Crystal Structure , Hirshfeld Surface Analysis and Biological Activities of trans-Dipyridinebis ( 3-acetyl-2-oxo-2 H-chromen-4-olato ) cobalt ( II )

The novel cobalt(II) complex, trans-dipyridinebis(3-acetyl-2-oxo-2H-chromen-4-olato)cobalt(II), was synthesized in ethanol. The coordination sphere of the cobalt cation was elucidated using single-crystal X-ray diffraction analysis and spectroscopic techniques (FT-IR, UV-Visible and fluorescence). Hirshfeld surface analysis indicates that hydrogen bond interactions, such as C–H···O hydrogen bonding between the oxygen of lactone group and the pyridine appear as a primary interaction between the complex’s molecules. The presence of π-π stacking was evident by the shape index and curvature. Analysis of 2D fingerprint plots confirm that intermolecular H···H, C···H and H···O interactions are well dominated and are in complement to the Hirshfeld surface. The metal-ligand coordination strongly influences the fluorescence intensity (the fluorescence quenching) and the offset of the emission wavelength. The metal complex was monitored for antimicrobial activity using the disk diffusion method and showed significant activity compared to the coumarin ligand.


Introduction
In recent decades, the new coumarin derivatives have received increased attention due of their important biological activities. 1 Coumarins exhibit biochemical and physical properties.So, they are used as improver's agents in cosmetics products, 2 fluorescent probes, 3 and indicators for biological research and as medicaments, 4 for the treatment of various clinical conditions. 5Research has also demonstrated the selective cytotoxicity of coumarins for tumor cells as well as their influence on the regulation of immune response, cell growth and differentiation. 6,7The physical, pharmacological and therapeutic properties of coumarins are adjusted by the substitution of a heterocyclic moiety as a substituent or as a fused component in the coumarin skeleton. 8he investigations of metal-based drugs have become an interesting subject for bioinorganic chemists. 9,102][13][14] The scientific research in this field has proven that the binding of a drug to a metal increases its activity.In some cases, the complex has an even more interesting activity than the parent compound. 15,16In particular, several metal-coumarin complexes have also been prepared and some show a biological activity superior to that of coumarin itself. 17,18obalt complexes have attracted the attention in the medical field because cobalt is an essential trace element in all animals.It is mainly present as vitamin B 12 which plays an important role in many biological processes.Thus, cobalt may be less toxic to the human body than other metals. 19,20ew studies are based on the investment of protocols for the synthesis of new coumarin derivatives prepared from 3-acetyl-4-hydroxycoumarin, in the form of complexes, 21 chalcones, 22,23 aminocoumarines, 24 Schiff bases, 25,26 and their complexes. 15,26We always try to highlight them through structural studies, physical properties and Bejaoui et al.: Crystal Structure, Hirshfeld Surface Analysis ... biological evaluation.In this context, we continue our studies on the synthesis of new Co(II) complexes from 3-acetyl-4-hydroxycoumarin and we succeeded to prepare trans-dipyridinebis(3-acetyl-2-oxo-2H-chromen-4-olato) cobalt(II), a new complex which was characterized by spectroscopic techniques such as FT-IR, UV-Visible and fluorescence.Its three-dimensional structure is determined by single-crystal X-ray diffraction.Characterization and quantification of its intermolecular interactions are performed by Hirshfeld surface analysis.This new complex was evaluated by its antioxidant and antimicrobial activities.

1. Analyses and Instrumentation
All analytical grade chemicals and solvent were commercially available purchased and used as received without further purification.The starting organic compound 3-acetyl-4-hydroxycoumarin was prepared as previously described. 27The IR spectra of ligand and its metal complex were recorded on a Bruker FT-IR spectrophotometer Tensor 27 by KBr pellet technique in the range 4000-400 cm -1 .The electronic absorption spectra were recorded on double beam UVD-3500 UV-Vis spectrometer in ethanol and dimethylsulfoxide (DMSO) in the region 200-900 nm.The fluorescence study of the complex was recorded on a SHIMADZU RF-5301PC spectrophotometer.Solutions of different concentrations (c 1 = 0.6 • 10 -3 mol/L, c 2 = 0.7 • 10 -4 mol/L, c 3 = 1.6 • 10 -5 mol/L, c 4 = 0.26 • 10 -5 mol/L, c 5 = 0.4 • 10 -6 mol/L) were prepared in DMSO and the experiments were carried out at room temperature.
The purity of the sample was also checked by the X-ray powder diffraction analysis.When the crystal structure was solved from the single crystal, the simple comparison of the measured with calculated powder diffraction pattern was made.It confirmed the phase purity of the synthesized sample.

3. X-Ray Diffraction
The single-crystal X-ray diffraction data were collected on a Supernova Atlas S2 CCD diffractometer with mirror-monochromated Cu-Kα radiation at 120 K. Crys-Alis PRO software was used for data reduction and correction of absorption. 28The crystal data, the data collection and the details of the structure are summarized in Table 1.The structure was solved by the direct method using Superflip software, 29 and was refined by least-squares calculations on F 2 with Jana2006. 30All hydrogen atoms present in the structure model were discernible in difference Fourier maps and could be refined to reasonable geometry.According to common practice, H atoms bonded to C were kept in ideal positions with C-H bond equals to 0.96 Å. and with U iso (H) = 1.2U eq .All non-hydrogen atoms were refined using harmonic refinement.The molecular and packing diagrams of new complex were generated using the software DIAMOND Version 3. 31 The ORTEP of the molecule with thermal ellipsoids was also generated. 32

4. Antimicrobial Assay
The antibacterial test was carried out by using standard paper disc method according to Ismail et al. 2010, 33 with slight modification.Briefly, 2 mg of each crude extract dissolved in 1 mL of sterile dimethyl sulfoxide (DMSO) was applied to sterile filter paper discs (6 mm).Then, discs were placed on tryptic soy agar (TSA, BIO RAD) plates, which were inoculated with 18 h cultured of the tested pathogen (10 6 bacteria/mL) in tryptic soy broth (TSB, BIO RAD).As negative control, a disc loaded with DMSO was simultaneously prepared.Plates were incubated overnight at 30 °C.After

1. Infrared Spectra
The comparison of the infrared absorption spectrum of the complex (Fig. 1) with that of the ligand shows the absence of frequency of the stretching vibration ν(OH) which appears around 3435 cm -1 in the spectrum of the uncoordinated coumarin ligand.This indicates that the carbonyl groups of the ligand were deprotonated during coordination with the cobalt(II) cations.The band around 1613 cm -1 is less intense and less wide compared to the analogous band in the coumarin ligand spectrum, this band corresponds to the lactone-carbonyl fragment which is a superposition of two stretching bands of the conjugated C=C and C=O.A decrease in the frequency of the stretch band of the C=O acetyl group is observed with respect to the starting coumarin ligand which is may be caused by the engagement of this group in the coordination sphere of cobalt(II) cation.The low frequency region shows the appearance of new low intensity bands in the spectrum of the cobalt complex at frequencies 473 cm -1 and 430 cm -1 , attributed to the vibration of the bonds Co-O(coumarin) and Co-N(pyridine).

2. UV-Visible Spectra
The UV-Vis spectra of the cobalt(II) complex were recorded at different concentrations in DMSO in Figure 2 (a).For high concentrations, these spectra display a large absorption band between 440 nm and 610 nm with maxima at 509 nm, this corresponds to the electronic transition d→d*.The position of the maximum is compatible with the octahedral configurations of cobalt(II) complexes. 34We also observed that the progressive decrease in the concentration of the complex is accompanied by a hypsochromic displacement in the low wavelength range of the absorption bands located between 270 nm and 350 nm, which are attributed to the π→π* electronic transition of coumarin as well as hypochromic effect on the intensity of absorption The electron absorption spectra of the complex in different solvents with different polarities ((DMSO) and ethanol) in order to examine the influence of the surrounding environment on the spectroscopic properties of the complex are presented in Fig. 2 (b).The absorption spectrum of the complex in DMSO solvent shows a redshift of the absorption maximum of the π→π* transition with λ max = 314 nm compared to the spectrum recorded in ethanol (λ max = 302 nm) and a hyperchromic effect.This is manifested by the dipole interaction between the complex  Bejaoui et al.: Crystal Structure, Hirshfeld Surface Analysis ... molecules and the surrounding molecules of the polar solvent, which causes a decrease of the energy difference of the HOMO-LUMO of electronic transition π→π* and consequently an increase of the wavelength.This result is evident when comparing the spectra of the complex studied in ethanol and DMSO.

Fluorescence Spectra
The analysis of the fluorescence spectrum of the complex in DMSO at concentration of 10 -5 M shows that the excitation peak of its possible fluorescent center is almost identical to its corresponding absorption band (recorded under the same conditions) and that the emission spectrum is approximately an inverted image of the absorption spectrum with a slight red shift (Fig. 3).The wavelength interval between the peak position of the absorption spectrum and the peak of the fluorescence spectrum of the same electron transition π→ π* is called the Stokes shift.Indeed, the probability that an electron re-turns to a particular vibrational energy level in the ground state is similar to the probability that this electron is in the ground state before excitation.That is, the same electronic transitions are the most favorable for absorption and emission.This concept is known as the mirror image rule.
The analysis of the emission spectra of the complex at different concentrations (Fig. 4) in the DMSO for a 330 nm excitation corresponding to the π→π* transition shows a broad band of fluorescence in the blue-green region with a maximum emission at about 416 nm.The increase in the concentration of the complex solution resulted in a significant decrease in the intensity of photoluminescence and a hypochromic shift towards the low wavelengths.This decrease is due to the solvation phenomenon, to the interaction between the solvent and the solute, as well as to the coordination effect of the co-  balt(II) ions.This effect has been observed in other cobalt(II) complexes. 21This phenomenon is known as fluorescence quenching.The comparison of the fluorescence spectra of the coordinated complex and uncoordinated coumarin (Fig. 4), recorded in the same solvent (DMSO) at concentration of 1.6 • 10 -5 M and an excitation at 303 nm reveals a significant decrease in emission intensity and a slight hypsochromic shift of the complex.Indeed, there seems to be a transfer of intramolecular photoinduced electrons (PeT) from coumarin to the cobalt(II) cation.The photoluminescence properties of the cobalt complex result mainly from the coumarin ligand.During excitation, an electron transfer takes place from the coumarin fluorophore excited at the LUMO energy level of the cobalt(II) complex, which leads to the quenching of the fluorescence.The decrease in the fluorescence intensity also indicates that the Co(II) ions have a quality of fluorescent quenching.The results of our study are consistent with works reports of Singh et al. 35

5. Hirshfeld Surface Analysis
The Hirshfeld surfaces were used for exploring intermolecular interactions in the studied crystal. 37,38 he molecular Hirshfeld surfaces calculations were performed using the CrystalExplorer 3.1 program, 39 which accepts a structure input file in the CIF format.The Hirshfeld surfaces of the title compound are illustrated in Fig. 8.This figure is showing surfaces that have been mapped over d norm (normalized contact distance) (Fig. 8 (1)), shape index (Fig. 8 (2)), curvedness (Fig. 8 (3)) and d e (distance from a point on the surface to the nearest nucleus outside the surface) (Fig. 8 (4)).The Hirshfeld surfaces of cobalt(II) complex were generated using a standard (high) surface resolution with 3D d norm surfaces mapped to a ranges -0.1829 to 1.0550.The d norm mapping indicates that strong hydrogen bond interactions, such as C-H • • • O hydrogen bonding between the oxygen of the lactone group and the pyridine appear as a primary interaction between the complex's molecules, seen as a bright red zone in the Hirshfeld surfaces (Fig. 8 (1)).
The size and shape of the Hirshfeld surface allow the qualitative and quantitative study and visualization of inter molecular close contacts in molecular crystals.The shape index and the curvature were used to identify packaging modes (planar stacking arrangements).In fact, the presence of π • • • π stack is evident as a flat region down on both sides of the molecules and is clearly visible on the curve surface.The pattern of the red and blue triangles on

D-H•••A D-H H•••A D•••A D-H•••A
C15-H1c15     the area of the shape index surface (Fig. 8 (2)) is characteristic of the π • • • π stacking.The blue triangles exhibit the convex regions due to the carbon atoms of the molecule inside the surface, while the red triangles represent the concave regions because of the carbon atoms of the stacked molecule π above. 40,41The C-H • • • π contact in the crystal shows a bright-orange spots on the d e surface (Fig. 8 (4)), 38 directly above the center of the C=C bonds and generates a distinct pattern of a pair of wings in the two-dimensional fingerprint plot (Fig. 9), it will become obvious that this is a characteristic feature of all C-H • • • π interactions.
The 2D fingerprint plots are deconstructed to highlight particular atom pair contacts.This allows the separation of the contributions of different types of overlapping interaction in the complete fingerprint.The analysis of 2D fingerprint plots for the title compound (Fig. 9

6. Antioxidant Activity
The antiradical activity of the extracts is determined by the method of reduction of the free radical of DPPH (1,1-diphenyl-2-picrylhydrazyl). 42The DPPH radical is a stable organic free radical with an adsorption band at 515-518 nm.The free radical scavenging mechanism is the transfer of the hydrogen atom from the test compound to DPPH, which is transformed into a stable molecule DP-PHH. 43The ability to reduce the DPPH radicals was determined by the decrease in its absorbance at 515 nm induced by antioxidants.The IC 50 (scavenging concentration 50%) allowing calculate the extract concentration needed to trap 50% of the DPPH radicals.It is determined graphically by linear regression.The graph was plotted with percent scavenging effects (% IDPPH) according to concentration (μmol/L) (Fig. 10).The metal complex showed good activities as a radical scavenger with IC 50 = 0.0195 μmol/L.A lower IC 50 value indicates greater antioxidant activity, compared to ascorbic acid (vitamin C) as a reference that has an IC 50 value of 21 μmol/L, 44 it is considered a good free radical trapper.In addition, it possesses significant antioxidant activity relative to its coumarin ligand, 21 and other similar cobalt(II) complexes.It is difficult to draw general conclusions about antioxidant structure-activity relationships with the limited number of antioxidant molecules.It may be suggested that metal ions significantly change the chemical properties of coumarin ligand.The complexation with the transition metal was carried out via the carboxylate and phenol groups of the two ligands of coumarin in equatorial positions, this can improve the antiradical activity. 45

7. Antimicrobial Activity
The antimicrobial activities of ligand and complex were screened against six Gram-negative bacteria: Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Vibrio alginolyticus, Pseudomonas fluorescens and Vib-   rio anguillarum and a Gram-positive bacterium: Staphylococcus aureus and against a fungus, ie Candida albicans, to evaluate their potential as an antimicrobial agent.The antimicrobial activities of the complex and its coumarin ligand under test against different strains of bacteria are shown in Table 4 and Figure 11.The most sensitive microorganisms to the Co complex were Escherichia coli, Vibrio alginolyticus and Vibrio anguillarum (gram negative) with inhibition diameters of 11.66, 13.33 and 12 mm, respectively.This complex also showed low activity against Pseudomonas aeruginosa, Salmonella typhimurium, Pseudomonas fluorescens, Candida albicans and Staphylococcus aureus.In contrast, the coumarin ligand 3-acetyl-4-hydroxycoumarin exhibits a broad antibacterial activity against all strains tested but was more active against Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Pseudomonas fluorescens, Candida albicans and Staphylococcus aureus with inhibition diameters of 11.33, 11, 11.66, 12.66, 11.33, and 11 mm, respectively.This relatively high antibacterial activity of uncoordinated coumarin may be due to its flat molecular structure that contributes to penetration across the membrane or bacterial cell wall. 46It is clear that the zones of inhibition may be larger or weak for metal chelates than for the coumarin ligand for certain microorganisms.In addition, the antibacterial actions of coumarin ligand can be significantly improved upon chelation with cobalt(II) ions. 47In particular cases, the increase in antimicrobial activity is due to the faster diffusion of metal complexes as a whole across the cell membrane or to the combined activity of the metal and the ligand. 48he minimum inhibitory concentration (MIC) was determined by the micro-dilution method according to the standard reference method for bacteria. 49Various concentrations of metal complex solutions (62.5, 125, 250, 500 μg/mL, and 1 mg/mL) were obtained by dissolving the compound in DMSO (2%) and then diluted to give serial two-fold dilutions.These solutions were added to each medium in the 96-well plates.The bacterial suspensions are added at the rate of 180 μL in suspension of 10 6 bacteria/mL to each well.The purpose of this method was to determine the exact concentration of the compound under study which will have an inhibitory effect on growth selected microorganisms.This concentration was considered as minimal inhibition concentration (MIC).The MIC values was determined against Escherichia coli, Vibrio algi-nolyticus and Vibrio anguillarum bacteria which have an inhibition diameter greater than 10 mm.As a negative control, DMSO did not affect the growth of bacterial strains at the concentrations used in this study.Values of MIC of complex are shown in Table 5.It should be noted that the cobalt(II) complex exhibited a good MIC result against Gram negative bacteria.

Conclusion
In this paper, a novel Co(II) complex trans-dipyridinbis(3-acetyl-2-oxo-2H-chromen-4-olato)cobalt(II) was synthesized and characterized by different spectroscopic techniques.The bonding of ligand to metal ion is confirmed by spectral studies (UV-Vis, IR, and fluorescence).The IR spectral analysis shows a decrease in the frequency of the stretch band of the C=O acetyl group with respect to the coumarin which may be due to the donor-acceptor interaction of this group with the cobalt(II) cation.The UV-Vis spectral studies suggested an octahedral geometry for the Co(II) complex.The metal-ligand coordination causes a decrease in the fluorescence intensity (the fluorescence quenching) and an offset of the emission wavelength.The crystal structure of the Co complex was studied by single-crystal X-ray diffraction; the cobalt(II) atom exhibits an axially elongated octahedral CoN 2 O 4 coordination geometry.In the packing of crystal lattice, all the complex molecules are connected by an extensive network of C-H • • • O hydrogen bonds and by interactions of type C-H • • • π and π-π.The molecular Hirshfeld surface and 2D fingerprint plots were used for quantitative mapping out of molecular interactions, revealing that close contacts are dominated by H • • • H, C • • • C and C • • • H interactions, these relatively weak contacts have clear signatures in the fingerprint plots.The new coumarin complex reveal potentially important antioxidant activity against free radicals DPPH compared to the coumarin derivative ligand and vitamin C. The antimicrobial studies suggested that the coumarin derivative ligand is biologically active and its metal complex exhibits significantly enhanced antimicrobial activity against some microbial strains in comparison to the free ligand.Thus, this new cobalt(II) complex can be used further in the pharmaceutical industry, as an antioxidant and antimicrobial agent, after testing its toxicity to humans.

Supplementary Material
CCDC reference number 1891314 for the cobalt complex contain the supplementary crystallographic data for this paper.These data can be obtained free of charge at www.ccdc.cam.ac.uk, or from Cambridge Crystallographic Data Center, 12, Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336 033; e-mail: deposit@ccdc.cam.ac.uk.

Figure 1 .
Figure 1.Comparison of the two IR spectra of the complex and ligand.
), the Co(II) cation is coordinated by two O atoms (O 1 , O 2 ) and two N atoms (N4, N5).The octahedral environment of cobalt(II) consists of 3-acetyl-2-oxo-2H-chromen-4-olato in equatorial sites in trans configuration and two pyridine molecules occupying the axial sites.The cobalt(II) atom exhibits an axially elongated octahedral CoN 2 O 4 coordination geometry.Compared to the other crystal structures in the literature (Table 2) 36 , the Co-O and Co-N distances as well as the O-Co-O, O-Co-N and N-Co-N angles are considered as the expected values of a cobalt(II) complex coordinated by four oxygen atoms in the equatorial plane and two nitrogen atoms in axial positions.
) and by interactions of type C-H • • • π and π-π.In fact, the inter molecular bond between the coumarin lactone group and the pyridine C15-H1c15 • • • O4, C18-H1c18 • • • O4 and C19-H1c19 • • • O3 creates a network of inter actions of type C-H • • • π which can be seen in the packing diagram where the complexes are connected in chains parallel to the axis [001] (Fig. 6).

Figure 5 .
Figure 5.A perspective view of complex showing the atom-labelling scheme and displacement ellipsoids at the 50% probability level, symmetry code: (i) -x, y, ½ -z.

Figure 6 .
Figure 6.Crystal packing of complex showing the formation of a chain along [001], showing the intermolecular C-H • • • O hydrogen bonds as thin green lines.Hydrogen atoms not involve in hydrogen bonding has been omitted for the sake of clarity.

Figure 7 .
Figure 7. Crystal packing of complex showing a weak offset π-π stacking interactions between coumarin ligands in (110) plane.The green dotted line represents π-π stacking interactions.Hydrogen atoms have been omitted for clarity.
) confirm that inter molecular H • • • H, C • • • H and H • • • O interactions are well dominated and are in complement to the Hirshfeld surface.The first main interaction of type H • • • H were represented by the largest region in the fingerprint plot with contribution 38.2%.A second main interaction of type C • • • H is due to hydrogen bonds C8 • • • H1c17-C17, and appears at the top left of the plot in the form of the characteristic wings with a contribution of 30.6%, these can also be identified by C-H • • • π type interactions.This deduction is compatible with the qualitative d e surface analysis.A third principal interaction of type H • • • O is manifested in the molecular structure by the C15-H1c15 • • • O4, C18-H1c18 • • • O4 and C19-H1c19 • • • O3 hydrogen bonds between axial pyridine and coumarin plane.

Figure 9 .
Figure 9. Two-dimensional fingerprint plots of the complex showing percentages of contacts contributing to the total Hirshfeld surface area of the molecule: (a) all interactions, and delineated into (b) H • • • H, (c) C • • • H/H • • • C, (d) O • • • H/H • • • O and (e) C • • • C interactions.

Figure 11 .
Figure 11.Antibacterial activity spectrum of the complex.

Table 1 .
Crystallographic details and structure refinement of complex

Table 4 .
Antimicrobial activities of the synthesized coumarin ligand and complex All data are the averages of the measurements in triplicate

Table 5 .
Minimum Inhibitory Concentration assay of metal complex against bacterial pathogens