Anion-Dependent Synthesis of Cu(II) Complexes with 2-(1H-Tetrazol-5-yl)-1H-indole: Synthesis, X-Ray Structures, and Radical Scavenging Activity

Two mononuclear Cu(II) complexes, [Cu(phen)2(HL)]ClO4·H2O·2DMF (1) and [Cu(phen)2(HL)2]·EtOH (2), comprising 1,10-phentantroline (phen) and 2-(1H-tetrazol-5-yl)-1H-indole ligand (H2L) ligands are reported. Analysis and characterization of the samples were performed using standard physicochemical techniques, elemental analysis, nuclear magnetic resonance, Fourier transform infrared spectroscopy, and UV-vis spectroscopy. Single-crystal X-ray crystallography revealed the formation of a pentacoordinate complex in 1 and a hexacoordinate complex in 2, in which the anionic ligand HL− has undergone monodentate coordination through the tetrazole unit. Furthermore, the crystal structure of H2L·MeOH is also discussed. The potential application of compounds 1 and 2 in bioinorganic chemistry was addressed by investigating their radical scavenging activity with the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and the results were supported also by theoretical calculations.


Introduction
Reactive oxygen species (ROS) and their physiological effects have been studied extensively since their discovery circa 60 years ago [1]. ese ROS can be divided into two groups: free oxygen radicals such as superoxide radical O ·− 2 , hydroxyl radical OH_, or organic peroxyl radicals ROO_and nonradical ROS such as ozone O 3 , hydrogen peroxide H 2 O 2 , and singlet oxygen 1 O 2 [2]. As the name suggests, these molecules are very reactive and are partly responsible for oxidative stress in cells leading to lipid peroxidation [3] and damage to DNA and proteins [4,5]. eir effect is generally considered toxic to the body and an increased level of these species has been linked to a number of pathologies such as inflammations [6], various cardiac diseases [7], and cancer [2]. e most common superoxide radical O ·− 2 is created in the electron transport chain, specifically in complexes I and III, as an unwanted by-product. Subsequently, this radical is released into the cytosol and in lesser degree to the mitochondrial matrix [8], where it then either reacts with nonenzymatic antioxidants, such as glutathione or ascorbic acid, or transforms into less damaging hydrogen peroxide or oxygen by metal-containing enzymes known as superoxide dismutases (SODs). e most common metal ions contained in these enzymes are Cu 2+ , Zn 2+ , and Mn 2+ ; these are also directly involved in the enzymatic reaction due to their ability to transfer the unpaired electron from the superoxide radical without forming yet another highly reactive radical [9]. Klug-Roth and Rabani et al. have shown that copper ion cycles between oxidation states (I) and (II), as shown by reactions (1) and (2), creating either hydrogen peroxide or oxygen molecule [10].
To improve the health conditions of patients affected by diseases linked to the increased oxidative stress, many research groups have tried to prepare complex compounds of low molecular weight that would mimic the activity of SODs; unfortunately, SODs themselves cannot be administered, as they would not pass the cell membrane and are quickly metabolized by kidneys [11,12]. In addition to the SOD-like activity of many copper compounds, many copper complexes are also known for their cytotoxic properties, most notably a group of ternary complexes called Casiopeínas. ese copper complexes are made of substituted 1,10-phenanthroline or 2,2′bipyridines and various anionic O,O-and N,O-ligands such as glycinate or acetylacetonate. e most prominent derivatives have been able to achieve values of IC 50 in the range of low micromolar concentrations on several tumor cell lines [13,14].
To mimic the copper coordination sphere in Cu,Zn-SOD, which consists of 4 histidine ligands bound to a copper center by imidazole nitrogen atoms [15], we have decided to use a ligand containing tetrazole, 2-(1H-tetrazol-5-yl)-1H-indole (H 2 L) (Scheme 1). Tetrazole is also a five-membered nitrogen-containing ring similar to that of imidazole, but tetrazoles can readily release proton and have acidic properties similar to those of carboxylic acid.
Several complexes containing tetrazole have already exhibited cytotoxic properties, most notably a dimeric Pt(II) complex prepared by Komeda et al., which was more potent and much less toxic than gemcitabine on PANC-1 tumor transfected mice [16]. Indole moiety is also known to be present in a plethora of used chemotherapeutics, such as Vinca alkaloids or panobinostat, which was approved for use on multiple myeloma in 2015 [17]. New complexes containing ligands with indole and tetrazole moiety could therefore exhibit interesting biological properties. To the best of our knowledge, 2-(1Htetrazol-5-yl)-1H-indole itself has not yet been tested for its cytotoxic properties and no complex comprising this ligand has been prepared yet. Additionally, inspired by the Casiopeínas group mentioned above, we chose 1,10phenanthroline (phen) as a coligand for our synthesis.

Materials and Methods
All solvents and chemicals were purchased from various commercial sources and used without further purification. Elemental analysis was performed on the ermo Scientific Flash 2000 analyzer. e infrared spectra of the complexes were measured on a Jasco FT/IR-4700 using ATR technique with a diamond plate in the range of 400-4000 cm −1 . e 1 H, 13 C, and 2D NMR spectra of the ligand were measured on a 400 MHz Varian spectrometer. UV/Vis spectra were measured on Cintra 3030 (GBC Scientific Instruments) double beam spectrometer.

Synthesis
e solution of 5 g (31 mmol) of 1H-indole-2-carboxylic acid in 25 ml of chloroform was mixed with 5 ml of thionyl chloride (SOCl 2 ) and three drops of dimethylformamide (DMF). e reaction mixture was then refluxed for 2 hours. e cooled solution was then poured into a slurry of 20 ml of aqueous ammonia and 20 g of ice. e mixture was then stirred for 2 hours at room temperature, during which a large amount of yellow precipitate appeared. e solid product was filtered off, washed with water, and dried in a vacuum desiccator. e product, 1H-indole-2-carboxamide, was used in the next step without further purification (yield: 4.4 g (89%)).
is mixture was refluxed for 30 minutes and subsequently it was poured onto 100 g of ice. e pH of the mixture was then adjusted to 8 by aqueous ammonia during which light brown product precipitated. is suspension was extracted 3 times with 50 ml of diethyl ether. e organic phase was dried over Na 2 SO 4 and filtered and the solvent was removed to dryness on a rotatory evaporator. e light brown product, 1H-indole-2-carbonitrile, was dried in a vacuum desiccator and used in the next step without further purification (yield: 4.12 g (74%)).
4.12 g (29 mmol) of 1H-indole-2-carbonitrile was dissolved in 25 ml of DMF. To this was then added 3.77 g (58 mmol) of NaN 3 and 1.55 g (29 mmol) of NH 4 Cl. is suspension was heated at 120°C for 18 hours. After it was cooled to room temperature, this mixture was poured into 100 ml of distilled water. e pH was adjusted to 1-2 with 2M HCl and the solution was extracted 3 times with 50 ml of ethyl acetate. e organic phase was washed with brine and dried over Na 2 SO 4 . After filtration, the solvent was removed on a rotatory evaporator to dryness. e resulting brown powder was recrystallized from methanol with a spoon of activated charcoal. e resulting off-white crystals of 2-(1Htetrazol-5-yl)-1H-indole (H 2 L) were suitable for X-ray diffraction analysis (yield: 2.84 g (45%)).
Anal. Calcd. (%) for C 10  H 2 O was added to 10 ml of ethanol together with 91.2 mg (460 μmol) of 1,10-phenanthroline monohydrate. After everything was dissolved, 50 mg (230 μmol) of H 2 L was added to the reaction mixture. e solution was mixed to complete dissolution of the ligand and the clear solution was left to evaporate at room temperature, leading to the formation of green crystals suitable for an X-ray diffraction analysis. e resulting product was filtered off, washed with diethyl ether, and dried in a vacuum desiccator (yield: 67 mg (35%)).

X-Ray Crystallography.
e data collection for H 2 L·MeOH (CSD number 2114450), 1 (CSD number 2114451), and 2 (CSD number 2114452) was carried out on SuperNova diffractometer from Rigaku OD equipped with Atlas2 CCD detector and Cu Kα sealed tube as source. CrysAlisPro version 1.171.41.93a was used for the data collection and for the cell refinement, data reduction, and absorption correction [18]. e molecular structure of the prepared compounds was solved by SHELXT [19] and subsequent Fourier syntheses using SHELXL [20]. Anisotropic displacement parameters were refined for all non-H atoms. e hydrogen atoms were placed in calculated positions and refined riding on their parent C atoms with C-H (aliphatic) bond length of 0.98Å and 0.99Å and with C-H (aromatic) bond length of 0.95Å in all three compounds. e hydrogen atoms of hydroxyl groups were also placed in  [21] was used for molecular graphics.

DPPH Scavenging
Activity. e DPPH scavenging assay was performed with some modifications according to a method reported by L. Tabrizi et al. [22]. In a cuvette, 150 μl of 1 mM solution of DPPH in methanol was mixed with 150/ 300/450 μl of 0.5 mM methanolic solution of a copper complex and the volume was adjusted to 3 ml with methanol. e solution was then mixed vigorously, and the absorbance was measured at 517 nm after 30 minutes. All experiments were carried out in triplicate. e resulting concentrations of the samples prepared this way were c DPPH � 50 μM and c complex � 25/50/75 μM. e radical scavenging activity was then determined by the following equation: where A is the measured absorbance of the sample and A 0 is the absorbance of pure DPPH.

Synthesis and General
Characterization. First, 2-(1Htetrazol-5-yl)-1H-indole (H 2 L) acting as a ligand was prepared by three-step chemical synthesis (Scheme 2), in which the 1H-indole-2-carboxylic acid was first converted to its amide using chlorination with SOCl 2 and subsequent reaction with aq. ammonia. e resulting 1H-indole-2-carboxamide was dehydrated in the second step to a nitrile. e 1H-indole-2-carbonitrile was then transformed into a tetrazole derivative H 2 L with the help of in situ generated HN 3 [23]. e formation of H 2 L was confirmed by the elemental analysis and 1 H and 13 C NMR ( Figures S1 and S2). NMR spectra were compared with already published ones [24], which confirmed that synthesis of 2-(1H-tetrazol-5-yl)-1Hindole has been successful. In 1 H NMR spectrum, we have additionally also observed a characteristic peak of CH 3 group of methanol with a chemical shift δ � 3.14 ppm, which we at first assumed was residual solvent peak [25]. However, subsequent elemental analysis, as well as crystal structure determination, indeed showed that one molecule of methanol is present within the crystal structure, which is in accordance with the measured NMR spectra. Proton signals were then assigned with the help of COSY ( Figure S3 Figure S9) [26]. e spectrum of 1 also comprises intensive peak at 1655 cm −1 that belongs to C=O stretching vibration of uncoordinated molecules of DMF. Furthermore, the presence of the noncoordinated perchlorate anion ClO − 4 in 1 is clearly demonstrated by peak at 1084 cm −1 [27].

Description of the Crystal Structures.
First, the crystal structure of the ligand H 2 L·MeOH is discussed (Figure 1). e prepared single crystals belong to the monoclinic crystal system with the space group C 2/c ( Table 1). As the data from elementary analysis and NMR spectroscopy suggest, a molecule of methanol is indeed present in the crystal structure, forming two types of hydrogen bonds with tetrazole rings. e first type is the hydrogen bond O1-H1· · ·N3 having bond distance d(O1· · ·N3) � 2.7952(27)Å (Figure 1(b)). e second type is formed between protonated tetrazole and methanol with d(N1· · ·O1) � 2.6820 (22)Å (Figure 1(b)). ere are also the intermolecular hydrogen bonds between H 2 L molecules formed between indole and tetrazole units with d(N5· · ·N4) � 2.9466 (23)Å (Figure 1(c)). e detailed information about these hydrogen bonds is listed in Table 2.
Compound 1 crystallized in a triclinic crystal system with a space group P -1 (Table 1). e asymmetric unit of 1 contains a [Cu(phen) 2 (HL)] complex, perchlorate anion, two molecules of DMF, and one molecule of water. e copper atom is coordinated by two phen ligands in bidentate fashion, and the anionic HL − ligand is coordinated through the nitrogen atom of the tetrazole unit, with the respective Cu-N distances listed in Table 3. us, the coordination number is 5 for {CuN 5 } chromophore, and the Addison parameter [28] is equal to 0.83, which means the coordination polyhedron is close to a trigonal bipyramid, as it is also evident in Figure 2. e shortest distance between oxygen of perchlorate anion and copper atom is d (Cu1· · ·O3C) = 3.9293 (17)Å, which means that the perchlorate anion is not coordinated. e cocrystalized solvents form net of the hydrogen bonds together with the indole part of the anionic ligand HL (Figure 2(b) (19)Å. Furthermore, there is π-π stacking interaction within the crystal structure of 1 in which 1,10-phenanthroline ligands are involved and the distance between their centroids is of 3.6182Å (Figure 2(c) and Table 4). Crystal system of compound 2 was also triclinic with a space group P-1. e asymmetric unit of 2 contains a neutral complex [Cu(phen) 2 (HL) 2 ] and a molecule of ethanol ( Figure 3). e copper atom is coordinated by two bidentate phen ligands and two monodentate anionic HL − ligands, thus forming {CuN 6 } chromophore with the respective Cu-N distances listed in Table 3. Due to Jahn-Teller effect, complex [Cu(phen) 2 (HL) 2 ] of 2 shows elongated square-bipyramidal geometry, in which the axial positions are occupied by nitrogen atoms of two phen ligands (Figure 3(a)). Moreover, the formation of supramolecular dimers is observed in the crystal structure of 2. ese supramolecular dimers are stabilized by hydrogen bonds between nitrogen atom of tetrazole ring and protonated nitrogen of indole of the second complex with following donor-acceptor distances: d(N54· · ·N23) � 3.098 (2) A (Figure 3(b) and Table 2). Furthermore, the supramolecular dimer is additionally stabilized by π-π stacking interactions formed by neighboring 1,10-phenanthrolines and indoles with the shortest distance between benzene and indole centroids of 3.5806 (12)Å (Table 4 and Figure 3(b)). Lastly, a hydrogen bond is also present between nitrogen atom of tetrazole unit and ethanol molecule with d(O1E···N33) � 2.939 (2)Å (Figure 3(b)).

Radical Scavenging Activity.
Spectroscopic determination of DPPH radical quenching is one of the most widely used methods which readily and reliably provide information about the radical scavenging activity of studied    Symmetry transformations used to generate equivalent atoms. a i: −x + 1, y, −z + 3/2; ii: x, −y + 1, z−1/2, b i: −x + 1, −y + 2, −z + 1; ii: −x + 1, −y + 1, −z + 1.  Bioinorganic Chemistry and Applications compounds. In our case, this represents a measure of the ability of studied complexes to scavenge detrimental radicals present in the intracellular environment, such as the aforementioned OH_ radical. e DPPH scavenging activity for 1 and 2 was measured by UV-Vis spectroscopy at 517 nm in triplicate and the values were averaged for each concentration (Figure 4). Conveniently, the studied complexes show no significant absorption in this region ( Figures S10 and S11). Evidently, both prepared complexes 1 and 2 possess DPPH radical scavenging activity, which increases with the increasing concentration of the copper complex ( Figure 4). For comparison, we also measured the DPPH radical scavenging activity of ascorbic acid, which is much larger than those of both of our complexes. Compound 2 shows slightly higher activity than complex 1. As the composition of both complexes is very similar, a possible reason for activity discrepancy between complexes 1 and 2 might be caused by the difference in their coordination polyhedra. Different geometries produce different ligand fields, which in turn affect energetic levels and splitting of d-orbitals. is consequently affects both the kinetic and thermodynamic behavior of the DPPH quenching reaction [22,29,30].
e thermochemistry data were calculated as implemented in ORCA at 298.15 K and Gibbs free energies were corrected by where the factor of 1.89 kcal/mol is due to the change in standard state from gas phase to solution phase [44].
First, the hydrogen atom transfer (HAT) mechanism was evaluated with the help of the following reactions: showing the formation of supramolecular dimers through π-π stacking, the hydrogen atoms were omitted for the sake of clarity. Table 4: Short ring interactions with respective distances (Å) and angles (°) for 1 and 2 a . (10) 3.4624 (7) 3.5106 (7) 16.9 Cg2 Cg1 (10) 3.5107 (7) 3.4623 (7) 14.0 2 c Cg1 Cg2 (    Evidently, the only spontaneous reaction (exergonic reactions) is attributed to SET in which the electron is donated to the complexes, which resulted in the reduction of Cu II to Cu I . Moreover, the Δ r G � −87.26 kcal/mol for [Cu(phen) 2 (HL)] + of 1 and the value of Δ r G � −81.58 kcal/ mol for [Cu(phen) 2 (HL) 2 ] of 2 are similar in agreement with the comparable radical scavenging activity of these compounds.

Conclusions
e impact of different copper salts on the preparation of metal complexes with 2-(1H-tetrazol-5-yl)-1H-indole ligand (H 2 L) was investigated. e single-crystal X-ray analysis confirmed formation of pentacoordinate [Cu(phen) 2 (HL)] ClO 4 ·H 2 O·2DMF (1) and hexacoordinate [Cu(phen) 2 (HL) 2 ]·EtOH (2) compounds. In both compounds, the anionic HL − ligand acts as a monodentate N-donor ligand attached to the central atom through the tetrazole unit. e investigation of the interaction of these complexes with DPPH radical in their methanolic solution revealed moderate radical scavenging activity. e subsequent theoretical DFT calculations proposed that the dominant mechanism is the single electron transfer to the studied complexes.

Data Availability
e data used to support the findings of this study are included within the article and the supplementary information file.

Conflicts of Interest
(1) Figure 4: DPPH radical scavenging activity of complexes 1 and 2 and ascorbic acid for three different concentrations.