction analysis.A mixture of pyridine-piperidine was used as a solvent, since similar compounds were previously obtained by adding pyridine and triphenylphosphine to a white precipitate, which indicates the need for the presence of strong bases of a donor nature to convert the resulting compound into a soluble form [63].The reaction proceeds in air without any precautions about daylight.Decanted and twice washed with cold distilled water, the major product was isolated in a 90% yield.
Scheme 2. Synthesis route of compound I, solvate molecules of water and pyridine have been omitted for clarity.
The 1 H NMR spectrum of I (Figure 1) is in agreement with the structural data.There is no signal of an SH proton in the 1 H NMR spectrum of I, which confirms the deprotonation of the ligand.The signal of CH2 protons is upfield shifted as compared to that one for the free ligand (3.65 and 3.86 ppm, respectively-see also Supplementary Materials).The signals from CH of pyridine (7.6-9.0 ppm) and the CH2, NH groups of piperidine (1.2-3.1 ppm) are also observed in the 1 H NMR spectrum.The 13 C NMR spectrum of I could not be recorded due to the low solubility of the compound in the common deuterated solvents.When trying to accumulate data to record a 13 C NMR spectrum over a 24 h period, we also discovered that a white precipitate formed in the NMR ampoule.It is likely that deuterated dimethyl sulfoxide molecules replace pyridine and piperidine, reducing the solubility of complex I. Scheme 2. Synthesis route of compound I, solvate molecules of water and pyridine have been omitted for clarity.
The 1 H NMR spectrum of I (Figure 1) is in agreement with the structural data.There is no signal of an SH proton in the 1 H NMR spectrum of I, which confirms the deprotonation of the ligand.The signal of CH 2 protons is upfield shifted as compared to that one for the free ligand (3.65 and 3.86 ppm, respectively-see also Supplementary Materials).The signals from CH of pyridine (7.6-9.0 ppm) and the CH 2 , NH groups of piperidine (1.2-3.1 ppm) are also observed in the 1 H NMR spectrum.The 13 C NMR spectrum of I could not be recorded due to the low solubility of the compound in the common deuterated solvents.When trying to accumulate data to record a 13 C NMR spectrum over a 24 h period, we also discovered that a white precipitate formed in the NMR ampoule.It is likely that deuterated dimethyl sulfoxide molecules replace pyridine and piperidine, reducing the solubility of complex I.

Molecular Structure of I
Complex I crystallizes in the monoclinic space group Pc in a form of crystal solvate [Ag 8 L 4 (Py)(Pype)]•4Py•11H 2 O. Since the solvate molecules of pyridine in the crystal are in strong thermal vibrations, only two solvate pyridine molecules were clearly identified, while two other molecules were taken into account as diffuse scattering using the SQUEEZE procedure [64].Complex I is a dimer of two butterfly-type silver-metal clusters (Figures 2 and 3), in which all Ag•••Ag distances lie in the range of 2.969(2)-3.254(2)Å.These distances are significantly greater than the sum of the covalent 2.7 Å radii of silver and can apparently be considered as weak secondary interactions (argentophilic) [65,66].
This molecule is pseudocentrosymmetric with the center of inversion at the center of mass of the silver atoms.The structural similarity of the two butterflies that make up the dimer is especially clearly visible when they are superimposed on each other (Figure 4).There is an almost complete coincidence of the positions of all atoms, with the exception of some differences in the orientation of the terminal benzyl substituents, as well as the difference between Py and Pype, which are coordinated by silver atoms.Four silver atoms of one part of the molecule coordinate four ligands, with each ligand bonded to three metal atoms: the nitrogen atom is bonded to one metal atom (Ag-N 2.275(13)-2.358(15)Å), and a sulfur atom connects two other metal atoms (Ag-S 2.421(4)-2.670(4)Å).The silver atoms at the base of the butterfly have a planar trigonal coordination AgNS2 (CN = 3), which is typical for silver clusters formed by a ligand containing an SCN fragment, where the sulfur atom is exocyclic and the CN is endocyclic, while the silver atoms at the tops of the butterfly wings, due to an additional coordination with the N-donor fragments, have a tetrahedral coordination AgN2S2 (CN = 4)."Butterfly" clusters are not typical for silver with this type of ligands, and in CCDC, there is only one similar structure obtained by Robert P. Lattimer et al. [63].Much more often a six-nuclei "Cyclohexane-Chair"-type cluster is formed [67,68].

Luminescent Properties
To the best of our knowledge, the luminescent properties of known silver(I) complexes have been studied mainly for square-planar polyhedrals [69], while the studied I contains two types of Ag + cations with different environments-triangular and distorted tetrahedral.Four silver atoms of one part of the molecule coordinate four ligands, with each ligand bonded to three metal atoms: the nitrogen atom is bonded to one metal atom (Ag-N 2.275(13)-2.358(15)Å), and a sulfur atom connects two other metal atoms (Ag-S 2.421(4)-2.670(4)Å).The silver atoms at the base of the butterfly have a planar trigonal coordination AgNS 2 (CN = 3), which is typical for silver clusters formed by a ligand containing an SCN fragment, where the sulfur atom is exocyclic and the CN is endocyclic, while the silver atoms at the tops of the butterfly wings, due to an additional coordination with the N-donor fragments, have a tetrahedral coordination AgN 2 S 2 (CN = 4)."Butterfly" clusters are not typical for silver with this type of ligands, and in CCDC, there is only one similar structure obtained by Robert P. Lattimer et al. [63].Much more often a six-nuclei "Cyclohexane-Chair"-type cluster is formed [67,68].

Luminescent Properties
To the best of our knowledge, the luminescent properties of known silver(I) complexes have been studied mainly for square-planar polyhedrals [69], while the studied I contains two types of Ag + cations with different environments-triangular and distorted tetrahedral.
The excitation spectrum of solid ligand L at room temperature at λ em = 490 and 510 nm gives a set of bands of 200-430 nm with a maxima at 238, 253, 281, and 400 nm, respectively (Figure S6a).In the emission spectra at λ ex = 240, 250 and 280 nm, a set of bands is observed in the region of 350-650 nm (Figure S6a), where the most intense bands have a maxima at 491, 509, and 530 nm.The excitation spectrum (λ em = 400 nm) of I under similar conditions is represented by broad bands in the region of 200-350 nm with a maxima at 240, 255, and 335 nm (Figure S6b).The emission spectrum of I at λ ex = 255 nm contains wide bands in the region of 300-600 nm with a maxima at 405, 422, and 487 nm.An analysis of the photophysical properties showed that the transition from ligand L to complex I is accompanied by a change in emission as a result of coordination of the ligand to the metal and/or transition to the anionic form.The relative emission intensity of the ligand, as well as its complex, is low.

Biological Activity
The biological activity of L and I was studied using the nonpathogenic mycobacterial strain Mycolicibacterium smegmatis.In mycobacterial strains, the resistance to chemical treatment agents is attributed to the low permeability of its cell wall due to its unusual structure.M. smegmatis is a rapidly growing nonpathogenic bacterium, which is therefore used as a model for slowly growing Mycobacterium tuberculosis and for the screening of antituberculosis drugs [70].The M. smegmatis test system exhibits higher resistance to antibiotics and antituberculosis agents than M. tuberculosis; therefore, the concentration <100 nmol/disc in contrast to M. tuberculosis (<4 µg/mL) is used as the selection criterion [71].All the results obtained for the in vitro biological activity of the compounds under study were compared with the activity of the first-line antituberculosis drug, Rifampicin (Rif), under the selected conditions of the experiment.The results of the antimycobacterial activity study are presented in Table 1.The data shown in Table 1 demonstrate that ligand L does not exhibit significant biological activity against the strain of mycobacterium.In turn, the efficiency of complex I is increased by almost twenty times in comparison with the free ligand.Similar effect-an increase in biological activity after complex formation is observed quite often [62,72].The effectiveness of strain suppression-like the reference drug Rif, I-has a bactericidal effect; unlike the previously obtained silver complex [62], it exhibits a bacteriostatic effect (most likely associated with the development of resistance genes of mycobacterium).
The IR spectrum of I was recorded in the range of 400-4000 cm -1 using a PerkinElmer (Waltham, MA, USA) Spectrum 65 spectrophotometer equipped with a Quest ATR Accessory (Specac (Orpington, Kent, UK)).The method of attenuated total reflection (ATR) was used.Excitation and emission spectra of the solid samples were recorded at room temperature in the visible range of the spectrum using a PerkinElmer LS-55 spectrometer.
The 1 H NMR spectra was registered on a Bruker AVANCE (300 MHz) spectrometer with TMS as the internal reference and DMSO-d 6 as the solvent.
To study the biological activity of the free ligand L and its complex I, the paper disk method was employed using the nonpathogenic M. smegmatis mc 2 155 strain.A solution of each substance was prepared as follows: a sample of the substance was dissolved in dimethyl sulfoxide to a concentration of 100 mmol or less, depending on the solubility of the substance.Then, tenfold and twofold dilutions of the resulting solution were made and applied to paper disks in an amount of 5 µL/disk.The test method involved measuring the size of the inhibition zone around paper discs containing a compound, which were placed on an agar plate and then seeded with the strain.The concentration of the compound was varied to determine its effect on the growth of the strain.The bacteria washed off Petri dishes with the Trypton-soy agar M-290 medium (Himedia, Mumbai, India) were grown overnight in the Lemco-TW liquid medium (Lab Lemco' Powder 5 g•L −1 (Oxoid, Basingstoke, UK), Peptone special 5 g•L −1 (Oxoid), NaCl 5 g•L −1 , Tween-80) at +37 • C until the average logarithmic growth phase occurred at optical density OD600 = 1.5, which was then mixed with a molten agar medium M-290 in a ratio of 1:9:10 (culture:Lemco-TW:M-290).The mixture was poured into Petri dishes containing pre-solidified agar medium (M-290) at a concentration of 5 mL/dish.After the top layer of agar had solidified, paper discs impregnated with a solution containing the test compound were placed on top of the plate.The culture was incubated for 24 h at +37 • C. The inhibition zone diameters of M. smegmatis mc 2 155 growth around the paper disks contained the free ligand L and its complex I were determined.The Minimum Inhibiting Concentration (MIC) was equal to the concentration of L and I that provided the smallest growth inhibition zone.
X-ray diffraction data for I were collected at 150 K with a Bruker Quest D8 CMOS diffractometer, using graphite monochromated Mo-Kα radiation (λ = 0.71073 Å, ω-scans).The crystal structure of I was solved using Intrinsic Phasing with the ShelXT [74] in Olex2 [75] and then refined with the ShelXL [76] refinement package using Least-Squares minimization against F 2 in the anisotropic approximation for non-hydrogen atoms.Hydrogen atoms were calculated and they all were refined in the isotropic approximation within the riding model.The contribution of highly disordered solvent molecules in structural voids was treated as diffuse using the SQUEEZE procedure implemented in the PLA-TON software, version 2023.1 [64].The electron count within cavities (V1 = 726 Å3 and V2 = 688 Å3) measured 170 and 223 e − , respectively; per formula unit of I, their sum corresponds to 11 water and 4 pyridine molecules.The crystal data and structure refinement parameters are given in Table 2. CCDC 2300382 contains the supplementary crystallographic data for this manuscript.

Conclusions
Here, we have presented a method for preparing a new silver(I) complex with 3-benzyl-4-phenyl-1,2,4-triazol-5-thiol and determined its crystal structure.Photophysical studies of L and its complex I showed weak emission of the organic ligand in neutral and anionic form; the complexation is accompanied by the changes in the emission spectra.The study of the biological activity of L and I have determined that the free ligand does not exhibit significant bactericidal action against Mycobacteria smegmatis, whilst complexation with silver has increased the biological activity of the ligand by 20 times.We believe that our results may encourage chemists to synthesize novel bioactive silver complexes of 1,2,4-triazole derivatives via a facile method in water-methanol mixtures to prepare advanced pharmaceutical products.

13 Figure 1 .
Figure 1. 1 H NMR spectra of I (blue line) and free ligand (red line).2.2.Molecular Structure of I Complex I crystallizes in the monoclinic space group Pc in a form of crystal solvate [Ag8L4(Py)(Pype)]•4Py•11H2O.Since the solvate molecules of pyridine in the crystal are in strong thermal vibrations, only two solvate pyridine molecules were clearly identified,

Figure 1 .
Figure 1. 1 H NMR spectra of I (blue line) and free ligand (red line).

Figure 1 .
Figure 1. 1 H NMR spectra of I (blue line) and free ligand (red line).

Figure 2 .
Figure 2. Molecular structure of I. Thermal ellipsoids are drawn at 30% probability level.Hydrogen atoms are omitted for clarity.Argentophilic interactions are shown in dotted lines.

Figure 2 . 13 Figure 3 .
Figure 2. Molecular structure of I. Thermal ellipsoids are drawn at 30% probability level.Hydrogen atoms are omitted for clarity.Argentophilic interactions are shown in dotted lines.Molecules 2024, 29, x FOR PEER REVIEW 5 of 13

Figure 3 .
Figure 3. Molecular structure of I. Hydrogen atoms, solvate pyridines, and benzyl and phenyl substituents of the ligand have been omitted for clarity.Argentophilic interactions are shown in dotted lines.

Figure 4 .
Figure 4. Superposition of a four-nuclear fragment of dimer I onto another four-nuclear fragment via an inversion pseudocenter; the standard deviation for silver and sulfur atoms is 0.09 Å. Superposition of a four-nuclear fragment of dimer I (solid line) onto another four-nuclear fragment (dashed line) via an inversion pseudocenter; the standard deviation for silver and sulfur atoms is 0.09 Å.

Figure 4 .
Figure 4. Superposition of a four-nuclear fragment of dimer I (solid line) onto another four-nuclear fragment (dashed line) via an inversion pseudocenter; the standard deviation for silver and sulfur atoms is 0.09 Å.

Table 1 .
The results of antibacterial activity study of free ligand L and complex I against M. smegmatis.The diameter of the paper disk is 6 mm.* The M. smegmatis growth inhibition zone is transparent, there is no weak background growth of the culture (bactericidal effect).** M. smegmatis growth inhibition zone overgrows (bacteriostatic effect).0-no growth inhibition zone.

Table 2 .
Crystal data and structure refinement for I.