Synergism antibacterial activity for novel synthesized Schiff base ligands and semi-thiosemicarbazones with ß-diketones and 4-aminoanti-pyrine

. Schiff base ligands were synthesised in this work, the first is (Z)-2-((Z)-3-((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)imino)-1,3-diphenylpropylidene) hydrazine-1-carboxamide, the second is (Z)-2-((1E,5Z,6E)-5-((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)imino)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-dien-3-ylidene)hydrazine-1-carbothioamide. The Schiff base ligands both were synthesised using the same method (the single pot reaction), using EtOH as the reaction medium and GAA as the catalyst in fixed temperature at 70 0C and reflux for 6 hrs. The starting materials of HL1 were semicarbazide,4-aminoantipyrine and dibenzoyl methane, while the starting materials of HL2 were TSC, 4-AAP and Curcumin. The ligands proved to be bi-dentate ligands that coordinate from the azomethane groups. FT-IR, U.V-Visible, 1H and 13C-NMR, molar conductivity, and magnetic susceptibility were used to identify all the compounds produced. The metal ions used in preparing the complexes in this work were Co(II), Cu(II) and Cr(III). Schiff base and its complexes were evaluated for antibacterial activity against four bacterial strains of Gram-negative (Escherichia coli and Pseudomonas aeruginosa), Gram-positive (Staphylococcus aureus and Bacillus Subtilis) and two types of fungi (Candida albicans and Rhizopus Sporium), the results were positive for all compounds tested.


Materials and methods
During the past few decades, the Schiff base metal complexes have been under attention due to their role in coordination chemistry, easy preparation, and the diversity of their structure. Schiff base is considered a bioactive compound; for example, these compounds behave as anti-cancer agents. The free ligands have a weaker biological effect when compared to their metal complexes 1,2 . Antipyrines played an essential role in drug development fields; also, they helped in monitoring and observing patients with chronic liver illness (Hepatitis B virus (HBC), hepatitis C virus (HCV) and alcohol-related disease) 3,4 . 4-aminoantipyrine condensation with suitable compounds such as ketones, aldehydes, thiosemicarbazones, carbazones or compounds with a similar structure results in flexible ligands which can coordinate with various metal ions 5 . β-diketones based Schiff base ligands have an important role in developing coordination chemistry; also, they play an important role in biochemical systems 6 . In this study, the β-diketones used are Curcumin and dibenzoyl methane; both are known for their biological activity and behavior as antihyperglycemic, anti-inflammatory, anti-cancer, insecticidal, anti-Alzheimer's, gastroprotective, antifungal, antibacterial agents 7 . Also significant are semicarbazones and thiosemicarbazones; they are made up of the scaffolds NH 2 -CO-NH-NH-and NH 2 -CS-NH-NH-respectively.
Thiosemicarbazones' and semicarbazones' capacity to coordinate with transition and main group metallic cations is due to the extended delocalization of electron density over their skeleton, which is increased by substitution at the N (4)-position 8,9 . The goal of this study was to synthesize and characterize Schiff bases with two azomethine groups derived from diketones (Curcumin and dibenzoylmethane), 4-amino antipyrine, semicarbazide and thiosemicarbazide, in order to test the ligand and its complexes' efficient biological activity against various types of bacteria and fungi.
The device used to measure the melting point is (MPA160 Digi Melt); a device type (jew wary, model 4070) was used to measure the molar conductivity of the metal complexes. The FT-IR spectroscopy was evaluated using (Shimadzu FT-IR 8400S) spectrophotometer. The UV-Vis spectra of the ligands and their transition metal complexes were recorded in DMSO at 0.001M using (the UV 160A Shimadzu apparatus). Balance Johnson Mattey device was used to measure the magnetic susceptibility of the metal complexes, and NMR spectroscopy was recorded using device type (Inova 500). Finally, The Mass spectrum was recorded using (Sciex Esi) spectrometer.

Synthesis of Schiff base ligand HL 1
For the preparation of HL 1 a mixture of 4-aminoantipyrine, semicarbazide and dibenzoylmethane was refluxed for six hrs. with stirring at a fixed temperature of 70 0 C. after that, the solution was left to evaporate for a day the resulted ligand was a yellow-colored, needle-shaped crystal and odorless.
The structure was confirmed by the data collected from elemental analysis, FT-IR, 1 H-NMR and 13 C-NMR; the synthesis of the ligand and it complexes is illustrated in Figure 1

Synthesis of Schiff base ligand HL 2
Curcumin (0.368 gm, 1 mmol) was dissolved in 20 ml ethanol, then adding four drops of GAA as a reaction catalyst, 0.204 grams (1mmol) 4-aminoantipyrine dissolved in 20 mL ethanol and 0.09 gm (1mmol) thiosemicarbazide dissolved in 20 mL ethanol were all progressively added at the same time. This mixture was refluxed by stirring at 70 °C for 6 hours; TLC was used to monitor the reaction. A dark brown precipitate was isolated and recrystallized from hot ethanol. The structure was confirmed by the data collected from elemental analysis, FT-IR, 1 H NMR and 13 C NMR. The synthesis of the ligand is illustrated in Figure 2.
was added progressively to the mixture. The residues that formed were separated, rinsed with cold ethanol multiple times, and then recrystallized from hot ethanol. The proposed geometry of the recrystallized structure is shown in Figure 2.

Results
All complexes were generally synthesized by reacting the metal salts with the Schiff base in a 1:2 mole ratio (metal salts: Schiff base). All of the complexes were colored solid compounds. Table 1 shows that the analytical findings corresponded with the proposed formula of a 1:2 metal: Schiff base molar ratio. The complexes synthesized using HL 1 were soluble in dimethylformamide and dimethyl sulfoxide, and it has partial solubility in chloroform, ethanol, methanol and water. In contrast, the complexes of HL 2 were soluble in water, DMSO and DMF and had a partial solubility in ethanol and methanol. Some of the physical and chemical characteristics are shown in Table 1. Table 1. Some of the physical and chemical characteristics of the synthesized compounds

FT-IR spectra
The spectrum of Schiff base ligands showed a band at 1624 cm-1 that referred to the imine group in HL 1 and 1627 cm -1 for HL 2 ligand, also a band appeared at 1670 cm -1 that assigned to the carbonyl group (C=O), the thione band was assigned at 817 cm -1 for HL 2 . The NH indicating band is set for HL 1 at 3441 cm -1 and 3417 cm -1 for HL 2 , and the OH group band of [HL 2 ] appears at 3525 cm -1 10-13 . Otherwise, the spectrum of the complexes showed shifting in the imine band, and the shifting range was 1600-1624cm -1 ; the carbonyl group and the thione group did not show any noticeable shifting, which means the two groups did not participate in the coordination with the metal ions. Also, new bands for stretching frequencies of M-N have been allocated at 460-420 cm -1 14 . Another band was observed at a range of 243-260 cm -1, indicating the formation of the M-Cl coordination bond 11 . The data are listed in Table 2, and the FT-IR spectrum is shown in Figures 3 and Figure 4.

Electronic spectra
The ligands' absorption spectra revealed peaks at 270-290 nm due to the π →π* and n→π* peaks around 340 nm; Figure 3 shows the ranges of the ligands. The complexes revealed intra-ligand fields at 267-289 and 340-347 nm; this slight shift is considered to approve the ligand→metal coordination, charge transfer peaks at 363 nm [13][14][15] , and d-d transition peaks as follows; The electronic spectra of ligand's HL 1 metal complexes confirmed an octahedral geometry of the synthesized complexes where the peaks Table 2. FT-IR selected data of the ligands and their metal complexes Table 3. Electronic spectral data of the ligands and their complexes exhibited was like the following; Co(II) complex exhibited a peak at 782nm that is related to the electronic transition 4 T 1 g→ 4 T 2 g, Cr(III) complex showed a peak at 779 nm which refers to the electronic changes 4 A 2g , The Cu(II) complex d-d transitions display two peaks at the d-d region at 778nm and 760 nm which are related to 2 B 1g → 2 A 2g 13-17 . The HL 2 complexes spectral data were as follows; Co(II) complex displayed peaks at the d-d region at 700 nm related to the electronic transition 4 , Synergism antibacterial activity for novel synthesized Schiff base ligands and semi-thiosemicarbazones with ß-diketones and 4-aminoantipyrine the complex's magnetic moment equals 3.88 BM, the data suggested an octahedral geometry. The Cr(III) complex spectra exhibited a peak at 803 nm related to 4 A 2g → 4 T 2g; the electronic spectrum and the magnetic moment of the complex, which equals 2.91 BM confirm an octahedral geometry structure. According to the electronic spectrum data collected, an octahedral geometry was suggested for the Cu(II) complex, where peaks were detected at 626 nm and 682 nm that are referring to 2 Eg→ 2 T 2 g and 2 B 1 g→ 2 A 1 g, and the magnetic moment of the complex is 2.15 BM [13][14][15][16][17] The molar conductivity of the ligands and its complexes measured in (S.cm2 mol-1) were as following: HL1 (

Mass spectroscopy
In the Schiff base ligands, mass spectrum Figure 4 the HL1 spectrum has a well-defined molecular ion peak at m/z = 467 amu, which correlates (M + 1) with the Schiff base ligand's molecular formula (C27H26N6O2). The spectra of HL1 ligand reveal a succession of peaks at m/z 409, 332, 290,199, 184. 105, 70 and 56, amu, which correspond to the fragments. The strength of these peaks indicates the pieces' stabilities. At the same time, the Mass of HL2 exhibited peaks at m/z+=627 amu attributed to

The 1 H and 13 C -NMR spectra of the ligands
According to a literature review, NMR spectroscopy is required to determine the structure of various compounds; the NMR spectra were recorded in DMSO-d 6 (dimethyl sulfoxide) using TMS (tetramethyl silane) as standard.  [19][20][21] . The spectra are shown in Figure 6 and Figure 7.   Figure 9.  Schiff compounds were synthesized were characterized using multiple techniques, and were approved to have better biological activity than the antibiotic used as a standard and the physical activity The Schiff base ligand was synthesized by reacting dibenzoyl methane 1mmol with 4-aminantipyrine 1mmol and semicarbazide 1mmol (HL 1 ). The imine group atoms (N) performed it as bidentate coordinates. The Schiff base ligand (HL 2 ) was synthesized by reacting curcumin 1mmol with 4-aminantipyrine 1mmol and thiosemicarbazide 1mmol. It turned out to be a bidentate ligand coordinate from the imine group. All complexes were produced based on available data such as FT-IR, UV-Visible, 13C,1H-NMR, molar conductivity, and magnetic susceptibility to adopt an octahedral geometric form. When tested for biological effectiveness against four types of bacteria and two types of fungi, all compounds gave a positive result; the synthesized compounds showed adequate inhibition in various ranges against the bacterium spices and the effect on the fungi spices.
Author Contributions: The experimental part of the research, measurements and preparation of the research draft were done by Reham H. Najem. The work was supervised and corrected by professor Hasan A. Hasan.