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Soc.00(0)1-13 (2023) Original scientific paper JSCS–12258 Published DD MM, 2023 1 Binuclear azide-bridged hydrazone Cu(II) complex: synthesis, characterization and evaluation of biological activity TEODORA VITOMIROV1, BOŽIDAR ČOBELJIĆ1, ANDREJ PEVEC2, DUŠANKA RADANOVIĆ3, IRENA NOVAKOVIĆ3, MILICA SAVIĆ3, KATARINA ANĐELKOVIĆ1, MAJA ŠUMAR-RISTOVIĆ1* 1University of Belgrade – Faculty of Chemistry, Studentski trg 12–16, 11000 Belgrade, Serbia, 2Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1000 Ljubljana, Slovenia, and 3University of Belgrade, Institute of Chemistry, Technology and Metallurgy, Department of Chemistry, Njegoševa 12, 11000 Belgrade, Serbia (Received 23 June; Revised 27 June; Accepted 21 July 2023) Abstract: The condensation product of 7-acetyl-6-azaindole and Girard's T reagent ((E)-2-(2-(1-(1H-pyrrolo[2,3-c]pyridin-7-yl)ethylidene)hydrazineyl)- N,N,N-trimethyl-2-oxoethan-1-aminium, HL ligand) was used as a ligand in the reaction with Cu(BF4)2·6H2O and NaN3. The reaction led to the formation of a binuclear Cu(II) complex containing two end-to-end (di--1,3-N3) azide bridges, as well as two NNO-donor hydrazone ligands, forming an axially elongated square pyramidal geometry around each Cu(II) center. This end-to-end (di--1,3- N3) azide bridge binding mode has not yet occurred in Cu(II) complexes containing the NNO-donor hydrazone ligands, which makes the structure of the complex even more interesting for further studies. The complex was characterized by elemental analysis, IR spectroscopy and X-ray crystallography, and it was found that it crystallizes in the triclinic space group P–1 with the asymmetric unit comprising one Cu(II) centre, zwitterionic ligand L, one azide (N3 −) ligand, and BF4 − counter anion. Examination of antimicrobial activity of the complex shows higher antifungal and antibacterial activity towards tested Gram-positive bacteria in comparison to the hydrazone ligand, with the antifungal activity of the complex even being comparable to the activity of amphotericin B. Keywords: Girard's T reagent; X-ray crystallography; antibacterial activity; antifungal activity. *Corresponding authors E-mail: majas@chem.bg.ac.rs https://doi.org/10.2298/JSC230623044V A cc ep te d m an us cr ip t mailto:majas@chem.bg.ac.rs https://doi.org/10.2298/JSC230623044V 2 VITOMIROV et al. INTRODUCTION Azide-bridged binuclear copper(II) complexes have been the subject of considerable research in the field of coordination chemistry due to their interesting structural and magnetic properties.1–3 These complexes consist of two copper(II) ions bridged by an azide group via either end-on (EO) or end-to-end (EE) coordination mode, forming single or double ligand bridges: µ1,1-N3 (EO) and µ1,3- N3 (EE).4 The azide group provides a unique building block for the construction of complex architectures and is a versatile bridging ligand that can adopt different coordination modes, such as mono-, bi- or tri-dentate,1 depending on the nature of the metal ion and the reaction conditions. Furthermore, it also acts as a strong magnetic coupler facilitating ferromagnetic and antiferromagnetic coupling between metal ions within a binuclear complex.2 On the other hand, the binuclear copper(II) complexes are known to exhibit either antiferromagnetic or ferromagnetic behavior, depending on the nature of the bridging ligand, the Cu– X–Cu angle and the geometry of the complex.3,5–9 In recent years, there has been growing interest in the biological activity of binuclear copper(II) complexes due to their potential applications as anticancer10,11 and antibacterial12–14 аgents. The ability of these complexes to catalyze the formation of reactive oxygen species (ROS) and to interact with DNA and proteins makes them attractive candidates for the development of new therapeutic agents. When discussing the azide-bridged binuclear Cu(II) complexes, several studies have reported the potential antibacterial activity of these complexes, all containing a tridentate NNO-donor hydrazone ligand, against various strains of bacteria.10,15,16 The mechanism of action is believed to be the disruption of the bacterial cell membrane due to the reduced polarity of the metal ion and increased lipophilicity of the formed complex, compared to lone metal ions and ligands.17 In addition to their biological applications, azide-bridged binuclear copper(II) complexes have also shown promising catalytic properties under sustainable and user-friendly conditions. These complexes have been reported to exhibit catalytic activity towards various reactions, including the N-arylation of imidazole and benzimidazole18 as well as the synthesis of 1,2,3-triazoles.19 The catalytic activity of these complexes is attributed to the redox properties of copper(II) ions and the Cu-N3-Cu bridge, which can facilitate electron transfer processes. In this study, we have synthesized and fully characterized a new binuclear copper(II) complex containing double end-to-end (di-µ1,3-N3) azide bridge, along with NNO-donor hydrazone ligands, and investigated its antibacterial and antifungal activities. The results of our study provide a detailed characterization of a binuclear copper(II) complex, as well as an overview of the potential of this complex as a new therapeutic agent. A cc ep te d m an us cr ip t BINUCLEAR AZIDE-BRIDGED HYDRAZONE Cu(II) COMPLEX 3 EXPERIMENTAL Materials and methods All chemicals and solvents (reagent grade) were obtained from commercial suppliers (NaN3 from Riedel-de Haën; all other chemicals from Sigma-Aldrich) and used without further purification. Elemental analyses (C, H, and N) were performed by standard micro-methods using the ELEMENTAR Vario ELIII C.H.N.S.O analyzer. IR spectra were recorded on a Nicolet 6700 FT-IR spectrometer using the ATR technique from 4000 – 400 cm-1 (strong–s, medium–m, weak–w). NMR spectra were recorded with a Varian 400/54 PS spectrometer in deuterated dimethyl sulfoxide (DMSO-d6). Ligand synthesis The ligand synthesis was carried out in two steps – the first step was obtaining 7-acetyl-6- azaindole using 7-bromo-6-azaindole as a starting compound. The reaction was performed by adding 0.75 mmol (18.2 mg) of Mg to 0.75 mmol (147 mg) of 7-bromo-6-azaindole in anhydrous diethyl ether and making a Grignard’s reagent, which then reacted with equimolar amount (0.75 mmol, 58.9 mg) of acetyl chloride and a small amount (2 mol %) of FeCl3 as a catalyst. This reaction was carried out at -60 °C using dry ice as a cooling bath. The next step was to synthesize the condensation product of 7-acetyl-6-azaindole and Girard's T reagent, ((E)-2-(2-(1-(1H-pyrrolo[2,3-c]pyridin-7-yl)ethylidene)hydrazineyl)- N,N,N-trimethyl-2-oxoethan-1-aminium). The reaction was carried out by dissolving 0.5 mmol (80 mg) of 7-acetyl-6-azaindole in methanol and adding 0.5 mmol (83.8 mg) of Girard’s T reagent to the reaction mixture, which was then refluxed for 3 hours. After cooling down to room temperature, the bright yellow precipitate was filtered and rinsed with ethanol. The reaction yield was 81 % (125.2 mg) and the ligand was then characterized by elemental analysis, IR and NMR spectroscopy. Labelling of C and H atoms described with NMR spectroscopy is presented in Fig. 1. Elemental analysis for C14H20ClN5O (%), Calculated: C 54.28, H 6.51, N 22.61. Found (%): C 54.68, H 7.09, N 22.13. IR (ATR, cm-1) selected peaks: 3377.4 (m), 3068.5 (m), 3019.4 (m), 2976.5 (m),1703.4 (s), 1618.3 (w), 1571.8 (m), 1550.4 (m), 1491.2 (m), 1431.9 (m), 1398.4 (s), 1333.1 (m), 1280.2 (m), 1231.0 (m), 1164.0 (m), 1125.8 (m), 987.3 (m), 947.3 (m), 919.3 (m), 858.8 (w), 818.5 (m), 800.1 (m), 710.9 (m), 655.4 (m), 609.1 (w). 1H NMR (400 MHz, DMSO-d6), δ (ppm): 11.66 (s, 1H, N2-H), 11.50 (s, 1H, N4-H), 8.15 – 7.72 (4H, C1-H, C2-H, C4-H, C5-H),4.91 (s, 2H, C11-H), 3.34 (t, 9H, C12-H), 2.32 (s, 3H, C9-H). 13C NMR (125 MHz, DMSO-d6), δ (ppm):167.24 (C10), 161.48 (C8), 156.29 – 120.21 (C1, C2, C3, C4, C5, C6, C7), 63.21 (C11), 53.74 (C12),12.73 (C9). Complex synthesis The synthesis of the complex was performed by dissolving 0.25 mmol (77.3 mg) of ligand in 20 mL of methanol and then adding 0.25 mmol (86.3 mg) of Cu(BF 4)2·6H2O, previously dissolved in 5 mL of H2O, and 1 mmol (65 mg) of NaN3 directly into the reaction mixture. The mixture was refluxed for 2 hours, and after 10 days dark green monocrystals of complex were obtained and filtered from the solution. The reaction yield was 73 % (169.6 mg) and the complex was characterized by elemental analysis, IR spectroscopy and X-Ray crystallography. Elemental analysis for C28H38B2Cu2F8N16O2(%), Calculated: C 36.11, H 4.11, N 24.06. Found (%): C 35.74, H 4.25, N 24.58. A cc ep te d m an us cr ip t 4 VITOMIROV et al. IR(ATR, cm-1) selected peaks:3350.3 (w), 3098.4 (w), 3044.3 (w), 2988.8 (w), 2063.4 (s), 1583.1 (s), 1557.8 (s), 1530.8 (s), 1501.3 (m), 1483.2 (s), 1441.4 (m), 1400.5 (m), 1347.7 (m), 1319.7 (m), 1296.1 (m), 1248.7 (m), 1185.3 (m), 1118.1 (m), 1034.8 (s), 997.2 (m), 972.1 (m), 926.3 (m), 910.6 (m), 815.7 (m), 733.2 (m), 655.2 (w). X-ray structure determination The crystal structure of compound [Cu2L2(1,3-N3)2](BF4)2 was determined by single- crystal X-ray diffraction methods. Crystallographic data and refinement details are given in Table I. Diffraction data were collected with Agilent SuperNova dual source diffractometer using an Atlas detector and equipped with mirror-monochromated MoKα radiation (λ = 0.71073 Å). The data were processed by using CrysAlis PRO.20 The structure was solved using SIR-9221 and refined against F2 on all data by full-matrix least-squares with SHELXL–2016.22 All non- hydrogen atoms were refined anisotropically. The nitrogen N2 bonded hydrogen atom was located in the difference map and refined with the distance restraints (DFIX) with d(N–H = 0.86 Å and with Uiso(H) = 1.2Ueq(N). All other hydrogen atoms were included in the model at geometrically calculated positions and refined using a riding model. The F4 fluorine atom in BF4 – is disordered over two orientations and was refined with the use of PART instruction. The occupancy of F4a and F4b refined to the ratio of 55 and 45%, respectively. CCDC 2271001 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. TABLE I. Crystal data and structure refinement details for [Cu2L2(1,3-N3)2](BF4)2 formula C28H38B2Cu2F8N16O2 Fw (g mol–1) 931.44 crystal size (mm) 0.500.400.10 crystal color green crystal system triclinic space group P –1 a (Å) 7.8476(3) b (Å) 9.8765(6) c (Å) 13.2990(9) α (º) 110.341(6) β (º) 103.742(5) γ (º) 92.524(4) V (Å3) 929.70(10) Z 1 calcd density (g cm-3) 1.664 F(000) 474 no. of collected reflns 8891 no. of independent reflns 4259 Rint 0.0430 no. of reflns observed 3639 no. parameters 279 R[I> 2σ (I)]a 0.0595 wR2(all data) b 0.1815 Goof, Sc 1.051 Δρmax/Δρmin (eÅ 3) +0.88/–0.80 A cc ep te d m an us cr ip t http://www.ccdc.cam.ac.uk/data_request/cif BINUCLEAR AZIDE-BRIDGED HYDRAZONE Cu(II) COMPLEX 5 a R = ∑||Fo| – |Fc||/∑|Fo|. b wR2 = {∑[w(Fo2 – Fc2)2]/∑[w(Fo2)2]}1/2. cS = {∑[(Fo 2 – Fc 2)2]/(n/p}1/2 where n is the number of reflections and p is the total number of parameters refined. Antimicrobial activity In vitro antibacterial and antifungal activity was tested against four Gram-positive bacteria(Bacillus subtilis ATCC 6633, Clostridium sporogenes ATCC 19404, Kocuria rhizophila ATCC 9341, Staphylococcus aureus ATCC 6538), four Gram-negative bacteria (Proteus hauseri ATCC 13315, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027,Salmonella enterica ATCC 13076), and three fungal strains (Aspergillus brasiliensis ATCC16404, Candida albicans ATCC 10231, Saccharomyces cerevisae ATCC 9763), by the double dilution method in microtiter plates 23. Antibacterial activity was determined using Mueller Hinton broth, whereas antifungal activity was determined using Sabouraud dextrose broth. One hundred microliters of fresh Mueller Hintonor Sabouraud dextrose broth were added to each well of the plate. Then, 100 µL of the compounds stock solution (10 mg/mL) prepared by dissolving compounds in DMSO was added. Each well was inoculated with 10 µL (106 cells per mL) of bacterial cultures and 10 µL (105 spores per mL) of fungal strains for antibacterial and antifungal determination, respectively. Bacterial strains were incubated at 37 °C for 24 h. Erythromycin was used as a positive control, while water served as a negative control. Fungal strains were incubated at 28 °C for 48 h. Amphotericin B was used as a positive control, while DMSO was used as a negative control. The bacterial growth was visualized by adding 20 μL of 0.5% 2,3,5-triphenyltetrazolium chloride (TTC) aqueous solution.24 The MIC was determined as the lowest concentration that resulted in inhibition of visible microbial growth. The brine shrimp test The brine shrimp test was performed against freshly hatched nauplii of Artemia salina.25 The compounds were dissolved in DMSO and various amounts (0.01–1 mg) were added to artificial sea water containing 10–20 nauplii. After 24 h illumination at room temperature, the number of dead and surviving nauplii were counted and statistically analyzed. All samples were tested in triplicate. LC50 was defined as concentration of compounds that caused the death of 50% of nauplii. Assessment of radical-scavenging activity Antioxidative activity of initial Cu(II) salt, appropriate ligand and the synthesized complex was determined using a DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay. All tested compounds were dissolved in DMSO (stock concentrations were 10 mg/mL). Foreach tested compound, two rows of the 96-well microplate were used: one for measuring the absorbance of the compounds themselves and the other for antioxidant activity. 50 μL of stock solutions of tested compounds were loaded into plate and double diluted by using multi-channel pipette. The rows used to measure the absorbance of the compounds were supplemented with 100 μL of pure methanol, while the other rows were supplemented with 100 μL of freshly prepared methanolic DPPH solution (6.58 × 10-5 M). In the control, 50 μL pure DMSO was loaded. Final concentrations of the compounds ranged from (0.5 mg per well) to (2.4∙10-4 mg per well). After 30 min of incubation at 37 °C in the dark, the absorption was measured at 517 nm. All measurements were done in triplicate. Free radical scavenging activity of compounds was measured using the equation: A cc ep te d m an us cr ip t 6 VITOMIROV et al. Activity = 100×(𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙− (𝐴𝑠𝑎𝑚𝑝𝑙𝑒−𝐴0)) 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 (1) where Acontrol is the absorbance of DPPH in the control probe, Asample is the absorbance of DPPH in the samples, and A0 is the absorbance of solutions of complexes 1 and 2 in DMSO, due to their intensive green colors. IC50 is defined as the concentration of antioxidant agent necessary to reduce the starting amount of DPPH by 50% and is calculated from the concentration-dependent free radical scavenging activity graph. Ascorbic acid was used as a control probe. RESULTS AND DISCUSSION The ligand synthesis was carried out in two steps, as presented in Fig. 1. After acetylation of 7-bromo-6-azaindole, the obtained 7-acetyl-6-azaindole reacted with Girard’s T reagent in a molar ratio 1:1 and the ligand was formed. The complex was then obtained in the reaction of Cu(BF4)2·6H2O, ligand and NaN3 in a molar ratio 1:1:4, as described in Fig. 2. Fig. 1. Step by step ligand synthesis Fig. 2. Complex synthesis IR spectra IR spectroscopy confirmed the coordination of the ligand via the pyrrole nitrogen of 7-acetyl-6-azaindole ring. The stretching vibrational mode of the N–H group appears in the IR spectrum of the ligand as a wide band at 3377.4 cm-1 and disappears in the spectrum of the complex. The ligand spectrum also shows a very intense band at 1703.4 cm-1, corresponding to the C=O stretching vibration of the carbonyl group. This peak does not appear in the spectrum of the complex, instead, A cc ep te d m an us cr ip t BINUCLEAR AZIDE-BRIDGED HYDRAZONE Cu(II) COMPLEX 7 an intense peak appears at 1034.8 cm-1, coming from the C–O stretching vibration. These all indicate the delocalization of the electron pair of the carbonyl group towards oxygen, which then favours the coordination of the ligand via the now negatively charged oxygen atom. The strong band appearing in the IR spectrum of the complex at 2063.4 cm-1 points out the presence of the N3– group in the complex structure. X-ray crystal structure determination Complex crystallizes in the triclinic space group P–1, with the asymmetric unit comprising one Cu(II) centre, zwitterionic ligand L, one azide (N3−) ligand, and BF4− counter anion. The crystal structure displays a centrosymmetric binuclear complex with the crystallographicaly independent Cu1 centre being coordinated to three donor atoms (N1, N3 and O1) of L and two N atoms (N6 and N8a where a = −x,−y,−z+1) from two azide ligands which bridge two Cu(II) centres by adopting a double end-to-end (di--1,3-N3) coordination mode. The Cu1Cu1a separation is 4.8232(6) Å. The molecular structure of the dimeric cationic moiety in the [Cu2L2(1,3-N3)2](BF4)2 complex and the atom-labelling scheme are shown in Fig. 3. Selected bond lengths and angles for the structure are given in Table S-I in the Supplementary material. The coordination polyhedron around Cu(II) is described as an axially elongated square pyramid with an index of trigonality (5) 26 of 0.05 [5 = (−)/60, where  and  are the two largest angles around the central atom]. The 5 is 0 for regular square based pyramidal geometry and 1.00 for regular trigonal bipyramidal geometry. The four in-plane bond distances are: Cu1–N1 1.972(3), Cu1–N3 1.975(3), Cu1–O1 1.930(3) and Cu1–N6 1.957(3) Å, and the apical distance Cu1–N8a (symmetry code a =−x,−y,−z+1)is 2.426(4) Å. The azide anion bridges in an asymmetric (basal–apical) fashion so that the in-plane and axial Cu–N(N3−) bond distances are significantly different. The tridentate NNO coordination of L to Cu(II) ion generates one six-membered chelate ring (Cu–N– C–C–C–N) and one five-membered chelate ring (Cu–N–N–C–O) fused along the Cu1–N3 bond. The chelate rings are non-coplanar, as indicated by the dihedral angle of 3.8 Å. In this complex the Cu–NAr and Cu–Nimine bonds are comparable in length (Cu1–N1 1.972(3) and Cu1–N3 1.975(3) Å, respectively). However, in Cu(II) complexes with two fused five-membered chelate rings generated by chelation of tridentate NNO donor hydrazone ligands the Cu–NAr bonds are longer than the Cu–Nimine bonds.27,28 Complex cations and BF4− anions generate a three- dimensional structure by means of intermolecular N–HF, C–HF and C–HN hydrogen bonds given in Table S-II. In addition, the complex cations are connected by means of intermolecular  bonds extending in [110] direction (Table S-III, Fig. S-1). A search of the Cambridge Structural Database (CSD)29 for the binuclear Cu(II)-azido complexes with hydrazone-based NNO-donor ligands revealed 12 crystal structures with double end-on (di--1,1-N3) coordination mode of bridging A cc ep te d m an us cr ip t 8 VITOMIROV et al. azide anions. No crystal structure of binuclear Cu(II)-azido complex with tridentate NNO-donor hydrazone ligand having double end-to-end (di--1,3-N3) coordination mode of bridging azides has been observed. The structure presented here is the first case. Details of CSD search are given in Table S-IV in the Supplementary material. Crystallographic programs ORTEP-3 for Windows30 and Mercury31 were used to prepare the drawings. Fig. 3. ORTEP presentation of the complex cation [Cu2L2(1,3-N3)2] 2+ in [Cu2L2(1,3-N3)2](BF4)2. Thermal ellipsoids are drawn at the 30% probability level. The unlabeled part of the dimeric molecule is generated by symmetry operation –x, –y, –z+1. Antimicrobial activity Antimicrobial activity of the complex and the starting compounds (Cu(BF4)2·6H2O and the ligand) was studied in DMSO solution by examining their minimum inhibitory concentration (MIC) on Gram-positive and Gram-negative bacteria and fungal strains. Erythromycin and amphotericin B were used as the standard drugs to compare the minimum inhibitory concentration values and the results are presented in Table II. Comparing the activities of the complex and the A cc ep te d m an us cr ip t BINUCLEAR AZIDE-BRIDGED HYDRAZONE Cu(II) COMPLEX 9 ligand itself, the complex was shown to have higher antibacterial activity against all tested Gram-positive bacteria and half of the tested Gram-negative bacteria. The most significant difference between the antimicrobial activities of the complex and the ligand is the one against S. aureus, where the activity of the complex is 3 times higher than that of the ligand alone. The antifungal activities of the complex are significantly higher than those of the ligand, especially in the case of S. cerevisiae, where the antifungal activity of the complex is even comparable to the activity of amphotericin B. TABLE II. Antimicrobial activity of the complex and the starting compounds (MIC values in mM) Microorganism Complex Ligand Cu(BF4)2·6H2O Standard a,b E. coli 0.671 1.011 1.821 0.038 P. aeruginosa 0.671 1.011 3.642 0.076 P. hauseri 1.342 1.011 3.642 0.038 S. enterica 0.671 0.505 3.642 0.038 S. aureus 0.671 2.022 3.642 0.076 C. sporogenes 0.671 1.011 3.642 0.076 K. rhizophila 1.342 2.022 3.642 0.076 B. subtilis 0.671 1.011 3.642 0.076 S. cerevisiae 0.084 2.022 3.673 0.011 C. albicans 0.168 4.044 7.346 0.022 A. brasiliensis 0.356 4.044 3.673 0.044 aStandard used for bacterial strains was erythromycin. bStandard used for fungal strains was amphotericin B. The brine shrimp test The brine shrimp lethality bioassay is an excellent predictive tool for the toxic potential of new bioactive compounds. The results of this test can be extrapolated to cell-line toxicity and anti-tumor activity.25,32 The results presented in Table III showed high toxicity of the ligand, compared to moderate toxicities of the complex and the Cu(II) salt. This moderate toxicity of the synthesized complex may indicate its potential as a new active drug. TABLE III. Results of the brine shrimp test (LC50, in mM) for the complex and the starting compounds Compound LC50(mM) Complex 0.613±0.051 Ligand 0.238±0.092 Cu(BF4)2·6H2O 1.174±0.155 K2Cr2O7 0.077±0.016 Assessment of radical-scavenging activity Assessment of radical-scavenging capacity was determined using a DPPH free radical scavenging assay and the results are presented in Table IV. According to A cc ep te d m an us cr ip t 10 VITOMIROV et al. the obtained results, the basic salt and ligand did not possess DPPH radical scavenging capacity, whereas the complex showed moderate antioxidant activity. TABLE IV. DPPH radical scavenging (IC50, in mM) of starting compounds and the complex Compound IC50 (mM) Complex 1.450±0.036 Ligand 7.834±0.126 Cu(BF4)2·6H2O >9.651 Ascorbic acid 0.079±0.003 CONCLUSION In this paper we have thoroughly described the synthesis and characterization of a new binuclear azide-bridged Cu(II) complex containing NNO-donor hydrazone ligands obtained in a condensation reaction between 7-acetyl-6- azaindole and Girard's T reagent. The X-ray crystallographic analysis of the complex revealed a binuclear structure, in which two Cu(II) ions are bridged by two end-to-end (di-µ1,3-N3) azide ligands, while the hydrazone ligands coordinate to each Cu(II) center in a tridentate manner forming an axially elongated square pyramidal geometry around each metal ion. The complex crystallizes in the triclinic space group P–1. Detailed research of the Cambridge Structural Database revealed there are no crystal structures of binuclear Cu(II)-azido complexes with tridentate NNO-donor hydrazone ligands having double end-to-end (di--1,3-N3) coordination mode of bridging azides, making this complex structure unique and rather interesting. Antimicrobial activity of the complex was also examined, and it was shown that the complex exhibits higher antibacterial activity towards all tested Gram-positive bacteria than the ligand itself, while the antifungal activity of the complex towards all tested fungal strains was not only higher than that of the ligand, but also comparable to the activity of the standard drug. Results obtained in the evaluation of antimicrobial activity of the complex may indicate its potential as an antifungal agent. Acknowledgment. This research has been financially supported by the Ministry of Science, Technological Development and Innovation of Republic of Serbia, contract numbers: 451-03- 47/2023-01/200026, 451-03-47/2023-01/200168 and 451-03-47/2023-01/200288, as well as by the Science Fund of the Republic of Serbia, #7750288, Tailoring Molecular Magnets and Catalysts Based on Transition Metal Complexes – TMMagCat, and by the Slovenian Research Agency (ARRS), grant number P1-0175. We thank the EN-FIST Centre of Excellence, Ljubljana, Slovenia, for the use of the SuperNova difractometer. SUPPLEMENTARY MATERIAL Supplementary Materials are available electronically from https://www.shd- pub.org.rs/index.php/JSCS/article/view/12452, or from the corresponding authors on request. A cc ep te d m an us cr ip t https://www.shd-pub.org.rs/index.php/JSCS/article/view/12452 https://www.shd-pub.org.rs/index.php/JSCS/article/view/12452 BINUCLEAR AZIDE-BRIDGED HYDRAZONE Cu(II) COMPLEX 11 И З В О Д ДИНУКЛЕАРНИ ХИДРАЗОНСКИ КОМПЛЕКС Cu(II) СА АЗИДНИМ МОСТОМ: СИНТЕЗА, КАРАКТЕРИЗАЦИЈА И ЕВАЛУАЦИЈА БИОЛОШКЕ АКТИВНОСТИ ТЕОДОРА ВИТОМИРОВ1, БОЖИДАР ЧОБЕЉИЋ1, АНДРЕЈ ПЕВЕЦ2, ДУШАНКА РАДАНОВИЋ3, ИРЕНА НОВАКОВИЋ3, МИЛИЦА САВИЋ3, КАТАРИНА АНЂЕЛКОВИЋ1, МАЈА ШУМАР-РИСТОВИЋ1,* 1Универзитет у Београду – Хемијски факултет, Студентски трг 12–16, 11000 Београд, Србија, 2Факултет за хемију и хемијску технологију, Универзитет у Љубљани, Вечна пот 113, 1000 Љубљана, Словенија, и 3Универзитет у Београду, Институт за хемију, технологију и металургију, Центар за хемију, Његошева 12, 11000 Београд, Србија Кондензациони производ 7-ацетил-6-азаиндола и Жираровог Т реагенса (лиганд HL) коришћен је као лиганд у реакцији са Cu(BF4)2·6H2O и NaN3. Реакција је довела до формирања бинуклеарног Cu(II) комплекса који садржи два азидна моста у „end-to-end“ (di--1,3-N3) моду, као и два NNO-донорска хидразонска лиганда који заједно формирају аксијално издужену квадратно-пирамидалну геометрију око сваког централног металног јона. Овај „end-to-end“ (di--1,3-N3) азидни мост се до сада није појављивао у структурама бакар(II) комплекса који садрже NNO-донорске хидразонске лиганде, што чини структуру комплекса још интересантнијом за будућа испитивања. Овај комплекс је окарактерисан елементалном анализом, ИЦ спектроскопијом и рендгенском структурном анализом и пронађено је да кристалише у триклиничној просторној групи P–1 са асиметричном јединицом која се састоји из једног Cu(II) центра, цвитер-јонског лиганда (L), једног азидног лиганда (N3−) и BF4− контра-јона. 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