Synthesis, characterization, and biological studies of some biometal complexes

Metal complexes Cu[C13H8O4N]22, Ni[Cl3H8O4N]23, and Co[C13H8O4N]24 of bioinorganic relevance have been synthesized with the Schiff base ligand 2-furylglyoxal–anthranilic acid (FGAA) [C13H9O4N] 1. All the complexes are well characterized by various spectral and physical methods. The antimicrobial activity of the complexes has been studied against some of the pathogenic bacteria and fungi. Results indicate that complexes have higher antimicrobial activity than the free ligand. This would suggest that chelation reduces considerably the polarity of the metal ions in the complexes which in turn increases the hydrophobic character of the chelate and thus enables permeation, through the lipid layer of microorganisms. All the complexes were assessed for their anticancer studies against a panel of selected cancer cells HOP62 and BT474 respectively. Results showed that the complexes are promising chemotherapeutic alternatives in the search of anticancer agents. The fluorescence quenching phenomenon is observed in the Schiff base metal complexes. The octahedral transition metal complexes 2, 3, and 4 have been obtained by treatment of ligand 2-furylglyoxal-anthranilic acid (FGAA) 1 with metal acetate. Complexes under investigations have shown antimicrobial, potential anticancer, and the DNA binding studies.


Background
The chemistry of transition metal complexes has received considerable attention largely due to their catalytic and bioinorganic relevance. Such complexes are also important due to their potential biological activities such as antibacterial, antifungal, antimalarial, and antitumor [1][2][3][4]. Medicinal inorganic chemistry is comparatively a new discipline which developed after the serendipitous discovery of the antitumor activity of cisplatin [5][6][7]. The clinical success of this platinum complex has stimulated considerable interest in the search for new metal complexes as modern therapeutics, diagnostic, and radiopharmaceutical agents. Copper, nickel, and cobalt complexes are used in the treatment of many diseases including cancer and as potential hypoxia-activated prodrugs [8][9][10][11][12][13][14]. Coordination compounds which form coordinate bonds via the sulfur, oxygen, and nitrogen donor atoms are well known and have a long history. The interest in preparation of new metal complexes gained the tendency of studying the interactions of metal complexes with DNA for their applications in biotechnology and medicine.
Deoxyribonucleic acid (DNA) is the primary target molecule for most anticancer and antiviral therapies according to cell biologists. Investigation on the interaction of DNA with small molecules is important in the design of new type of pharmaceutical molecule. Schiff base constitutes an important class of nitrogen donor ligands and occupy a prominent position among the recent achievement in the field of coordination chemistry. The azomethine which is the functional group of Schiff base is aided in forming a stable complex. The chemistry of Schiff base metal complexes is exploited in industries, technologies, and in medicinal fields. The present investigations deal with the synthesis, characterization, antimicrobial, anticancer, and DNA cleavage studies of Cu(II), Ni(II), and Co(II) metal complexes containing Schiff base ligand 2-furylglyoxal-anthranilic acid (FGAA).

Methods
All reagents used were of analytical grade and used as purchased commercially; however, the solvents were purified by the standard procedure [15]. The ligand 2furylglyoxal-anthranilic acid (FGAA) was prepared by the reported procedure [16][17][18][19][20]. C, H, and N were analyzed on Carlo-Erba microanalyzer. Metal contents were estimated by standard procedure [21]. FTIR was recorded on Thermo Nicolet Avater 370. Electronic spectra on Shimadzu UV-160A spectrophotometer. The conductance measurements were carried out on a metal CM-180 Eliodigital conductivity meter. Magnetic studies were done by a Guoy balance using Hg [Co (SCN) 4 ] as the calibrant. 1 H and 13 C NMR spectra in dimethyl sulfoxide (DMSO) were recorded on a Brucker WH 300 (200 MHz) and Varian Gemini (200 MHz) spectrometers using tetramethylsilane (TMS) as an internal reference.
The in vitro antimicrobial screening effects of the investigated compounds were tested against the bacterial species: Escherichia coli, (E. coli) and Klbsiella pneumoniae (K. pneumoniae), and fungal species: Aspergillus niger (A. niger) and Candida albicans (C. albicans) by using Kirby Bauer Disk diffusion method [22][23][24]. Chloramphenicol and nystatin were used as the standard antibacterial and antifungal agents. The tested compounds were dissolved in DMF solution (which has no inhibition activity) and solution soaked in filter paper disk of 5 mm diameter and 1 mm thickness. The disks were incubated 24 h for bacterial and 72 h for fungal species at 37°C. The minimum inhibitory concentration (MIC) value of the compounds was determined by the serial dilution method [25][26][27].
The in vitro cancer studies of all the compounds were assessed for their anti-proliferation test against a panel of selected human cancer cell lines such as HOP62 (lung) and BT 474 (breast) by using SRB (sulforhodamine B) assay [28][29][30][31][32][33][34][35][36][37] concentration of drug used 10, 20, 40, and 80 μg/mL ADR (adrimycin) was used as a positive control which controls cells with definite structure and clear cell wall without degeneration. Each drug was assayed inducing 50% growth inhibition (GI 50 ), total growth inhibition (TGI), and 50% cytotoxicity (LC 50 ) after a 48 h incubation period were calculated by linear interpolation from the observed data points. Fluorescence measurements were recorded on an F-7000 FL spectrophotometer at room temperature.

Results
All the metal complexes were colored, non-hygroscopic in nature, and stable at room temp. They were insoluble in common organic solvents but soluble in DMF and DMSO. The results of the elemental analysis are in good agreement with the calculated values. The molar conductance value indicates their non-electrolytic nature. Physical and analytical data of complexes are summarized in Table 1.
On the basis of analytical and spectral data, octahedral geometry has been assigned to the complexes. The results of antimicrobial activity and anticancer studies indicate metal complexes are much more active as compared to ligand fragments. Fluorescence quenching phenomena are observed in its metal complexes by fluorescence studies.

IR spectral studies
Infrared spectra of free ligand, a sharp band [38][39][40] appeared at 1615-1590 cm -1 ascribed to the stretching vibrations of azomethine group and was shifted to lower frequency region after complexation suggesting thereby the participation of imine nitrogen. A strong band appeared at 1735-1690 cm -1 in the IR spectra of ligand (FGAA) which is due to the presence of stretching vibration of carbonyl group coordination through this carbonyl oxygen to the central metal ion is confirmed by a negative shift in this frequency in the spectra of corresponding metal complexes. IR spectra of ligand displays a bond of medium intensity in the region of 3550-3490 cm -1 due to the -OH stretching vibration of free -CO 2 H group. Coordination of ligand as a consequence of deprotonation of -CO 2 H group is evident by the disappearance of the above band in the IR spectra of respective complexes [41,42]. Furthermore, the asymmetrical and symmetrical vibrations of COOgroup appeared at 1560-1535 cm -1 and 1340-1325 cm -1 Δν (as-s) value 220-210 cm -1 further indicate the coordination through unidentate carboxylate group. Some new bands appeared in the IR spectra of metal complexes at 550-530 cm -1 , 450-430 cm -1 , and 335-325 cm -1 are probably due to the formation of M-O, M-N, and M-S bonds respectively which further give additional evidences in favor of the coordination of metals  through azomethine nitrogen, carbonyl oxygen, and carboxylate group.

Electronic spectral and magnetic studies
Divalent copper having a d 9 configuration give rise to a 2D free ion term which split into a regular octahedral environment into a lower doublet 2 E g and an upper triplet 2 T 2g levels. In electronic spectra [43][44][45][46] of a true octahedral system, only one band due to 2 E g → 2 T 2g transitions is expected but true octahedral structures are not common. Therefore, instead of a one broad band due to 2 E g → 2 T 2g transition. These transitions from the ground state 2 B 1g → 2 A 1g → 2 B 2g and 2 E g are expected as a consequence of John-Tellers configuration stability. The 2 E g orbitals separate so that one goes up as much as the other goes down.
The T 2g orbitals separate in such a way that the doubly degenerate pair goes down only half as far as the single orbital goes up therefore in case of Cu (II); there is no net energy change for T 2g electrons since four are stabilized while two are destabilized due to which Cu(II) complex shows distortion in an octahedral geometry. The electronic spectra of Cu(II) complex displays three spectral bands in the region 10635, 14850, 16345 cm -1 which are in good agreement with the distorted geometry of complex under investigation. This geometry is further supported by the magnetic moment value 1.98 B.M. of the complex. In the Ni(II) complex, three bands in the range 10750, 1665, 25650 cm -1 corresponding to the transition 3 A 2g → 3 T 2g → 3 T 1g and 3 T 1g (P) are observed which clearly indicate the octahedral geometry. The theoretical value of ν 2 /ν 1 for octahedral Ni(II) complex is found 1.55. The observed value lies 1.60 which is in conformity with the distorted octahedral geometry of the ligand around central Ni(II) ion lowering the ratio of ν 2 /ν 1 may be attributed due to configuration interaction between T 1g (P) and T 1g (F) excited state. The octahedral geometry is further supported by their magnetic value 3.14 B.M. In octahedrally surrounded Co(II) ions, three bands in the region 8000, 15616, 18175 cm -1 are expected which may be assigned to 4 T 1g to 4 T 2g (F) (ν 1 ), 4 A 2g (F) (ν 2 ), and 4 T 1g (P) (ν 3 ) transitions respectively. The 4 A 2g

NMR spectral studies
In the 1 H NMR spectra of free Schiff base ligand, the signals were appeared in the range of 7.15-7.20 ppm due to (HC=N) proton [47]. However, in the spectra of Schiff base metal complexes of Cu(II), Ni(II), and Co(II), the signals were observed in the downfield regions of 8.0-9.0 ppm supporting the coordination of iminonitrogen atom to Cu (II)/Ni (II)/Co (II) [48] while the free ligand NMR spectra has a characteristic NMR signal for carboxyl group proton in the 10.5-12.5 ppm range, the disappearance of this signal in the 1 H NMR spectra of metal complexes indicating the involvement of carboxylate ion oxygen in chelation through deprotonation.
There is no appreciable change in the peak position corresponding to NH and aromatic protons. The 13 C-NMR signals for the metal, complexes are assigned by the comparison with the spectra of corresponding free Schiff base ligand. A downfield shift of CH = N group in the range of 150-160 ppm and for 175-182.5 ppm. In the complexes, NMR spectra indicate that the ligand coordinates through both the nitrogen atom of CH = N and the oxygen of COOion [49][50][51][52] (Fig. 1).

In vitro antimicrobial studies
The antibacterial and antifungal activity of the ligand and complexes [53,54] were assayed against some of the bacteria and fungi. DMF is used as negative control and chloramphenicol is used as a positive standard for antibacterial and nystatin for antifungal activities ( Fig. 2a  and b). The minimum inhibitory concentration (MIC) value of the compounds was determined by the serial dilution method and is given in Table 2.
The in vitro antimicrobial activity results revealed that complexes are more microbial toxic than the ligand. The activity order of the synthesized complexes and ligand are as follows 2 > 4 > 3 > 1.
Such increased activity of the metal chelates can be explained on the basis of Tweedy's chelation theory on chelation, the polarity of the metal ion will be reduced to a greater extent due to the overlap of the ligand orbital and partial sharing of the positive charge of the metal ion with donor groups. Further, it increases the delocalization of π electrons over the whole chelate ring and enhances the penetration of the metal complexes into lipid membranes and blocking of the metal-binding sites in the enzymes of microogranism. These complexes also disturb the respiration process of the cell and thus block the synthesis of proteins which restricts further growth of the organism. The complex 2 shows higher antimicrobial activity than other complex 3 and 4 complex. The variation in the effectiveness of different compounds against different organisms depends either on the impermeability of the cells of the microbes or on differences in ribosome of microbial cells. Further, lipophilicity which controls the rate of entry of molecules into the cells is modified by coordination so compounds 2, 3, and 4 can become more active than compound 1 compared to the standard compounds chloramphenicol and nystatin ( Fig. 2a and b); the present metal complexes are much less active against the representative strains of microorganism [55,56].

In vitro anticancer studies
The data obtained by the SRB assay show that metal complexes 2 and 3 have inhibitory effects on the growth of HOP62 (Tables 3 and 4) (Fig. 3) and BT474 (Tables 5 and 6) (Fig. 4). Cancer cells in dose-dependent manner. Complex 4 exhibits cytotoxic effect on BT474 cancer cells but no antiproliferative effect against cell line   Table 6 Parameters calculated from graph (Fig. 4)  HOP62. The antiproliferative effect of tested complexes is likely due to the lipophilicity of the complexes that alleviate the transport of metal complexes into the cell and posteriorly into the organelles where metal may possibly contribute to toxicity by inhibitory cellular respiration and metabolism of biomolecules [57][58][59]. The pure metals are inactive however the activity of metal cations varies on their bioavailability hence delivery methods/solubility and ionization of metal sources are significant parameters to deal metals in biological system [60][61][62]   Fluorescence studies Emission intensity of the three complexes increases on increasing the conc. of CT DNA. The enhancement of emission intensity is an indication of binding for the complexes to the hydrophobic pockets of DNA and complexes can be protected efficiently by the hydrophobic environment inside the DNA helix [63,64]. The high binding affinities of the metal complexes are probably attributed to the extension of the π system of the intercalated ligand due to the co-ordination of transition metal ions which also leads to a planar area greater than that of the free ligand and the coordinated ligand penetrating more deeply into and stacking more strongly with base pairs of DNA.

Conclusions
In the present study, novel metal complexes of Cu(II), Ni(II), and Co(II) were prepared and characterized by physico-chemical methods. Spectral studies demonstrate the ligand coordinating through azomethine nitrogen and carboxylate oxygen atoms and reveal octahedral geometry for Cu(II), Ni(II), and Co(II) complexes. The antibacterial and antifungal data given for the compound presented in this paper allowed us to state that the metal complexes generally have better activity than the ligands and less activity than standards. The metal complexes exerted growth inhibition on the human tumor cell lines showing promise as potential anticancer drugs deserving of further investigation. The Schiff base exhibits a strong fluorescence emission contrast to this partial fluorescence quenching phenomena is observed in its metal complexes.