Synthesis of Ni(II), Cu(II) and Zn(II) coumarin-3-carboxilic acid derivates and their and their physical-chemical properties

In the search for new drugs, coumarins are an important class of compounds due to their chemical and biological properties, such as their potential to reduce cancer, diabetes, and cardiovascular diseases. They are heterocyclic compounds that contain oxygen in their structure and are found in plants.To improve the chemical and biological properties of the coumarin-3-carboxilic acid, were prepared metal transition complexes of Cu(II), Ni(II) and Zn(II) of coumarin-3-carboxilic acid by a new synthetic route. All complexes were characterized by Ultraviolet (UV), Infrared (FTIR), and Raman spectroscopy; Scanning electronic microscopy (SEM); X-Ray diffraction (XRD), as well as conductivity and elemental analysis. Electron microscopy and X-ray analysis showed that the degree of crystallinity of the complexes changes when compared with the precursor, 3-carboxy-coumarin acid, and the degree of crystallinity depends on the nature of the metal ion attached to coumarin. In addition, the antioxidant action of the complexes was evaluated by the DPPH method, and the results showed a higher activity of the complexes when compared with the precursor, suggesting that these complexes may present biological properties of interest.


Introduction
Coumarin is found in plants such as cinnamon, guaco, lavender, and peppermint. It is a simple oxygen-containing heterocycle that has received significant attention due to its biological properties including in medicine, food, the chemical industry, and botany (Wang et al., 2013), (Song et al., 2017), (Wang et al., 2019). Coumarins play a key role in natural products and act as a secondary metabolite in plants (Elhusseiny, Aazam, Al-Amri, 2014). They can be found in different concentrations and are distributed in fruits, roots, stems, and leaves as well as in bacteria and fungi (Patil et al., 2015). In nature, more than 3400 coumarins have been found, and these are distributed in 160 families (Musa, Cooperwood  Khan., 2008). The coumarins stand out for their biological properties, which is attributed to the functional groups present in their structure (Lv et al., 2015).
The properties of coumarins may be related to the effects of antioxidant activities along with other mechanisms of action such as anti-inflammatory action and interactions with enzymes (Borges Bubols et al., 2013), (Creaven et al., 2006).
It is well known that a variety of coumarin-derived complexes can be obtained through different coordination modes with varying spectroscopic properties and potential applications in different areas especially when coordinated to d-block transition metal ions (de Alcantara et al., 2015).
The biological action of coumarin-3-carboxylic acid (HCCA) can be potentiated by complexing with metals through different coordination modes with spectroscopic properties and potential applications (Creaven et al., 2011), (Islas et al., 2018).
One example is an increase in the antimicrobial activity of the coordination of the silver (I)-HCCA complex versus free ligand as reported previously (Creaven et al., 2006). A series of complexes of HCCA with Zn, Co, Ni, and Mn ions were synthesized, and their molecular structure and spectroscopic studies were determined based on density functional theory (Creaven et al., 2011). However, more detailed analysis about the physicochemical properties and structure of the complexes for pharmaceutical applications has not been performed.

Material
All of the chemicals including coumarin and the acetate salt of diphenyl picrylhydrazyl radical (DPPH) were obtained from Sigma-Aldrich and Merck. All solvents used were of PA grade.

Analytical instruments
IR spectra were obtained in solid state via reflectance between 4000 and 400 cm -1 on a Nicolet-Nexus instrument, Raman spectra were collected using a Bruker RFS -100/S; a 1064 nm YAG laser was used as the excitation source at 400 mW.
The melting point was measured using QUIMIS equipment, and the UV/VIS spectra were obtained in a water solution between 200 and 500 nm with a Femto 800-XI spectrometer. The conductivity analysis was performed in DMF solution (3mM) and expressed in Ω -1 cm 2 mol -1 . Analysis with scanning electron micrographs (SEM) was performed with a Supra 35-VP instrument (Carl Zeiss, Germany). The samples were evenly distributed on SEM specimen stubs with double adhesive tape. The micrographs were obtained with an accelerating potential of 2 kV. Monochromatic Cu Ka radiation (wavelength =1.54056 Å) was produced by Rigaku-DMax/2500PC, Japan. The powdery samples were packed tightly in a rectangular aluminum cell prior to exposure to the X-ray beam. The scanning regions of the diffraction angle, 2θ, were 10 -80º, and radiation was detected with a proportional detector. in 20 mL of methanol at 50ºC. The mixture was stirred for 1 h. The solutions were stored at 5ºC for 24 hours until precipitation.

General Synthesis of complexes
The precipitate was filtered and washed with cold methanol.

Antioxidant Activity
The solution contained 1mL of DPPH (diphenyl picrylhydrazyl radical) (60 μM) and different concentrations of complexes were prepared. For coumarin-3-carboxylic acid (HCCA) the concentrations varied from 100 to 1000 µM, for Zn(II)-HCCA from 42 to 420 µM, for Ni(II)-HCCA from 42 to 420 µM and for Cu(II)-HCCA from 65 to 650 µM. Ethanol PA was added to each solution until the volume of 2 mL and DPPH concentration of 30 μM. The solutions were vigorously mixed and allowed to stand in the dark for 30 min at 25 ºC. The absorbance of the resulting solutions was measured at 517 nm against a blank sample containing only DPPH (the negative control). Rutin was the reference (Ikeda et al., 2015).

Synthesis and Characterization of Complexes
The  Table 1. Although the formation of binuclear structures was considered, the elemental analysis data for the three complexes correlated better with a mononuclear structure (Table 1). However, the Ni(II)-HCCA and Zn(II)-HCCA has a ratio of two coumarin molecules to one metal center; Cu(II)-HCCA is one coumarin molecule to one copper ion (Fig 3). The experimental data of conductivity for Cu(II)-HCCA, Ni(II)-HCCA, and Zn(II)-HCCA (Table 1)   The vibrational FTIR and Raman spectra of Cu(II)-HCCA, Ni(II)-HCCA, and Zn(II)-HCCA were measured and compared with the HCCA (Figure 2). In the region between 3300 and 2500 cm -1 , there were broad and weak bands corresponding to O-H stretching. Between 1440 and 1395 cm -1 there was medium C-O bending of the carboxylic acid group. These data indicate the permanence of the hydrogen atom in HCCA (Table 2); These results were confirmed by Raman spectroscopy (Figure   2), whose characteristic stretching frequency of O-H at 3080 cm -1 was observed for all HCCA binders and all complexes. Metal ions interact with coumarin molecules via the carboxylic and carbonyl groups, and those interactions promote a polarization of the bond leaving the C=O bond. This in turn promotes a shift of the C=O band to lower frequencies versus the HCCA bands ( Table 2)   The bands assigned to the C=O (stretching) and C=C aromatic (stretching) was also confirmed by the Raman spectra in  Raman and IR data for all complexes indicate coumarin-3-carboxy acid (HCCA) binder H carboxylic remains in the molecule after metallic coordination. According to the conductivity data (Table 1) all the synthesized complexes are neutral suggesting the presence of two negatively charged binders coordinated to Cu(II), Zn(II), and Ni(II) ions, forming the respective complexes.
Therefore, based on spectroscopic data (Table 2 and Figure 2), conductivity and elemental analysis (Table 1) it is suggested a octahedral structure for Ni(II)-HCCA and Zn(II)-HCCA. There is coordination of two molecules of coumarin-3-carboxy acid occurred, but there is deprotonation of only one HCCA and interaction of one molecule of ethyl acetate leaving the complexes neutral ( Figure 3A). For the Cu(II)-HCCA complex, it is suggested a square planar structure with coordination of only one molecule of coumarin-3-carboxy acid, which does not suffer deprotonation and the coordination of one molecule of acetate ( Figure 3B). into the coordination sphere . Although single crystal X-ray crystallography is the most precise source of information regarding the structure of a complex, the difficulty in obtaining suitable crystals in a proper symmetric form has rendered this method unsuitable for such a study. in Figure 5(a-h). Figure 5(a) shows that the HCCA particles have irregular sizes, but with a structure that is regularly scattered ( Figure 5b). Figure 5(c-d) shows that the Cu(II)-HCCA grains are small with similar sizes. The micrographs of Ni(II)-HCCA and Zn(II)-HCCA ( Figure 5(e-f, g-h)) indicated the presence of defined crystals free of any sign of the metal ion. In the metal complexes, crystals grew via agglomerates of plates of various sizes . Source: Authors.

Antioxidant activity of Complexes
The antioxidant potential of the complexes was evaluated via the DPPH method (Halli et al., 2012) at concentrations ranging from 100 to 1000 µM ( Figure 6). Cu(II)-HCCA had the best correlation between percentage of free radical inhibition and concentration (31% at 160 µM) while HCCA inhibits 2% at a concentration of 236 µM, Zn(II)-HCCA 22% at 529 µM and Ni(II)-HCCA 22% at 834 µM. However, when the antioxidant activity of the complexes is compared with the antioxidant activity of rutin, this is still much lower than the positive control, 95% of inhibition at 320 µM ( Figure 6).
According to the literature, the transition metal complexes derived from coumarins show greater antioxidant action than their respective precursors (de Alcantara et al, 2015). Comparing the antioxidant activity results obtained from Cu(II)-HCCA and Ni(II)-HCCA with Cu(II) and Ni(II) complexes derived from 3-aminicoumarin, the latter showed 1.6 and 5 times higher activity, respectively, than Cu(II)-HCCA and Ni(II)-HCCA complexes (Kadhum et al, 2011). However, no comparative study of the antioxidant action of 3-aminicoumarin and its complexes has been shown.
The increased antioxidant activity of these complexes can be attributed to the electron withdrawing effect of the Zn(II), Ni(II) and Cu(II) ions, which facilitates the release of hydrogen carboxylic to reduce the DPPH radical (Ejidike  Ajibade, 2015). The DPPH radical scavenging ability of the test samples can thus be ranked in the order Rutin > Cu(II)-HCCA > Ni(II)-HCCA > Zn(II)-HCCA > HCCA.

Conclusion
A new method has been developed for the synthesis of Cu(II)-HCCA, Ni(II)-HCCA, and Zn(II)-HCCA. The SEM and XRD analyses showed that the degree of crystallinity of the complex depends on the nature of the metal ion bound to HCCA.
Ni(II)-HCCA and Zn(II)-HCCA showed higher crystallinity than Cu(II)-HCCA. All complexes presented better antioxidant activity than the HCCA precursor.