Synthesis of a New Dinuclear Ag(I) Complex with Asymmetric Azine Type Ligand: X-ray Structure and Biological Studies

: Aspects of the molecular and supramolecular structure of the new dinuclear [Ag(L)(NO 3 )] 2 complex, where L is 2-(( E )-((( E )-1-(thiazol-2-yl)ethylidene)hydrazono)methyl)phenol, were discussed. The complex was crystallized in the monoclinic crystal system and P2 1 /n space group. The unit cell parameters are a = 10.3274(2) Å, b = 11.4504(3) Å, c = 12.7137(3) Å and β = 108.2560(10) ◦ . The asymmetric unit comprised one [Ag(L)(NO 3 )] formula in which the azine and nitrate ligand groups act as NN- and OO-bidentate chelates, respectively. The coordination environment of the Ag(I) is completed by one weak Ag-O bond with another [Ag(L)(NO 3 )] unit, leading to the dinuclear formula [Ag(L)(NO 3 )] 2 . This was clearly revealed by Hirshfeld analysis. Additionally, the Ag . . . C, O . . . H and C . . . C intermolecular interactions played an important role in the molecular packing of the studied complex. The antimicrobial, antioxidant and cytotoxic activities of the [Ag(L)(NO 3 )] 2 complex and the free ligand ( L ) were discussed. While the [Ag(L)(NO 3 )] 2 complex showed very weak antioxidant activity, the results of the antifungal and cytotoxic activities were promising. The inhibition zone diameters (IZD) and the minimum inhibitory concentration (MIC) values were determined to be 31 mm and 20 µ g/mL, respectively, against A. fumigatus , which is compared to 17 mm and 156 µ g/mL, respectively, for the positive control Ketoconazole. Generally, the Ag(I) complex has better antimicrobial activities than the free ligand against all microbes except for S. aureus , where the free ligand has higher activity. Additionally, the IC 50 value against colon carcinoma (HCT-116 cell line) was determined to be 12.53 ± 0.69 µ g/mL, which is compared to 5.35 ± 0.49 µ g/mL for cis -platin. Additionally, the Ag(I) complex displays better cytotoxicity than the free ligand ( L ) (242.92 ± 8.12 µ g/mL).


Introduction
The search for novel drugs for the treatment of diseases such as cancers and antibioticresistant microbes is still a challenge, and much effort has been devoted to the discovery of new chemotherapeutic agents. Transition metal complexes are continuously designed, synthesized, and assessed against different targets [1][2][3][4][5]. For example, cis-platin is used as an anticancer drug, although it still has limitations due to resistance and significant side effects [6]. This disadvantage encouraged many scientists to search for alternative transition metal complexes for different purposes [7]. Silver(I) complexes play a significant role in the treatment of many diseases such as tumors and bacterial infections [8][9][10][11][12][13][14][15]. The optimism surrounding silver-based drugs is due to their higher drug-tolerance profiles and higher selectivity to cancer cells rather than the non-cancerous cells compared to other 15]. The optimism surrounding silver-based drugs is due to their higher drug-tolerance profiles and higher selectivity to cancer cells rather than the non-cancerous cells compared to other metal complexes. These potential benefits make this area of research important and create the need for further exploration.
One important class of chelating ligands in coordination chemistry is Schiff bases, which are well known and have become important molecules in pharmaceutical and medicinal fields. They exhibit many biological effects, including anti-fungal [16,17], antibacterial [18], herbicidal [19], anti-HIV [16] antitubercular [20], anti-inflammatory [21] and anti-tumor effects [22]. Coordination between the metal and chelating ligands creates a new product with enhanced therapeutic efficiency [23][24][25]. Understanding and discovering the functions of metal ions in disease treatment is still a challenge in medicinal inorganic and bioinorganic chemistry [26]. In many cases, the donor sequence of the chelating ligands and the identity of the metal play a crucial role in pharmacological activity [27,28]. The thiazole-based Schiff base ligands and their analogues are an important class of chelating ligands in coordination chemistry [29]. The cytotoxic properties of some silver(I) complexes of Schiff bases derived from thiazole and pyrazine scaffolds were reported by X.-J. Tan et al. [30]. Additionally, Ag(I) thiazole-based coordination polymers have interesting photophysical properties [31]. Recently, our research group reported the synthesis of some Ag(I) complexes with symmetric azine-type ligands and explored their molecular, supramolecular and biological aspects [32][33][34].
Herein, we reported the synthesis, structural and biological evaluation of a new dinuclear Ag(I) complex with the asymmetric azine-type ligand shown in Figure 1. The new Ag(I) metal complex was evaluated for biological efficacy against different targets including anticancer, antimicrobial and antioxidant reactivities.

Synthesis and Characterizations
In our previous work, we examined the reaction of a number of hydrazones and hydrazides with Ag(I) salts [32][33][34]. Unexpectedly, these reactions proceeded to the formation of the corresponding azine, affording the Ag(I)-azine complexes as final products. Conversely, the reaction of AgNO3 with the asymmetric azine ligand L proceeded without hydrolysis and afforded the dinuclear [Ag(L)(NO3)]2 complex with high yield (Scheme 1). The product was characterized using elemental analysis, FTIR and XPS spectroscopic techniques, and then its structure was unambiguously determined with the aid of singlecrystal X-ray diffraction. The FTIR spectral band corresponding to the υC=N stretching vibration appeared at 1615 cm −1 in case of the [Ag(L)(NO3)]2 complex, while it appeared at 1619 cm −1 in the free ligand (L). This small spectral shift could be attributed to the coordination between the Ag(I) ion and the azine ligand via the N-atom of the C=N group. The υO-H and υC=C stretching vibrations appeared in both compounds at the same wavenumbers of 3432 and 1548 cm −1 , respectively. An intense band appeared at 1384 cm −1 only in the [Ag(L)(NO3)]2 complex, corresponding to the υN-O stretching vibration. This is good evidence of the presence of the nitrate group, which is not found in the FTIR spectra of the free ligand.

Synthesis and Characterizations
In our previous work, we examined the reaction of a number of hydrazones and hydrazides with Ag(I) salts [32][33][34]. Unexpectedly, these reactions proceeded to the formation of the corresponding azine, affording the Ag(I)-azine complexes as final products. Conversely, the reaction of AgNO 3 with the asymmetric azine ligand L proceeded without hydrolysis and afforded the dinuclear [Ag(L)(NO 3 )] 2 complex with high yield (Scheme 1). The product was characterized using elemental analysis, FTIR and XPS spectroscopic techniques, and then its structure was unambiguously determined with the aid of single-crystal X-ray diffraction. The FTIR spectral band corresponding to the υ C=N stretching vibration appeared at 1615 cm −1 in case of the [Ag(L)(NO 3 )] 2 complex, while it appeared at 1619 cm −1 in the free ligand (L). This small spectral shift could be attributed to the coordination between the Ag(I) ion and the azine ligand via the N-atom of the C=N group. The υ O-H and υ C=C stretching vibrations appeared in both compounds at the same wavenumbers of 3432 and 1548 cm −1 , respectively. An intense band appeared at 1384 cm −1 only in the [Ag(L)(NO 3 )] 2 complex, corresponding to the υ N-O stretching vibration. This is good evidence of the presence of the nitrate group, which is not found in the FTIR spectra of the free ligand. The X-ray photoelectron spectral analysis confirmed the elemental composition in [Ag(L)(NO3)]2 and highlighted the spin-orbital coupling for each element that is directly affected by their structure and oxidation states. Elemental composition and characteristic binding energies are reported in Table 1, while Figure 2 represents the binding energyintensity relationship for Ag, N, S, O and C in the studied complex [34][35][36].
Silver showed a characteristic doublet peak corresponding to the Ag(I) oxidation state as 3d3/2 and 3d5/2 with binding energies (B.E) of 374.50 and 368.49 eV, respectively, with spin-orbit splitting ΔE = 6.01 eV and an intensity ratio of 0.67. Nitrogen showed two peaks: N1s at 399.78 eV for covalent-coordinate nitrogen atoms and a characteristic peak of nitrate nitrogen N1sA at 406.71 eV. Sulphur in the thiazolyl ring showed a characteristic doublet peak at 165.92 and 164.76 eV, corresponding to S2p5/2 and S2p3/2, respectively, with ΔE = 1.16 eV and an intensity ratio of 0.55. Carbon showed three characteristic peaks as C1s, C1sA and C1sB at 284.68, 285.25 and 286.29 eV, respectively, confirming the presence of C-C, C-S and C-N/C-O bonds. Oxygen showed one broad peak centered at 532.6 eV.  The X-ray photoelectron spectral analysis confirmed the elemental composition in [Ag(L)(NO 3 )] 2 and highlighted the spin-orbital coupling for each element that is directly affected by their structure and oxidation states. Elemental composition and characteristic binding energies are reported in Table 1, while Figure 2 represents the binding energyintensity relationship for Ag, N, S, O and C in the studied complex [34][35][36].  The X-ray photoelectron spectral analysis confirmed the elemental composition in [Ag(L)(NO3)]2 and highlighted the spin-orbital coupling for each element that is directly affected by their structure and oxidation states. Elemental composition and characteristic binding energies are reported in Table 1, while Figure 2 represents the binding energyintensity relationship for Ag, N, S, O and C in the studied complex [34][35][36].
Silver showed a characteristic doublet peak corresponding to the Ag(I) oxidation state as 3d3/2 and 3d5/2 with binding energies (B.E) of 374.50 and 368.49 eV, respectively, with spin-orbit splitting ΔE = 6.01 eV and an intensity ratio of 0.67. Nitrogen showed two peaks: N1s at 399.78 eV for covalent-coordinate nitrogen atoms and a characteristic peak of nitrate nitrogen N1sA at 406.71 eV. Sulphur in the thiazolyl ring showed a characteristic doublet peak at 165.92 and 164.76 eV, corresponding to S2p5/2 and S2p3/2, respectively, with ΔE = 1.16 eV and an intensity ratio of 0.55. Carbon showed three characteristic peaks as C1s, C1sA and C1sB at 284.68, 285.25 and 286.29 eV, respectively, confirming the presence of C-C, C-S and C-N/C-O bonds. Oxygen showed one broad peak centered at 532.6 eV.  Silver showed a characteristic doublet peak corresponding to the Ag(I) oxidation state as 3d 3/2 and 3d 5/2 with binding energies (B.E) of 374.50 and 368.49 eV, respectively, with spin-orbit splitting ∆E = 6.01 eV and an intensity ratio of 0.67. Nitrogen showed two peaks: N1s at 399.78 eV for covalent-coordinate nitrogen atoms and a characteristic peak of nitrate nitrogen N1sA at 406.71 eV. Sulphur in the thiazolyl ring showed a characteristic doublet peak at 165.92 and 164.76 eV, corresponding to S2p 5/2 and S2p 3/2 , respectively, with ∆E = 1.16 eV and an intensity ratio of 0.55. Carbon showed three characteristic peaks as C1s, C1sA and C1sB at 284.68, 285.25 and 286.29 eV, respectively, confirming the presence of C-C, C-S and C-N/C-O bonds. Oxygen showed one broad peak centered at 532.6 eV.

X-ray Structure Description of [Ag(L)(NO 3 )] 2 Complex
The X-ray structure showing the asymmetric unit of the [Ag(L)(NO 3 )] 2 complex is shown in the upper part of Figure 3. This complex was crystallized in the monoclinic crystal system and P2 1 /n space group. The unit cell parameters were a = 10.3274 (2) (1) via one nitrogen atom from the thiazole moiety and another nitrogen atom from one of the two N-atoms of the azine group. The Ag1-N1 and Ag1-N2 distances were 2.218(5) and 2.603(4) Å, respectively ( Table 2). The bite angle of the azine ligand was 69.37 (16) • . Additionally, the Ag1-O2 and Ag1-O3 bonds with the nitrate group were not equidistant. The former was significantly shorter (2.347(6) Å) than the latter (2.631(6) Å). The bite angle in this case was only 50.4(2) • . Interestingly, the Ag1 formed a weak interaction with a neighboring complex molecule via the symmetry related O3# atom, where the Ag1-O3 # distance was found to be 2.781(7) Å. Hence, the molecular structure of this complex could be described by the dimeric structure [Ag(L)(NO 3 )] 2 shown in the lower part of Figure 3.
The packing of the [Ag(L)(NO 3 )] 2 complex is dominated by the weak non-covalent C-H . . . O interactions, as depicted in Table 3 Table 3.

Hirshfeld Analysis
It is well acknowledged that the molecules in the crystal are packed in a way that maximizes the crystal stability via complicated sets of intermolecular interactions. Hirshfeld analysis is considered a powerful tool for predicting all intermolecular contacts in the crystal. For the [Ag(L)(NO3)] complex unit, the different Hirshfeld surfaces are shown in Figure 5. There are three Hirshfeld surfaces: dnorm, shape index and curvedness. According to Spackman et. al., the dnorm is the normalized contact distance, shape index shows the shape of surface (concave (−1.0) to convex (+1.0)) and curvedness indicates the surface flatness (flat (−4.0) to singular (+0.4)) [37].  Table 3.

Hirshfeld Analysis
It is well acknowledged that the molecules in the crystal are packed in a way that maximizes the crystal stability via complicated sets of intermolecular interactions. Hirshfeld analysis is considered a powerful tool for predicting all intermolecular contacts in the crystal. For the [Ag(L)(NO 3 )] complex unit, the different Hirshfeld surfaces are shown in Figure 5. There are three Hirshfeld surfaces: d norm , shape index and curvedness. According to Spackman et al., the d norm is the normalized contact distance, shape index shows the shape of surface (concave (−1.0) to convex (+1.0)) and curvedness indicates the surface flatness (flat (−4.0) to singular (+0.4)) [37].
There are many red spots in the d norm map, and these refer to the short distance Ag The red spots close to the Ag1 and O3 atoms are related to the weak Ag1-O3 bond (2.782 Å), which confirms the dinuclear formula of this complex. Additionally, the presence of red/blue triangles combination in the shape index as well as the flat green area in curvedness reveals the presence of some π-π stacking interactions between the phenyl and thiazolyl aromatic moieties, where the shortest C . . . C contact distance is 3.408 Å and corresponds to the C2 . . . C10 contact. This interaction has a slightly longer distance than twice the vdWs radii of carbon atoms indicating relatively weak π-π stacking interactions between the phenyl and thiazolyl moieties. The rings centroid-centroid distance was calculated to be 3.697Å, which shows the importance of stacking interactions involving π-electrons of these aromatic rings in the molecular packing of the studied complex. All these interactions appeared as sharp spikes in the fingerprint plots, revealing short distances and strong interactions ( Figure 6). Inorganics 2022, 10, x FOR PEER REVIEW 7 of 14 There are many red spots in the dnorm map, and these refer to the short distance Ag…O, Ag…C and O…H contacts. The Ag1…C10 (3.389 Å) and Ag1…C11 (3.286 Å) as well as the O4…H1A (2.363 Å), O4…H5C (2.486 Å) and O4…H2 (2.375 Å) contacts have shorter distances than the vdWs radii sum of the interacting atoms. The red spots close to the Ag1 and O3 atoms are related to the weak Ag1-O3 bond (2.782 Å), which confirms the dinuclear formula of this complex. Additionally, the presence of red/blue triangles combination in the shape index as well as the flat green area in curvedness reveals the presence of some π-π stacking interactions between the phenyl and thiazolyl aromatic moieties, where the shortest C…C contact distance is 3.408 Å and corresponds to the C2…C10 contact. This interaction has a slightly longer distance than twice the vdWs radii of carbon atoms indicating relatively weak π-π stacking interactions between the phenyl and thiazolyl moieties. The rings centroid-centroid distance was calculated to be 3.697Å, which shows the importance of stacking interactions involving π-electrons of these aromatic rings in the molecular packing of the studied complex. All these interactions appeared as sharp spikes in the fingerprint plots, revealing short distances and strong interactions ( Figure 6).

Antimicrobial Activity
The antimicrobial activity of the studied Ag(I) complex on selected bacterial and fungal strains was determined in terms of the inhibition zone diameter (IZD) and the minimum inhibitory concentration (MIC). The antimicrobial activity results are depicted in Table 4. The IZDs are very small for the Gram-positive bacterial strain compared with Gram-negative bacteria. The sizes of the inhibition zones were 8 and 9 mm for S. aureus and B. subtilis, respectively, compared with 12 and 15 mm for E. coli and P. vulgaris, respectively. Hence, the studied Ag(I) complex is more potent against Gram-negative bacteria than Gram-positive bacteria. Moreover, the MIC value was lowest for P. vulgaris (625 µg/mL), which indicates the highest potency against this bacterium. In comparison with the antibacterial control Gentamycin, the studied Ag(I) complex is considered a weak antibacterial agent. In terms of antifungal activity, the studied [Ag(L)(NO 3 )] 2 complex inhibited both A. fumigatus and C. albicans. The IZDs were 31 and 18 mm for A. fumigatus and C. albicans, respectively, while the IZDs were 17 and 20 mm, respectively, for the standard Ketoconazole. The corresponding MIC values were 20, 625, 156 and 312 µg/mL. Hence, the studied Ag(I) complex showed the highest potency against A. fumigatus compared with the other microbes. Moreover, the Hirshfeld analysis gave accurate results for the percentages of the different intermolecular contacts between the surface and neighboring molecules (Figure 7). The most dominant contacts were the O…H and H…H interactions, which contributed to more than half of the whole observed contacts. Additionally, the percentage of Ag…O, Ag…C and C…C interactions were 3.7, 2.9 and 6.7%, respectively. Other contacts shown in Figure 7    Moreover, the Hirshfeld analysis gave accurate results for the percentages of the ferent intermolecular contacts between the surface and neighboring molecules (Figur The most dominant contacts were the O…H and H…H interactions, which contribute more than half of the whole observed contacts. Additionally, the percentage of Ag… Ag…C and C…C interactions were 3.7, 2.9 and 6.7%, respectively. Other contacts sho in Figure 7

Antimicrobial Activity
The antimicrobial activity of the studied Ag(I) complex on selected bacterial and gal strains was determined in terms of the inhibition zone diameter (IZD) and the m mum inhibitory concentration (MIC). The antimicrobial activity results are depicte Table 4. The IZDs are very small for the Gram-positive bacterial strain compared Gram-negative bacteria. The sizes of the inhibition zones were 8 and 9 mm for S. au and B. subtilis, respectively, compared with 12 and 15 mm for E. coli and P. vulgaris spectively. Hence, the studied Ag(I) complex is more potent against Gram-negative teria than Gram-positive bacteria. Moreover, the MIC value was lowest for P. vulgaris μg/mL), which indicates the highest potency against this bacterium. In comparison the antibacterial control Gentamycin, the studied Ag(I) complex is considered a weak tibacterial agent. In terms of antifungal activity, the studied [Ag(L)(NO3)]2 complex in ited both A. fumigatus and C. albicans. The IZDs were 31 and 18 mm for A. fumigatus C. albicans, respectively, while the IZDs were 17 and 20 mm, respectively, for the stand Ketoconazole. The corresponding MIC values were 20, 625, 156 and 312 μg/mL. He the studied Ag(I) complex showed the highest potency against A. fumigatus comp with the other microbes.
Additionally, the antimicrobial activities of the free L against the same microbes w examined, and the results were compared with those of the [Ag(L)(NO3)]2 complex (T 4). It is clear that the free ligand (L) is active only against S. aureus. It has greater act against this microbe than the [Ag(L)(NO3)]2 complex. The inhibition zone diameters a and 11 mm, respectively. In contrast, the free ligand is not active at the applied concen tion against any other microbe, while the [Ag(L)(NO3)]2 complex showed diverse anti terial and antifungal activities.   Additionally, the antimicrobial activities of the free L against the same microbes were examined, and the results were compared with those of the [Ag(L)(NO 3 )] 2 complex (Table 4). It is clear that the free ligand (L) is active only against S. aureus. It has greater activity against this microbe than the [Ag(L)(NO 3 )] 2 complex. The inhibition zone diameters are 8 and 11 mm, respectively. In contrast, the free ligand is not active at the applied concentration against any other microbe, while the [Ag(L)(NO 3 )] 2 complex showed diverse antibacterial and antifungal activities.

Anticancer and Antioxidant Activities
The results of the cytotoxicity test for the [Ag(L)(NO 3 )] 2 complex against colon carcinoma are presented in Table 5. The %cell viability reached only 1.28 ± 0.46 at 500 µg/mL, and the concentration required to cause toxic effects in 50% of intact cells (IC 50 ) was determined to be 12.53 ± 0.69 µg/mL. This indicates high cytotoxic activity against this cell line. For the free ligand (L), the IC 50 value was determined to be 242.92 ± 8.12 µg/mL, which indicates lower cytotoxic effect of the free ligand and confirms the enhancement in cytotoxic activity as a result of the complexation between the ligand L and Ag(I) ion. The corresponding value for cis-platin as positive control was 5.35 ± 0.49 µg/mL in the same experimental conditions. Hence, the studied [Ag(L)(NO 3 )] 2 complex has promising cytotoxic activity against the examined cell line.
The results of the antioxidant activity of the [Ag(L)(NO 3 )] 2 complex, determined using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay [38], is reported in Table 6. The %DPPH scavenging at 1280 µg/mL was only 75.18%, indicating low antioxidant activity of the [Ag(L)(NO 3 )] 2 complex. Moreover, the concentration required to inhibit DPPH radical by 50% (IC 50 ) was determined to be 626.91 ± 10.87 µg/mL, which further confirms the low antioxidant activity of the Ag(I) complex. The corresponding values for the free ligand L and ascorbic acid as positive control were 156.48 ± 3.66 and 12.3 ± 0.51 µg/mL, respectively. These results indicated low antioxidant activity of the [Ag(L)(NO 3 )] 2 complex compared with the positive control and the free ligand.

Materials
All chemicals were purchased from Aldrich chemical company.

Instruments
Instruments including the X-ray diffractometer used for the single crystal structure measurement and solution structure details [39,40] are given in the Supplementary Data. The crystallographic data of the [Ag(L)(NO 3 )] 2 complex are listed in Table 7.

Synthesis of [Ag(L)(NO 3 )] 2 Complex
The azine ligand (L) was synthesized using the method described in the Supplementary Data and following procedures present in the literature [41,42]. An ethanolic solution of L (0.1 mmol in 10 mL) was added to 0.1 mmol of AgNO 3 in 5 mL of distilled water. An immediate yellow precipitate was formed, which was dissolved by adding 5 mL of acetonitrile. The solution was filtered, and the clear solution was then left at room temperature to slowly evaporate. After 5 days, a yellow crystalline product was obtained for the [Ag(L)(NO 3

Hirshfeld Calculations
The Hirshfeld topology analyses were performed using the crystal explorer 17.5 program [37].

Biological Studies
The bioactivities of the [Ag(L)(NO 3 )] 2 complex were determined according to the biological activity methods listed in the Supplementary Data [38,43,44].

Conclusions
A new dinuclear [Ag(L)(NO 3 )] 2 complex of the asymmetric azine-type ligand (L) was synthesized by a self-assembly method. The azine ligand L acts as a bidentate chelate via the thiazole and azine nitrogen atoms. Additionally, the nitrate ion is a bidentate ligand via two non-equidistant Ag-O bonds. The bite angles of the two bidentate chelates are 69.37 (16) • and 50.4(2) • , respectively. The coordination sphere of Ag(I) is completed by one weak Ag-O bond with an oxygen atom from a neighboring nitrate group in another [Ag(L)(NO 3 )] unit. Hence, the coordination number of the silver is five, and the structure could be represented by the dinuclear formula [Ag(L)(NO 3 )] 2 . Hirshfeld analysis of the [Ag(L)(NO 3 )] complex revealed the importance of the Ag . . . C, O . . . H and C . . . C contacts in molecular packing. The studied Ag(I) complex showed promising antifungal activity against A. fumigatus. Although the studied Ag(I) complex showed very weak antioxidant activity, the cytotoxicity results were promising. The IC 50 value against colon carcinoma (HCT-116 cell line) was determined to be 12.53 ± 0.69 µg/mL, which is compared with 5.35 ± 0.49 µg/mL for cis-platin. In comparison with the free ligand, the Ag(I) complex has higher cytotoxicity (242.92 ± 8.12 µg/mL). Additionally, the Ag(I) complex has greater antimicrobial activity than the free ligand against all microbes except S. aureus.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/inorganics10110209/s1; Figure S1, FTIR spectra of L (upper) and [Ag(L)(NO 3 )] 2 (lower) complex; Figure S2, NMR spectra of L; instrumental details; biological activity methods and synthesis of L; Table S1, the cytotoxicity of the free ligand (L) against colon carcinoma; Table S2, DPPH scavenging assay for of the free ligand (L).