Coordination Modes of a Schiff Base Pentadentate Derivative of 4-Aminoantipyrine with Cobalt(II), Nickel(II) and Copper(II) Metal Ions: Synthesis, Spectroscopic and Antimicrobial Studies

Transition metal complexes of Co(II), Ni(II) and Cu(II) metal ions with general stoichiometry [M(L)X]X and [M(L)SO4], where M = Co(II), Ni(II) and Cu(II), L = 3,3’-thiodipropionic acid bis(4-amino-5-ethylimino-2,3-dimethyl-1-phenyl-3-pyrazoline) and X = NO3−, Cl− and OAc−, have been synthesized and structurally characterized by elemental analyses, molar conductance measurements, magnetic susceptibility measurements and spectral techniques like IR, UV and EPR. The nickel(II) complexes were found to have octahedral geometry, whereas cobalt(II) and copper(II) complexes were of tetragonal geometry. The covalency factor (β) and orbital reduction factor (k) suggest the covalent nature of the complexes. The ligand and its complexes have been screened for their antifungal and antibacterial activities against three fungi, i.e. Alternaria brassicae, Aspergillus niger and Fusarium oxysporum and two bacteria, i.e. Xanthomonas compestris and Pseudomonas aeruginosa.


Introduction
The transition metal complexes of 4-aminoantipyrine and its derivatives have been extensively examined due to their wide applications in various fields like biological, analytical and therapeutical [1−4]. Further, they have been investigated due to their diverse biological properties as antifungal, antibacterial, analgesic, sedative, antipyretic and anti-inflammatory agents [5][6][7]. In recent years, a number of research articles have been published on transition metal complexes derived from 4-aminoantipyrine derivatives with aza or aza-oxo donor atoms [8−11]. We were interested in examining the biological activities of NS-donor Schiff's bases and their transition metal complexes, thus, in this article, we report the antifungal and antibacterial activities of the pentadentate (NNSNN-donor) Schiff's base ligand 3,3'-thiodipropionic acid bis(4-amino-5-ethylimino-2,3-dimethyl-1-phenyl-3pyrazoline) and its complexes with Co(II), Ni(II) and Cu(II) metal ions. The ligand and its complexes were characterized by physicochemical and spectral studies.

Mass spectra
Mass spectra provide a vital clue for elucidating the structure of compounds. The ESI mass spectrum of ligand L is given in Figure 1. The spectrum shows the molecular ion peak at m/z = 602 and the isotopic peak at m/z = 603 (M + +1) due to 13 C and 15 N isotopes. The base peak at m/z = 214 is due to the ethylimino-2,3-dimethyl-1-phenyl-3-pyrozoline (C 13 H 16 N 3 ) + ion. Another intense peak at m/z = 589 is due to a (C 31 H 39 N 8 SO 2 +2H) + ion. The different competitive fragmentation pathways of ligand give the peaks at different mass numbers at 28, 29, 43, 60, 77, 88, 108, 131, 174, 282, 388, 390, 467, 544 and 573. The intensity of these peaks reflects the stability and abundance of the ions [13].  NMR spectra NMR data of the ligand are given in Table 2. Its 1 H-NMR spectrum ( Figure 2) displays a triplet at ca. δ 1.136-1.171 ppm (s, 6H, 2H 3 C-C), due to the six protons of two methyl groups attached to the CH 2 groups, two singlets at ca. δ 1.601 ppm (s, 6H, 2H 3 C-C) and ca. δ 1.714 ppm (s, 6H, 2H 3 C-N) due to protons of methyl groups attached to the pyrazoline rings, two multiplets at ca. δ 5.7−5.9 ppm (m, 12H, 6CH 2 ) due to the protons of six methylene groups and at ca. δ 7.1−7.9 ppm (m, 10H, aromatic) due to the protons of two phenyl rings and a broad signal at ca. δ 9.6 ppm (s, br, 2H, 2NH), corresponding to the two protons of two NH groups [14].
The 13 C-NMR spectrum ( Figure 3) displays the signals corresponding to the different non-equivalent carbon atoms at different values of δ as follows: at ca. δ 10.07 ppm (H 3 C-C), 15.87 ppm (H 3 C-H 2 C) and 18.97 ppm (H 3 C-N) corresponding to carbon atoms of methyl groups; at ca. δ 25.87 ppm (H 2 C-S), 27.55 ppm (H 2 C-N) and 97.13 ppm (MeH 2 C-N) due to methylene carbon atoms; at ca. δ 117.73, 119.92, 121.07 and 125.81 ppm due to the aromatic carbon atoms; at ca. δ 143.23 and 151.07 ppm (H 3 C-C and H 3 C-N) due to carbon atoms of pyrazoline rings; at ca. δ 153.97 ppm (C=N) due to carbon atoms azomethine groups and at ca. δ 165.15 ppm (C=O) due to carbon atom of carbonyl groups [15,16].

IR spectra
Selected IR bands of the ligand and its complexes are listed in Table 3. The IR spectrum of the ligand displays bands at 1647, 1621 and 1532 cm −1 , which may be assigned to the ν(C=O) amide I, ν(C=N) (azomethine linkage) stretching vibration and δ NH (NH in-plane-bending) (amide III) vibrations. The band appearing at 768 cm -1 in the spectrum corresponds to the C-S stretching vibration. The C-S group is less polar in comparison to a C=O group and has a considerably weaker bond, so consequently the corresponding band appeared at a lower frequency. The bands corresponding to the C=N stretching, NH in-plane-bending and C-S stretching vibrations show the downward shift upon coordination which indicates that the nitrogen atoms of azomethine and NH groups and sulfur atom of C-S group are coordinated to the metal atom. However, the band corresponding to the C=O group (amide I) remains almost unchanged on complexation, which indicates that the carbonyl group oxygen atom is not involved in coordination. This discussion suggests that the ligand coordinates to metal atom in quinquedentate fashion (NNSNN) [17][18][19][20][21].  The coordination behavior of the ligand is also verified by the appearance of new IR bands in the spectra of complexes in the 384-501 and 313-351 cm −1 range (Table 3). These bands may be assigned to ν(M−N) and ν(M−S) stretching vibrations, respectively. In addition, the IR spectra of complexes also display the bands due to anions. The nitrato complexes show the IR bands in the range 1397−1407 In the sulphato complexes, the two medium intensity bands in the range 951−964 cm −1 (ν 1 ) and 439−448 cm −1 (ν 2 ) and a strong band 1037−1137 cm −1 (ν 3 ) are appeared. The splitting of ν 3 band in to two bands suggests the coordination of SO 4 −2 ion in unidentate manner [22]. Table 3. Selected IR bands of Schiff's base ligand and its complexes. Abbreviations: vs = very strong, s = strong, ms = medium strong, m = medium, mw = medium weak, w = weak, br = broad, sh = sharp

Electronic spectra
The electronic spectra of the complexes were recorded in DMSO solutions. The electronic spectral data of the complexes are given in Table 4  The ligand field parameters like Racah inter-electronic repulsion parameter B, ligand field splitting stabilization energy 10 Dq, covalency factor β and ligand field stabilization energy (LFSE) have been calculated for the Co(II) and Ni(II) complexes. The values of B and Dq of Co(II) complexes were calculated from the transition energy ratio diagram using ν 3 /ν 1 ratio. The value of β for the complexes under study accounts for the covalent nature of the complexes. The evaluated parameters are listed in Table 4.

EPR spectra
The X−band EPR spectra of the Co(II) complexes were recorded at liquid nitrogen temperature in polycrystalline form. The line shaped EPR spectra of Co(II) complexes with g iso = 2.10−2.14 ( Table 5) correspond to the tetragonal symmetry around the Co(II) atoms.
As a consequence of the fast spin-relaxation time of high-spin cobalt(II) ion, the signals are observed only at low temperature. The polycrystalline powder EPR signals for the Co(II) complexes are broad. The spectra are consistent with an S = 3/2 spin state. No hyperfine splitting of the transitions is detected since it is difficult to resolve this splitting in nonmagnetically diluted Co(II) complexes. The line shapes are mostly dominated by the unresolved hyperfine interactions and by a distribution of E/D, where E and D describe the axial and rhombic Zero field splitting (ZFS) constants, respectively. The spread of E/D results in a spread of g-values (g-strain). The dominant broadening effect is realized when the g-strain is converted in <B-strain> with the relation ΔB = -(hν/β)(Δg/g 2 ), where the parameters have their usual definitions. Thus, the largest and smallest g-values of the S = 3/2 spectrum have field widths that differ by an order of magnitude, rationalizing why the high field features of the spectra are so broad [25,26].  The X−band EPR spectra of copper(II) complexes were recorded at room temperature in polycrystalline form. The spectra show only one broad signal at g iso = 2.08 -2.10 ( Figure 4). The spectral studies reveal that the Cu(II) ion in the present complexes is in tetragonal field and shows the D 4h symmetry. The calculated values of g || and g ⊥ for the complexes show the order as g || > g ⊥ > 2.0023 (Table 5), which is consistent with the 2 2 x y d − ground state [27,28]. The odd electron is located in the B 1g antibonding orbital.
The geometric parameter G i.e. the measurement of exchange interaction between the copper centres in the polycrystalline compounds, is calculated by using the expression: The complexes in the present study show the G values greater than 4 (Table 5), which suggest that the interaction between metal centres is negligible [24].
For the copper(II) complexes, the EPR parameters and the d-d transition energies are used to evaluate, the orbital reduction factor k by using the expression: k 2 = k || 2 + 2k ⊥ 2 /3, where k || and k ⊥ are the parallel and perpendicular components of the orbital reduction factor. The low values of k (0.61-0.74) indicate the covalent nature of the complexes (Table 5). On the basis of above discussion, the structures of complexes are given in Figure 5. The data of the antifungal and antibacterial activities of ligand and complexes are given in Tables 6  and 7. The data reveal that the complexes have higher activities than the free ligand ( Figure 6). This enhancement of the activity of ligand on complexation can be explained by Overtone's Concept and Chelation Theory [29]. The theory states that chelation reduces the polarity of the metal atom by the partial sharing of its positive charge with donor groups and possible π-electron delocalization over the whole ring. This results with increasing of the lipophilic character of the complex and favor the permeation of the complex through the lipid layer of cell membrane. The complex blocks the metal binding sites in the enzymes of microorganisms. Consequently, the complex disturbs the metabolism pathways in cell and as a result microorganisms die.

Conclusions
The ligand 3,3'-thiodipropionic acid bis(4-amino-5-ethylimino-2,3-dimethyl-1-phenyl-3pyrazoline, characterized on the basis of elemental analysis, IR, Mass, 1 H-NMR and 13 C-NMR spectral studies, coordinates to Co(II), Ni(II) and Cu(II metal ions in a pentadentate (NNSNN) manner. The value covalency factor (β) and orbital reduction factor (k) suggest the covalent nature of the complexes. The screening of biological activities of ligand and its complexes against the fungi Alternaria brassicae, Aspergillus niger and Fusarium oxysporum and the pathogenic bacteria Xanthomonas compestris and Pseudomonas aeruginosa indicates that the complexes show the enhanced activity in comparison to free ligand. The stoichiometric analyses were carried out on a Carlo-Erba 1106 analyzer. Metal contents were estimated on an AA-640-13 Shimadzu flame atomic absorption spectrophotometer in solution prepared by decomposition of the complex in hot concentrated HNO 3 . The 1 H-NMR spectrum was recorded with a model Bruker Advance DPX-300 spectrometer operating at 300 MHz using EtOD as a solvent and TMS as an internal standard. IR spectra were recorded as KBr pellets and CsI pellets (for chloro complexes) in the region 4,000−200 cm −1 on a FT-IR spectrum BX-II spectrophotometer. Electron spray ionization mass spectrum was recorded on a model Q Star XL LCMS−MS system at source temperature 300°C and voltage with +ve mode 5,500 V and -ve mode 4,500 V. The electronic spectra were recorded on Shimadzu UV mini-1240 spectrophotometer using DMSO as a solvent. EPR spectra were recorded for solids on an E4-EPR spectrometer at room temperature and liquid nitrogen temperature operating at X-band region with 100 KHz modulation frequency, 5 mw microwave power and 1 G modulation amplitude using DPPH as standard. The molar conductance of complexes was measured in DMSO at room temperature on an ELICO (CM 82T) conductivity bridge. The magnetic susceptibility was measured at room temperature on a Gouy balance using CuSO 4 .5H 2 O as callibrant.

Synthesis of the complexes
To a hot solution of Schiff's base ligand (1 mmol) in acetonitrile (15 mL), a hot solution of corresponding metal salt like nitrate, chloride, acetate or sulphate (1 mmol) in acetonitrile (10 mL) was added slowly with constant stirring. The resulting mixture was refluxed for 8−10 h at 70−80°C. On cooling the mixture overnight at 0°C, the colored product which separated out was filtered, washed with acetonitrile and dried under vacuum over P 4 O 10 .

Biological activities
The Disc Diffusion Method and Food Poison Technique [30,31] were employed for screening the antibacterial and antifungal activities, respectively, of the ligand and its complexes. The compounds were screened for their antifungal and antibacterial properties using three fungi -Alternaria brassicae, Aspergillus niger and Fusarium oxysporum -and two bacteria -Xanthomonas compestris and Pseudomonas aeruginosa.
The antibacterial activity was determine with the Disc Diffusion Method. Stock solutions were prepared by dissolving the compounds in DMSO and serial dilutions of the compounds were prepared in sterile distilled water to determine the Minimum Inhibition Concentration (MIC). The nutrient agar medium was poured into Petri plates. A suspension of the tested microorganism (0.5 mL) was spread over the solid nutrient agar plates with the help of a spreader. Fifty μL of the stock solutions was applied on the 10 mm diameter sterile disc. After evaporating the solvent, the discs were placed on the inoculated plates. The Petri plates were sealed with Parafilm ® and first placed at low temperature for two hours to allow the diffusion of a chemical and then incubated at a suitable optimum temperature (29 ± 2°C) for 30-36 hours. The diameter of the inhibition zones was measured in millimeters. DMSO was used as control and streptomycin as a standard drug.
The Food Poison Technique was used to determine the antifungal activity of the compounds. The stock solution of the compound was directly mixed into the PDA (Potato Dextrose Agar) medium at the tested concentration. A disc of 5 mm of test fungal culture of a specific age growing on solid medium was then cut with a sterile cork borer and was placed at the center of the solid PDA plate with the help of inoculums' needle. The plates were sealed with Parafilm ® and incubated at 29 ± 2°C for 7 days. DMSO was used as a control and Captan as a standard fungicide. The inhibition of the fungal growth expressed in percentage terms was determined from the growth in the test plate relative to the respective control plate as given below: Inhibition (%) = (C-T) 100 / C where C = diameter of fungal growth in the control plate and T = diameter of fungal growth in the test plate.