Original article revista.iq.unesp.br | Vol. 48 | n. 3 | 2023 | 36 Eclética Química, vol. 48, n. 3, 2023, 36-53 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.3.2023.p36-53 Novel organophosphorus Schiff base ligands: Synthesis, characterization, ligational aspects, XRD and biological activity studies Yasmin Mos’ad Jamil1+ , Fathi Mohammed Al-Azab1 , Nedhal Abdulmawla Al-Selwi1 1. Sana’a University , Faculty of Science, Sana’a, Yemen. +Corresponding author: Yasmin Mos’ad Jamil, Phone: +967 771952842, Email address: y.jamil@su.edu.ye ARTICLE INFO Article history: Received: November 03, 2022 Accepted: May 05, 2023 Published: July 01, 2023 Keywords: 1. antimicrobial activity 2. antioxidant activity 3. nanoparticles 4. X-ray diffraction Section Editors: Assis Vicente Benedetti ABSTRACT: Six complexes have been synthesized from Cu(II), Ni(II), and Co(II) with new bidentate N2 donor Schiff base ligand (2-methoxybenzalidene-1-phenylsemicarbazide L1) and tridentate N2O donor organophosphorus Schiff base ligand (2-methoxybenzalidenediphenylphosphate-1- phenylsemicarbazide L2). Both ligands were synthesized and characterized by metal analysis, infrared (IR), ultraviolet visible (UV-Vis), and nuclear magnetic resonance (NMR) spectral studies. The chemical structures of the synthesized complexes were characterized using their metal analysis, magnetic susceptibility, molar conductance, IR, and UV-Vis spectra. According to molar ratio studies, the complexes have the composition of ML2 for L1 and ML for L2. The X-ray diffraction (XRD) studies showed that the particle size of ligands and L1 complexes were in nano-range. The ligands and their metal complexes have been screened for their antioxidant, antibacterial and antifungal activity. https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.3.2023.p36-53 mailto:y.jamil@su.edu.ye https://orcid.org/0000-0003-1585-3770 https://orcid.org/0000-0002-5578-2981 mailto:f.alazab@su.edu.ye https://orcid.org/0000-0003-1575-3673 mailto:n.salwi@su.edu.ye https://ror.org/04hcvaf32 Original article revista.iq.unesp.br 37 Eclética Química, vol. 48, n. 3, 2023, 36-53 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.3.2023.p36-53 1. Introduction Schiff base compounds are commonly used as ligands in inorganic chemistry to create stable complexes with different transition metal ions (Abu-Dief et al., 2021). They are prepared by a condensation reaction of aromatic/aliphatic aldehydes and amines to form an azomethine group (CH=N) (Abu-Yamin et al., 2022). In previous research, many biological activities of Schiff base compounds have been documented including antibacterial, antifungal, antimalarial, antiproliferative, analgesic, anti-inflammatory, antiviral, antipyretic, and anticancer properties (Camellia et al., 2022; Ceramella et al., 2022; Ibeji et al., 2022; Jamil et al., 2023; Kaczmarek et al., 2018; Kafi-Ahmadi and Marjani, 2019; Song et al., 2020). A survey of the literature reveals a work devoted to the synthesis, characterization, and biological activities of many metal complexes of Schiff base from 2-methoxybenzaldyde (Camellia et al., 2022; Ibeji et al., 2022; Sani and Iliyasu, 2018; Yusof et al., 2015) In addition, organophosphorus compounds with transition metal ions have been the subject of several studies since complexes of such compounds exhibit biological activity (Abd El-Wahab and El-Sarrag, 2004; Brzezińska- Błaszczyk et al., 1996; El-khazandar, 1997; Ochocki et al., 1995). Hence, in the present study, a new Schiff base of 2-methoxybenzaldyde and 1-phenylsemicarbazide, its organophosphorus Schiff base, and their complexes with Cu2+, Ni2+, and Co2+ were prepared and characterized. The biological activities were investigated for these compounds, such as their antibacterial and antioxidant. 2. Experimental 2.1 Materials The chemicals, 1-phenylsemicarbazide (Riedel-de Haën), 2-methoxybenzaldyde (Aldrich), diphenyl chlorophosphate (Riedel-de Haën), Triethyl amine (Riedel-de Haën), bipyridine (Aldrich), ferric chloride (Aldrich), ascorbic acid (BDH), sodium acetate trihydrate (BDH), glacial acetic acid (BDH) and metals chloride hydrate (CuCl2.2H2O, NiCl2.6H2O, and CoCl2.6H2O) were purchased from BDH. The solvents used were of spectroscopic grade. 2.2 Physical measurements Elemental analysis for C, H, and N was performed with a Vario EL Fab. CHN Nr, at Central Laboratory, Faculty of Science, Cairo University, Giza, Egypt. Conductivity measurements were done with a Jenway conductivity meter model 4510 in dimethylformamide (DMF). The magnetic moments were determined by Gouy’s method using a magnetic susceptibility balance from Johnson Metthey and Sherwood model. Ultraviolet visible (UV-vis) spectra were measured with (Specord200, Analytilk Jena, Germany) in the range of 200–900 nm, at Sana’a University. Fourier-transform infrared (FT-IR) spectra were recorded in transmission mode by an (FT/IR-140, Jasco, Japan) spectrophotometer in the wavenumber range of 4000– 400 cm–1 (KBr was used as a matrix material for pellets). 1H and 13C NMR spectra for ligands were performed in d6-DMSO solvent using tetramethylsilane as an internal standard, at Cairo University, Giza, Egypt. All melting points reported for the compounds are measured in glass capillary tubes in Celsius degrees. Chloride was determined gravimetrically by silver nitrate (Vogel, 1961). The amount of coordinated and uncoordinated water molecules was determined gravimetrically using the weight loss method (Vogel, 1961). The X-ray diffraction (XRD) patterns were obtained using XD-2 (Shimadzu ED-720) powder X-ray diffractometer at a voltage of 35 kV and a current of 20 mA using CuK(α) radiation in the range of 5° ˂ 2θ ˂ 70° at 1° min–1 scanning rate and a wavelength 1.54056 Å at Yemen Geological Survey and Mineral Resources Board. 2.3 Synthesis of ligands 2.3.1 Synthesis of Schiff base (2- methoxybenzalidene-1-phenylsemicarbazide L1) The reaction mixture, including an ethanolic solution of amine (1-phenylsemicarbazide) (7.55 g, 0.05 mol) with an ethanolic solution of aldehyde (2- methoxybenzaldyde) (6.80 g, 0.05 mol) was refluxed on hot reflux under constant stirring for 2–3 h, then the product was separated on cooling, it was filtered, washed several times with ethanol and ether, then the obtained crystals product was recrystallized by ethanol to give the corresponding Schiff base. The reaction is given in Fig. 1. https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.3.2023.p36-53 Original article revista.iq.unesp.br 38 Eclética Química, vol. 48, n. 3, 2023, 36-53 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.3.2023.p36-53 Figure 1. Preparation of Schiff base 2-methoxybenzalidene-1-phenylsemicarbazide L1. 2.3.2 Synthesis of organophosphorus Schiff base (2- methoxybenzalidenediphenylphosphate-1- phenylsemicarbazide L2) The organophosphorus Schiff base L2 was prepared by condensation between a solution of Schiff base (4.03 g, 0.015 mol) in dry benzene (50 mL) with a solution of diphenyl chlorophosphate (3.69 g, 0.015 mol) in dry benzene (50 mL) in 1:1 molar ratio in presence of triethyl amine (Fig. 2). After complete addition, the reaction mixture was heated under reflux for 2 h. The formed solid (triethyl amine hydrochloride) was filtered and the product was obtained after evaporation in a water bath. Figure 2. Preparation of organophosphorus Schiff base 2-methoxybenzalidenediphenylphosphate-1- phenylsemicarbazide L2. + (C2H5)3N-HCl https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.3.2023.p36-53 Original article revista.iq.unesp.br 39 Eclética Química, vol. 48, n. 3, 2023, 36-53 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.3.2023.p36-53 2.4 Synthesis of metal complexes 2.4.1 Synthesis of Schiff base metal complexes A hot ethanolic solution of metal chloride was added dropwise to an ethanolic solution of Schiff base (2 L:1 mol L–1) molar ratio. The mixture was refluxed on a hot plate with stirring for 2–3 h. After evaporation of the solvent, the solid product was washed several times with ethanol and dried over anhydrous CaCl2. 2.4.2 Synthesis of organophosphorus Schiff base metal complexes A solution of metal chloride in 20 mL absolute ethanol was added dropwise to the solution of organophosphorus Schiff base in warm absolute ethanol (1 L: 1 mol L–1). After the addition, the mixture was heated under hot reflux for three hours. The solution was evaporated, and the solid complexes were collected using ether. 2.5 Determination of the stoichiometry of the formed complexes of molecular structure (Molar ratio method) The molar ratio method was described by the Molar ratio method (Yoe and Jones, 1944). The concentrations of metal ions (Cu2+, Ni2+, and Co2+) were kept constant at (1×10–3 mol L–1) in methanol and the concentration of ligands (L1 and L2) was regularly varied in methanol. The absorbance of the prepared solutions was measured at the constant wavelength (λmax). The absorbance values were plotted versus the molar ratio [ligand] / [metal ion]. The intersections of the obtained straight lines indicate the molar ratio of the stable complexes. 2.6 Crystallinity and particle size from XRD The percentage of crystallinity, XC (%) was calculated based on the integrated peak areas of the principal peaks (Shah et al., 2006). The crystallinity of the complexes is calculated relative to the crystallinity of the ligands as a ratio (Eq. 1): 𝑋𝐶 (%) = 𝐴𝑐𝑜𝑚𝑝𝑙𝑒𝑥 𝐴𝑙𝑖𝑔𝑎𝑛𝑑 × 100 (1) where Acomplex and Aligand are the areas under the principal peaks of the complex and ligand sample, respectively. X-ray diffraction was also used to determine the average particle size (D) which was estimated by the Scherrer equation (Akhtar et al., 2015; Patterson, 1939) (Eq. 2): 𝐷 = 𝐾 𝜆 𝛽 𝑐𝑜𝑠𝜃 (2) where K is Scherrer constant and equals 0.94, λ is the X- ray wavelength of Cu-Kα radiations (1.5405 Å), β is full width at half maximum (FWHM) and θ is Bragg diffraction angle in degrees. 2.7 Antioxidant activity The total antioxidant activity of the compounds has been studied using ferric-bipyridine reducing capacity (FBRC) (Al-Azab et al., 2023). It was taken (1 mL, 10–2 mol L–1 FeCl3.6H2O) to 10 mL volumetric flask different volumes of the standard antioxidant ascorbic acid (0.1 g L–1) were added (0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2 mL). This was followed by 2.0 mL 0.3 mol L–1 acetate buffer (pH 4) and 1.0 mL bipyridine (6.4 × 10–3 mol L–1). The volume was completed to the mark with deionized water. After 10 min of incubation at room temperature, the absorbance was recorded against a blank at 535 nm. The absorbance values were plotted against the concentration of the various antioxidant solutions (Fig. 3). Similarly, 0.02 mL of (0.1 mg mL–1 methanol) of each tested compound was reacted with 1 mL, 10–2 mol L–1 FeCl3.6H2O solution, (6.4 × 10–3 mol L–1) bipyridine and 2.0 mL 0.3 mol L–1 acetate buffer (pH 4). This mixture was diluted to 10 mL with deionized water for the antioxidant assay. The fixed reaction time and fixed state measurement method were used to find out the antioxidant activity of these compounds in methanol as a solvent. All spectrophotometric measurements were measured using UV-vis spectrophotometer (Specord200, Analytikjena, Germany) at Sana’a University. Figure 3. Calibration curve of ascorbic acid with Fe-Bp complex. https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.3.2023.p36-53 Original article revista.iq.unesp.br 40 Eclética Química, vol. 48, n. 3, 2023, 36-53 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.3.2023.p36-53 2.8 Antimicrobial activity The synthesis compounds were exanimated for their antimicrobial activity in the Laboratory of Microbiology at Sana’a University. They Germinated four types of bacteria (Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa) and one fungus (Candida albicans) on nutrient agar and Sabouraud agar solid media respectively. Using the filter paper disc method (Ericsson et al., 1960), it was prepared one concentration from each compound (1000 µg mL–1) in DMSO. Then added (100 µL) from each prepared compound on a filter paper (Whatman No. l filter paper, 5 mm diameter) which contained the bacteria with agar solid media, all the Petri dishes were put in an incubation period at 37 °C for 24 h. Gentamicin 120 μg mL–1 was used as a reference substance for bacteria and Mycostatin 30 µg mL–1 for fungi. The results were registered by calculating the diameter of the inhibition zone (mm). 3. Results and discussion The Schiff base ligand (L1) was prepared by the condensation of 1-phenylsemicarbazide with 2- methoxybenzaldyde in the molar ratio 1:1. The organophosphorus ligand (L2) was prepared by the condensation of diphenylchlorophosphate with the prepared Schiff base (L1) in the molar ratio 1:1. The ligand ratio of (L1) with Cu(II), Ni(II), and Co(II) complexes was found to be 1:2, and the ligand ratio of (L2) with Cu(II), Ni(II), and Co(II) complexes were found to be 1:1. The molar conductance data of the complexes of L1 are 18.44, 20.31, and 21.09 Ω–1 cm2 mol–1 in DMF (1 × 10–3 mol L–1). These results show that these complexes are non-electrolytes (Aderoju and Sherifah, 2015; Geary, 1971), so the complexes of L1 are insoluble in water and common organic solvents but soluble in polar solvents such as DMF and DMSO. The non-electrolytic nature of these complexes suggests that the chloride anions of the three salts have coordinated with the L1 chelate of metal complexes. The reaction of the synthesized L1 complexes with silver nitrate have been not given a white precipitate, and after digestion with nitric acid, the silver nitrate test gave a positive result. Furthermore, the results of molar conductance of L2 complexes (73.75, 84.93, and 77.79) show that these complexes are 1:1 electrolytes in DMF (1 × 10–3 mol L–1) (Geary, 1971; Shakdofa et al., 2017). The complexes of L2 are slightly soluble in water and soluble in common organic solvents. In the case of L2 complexes, the precipitation was observed upon the addition of silver nitrate to the solution of the complexes (Vogel, 1961). L1 and L2 and their complexes were characterized by elemental analyses, FT-IR, 1HNMR, and electronic spectra which are compliant with the proposed structures as shown in Fig. 4a and b. Table 1 lists some physical and analytical data on the ligands and their complexes. Figure 4. Proposed structure of (a) L1 complexes; (b) L2 complexes. https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.3.2023.p36-53 Original article revista.iq.unesp.br 41 Eclética Química, vol. 48, n. 3, 2023, 36-53 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.3.2023.p36-53 Table 1. Some physical properties and elemental analysis of the ligands and their complexes. Compound Color (Yield) M.P . (°C) Ʌm (Ω–1 cm2 mol–1) F.Wt (g mol–1) Elemental analysis calculated% (found) C H N P M C12H11N3O2(L1) White (70%) 174 - 269.30 66.90 (67.04) 5.61 (5.78) 15.60 (15.27) - - C23H10N3O5P(L2) Brown (75%) 153 - 479.36 65.15 (64.96) 2.94 (3.19) 8.76 (8.90) 7.05 (6.89) - [Cu(L1)2Cl2]∙2H2O Green (70%) 240 18.44 709.08 50.81 (51.30) 4.83 (4.88) 11.85 (12.08) - 8.96 (9.00) [Ni(L1)2Cl2]∙2H2O Green (50%) 250 20.31 704.22 51.16 (50.99) 4.87 (5.02) 11.93 (12.20) - 8.33 (8.75) [Co(L1)2Cl2]∙2H2O Dark blue (57%) 220 21.09 704.46 51.14 (51.36) 4.86 (4.33) 11.92 (11.68) - 8.36 (8.13) [Cu(L2)Cl (H2O)2].Cl Dark red (17%) 130 73.75 649.84 48.05 (47.85) 2.79 (2.86) 12.93 (13.18) 4.76 (4.55) 9.77 (9.53) [Ni(L2)Cl (H2O)2].Cl Red (19%) 108 84.93 644.98 48.41 (48.47) 2.82 (3.00) 13.02 (12.79) 4.80 (4.70) 9.09 (9.00) [Co(L2)Cl (H2O)2].Cl Red (23%) 112 77.79 645.22 48.39 (48.01) 2.81 (2.57) 13.02 (13.24) 4.79 (4.99) 9.13 (8.98) 3.1 Nuclear magnetic resonance spectral studies 3.1.1 1HNMR spectra of the ligands The 1HNMR spectra of the ligands L1 and L2 (Figs. S1 and S2 see Supplementary Material) exhibited signals which are consistent with the proposed structure. The peaks at 6.019, 6.713, and 5.997, 6.692 ppm are assignable to the protons of the NH groups in the ligands L1 and L2 respectively (El-Tabl et al., 2007; Shakdofa et al., 2017). Azomethine proton appeared as a singlet signal at 8.656 ppm (s, 1H) in L1 (Çakır et al., 2003) and this signal disappeared in the L2 spectrum due to a replacement reaction on this group with eliminated HCl and forming a P-C bond. The signals at 3.908 and 3.050 were assigned to (s, 3H, OCH3) L1 and L2, respectively (Çakır et al., 2003). The peaks appearing in the range 6.742–7.831 and 6.717–7.842 ppm may correspond to protons of the aromatic hydrogen of L1 and L2 respectively (Galil et al., 2015). 3.1.2 13CNMR Spectra of the ligands The 13CNMR spectra (Figs. S3 and S4 see Supplementary Material) showed singlet signals at 161.688 and 160.421 ppm that attributed to carbonyl carbon C=O of the ligands L1 and L2 respectively (Carrasco et al., 2020; Galil et al., 2015). Also, the spectra exhibited two peaks at δ 149.517 and δ 149.623 ppm that were able to be appointed to azomethine carbon C=N of the ligands L1 and L2, respectively (Shakdofa et al., 2017). The peaks between 111.241–130.917 and 112.189–130.143 ppm is attributed to aromatic carbon in L1 and L2, respectively (Galil et al., 2015). The carbon of the methoxy group appeared at 55.534 and 45.381 ppm in ligands L1 and L2, respectively (Yusof et al., 2015). 3.2 IR spectra of the ligands and their complexes Important IR bands of the ligands and their complexes are given in Table 2 and Fig. 5a. The presence of IR bands at 1,655 and 1,602 cm–1 of ligand L1 assigned to υ(C=O) and υ(C=N) respectively (Hossain et al., 2019). The strong bands at 3,334 and 3,397 cm–1 are assignable and attributed to υ(NH) of this ligand (Nakamoto, 1998). The bands showed at 3,057, 2,958, and 1,267 cm–1 belong to aromatic υ(C-H), aliphatic υ(C-H), and υ(-C-N) stretching mode of vibrations, respectively (Jassem et al., 2013). The band at 2,853 cm–1 in its spectrum can be appointed to υ(-O-CH3) (Mohapatra et al., 2011). The IR spectra of the complexes (Fig. 5a) are characterized by the appearance of broad bands at 3,424, 3,346, and 3,324 cm–1 due to the water molecules in L1(Cu), L1(Ni), and L1(Co) complexes, respectively (González-García et al., 2016). In the spectra of all the complexes, no change in the frequency of (C=O) but the 1,602 cm–1 band was shifted to a lower frequency indicating the involvement of the N atom of the azomethine group in coordination (Table 2). The bands of υ(NH, NH) disappeared in complexes due to the broad bands of water. New vibrations at 400–600 cm–1 that are not present in the free ligand are attributed to the existence of υ(M-N) and υ(M-NH) (Yassin et al., 2020). Based on the observations, it has been suggested that the ligand L1 acts as bidentate. Although the presence evidence of υ(M-Cl) could not be brought in the IR data due to instrumental limitation, the insolubility of L1 complexes in water and their non- https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.3.2023.p36-53 Original article revista.iq.unesp.br 42 Eclética Química, vol. 48, n. 3, 2023, 36-53 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.3.2023.p36-53 electrolytic nature evinced that 2Cl– coordinated with metal in L1 complexes . In the ligand L2 spectrum (Fig. 5b), the bands observed at 1,654 and 1,591 cm–1 corresponds to υ(C=O) and υ(C=N), respectively (Hossain et al., 2019). The presence of υ(P=O) and υ(P-O-C) is indicated by the appearance of bands at 1,188 and 1,072 cm–1, respectively (El- khazandar, 1997). The coordinated water molecules with metal ions in the complexes were proved by the bands 3,347, 3,407, and 3,312 (Aderoju and Sherifah, 2015). The IR spectra of the metal complexes of ligand L2 (Fig. 5b) proposed that this ligand tridentate with the phosphoryl-oxygen, amine- nitrogen, and azomethine-nitrogen due to the shift in the position of υ(P=O), υ(-NH) and υ(C=N) (Table 2). Table 2. The main IR absorption bands of the ligands and their complexes. Compound υ(H2O) υ(NH, NH) υ(-O-CH3) υ(C=N) υ(C=O) υ(P=O) υ(P-O-C) υ(M-N) υ(M-NH) C16H14N4O(L1) - 3398 3335 2853 1602 1655 - - - - C27H13N4O4P (L2) - 3450 3347 2878 1591 1654 1188 1072 - - [Cu(L1)2Cl2]∙2H2O 3424 3424 2850 1580 1651 - - 527 428 [Ni(L1)2Cl2]∙2H2O 3346 3346 2849 1571 1649 - - 558 414 [Co(L1)2Cl2]∙2H2O 3324 3324 2855 1577 1653 - - 574 424 [Cu(L2)Cl (H2O)2].Cl 3421 3467 3452 2866 1560 1650 1165 1072 516 410 [Ni(L2)Cl (H2O)2].Cl 3407 3407 2850 1617 1655 1160 1067 520 458 [Co(L2)Cl (H2O)2].Cl 3312 3365 3312 2846 1603 1651 1161 1068 517 467 Figure 5. Infrared spectra. (a) L1 and its complexes; (b) L2 and its complexes. 3.3 Electronic spectra and magnetic measurements The absorption spectra of the ligands L1 and L2 and their metal complexes were recorded in DMSO in the range of 200–900 nm (Table 3). The ligands L1 and L2 exhibited two absorption bands (Figs. 6a and b) at 37,037.03, 35,714.28 and 33,333.33, 32,258.06 cm–1 that have been allocated for the π-π* and n-π* transitions, respectively (Mohammed et al., 2019). In the metal complexes, the transition of n-π* has been seen but they are shifted to another range. The shift in the transitions has been accounted for by the complexation between ligands with the metal ions. The Cu complexes of L1 and L2 exhibited single broad absorption peaks at 13,513.51 and 13,333.33 cm–1 (Figs. 6a and b), respectively, attributable to the 2Eg→2T2g transition, indicating a distorted octahedral structure (Al- Maydama et al., 2008; Gup and Kirkan, 2005). The electronic spectra of the L1(Ni) and L2(Ni) complexes (Figs. 5a and b) showed absorption bands at 18,181.81, 15,151.51 and 17,543.85, 15,151.51 cm–1, respectively, attributable to the 3A2g → 3T1g(P) and 3A2g → 3T1g(F) transitions, which is compatible with this complex having the octahedral structure (Gup and Kirkan, 2005). https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.3.2023.p36-53 Original article revista.iq.unesp.br 43 Eclética Química, vol. 48, n. 3, 2023, 36-53 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.3.2023.p36-53 The electronic spectra of the L1 and L2 complexes with Co (Figs. 6a and b) exhibited characteristic bands at 20,833.33, 15,873.01, 13,888.88 and 21,739.13, 20,000, 17,543.85 cm–1 assigned to 4T1g→ 4T1g(P), 4T1g→ 4A2g and 4T1g→4T2g(F) transitions. These bands are associated with the octahedral structures (Gup and Kirkan, 2005). This information with the effective magnetic moment (µeff) data (Table 3) of all complexes helped to support the suggested octahedral geometry (Al-Hakimi et al., 2011; Fouda et al., 2008). Table 3. Electronic spectral and magnetic moment data of the ligands and their complexes. Compound µeff (B.M.) (π→π*), transition (n→π*) transition d-d transition band (cm–1) Assignments Supposed structure C16H14N4O(L1) - 37,037.03 33,333.33 - - - C27H13N4O4P(L2) - 35,714.28 32,258.06 - - - [Cu(L1)2Cl2].2H2O 1.88 34,482.75 30,303.03 13,513.51 2Eg→2T2g Distorted Octahedral [Ni(L1)2Cl2]∙2H2O 2.75 35,714.28 32,258.06 18,181.81 15,151.51 3A2g → 3T1g(P) 3A2g → 3T1g(F) Octahedral [Co(L1)2Cl2]∙2H2O 4.19 35,714.28 32,258.06 20,833.33 15,873.01 13,888.88 4T1g→ 4T1g(P) 4T1g→ 4A2g 4T1g→4T2g(F) Octahedral [Cu(L2)Cl (H2O)2].Cl 1.61 37,037.03 33,333.33 13,333.33 2Eg→2T2g Distorted Octahedral [Ni(L2)Cl (H2O)2].Cl 2.39 35,714.28 31,250.00 17,543.85 15,151.51 3A2g → 3T1g(P) 3A2g → 3T1g(F) Octahedral [Co(L2)Cl (H2O)2].Cl 4.77 35,714.28 31,250.00 21,739.13 20,000.00 17,543.85 4T1g→ 4T1g(P) 4T1g→ 4A2g 4T1g→4T2g(F) Octahedral Figure 6. UV-Vis spectra. (a) L1 ligand and its complexes; (b) L2 ligand and its complexes. 3.4 Molar ratio (stoichiometry) of the studied complexes Investigation of the molecular structure of the complexes formed between the metal ions of (Cu2+, Ni2+, and Co2+) with ligands in L1 and L2 in methanol using molar ratio (Figs. 7 and 8) revealed the formation of (1:2) (M:L) complexes for L1 and (1:1) (M:L) complexes for L2 under investigation. The results are depicted in Table 4. https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.3.2023.p36-53 Original article revista.iq.unesp.br 44 Eclética Química, vol. 48, n. 3, 2023, 36-53 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.3.2023.p36-53 Figure 7. Molar ratio plot for the complexes between metal ions of (a) Cu, (b) Ni and (c) Co with L1 in methanol. Figure 8. Molar ratio plot for the complexes between metal ions of (a) Cu, (b) Ni and (c) Co with L2 in methanol. Table 4. Molar ratios for determination of stoichiometry of the L1 and L2complexes in methanol. L1 [L]/[M] Absorbance of complexes Cu(II) λ = 330 Ni(II) λ = 310 Co(II) λ = 310 0.5:1 0.121 0.067 0.043 1:1 0.170 0.099 0.077 1.5:1 0.195 0.143 0.117 2:1 0.230 0.181 0.159 2.5:1 0.232 0.185 0.174 3:1 0.222 0.187 0.184 3.5:1 0.218 0.182 0.190 4:1 0.220 0.184 0.196 L2 [L]/[M] Absorbance of complexes Cu(II) λ = 300 Ni(II) λ = 320 Co(II) λ = 320 0.25:1 0.290 0.200 0.313 0.5:1 0.369 0.326 0.399 0.75:1 0.450 0.480 0.483 1:1 0.495 0.531 0.511 1.25:1 0.492 0.555 0.530 1.5:1 0.499 0.558 0.541 1.75:1 0.501 0.563 0.538 2:1 0.509 0.571 0.544 3.5 X-ray diffraction The XRD patterns of the L1 and its complexes are shown in Fig. S5a–d (see Supplementary Material). The decrease of the XRD peak intensities of L1 complexes can be attributed to the reduction in crystallinities due to the complexation. This is in good agreement with the lowering in relative crystallinity calculated for these complexes (the crystallinity calculation in Table 5 is based on the integrated area of principal peaks of the complex to the L1 ligand obtaining a relative https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.3.2023.p36-53 Original article revista.iq.unesp.br 45 Eclética Química, vol. 48, n. 3, 2023, 36-53 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.3.2023.p36-53 crystallinity) (Shah et al., 2006). The particle size calculated for L1 and its complexes (2.074–5.552 nm) in the range of nanoparticle size—the nanoparticle size reported in the literature ranges between 1–100 nm (Boverhof et al., 2015). The particle size of L2 ligand was found within nanometric range (10.555 nm), nevertheless its complexes appeared as an amorphous character (Fig. S5e–h, see Supplementary Material). From this change, it is expected to improved properties of L2 complexes as compared with L2 ligand. Table 5. Data of the principal values of intensity of the ligands and L1 complexes from XRD spectra. Compound 2θ β D (nm) Mean D XC (%) L1 6.780 13.720 18.880 19.960 23.920 28.220 29.900 0.340 0.419 0.459 0.405 0.379 0.295 0.337 4.266 3.480 3.154 3.630 3.905 5.061 4.447 3.991 100 L2 12.799 16.740 25.441 31.040 0.120 0.153 0.121 0.180 12.142 9.464 12.268 8.349 10.555 100 [Cu(L1)2Cl2]∙2H2O 6.719 13.460 18.599 20.919 31.659 0.352 0.562 0.229 0.237 0.178 4.120 2.576 6.407 6.213 8.455 5.552 10.392 [Ni(L1)2Cl2]∙2H2O 16.279 32.761 33.579 0.439 0.240 0.232 3.332 6.033 6.241 5.202 18.506 [Co(L1)2Cl2]∙2H2O 14.600 35.520 57.199 57.579 0.603 0.762 0.745 0.868 2.421 1.995 2.213 1.668 2.074 3.488 3.6 Antioxidant activity The ligands and L2 complexes were investigated using FBRC technique to determine the antioxidant activity (Al- Azab et al., 2023). Fe(II)-bipyridine complex produced by the reaction of Fe(III) with antioxidant followed by bipyridine exhibited maximum absorption at 535 nm (Fig. 9). Total antioxidant activity is based on the redox reaction between compounds and Fe(III) at room temperature. The initial antioxidant concentration is indicated by the concentration of the oxidizing Fe(III). Figure 9. Stoichiometry outline of FBRC method. https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.3.2023.p36-53 Original article revista.iq.unesp.br 46 Eclética Química, vol. 48, n. 3, 2023, 36-53 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.3.2023.p36-53 The results in Table 6 showed that these compounds were found to possess high potent antioxidant activity due presence of a combination of donor sites such as amide oxygen and imine nitrogen (Kostova and Saso, 2013). Table 6. FBRC values express antioxidant activity as mg L–1 for prepared compounds to 1 mg L–1 of ascorbic acid FBRC (mg L–1) Sample 0.0248 ± 0.0001 L1 0.0281 ± 0.0004 L2 0.0201 ± 0.0002 [Cu(L2)Cl (H2O)2].Cl 0.0183 ± 0.0002 [Ni(L2)Cl (H2O)2].Cl [Ni(L2)Cl∙(H2O)2].Cl 0.0187 ± 0.0005 [Co(L2)Cl (H2O)2].Cl [Co(L2)Cl∙(H2O)2].Cl 3.7 Antibacterial and antifungal screening for L1, L2 and their complexes The synthetic ligands and their metal complexes havbeen examined for their antibacterial and antifungal activities using the disk diffusion method (Ericsson et al., 1960) against four types of Bacteria (Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa) and one fungus (Candida albicans) on nutrient agar and Sabouraud dextrose agar (SDA) solid media, respectively. The inhibition zone diameter of the compounds is shown in Table 7 and Fig. 10a–d. From these results, it was noted that: 1- No inhibition zone was observed for ligands and their metal complexes against the fungus (Candida albicans); 2- The metal complexes are more potent bactericides than the ligands; 3- The order of antibacterial activity for the compounds with Bacillus subtilis was L1(Cu) = L2(Ni) > L1(Ni) > L2(Cu) > L2 > L1 > L1(Co) = L2(Co), with Staphylococcus aureus was L2(Ni) > L1(Cu) > L1(Co) > L2(Co) > L2 > L1 > L1(Ni) = L2(Cu), with Escherichia Coli was L1(Co) > L1(Cu) > L2(Cu) > L2(Co) > L2 > L1 > L1(Ni) = L2(Ni) and with Pseudomonas aeruginosa was L1(Co) = L2(Co) > L1(Cu) > L2(Ni) > L2 > L1 > L1(Ni) = L2(Cu). In general, the complexes showed good antibacterial compared to the free ligands. Based on the chelation theory (Franklin and Snow, 1989), the increased inhibition activity of the metal complexes may be explained on the basis that their structures mainly possess C=N bonds. Besides, the coordination decreases the polarity of the metal ion due to the partial sharing of its positive charge with donor groups and possible π-electron delocalization inside the chelate ring-shaped during coordination that makes the complexes more lipophilic. This increased lipophilicity enhances the penetration of the metal complexes into lipid membranes, blocks the metal binding sites in the enzymes, and limits the further development of the organisms. Table 7. Biological activities of the ligands and their metal complexes against bacteria and fungus (zone of inhibition in mm). Compound (1000/mL–1) Bacteria Fungi gram-positive gram-negative Candida albicans Bacillus subtilis Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa L1 5 3 4 3 0 L2 6 5 6 5 0 [Cu(L1)2Cl2].2H2O 11 8 9 10 0 [Ni(L1)2Cl2].2H2O 10 0 0 0 0 [Co(L1)2Cl2].2H2O 0 7 10 15 0 [Cu(L2)Cl (H2O)2].Cl [Cu(L2)Cl∙(H2O)2].Cl 7 0 8 0 0 [Ni(L2)Cl (H2O)2].Cl [Ni(L2)Cl∙(H2O)2].Cl 11 10 0 7 0 [Co(L2)Cl (H2O)2].Cl [Co(L2)Cl∙(H2O)2].Cl 0 6 7 15 0 Gentamicin (120 μg/ml) 29 25 23 28 - Mycostatin (30 μg/ml) - - - - 18 https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.3.2023.p36-53 Original article revista.iq.unesp.br 47 Eclética Química, vol. 48, n. 3, 2023, 36-53 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.3.2023.p36-53 Figure 10. Antibacterial activity of the ligands and their metal complexes against gram-positive bacterium (a) Bacillus subtilis; (b) Staphylococcus aureus; (c) Pseudomonas aeruginosa; and gram-negative bacterium (d) Escherichia coli. 4. Conclusions In summary, the synthesis and physicochemical analysis were done for new ligands L1 and L2 with their complexes. In complexes, the ligand L1 acts as neutral- bidentate and L2 acts as neutral-tridentate around the metallic ion. Electronic properties and magnetic susceptibility suggested octahedral geometry for all complexes. According to XRD, the ligands and L1 complexes were in the nanoscale (2.074–10.555 nm). The ligands and L2 complexes gave high antioxidant activity using FBRC technique. All compounds have been screened for their antibacterial and antifungal activity. Authors’ contribution Conceptualization: Jamil, Y. M.; Al-Azab, F. M. Data curation: Al-Selwi, N. A. Formal Analysis: Jamil, Y. M.; Al-Selwi, N. A. Funding acquisition: Not applicable. Investigation: Jamil, Y. M.; Al-Azab, F. M.; Al-Selwi, N. A. Methodology: Jamil, Y. M.; Al-Azab, F. M.; Al-Selwi, N. A. Project administration: Jamil, Y. M.; Al-Azab, F. M. Resources: Jamil, Y. M.; Al-Azab, F. M.; Al-Selwi, N. A. Software: Al-Selwi N. A. Supervision: Jamil, Y. M.; Al-Azab, F. M. Validation: Jamil, Y. M.; Al-Azab, F. M. Visualization: Jamil, Y. M.; Al-Azab, F. M.; Al-Selwi, N. A. Writing – original draft: Al-Selwi, N. A. Writing – review & editing: Jamil, Y. M. https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.3.2023.p36-53 Original article revista.iq.unesp.br 48 Eclética Química, vol. 48, n. 3, 2023, 36-53 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.3.2023.p36-53 Data availability statement All data sets were generated or analyzed in the current study Funding Not applicable. Acknowledgments Not applicable. References Abd El-Wahab, Z. H.; El-Sarrag, M. R. Derivatives of phosphate Schiff base transition metal complexes: synthesis, studies and biological activity. Spectrochimica Acta Part A. 2004, 60, 271–277. https://doi.org/10.1016/S1386- 1425(03)00216-6 Abu-Dief, A. M.; El-Metwaly, N. M.; Alzahrani, S. O.; Bawazeer, A. 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Figure S2. 1HNMR spectrum of L2. https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.3.2023.p36-53 Original article revista.iq.unesp.br 52 Eclética Química, vol. 48, n. 3, 2023, 36-53 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.3.2023.p36-53 Figure S3. 13CNMR spectrum of L1. Figure S4. 13CNMR spectrum of L2. https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.3.2023.p36-53 Original article revista.iq.unesp.br 53 Eclética Química, vol. 48, n. 3, 2023, 36-53 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.3.2023.p36-53 Figure S5. XRD pattern of (a) L1; (b) L1(Cu); (c) L1(Ni); (d) L1(Co); (e) L2; (f) L2(Cu); (g) L2(Ni); (h) L2(Co); https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.3.2023.p36-53