Fig. 1 FTIR spectra of (a) azo Schiff-base ligand and (b) Cu (II) complex.

Mass spectra

The mass spectra of synthesized azo Schiff-base ligand and its Cu(II) complex were recorded at room temperature. The obtained molecular ion peaks confirmed the proposed formulae for the synthesized compounds. The mass spectrum of the ligand shows the molecular ion peak at m/z 788.8 for the compound C48H40N10O2, confirming the proposed formula for the synthesized compound. Also The mass spectrum of the Cu(II) complex exhibited the molecular ion peak at m/z 922.5 for the molecular formula Cu(C48H40N10O2)Cl2, which was consistent with the molecular weight of the Cu(II) complex and in good agreement with their formula as expressed from micro analytical data. The mass spectral data fragmentation of the ligand and Cu(II)-complex are shown in Scheme 3, 4, and Fig. 2.

Scheme 3 Mass spectrum fragmentation of azo Schiff-base ligand.

Scheme 4 Mass spectrum fragmentation of Cu- complex.

Fig. 2 Mass spectrum of (a) azo Schiff-base ligand and (b) Cu (II) complex.

1H-NMR spectra                                                                                            

The 1H-NMR spectrum of the ligand showed the following signals: Phenyl multiples at 6.8-8.7 ppm, =C-CH3 at 2.7 ppm, -N-CH3 at 3.3 ppm, and -OH at 10.6 ppm [6, 22]. This peak was noted in the spectra of complexes indicated that the –OH proton did not contribute to the complexity. There was no appreciable change in all other signals in the complexes, as shown in Fig. 3.

Fig. 3 1H-NMR spectrum of (a) azo Schiff-base ligand and (b) Zn (II) complex.

13C-NMR spectra

The 13C-NMR spectra of the azo Schiff-base ligand were measured at room temperature in DMSO-d6 as a solvent. The spectra of the ligand are shown in Fig. 4. The 13C-NMR spectrum of the ligand displayed characteristic signals at 11.3, 39.3,89 and 155.3 ppm due to >C–CH3, >N-CH3, >C-N=N- antipyrine ring and >C-OH naphthol ring of the Schiff base-azo ligand, respectively [8, 23]. The peak at δ = 163.3 ppm was due to azomethine carbon of the ligand [8]. Moreover, the spectrum of the ligand showed peaks in the region of 108.2, 109.9, 111.09, 122.1, 126.6, 127.6, 129.01 and 140.9 ppm due to aromatic carbon atoms.

Fig. 4 13C-NMR spectrum of the azo Schiff-base ligand.

Electronic spectra

Electronic spectra provided the most detailed information about the electronic structure. The UV-Vis spectrum of the azo Schiff-base ligand exhibited two charge transfer (CT) bands at 316 nm 31645/cm and 412 nm 24271/cm, which was attributed to π–π* and n– π* transitions within the azo Schiff-base ligand. In the spectrum of the complexes, the CT band at 316 nm remained as such, in agreement with the π–π* transition of the azo Schiff-base ligand. The band observed at 412 nm in the spectrum of the free ligand (HL) was red-shifted to 449-549 nm in the complexes due to ligand to metal charge transfer (LMCT) transition [24], suggesting an octahedral geometry around metal(II) in the complexes [25]. The electronic spectra of the ligand and the Cu(II) complex are shown in Fig. 5. The electronic transitions, magnetic properties and conductivity values of the ligand and its complexes are listed in Table 4.

Fig. 5 Electronic spectrum of (a) ligand and (b) Cu(II) complex.

Magnetic measurements

The Fe(III) complex showed magnetic value at magnetic effect (μeff) = 5.4 B.M, which was consistent with an octahedral geometry [26]. The Co(II) complex had a magnetic moment of 4.91 BM, which was in agreement with the reported value for octahedral Co(II) complexes [27]. The present Ni(II) complex showed a magnetic moment value of 3.1 within the range of 2.9-3.3 BM [25, 28], suggesting an octahedral environment. The Cu(II) complex showed a magnetic moment value of 1.82 BM, higher than the spin-only value 1.73 BM as expected for one unpaired electron, which was monomeric and consistent with a distorted octahedral geometry [29]. The Zn(II), Cd(II), Hg(II), Au(III) were diamagnetic and according to the empirical formulae of complexes. An octahedral geometry was proposed [30], except the Au(III)  complex suggested a square planar geometry around the central metal ion [31]. Based on the above results, we could deduce the probable structures of the complexes as shown in Fig. 6.

Table 4 Electronic spectra, conductivity and magnetic moment of complexes

Fig. 6 Proposed structural formulae of the complexes.

Anticancer activity

Evaluation of newly synthesized complexes in cancer therapy were studied. In this study the antitumor activities of the synthesized Au(III) complex were tested against colon cancer (disease: Dukes' type B, colorectal adenocarcinoma) LS174 cell line. The results showed that the highest inhibitory effect was reported for complex, giving the IC50 value as of 465. The cell cytotoxic effect of the tested Au(III) complex was calculated. The optical density was measured with the micro plate reader to determine the number of viable cells, and the percentage of viability was calculated as

[1-(ODt/ODc)] × 100%, (1)

where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells. The relation between surviving cells and drug concentration was plotted to get the survival curve of each tumor cell line after treatment with the specified complex. The 50% inhibitory concentration (IC50), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose response curve for each concentration using Graph pad Prism 6. The anticancer activity of the synthesized Au(III)complex was determined against an colon cancer LS-174 cell line using different concentrations, evaluated and compared with the standard drug doxorubicin (DOX). The gold complex exhibited good results compared with the standard DOX. The in-vitro inhibitory activity of the tested gold complex against colorectal carcinoma LS-174 cells was expressed as IC50 value (μg/mL). The results are shown in Table 5, 6, Fig. 7 and 8. The cytotoxicity of Au(III)  complex on normal cells, i.e. rat embryo fibroblast cell line (REF) was studied using methyl thiazolyl tetrazolium (MTT) assay. The results indicated the effect of toxicity was very low. The results are shown in Fig. 9. The positive charge of the metal increased the acidity of coordinated ligand that bore protons, leading to stronger hydrogen bonds which enhanced the biological activity [32]. Moreover, Gaetke and Chow had reported that, metal has been suggested to facilitate oxidated tissue injury through a free-radical mediated pathway analogous to the Fenton reaction [33]. By applying the electron spin resonance (ESR trapping technique, evidence for metal-mediated hydroxyl radical formation in vivo has been obtained [34]. Reactive oxygen species (ROS) was produced through a Fenton-type reaction as follows:

LM(II) + H2O2 → LM(I) + .OOH + H+, (2) and

LM(I) + H2O2 → LM(II) + .OH + OH-, (3)

where L refers to the organic ligand.

Also, metal could act as a double-edged sword by inducing DNA damage and also by inhibiting their repair [35]. The OH radicals reacted with DNA sugars and bases, and the most significant and well-characterized of the OH reactions was hydrogen atom abstraction from the C4 on the deoxyribo to yield sugar radicals with subsequent β-elimination (Scheme 5). By this mechanism strand break occurred as well as the release of the free bases. Another form of attack on the DNA bases was by solvated electrons, probably via a similar reaction to those discussed below for the direct effects of radiation on DNA [35].

Scheme 5 Mechanism of Fenton reaction.

Table 5 The percentage of inhibition and Log x for the Au(III) complex

Note: Conc. = concentration; y = statistical value; IC50 = 50% inhibitory concentration.

Table 6 The percentage of inhibition and Log x for the DOX drug

Note: Conc. = concentration; y = statistical value; IC50 = 50% inhibitory concentration.

Fig. 7 Curve with plotting of lC50 of (a) doxorubicin drug and (b) Au(III) complex.

Fig.  8 (a) Untreated cell with MTT; (b) untreated cells; (c) and (d) treated cells with gold complex in conc. = 1000 and 2000 µg/mL.

Fig. 9 Determination of the cytotoxic effect on REF.  

Conductivity measurement

Most of the chelate complexes prepared in this work showed the conductivity values ranged between 73-80 S cm2/mol in DMSO at room temperature, which were very low values [25] compared with the high values of conductivity of both Au(III) and Fe(III) complexes [31]. This could support the electrolytic nature of the metal complexes. According to these results, the structural formulae of these ligand and complexes may be proposed in Fig. 6.

Conclusions

In this paper we explored the synthesis and coordination chemistry of some monomeric complexes obtained from the reaction of the tetra dentate ligand (L) with some metal ions as shown in Fig. 6. The mode of bonding and the overall structure of the complexes were determined through physico-chemical and spectroscopic methods. Complex formation study via molar ratio was investigated and results were consistent with those found in the solid complexes with a ratio of M : L = 1 : 1. The biological screening effect of the gold complex was tested against human colon cancer cell line LS-174. The results showed the highest inhibitory effect for the complex.

Conflict of Interests

The authors declare that no competing interest exists.

References

1. A.A. Al-Hamad, S.A. Shaker, Synthesis, characterization, structural studies and biological activity of a new Schiff base-azo ligand and its complexation with selected metal ions. Oriental Journal of Chemistry, 2011, 27(3): 835-845.
2. I.N. Witwit, H.A. Mohamad, Synthesis and spectral study of new tetradentate Azo-methine ligand as adrevitave of 4, 5-diphenyl Imidazole and its complexes with some of metal Ions. Journal of the Faculty of Basic Education, 2016, 95: 1-12.
3. L.A. Mohamad, R.A. Albaki, H. Mohseen, et al., Synthesis, characterization, structural studies and Biology activity of a new macrocyclic Schiff base ligand and it's complexation with selected metal Ions. Journal of scientific Research in Pharmacy, 2013, 2(3): 7-13.
4. P. Pattan Ayak, D. Patra, J. Pratihar, et al., Osmium and cobalt complexes in corporating facially coordinated N,N,O donor azo-imine ligands: Redox and catalytic. Properties. J. chem. Sci, 2013, 125(1): 51-62.
5. D.M. Jamil1, A.K. Al‑Okbi, S.B. Al‑Baghdadi, et al., Experimental and theoretical studies of Schiff bases as corrosion inhibitors. Chemistry Central Journal, 2018, 12(7): 1-7
6. C. Anitha, S. Sumathi, P. Tharmaraj, et al., A new azo-schiff base: Synthesis, chaactrization biological activity and theoretical studies of its complexes. Applie Organo Metallic Chem, 2018, 32: 1 -15.
7. M.A. Karam, H.D. Salman, Synthesis, characterization and antimicrobial studies of transition metal complexes with azo ligand derivative from 4-amino antipyrine. Mesopo. Environ. J., 2017, Special Issue C: 83-91
8. N. Srividhya, A. Xavier, Antimicrobial activities of Schiff base metal complexes of 4- amino antipyrine. International Journal of Research in Engineering and Advanced Technology, 2017, 4(6): 28-35.
9. N.G. El-Kholy, Synthesis, spectroscopic characterization, antimicrobial, antumor properties of new 4-amino- 2,3 dimethyl -1-pheny-3-pyrazolone-5-one (antipyrine) Schiff bases and its transition metal complexes. Journal of American Science, 2017, 13(2): 132-145.
10. G. Turan-Zitouni, M. Sivaci, Synthesis of some trizolyl-antipyrin derivatives and investigation of analgesicactivity. Eur. J. Med. Chem., 2001, 36(8): 685-689.
11. A.N. Lutsevich, K.I. Bender, and O.V. Reshetko, The relationship between antipyrine kinetics, the seromucoid content and the xanthine oxidase activity in the plasma of rats with acute and chronic in flammation. Ekspklin Farmakol, 1995, 59: 51-55.
12. M.S. Karan, D. Arish, and J. Johnson, Synthesis, characterization and biological studies on some metal complexes with Schiff base ligand containing pyrazolone moity. J. of Saudi Chem. Soc., 2016, 20: 591-598.
13. M.A. Metwally, M.A. Gouda, A.N. Harmal, et al., Synthesis, antitumor, cytotoxic and antioxidant evaluation of some new pyrazolo triazines attached to antipyrine moiety. Eur. J. Med. Chem., 2012, 56: 254-262.
14. A. Accardo, L. Alo, and M. Aurilio, Receptor binding peptides for target-selective delivery of nano particles encapsulated drugs. Int. J. Nano Medicine, 2014: 1537-1557.
15. S.R. Jumar, Anticancer activity of eco-friendly gold nano particles against lung and liver cancer cells, Journal of Genetic Engine erring and Biotechnology, 2016, 14: 195-202.
16. K.J. Al-Adilee, Preparation and characterization of some transition metal complexes with novel Azo-schiff base ligand derived from 2(E)-(1H-benzo) [d] imidazole-2-ylydiazenyl)-5- (E)-benzylidene imino) phenol (BIADPA). Pharmaceutical, Biological and Chemical Sciences, 2015, 6(5): 1297-1285.
17. M.B. Ummathur, P. Sayunderi, and K. Kuutty, Schiff bases of 3-[2-(193-Benzothiazol-2-yl] hydrazinyliene] pentane-2,4-dione with aliphatic diamines and their metal. Journal of the Argentine Chemical Society, 2009, 97(2): 31-39.
18. D. Çanakçi, O.Y.  Saribiyik, and S. Serin, Synthesis, structural characterization of Co(II), Ni(II) and Cu(II) complexes of azo dye ligand derived from dihydroxy naphthalene. International Journal of scientific Research and Inovative Technology, 2014, 1(2): 91-107
19. N. Raman, S. Thalamuthu, J. Dhaveethuraja, et al., DNA cleavage and antimicrobial activity studies on transition metal (II) complexes of 4-amino-antipyrine derivative. J. Chil. Chem. Soc, 2008, 53(1).
20. B.K. Al-Salami, R.A. Gata, and K.A. Asker, Synthesis spectral, thermal stability and bacterial activity of schiff base derived from selective amino acid and their complexes. Pelagia Research Libray, 2017, 8(3): 4-12.
21. S.D. Dakore, V. Kamble, and P. Bisal, Synthesis and characterization of biologically active N2O2 type of Novel Schiff base metal complexes derived from 4-aminoanty pyrine using TEA. J. Chem Studies, 2017, 5(1): 110-113.
22. M.Y. Nassar, I.S. Ahmed, H.A. Dessouki, et al., Synthesis and characterization of some Schiff base complexes derived from 2, 5-dihydroxyacetophenone with transition metal ions and their biological activity. Environ. Sci., 2018, 5: 60-71
23. L.M. L. Ogboji, O.Z. Esezobor, Synthesis of 4,4'-dihydroxyazo benzene and 4'dihydroxyphenyl azo-2-naphthol from diazotised aniline and anthocyanins from diazotised aniline and anthocyanins from delonix regia flower. Chemical Science International Journal, 2017, 20(1): 1-5
24. Z.J. Mohamed, A.H. Al-Khafagy, Characterization and Biological study of heterocyclic azo-schiff base compound and some of its metal complexes. International Journal of Current Research, 2013, 5(12): 3705-3710.
25. M.S. Mohamad, Some transition metal complexes with new Schiff base ligand hexa dentate. Acta Chimica Pharm Indica, 2013, 3(2): 140-148.
26. M.K.P. Shree, S. Vats, Schiff base and Transition metal complexes. J. Biol. Chem. Sci., 2016, 3(2): 265-300.
27. S.H. Kadhim, I.Q. Abd-Alla, and T.J. Hashim, Synthesis and characteris study of Co(II), Ni(II), and Cu(II) complexes of new Schiff base derived from 4-amino antipyrine. Inte. J. Chem. Sci., 2017, 15(1): 107.
28. R.M. El-Ferjani, M. Ahmad, F.W. Harum, et al., Synthesis, characterization and antibacterial activity of Schiff base derived from 4-Dimethyl amino benzaldehyde with some amino acids and 4-amino antipyrine toward Cu(II), (Ni(II), Co(II), Cd (II) and Mn (II) ions. Journal of Applied Chemistry, 2017, 10(6): 6-13.
29. E. Yousif, A. Majed, and K. Al-Summarrae, Metal complexes of Schiff base: preparation, characterization and antibacterial activity. Arabian Journal of Chemistry, 2013: 2006-2012.
30. M. Suresh, V. Prakash, Preparation and characterization of Cr(II), Mn(II), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) chelates of Schiff bases derived from vanillin and 4- amino antipyrine. Inter. J. Physical Sci., 2010, 5(4): 2203-2211.
31. P. Deshmukh, P.K. Soni, A. Kankoriya, et al., 4-Amino antipyrine: A significant tool for the synthesis of biologically active Schiff base and metal complexes. Int. J. Pharm. Sci, Rev., 2015, 34(1): 162-170.
32. B. Bauer-Siebenlist, F. Meyer, E. Farkas, et al., Effect of Zn...Zn separation on the hydrolytic activity of model dizinc phosphodiesterases. J. Chem. Eur., 2005, 11: 4349-4360.
33. L.M. Gaetke, Copper toxicity, oxidative stress, and antioxidant nutrients. J. Toxicology, 2003, 189: 147-163.
34. D.E. Nikles, M.J. Powers, Copper(II) complexes with tetradentate bis(pyridyl)-dithioether and bis(pyridyl)-diamine ligands. Effect of thio ether donors on the electronic absorption spectra, redox behavior, and EPR parameters of copper(II) complexes, Inorganic Chem., 1983, 22: 3210-3217.
35. C.A. Rouzer, Some transition metal complexes with new Schiff base ligand hexa dentate. Acta Chimica J. Chem. Res. Toxicol., 2010, 23: 1517-1518.

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