Synthesis, crystal structure and biological activity of copper(II) complex with 4-nitro-3-pyrazolecarboxylic ligand

: Reaction of 4-nitro-3-pyrazolecarboxylic acid and Cu(OAc)2H 2 O in ethanol resulted in new coordination compound [Cu 2 (4nitro-3pzc) 2 (H 2 O) 6 ]2H 2 O, (4nitro-3pzc = 4-nitro-3-pyrazolecarboxylate). The compound was investigated by means of single-crystal X-ray diffraction and infrared spectroscopy. Biologi- cal activity of the complex was also tested. In the crystal structure of [Cu 2 (4nit- 3pzc) 2 (H 2 O) 6 ]2H 2 O, the Cu(II) ion is in a distorted [4+2] octahedral coordination due to the Jan-Teller effect. A survey of the Cambridge Structural Database shows that the octahedral coordination geometry is generally rare for the pyrazole bridged Cu(II) complexes. In the case of Cu(II) complexes with the 3-pyrazole-carboxylato ligands, no complexes with the similar, octahedral coordination geometry have been reported. Biological research based on determining the inhibition effect of commercial fungicide Cabrio top and newly synthesized complex on Ph. viticola have been carried using the phytosanitary method. Cu(II) complexes with the unsubstituted 3-pyrazolecarboxylato ligands . In the case of Cu(II) complexes with the parent 3-pyrazolecarboxylato ligand, the CSD search reveals mononuclear (CSD refcode: DABRUW 1 ), binuclear (CSD refcodes: BEQGIQ 2 , BOYBOI 3 and RUNXOO 4 ) and polynuclear complexes (CSD refcodes: BOYBUO 3 , LAGNIT 5 , QOFLAA 6 ); only in mononuclear DABRUW 1 the Cu(II) is found in an octahedral coordination environment. In all binuclear complexes the Cu(II) is placed in a square pyramidal coordination environment, where fully deprotonated ligands bridge and chelate the pair of Cu(II) centers to form the square base of a polyhedron. Among these complexes the binuclear BEQGIQ complex, can be considered as a five-coordinated analogue of the title octahedral complex, with one axial H 2 O ligand less in Cu(II) coordination sphere. 2 In


INTRODUCTION
Pyrazole (pz) and pyrazole derivatives bearing different functional groups belong to an important class of ligands, capable of a variety of coordination modes with different metal ions. 1,2Apart from the confirmed biological signific-886 KOSOVIĆ et al.   ance of pz-based ligands and their metal complexes, 3,4 these compounds are recognized as valuable components of novel materials (magnetic, 5 energetic, 6 luminescent 7 and catalytic 7 ) and metal organic frameworks. 8,9The properties of these materials are frequently based on the ability of the pz ligands to produce biand poly-nuclear metal complexes coordinated by the two ortho positioned N donors. 1,2It is well known that the presence of substituents on the pz ring could significantly increase the ligand denticity and also influence the coordination geometry of metals.Among the pz-based bridging ligands, the 3,5-disubstituted pz derivatives are the most commonly investigated.Recently, the structural properties of Cu(II) and Co(II) complexes have been reported in which the dianion of 4-nitro-3-pyrazolecarboxylic acid (Scheme 1a) was used as a bridging ligand. 10These compounds are rare examples of metal complexes comprising a 3,4disubstituted pz derivative as a bridge and also the first crystal structures of the transition metal complexes with ligands derived from 4-nitro-3-pyrazolecarboxylic acid (Scheme 1a).Continuing interest in pyrazole-derived molecules, [10][11][12] and particularly the bridging and structural behaviour of the 4-nitro-3-pyrazolecarboxylate ligand, 10,11 a new binuclear complex [Cu 2 (4nitro-3pzc) 2 (H 2 O) 6 ]2H 2 O, Scheme 1b, was synthesized.Here, the Cu(II) ion is found in a tetragonally distorted octahedral environment, dissimilar to previously reported five-coordinated Cu(II) complex with the same ligand. 10The novel complex was characterized by singlecrystal X-ray diffraction and infrared spectroscopy.The antifungal activity of the compound was also tested for the fungi Ph. viticola using the phytosanitary method.

Synthesis
The reaction of a warm ethanol (5 cm 3 ) solution of Cu(OAc) 2 H 2 O (0.25 mmol, 0.0499 g) with 4-nitro-3-pyrazolecarboxylic acid (0.5 mmol, 0.08 g, dissolved in 6 cm 3 ethanol) gave a green solution of the complex compound.The solution was allowed to crystallize.After two days, the green prism crystals of the complex of formula [Cu  O, also confirmed by X-ray structural analysis.

Methods
Infrared spectroscopy.Fourier-transform infrared (FTIR) attenuated total reflection (ATR) spectra of [Cu 2 (4nitro-3pzc) 2 (H 2 O) 6 ]2H 2 O were recorded using a Bruker Tensor 27 FTIR spectrometer, equipped with a mid-IR glo(w)bar light source, a KBr beam splitter, a DLaTGS detector, and a Harrick MVP2 diamond ATR accessory at the Department of Mineralogy and Crystallography, University of Vienna.A total of 32 scans were accumulated between 4000 and 350 cm -1 at a spectral resolution of 4 cm -1 .As the ATR spectra probe the complex index of refraction instead of pure absorption, a slight red shift of the band positions has to be considered. 13-Ray diffractometry and crystal structure solution.Single-crystal X-ray diffraction data for [Cu 2 (4nitro-3pzc) 2 (H 2 O) 6 ]2H 2 O were collected on a Nonius Kappa CCD single-crystal four-circle diffractometer (MoK α -radiation, graphite monochromator), equipped with a 300 mm diameter capillary-optics collimator at the Department of Mineralogy and Crystallography, University of Vienna.The unit cell parameters were determined with HKL SCALEPACK (Nonius, 2005-2007).A complete sphere of reciprocal space (ϕ and ω scans) was measured.
The intensity data were processed with the Nonius program suite DENZO-SMN 14 and corrected for absorption by the multi-scan method. 15A monoclinic unit cell with a primitive Bravais lattice and space group symmetry P2 1 /n were derived from the reflection conditions and intensity statistics, both confirmed by consecutive structure refinement.The crystal structure was solved by direct methods incorporated in SHELXS and refined on F 2 by full-matrix least-squares using the SHELXL program. 16Anisotropic displacement parameters were allowed to vary for all non-hydrogen atoms.The H atom of the pyrazole C atom was placed at the geometrically calculated position and refined using the riding model.H atoms belonging to coordinated and solvent water molecules were determined by the CALC-OH 17 program based on hydrogen-bonding interactions.These H atoms were then treated as riding with O−H distances fixed to 0.85 Å and the isotropic thermal parameters tied to the parent oxygen U iso (H) = 1.2U eq (O).Relevant crystallographic details are listed in Table S-I of the Supplementary material to this paper.

Biological activity
The biological activity based on the determination of the inhibition effect of the commercial fungicide Cabrio top and the newly synthesized complex [Cu 2 (4nitro-3pzc) 2 (H 2 O) 6 ]2H 2 O on Ph. viticola were determined using the phytosanitary method.The diameters of fungal mycelium Ph. viticola as parameters of the inhibition effect were processed using variance analysis, while the testing was realized using the LSD test. 18

Syntheses and characterization
The [Cu 2 (4nitro-3pzc) 2 (H 2 O) 6 ]2H 2 O complex was obtained in the reaction of ethanolic solutions of Cu(OAc) 2 H 2 O and 4-nitro-3-pyrazolecarboxylic acid.The same product was obtained when Cu(NO 3 ) 2 3H 2 O was used instead of Cu(OAc) 2 H 2 O.The FTIR ATR spectrum band positions and band assignments of [Cu 2 (4nitro-3pzc) 2 (H 2 O) 6 ]2H 2 O (by comparison with literature) are summarised in Table S-II of the Supplementary material.A broad absorption band in the wavenumber region between ≈3600 and 2800 cm −1 is assigned to the stretching vibrations of H 2 O groups and is in agreement with the presence of medium strong to weak hydrogen bonds observed in the X-ray structure analysis.Using the distance-frequency correlation diagram for H bonds of Libowitzky, 19 an even wider range of donor-acceptor distances, i.e., ≈3.20 to 2.60 Å is expected.If, however, the wavenumber range above is constrained to the most intense region of the broad band, almost perfect agreement is obtained.Although the two superimposed narrow features at 3507 and 3157 cm -1 may also well originate from O−H stretching vibrations of H 2 O, an assignment to the N−H stretching vibration of pyrazole 20 for the former, and a C−H stretching vibration for the latter 21 is even more tempting.
The spectral region between 1700 and 1000 cm -1 and the hump below 900 cm -1 contains the bending/deformation mode of the H 2 O molecules (1604 cm -1 with asymmetric wing towards higher values) and a number of sharp bands that are characteristic of the vibrations of the pyrazole ring, the nitro group and the carboxylic unit.The latter shows the C=O stretching vibration as a shoulder at ≈1675 cm -1 and the C−O stretching band at 1298 cm -1 . 22The C−O band position, however, is very tentative, as the direct coordination to Cu is assumed to influence the bond forces.The bending mode of the carboxylic group is assigned to the band at 806 cm -1 . 23The NO 2 nitro group reveals symmetric and antisymmetric stretching vibrations at 1354 and 1506 cm -1 , respectively, whereas the bending mode is observed at 858 cm -1 . 24For comparison, in pure 4-nitro-3-pyrazolecarboxylic acid these features are at 1370, 1510 and 860 cm -1 . 24The remaining bands (Table S-II) are assigned to the ring stretching modes (N−N, N−C and C−C) and the C−H bending modes of the pyrazole unit, 21,22 i.e., at 1535, 1461, 1401, 1391, 1207, 1137, 1110, 1007 and 750 cm -1 .

Description of crystal structure
The crystal structure of [Cu 2 (4nitro-3pzc) 2 (H 2 O) 6 ]2H 2 O consists of binuclear complex units with copper(II) ions linked by a pair of dianionic pz ligands, Fig. 1a.The pz ligands simultaneously behave as N,O-chelating and N,Nbridging, giving rise to a five-membered chelate adjacent to the pz ring and to an approximately planar six-membered Cu 2 N 4 metalocycle.Each copper centre is placed in a distorted octahedral environment, which exhibits pronounced elongation in the axial direction due to the Jahn-Teller effect (Fig. 1b).The coordination bonds to axial H 2 O ligands are 2.5264(12) and 2.4123(11) Å for Cu-O6 and Cu-O7, respectively.The equatorial plane of this coordination polyhedron is formed by the NNO-donor set from the pz ligand and an additional H 2 O ligand (Fig. 1).The lengths of equatorial coordination bonds (Table I) are comparable to those found in the previously reported square-pyramidal [Cu 2 (4nitro--3pzc) 2 (dmf)(H 2 O)] complex comprising the same pz ligand. 10 In the recently reported crystal structure of [Co 2 (4nitro-3pzc) 2 (H 2 O) 6 ]2H 2 O complex with the same pz bridge, 10 the Co(II) was found in an octahedral coordination environment equivalent to that of the Cu(II) ion in the title complex.The comparison of the geometrical features of the two octahedral complexes is given in the Supplementary material; here it is noted that their crystal arrangements are characterized by the same structural motifs, based on strong O-H⋅⋅⋅O hydrogen bonds (Table II).In both crystal structures, the presence of solvent water along with the coordinated water molecules significantly increases the complexity of the hydrogen bonding networks.Considering only the direct hydrogen bonding between the complex units, each crystal structure is dominated by two interactions O5-H5a⋅⋅⋅O2 and O6-H6a⋅⋅⋅O1 (Table II).These interactions, formed between the coordinated water donors and carboxyl O acceptors, connect the complex units into corresponding zigzag chains (Fig. 2).An additional chain of directly bonded complex units is formed by the O7-H7b⋅⋅⋅O6 hydrogen bond that involves only the axially coordinated water ligands (Fig. 2).Similar to the Co(II) complex, this is the only case where the coordinated water acts as a hydrogen bonding acceptor, while all coordinated water molecules form significant hydrogen bonds as H-atom donors (all H atoms engaged).Contrary, the solvent water molecule simultaneously serves as donor and acceptor in four hydrogen bonds (Table II).These interactions connect the chains of directly bonded complex units into the three-dimensional crystal structure of [Cu 2 (4nitro-3pzc) 2 (H 2 O) 6 ]2H 2 O (Table II).Due to ability of pz ring to closely coordinate a pair of metal ions and support potential metal•••metal interaction, pz-based ligands are widely used in the synthesis of polynuclear metal complexes.Among 1136 pz-bridged metal complexes deposited in the Cambridge Structural Database 25 (CSD, version 5.40), those comprising at least one Cu centre represent the largest subgroup with 251 crystal structures.Interestingly, the Cu ion in these complexes is rarely found in an octahedral coordination environment.Thus, the CSD search for pz-bridged metal complexes comprising at least one Cu centre revealed 16 crystal structures with a six-coordinated Cu centre, 169 structures with a five-coordinated and 70 structures with a four-coordinated Cu centre.
The CSD does not contain binuclear Cu(II) complexes with 4-substituted-3--pyrazolecarboxylate ligands.In the case of Cu(II) complexes with the parent 3-pyrazolecarboxylato ligand, the CSD search revealed mononuclear (CSD refcode: DABRUW 26 ), binuclear (CSD refcodes: BEQGIQ, 27 BOYBOI 28 and RUNXOO 29 ) and polynuclear complexes (CSD refcodes: BOYBUO, 28 LAGNIT, 30 QOFLAA 31 ); only in the mononuclear DABRUW 26 was the Cu(II) found in an octahedral coordination environment.In all binuclear complexes, the Cu(II) is placed in a square pyramidal coordination environment, where fully deprotonated ligands bridge and chelate the pair of Cu(II) centres to form the square base of a polyhedron.Among these complexes, the binuclear BEQGIQ could be considered as a five-coordinated analogue of the title octahedral complex, with one axial H 2 O ligand less in the Cu(II) coordination sphere. 27In general, regardless of different coordination geometry, the binuclear, octahedral [Cu 2 (4-nitro-3pc) 2 (H 2 O) 6 ]2H 2 O complex and the extracted square pyramidal 3-pyrazolecarboxylato Cu(II) complexes exhibit a number of common features.The comparison of the structural features of binuclear metal complexes with the 3-pyrazolecarboxylato ligands can be found in the Supplementary material to this paper.

Biological activity
A study on the biological efficacy of the applied compound under laboratory conditions was performed on nutrient medium potato dextrose agar (PDA), in Petri dishes of 90 mm in diameter.The solutions of the complex and a commercial fungicide (Table III) were poured into the nutrient medium at a temperature of 60 °C.The test was performed with five different concentrations of each compound and each was repeated four times.The initial concentration of all compounds was 0.12 %, and each subsequent composition was two times lower.Petri dishes with nutrient medium without added compounds served as controls.After the homogenization of the medium with the compound and agar solidification, mycelial fragments (6 mm in diameter) obtained from 10 days old pure cultures of Ph. viticola were placed in the centre of the Petri dishes.The inoculated Petri dishes were maintained in an incubator at 25 °C.Mycelia growth of the fungus 892 KOSOVIĆ et al.
was measured when the colony in a control had covered about 2/3 of Petri dish diameter, which was the case after 10 days.The commercial fungicide expressed noticeably better inhibition effects than the applied complex.All concentrations of commercial fungicide showed statistically significant inhibition to colony growth of Ph. viticola, in comparison to the control.The studied complex did not express such a distinguished inhibition as a commercial fungicide, although there were concentrations among them that demonstrated a certain activity.
In contrast to the commercial fungicide that expressed a high inhibitory effect at all tested concentrations, the ligand showed a weak fungicidal effect. 32amely, some tested pyrazole derivatives, depending on their structure and concentration, show a statistically significant inhibitory effect 33 or a large fungicidal effect on pathogenic fungi that causes Phomopsis cane and leaf spot disease (patent application pending).Unlike the Cu(OAc) 2 H 2 O ligand, which at low concentrations (0.015 and 0.0075 %) shows a statistically significant inhibitory effect, but significantly lower than the commercial fungicide, while high concentrations of ligand (0.12 and 0.06 %) led to the increase in the diameter of the mycelium of the fungus.The weak inhibitory effect of the ligand and Cu(OAc) 2 H 2 O results in the present case also to the weak inhibitory effect of the complex compound on the examined fungus.

CONCLUSIONS
The dinuclear copper(II) complex comprising the dianion of 4-nitro-3-pyrazolecarboxylic acid as a bridge was synthesized by slow evaporation from ethanol at room temperature.The crystal structure of [Cu 2 (4nitro-3pzc) 2 (H 2 O) 6 ]2H 2 O contains Cu(II) ion in an octahedral coordination environment, significantly distorted due to the Jan-Teller effect.The three-dimensional arrangement of [Cu 2 (4nitro-3pzc) 2 (H 2 O) 6 ]2H 2 O is governed by strong intermolecular O-H⋅⋅⋅O hydrogen bonds involving the pyrazole ring substituents, coordinated water and the lattice water molecules.A CSD search revealed only rare cases of complexes in which pz-bridged Cu(II) ions display octahedral coordination geometry.In binuclear complexes of Cu(II) with the parent 3-pyrazolecarboxylato ligand, the metal ion is usually placed in a square pyramidal coordination environment.A commercial fungicide expressed noticeably better inhibition effects than the applied complex compound.All applied concentrations of the commercial fungicide showed statistically significant inhibition to colony growth of Ph. viticola, in comparison to the control.
2 (C 4 HN 3 O 4 ) 2 (H 2 O) 6 ]2H 2 O, measuring about 0.20 mm in size, were filtered and washed with a small amount of ethanol.Repeated synthesis under the same conditions and with the same ligand, where Cu(NO 3 ) 2 3H 2 O was used instead of Cu(OAc) 2 H 2 O, resulted in a complex of the same structural formula [Cu 2 (C 4 HN 3 O 4 ) 2 (H 2 O) 6 ]2H 2

TABLE II
2 O.The three strongest hydrogen bonds formed between the complex units are presented by dashed lines.________________________________________________________________________________________________________________________ Available on line at www.shd.org.rs/JSCS/(CC) 2020 SCS.