Co(II), Ni(II), Cu(II) and Zn(II) complexes of 1,8-naphthalimide-based ligand: syntheses, characterizations and evaluation of antibacterial activities

Abstract Mononuclear later first-row transition metal complexes, [M(L)2] (1–4), where M = Co(II) (1), Ni(II) (2), Cu(II) (3), and Zn(II) (4) incorporating 1,8-naphthalimide based ligand were designed. The complex preparation was achieved by reacting metal nitrates with ligand (L) in 1:2 stoichiometry. The ligand and complexes were characterized with analytical and photophysical techniques. Single crystal X-ray diffraction determines that 3 is square planar. The complexes are highly fluorescent compared to the free ligand. The synthesized ligand (L) and complexes (1–4) were screened for their antibacterial activities against gram-positive [Streptococcus mutans (SM), Staphylococcus aureus (SA)] and gram-negative [Escherichia coli (EC), Klebsiella pneumoniae (KL)] bacterial strains. Ni(II) and Cu(II) complexes exhibited good activity against K. pneumonia and S. aureus compared to the free ligand. Particularly effective against gram-negative bacteria (Klebsiella pneumoniae), Ni(II) complex (2) has a MIC value of 6.25 µM and exhibits similar activity to standard ciprofloxacin. Cu(II) complex (3) is two times more effective than the common antibiotic ciprofloxacin with a MIC value of 3.125 µM against gram-positive bacteria (S. aureus).


Introduction
Metal ions play an important role in many biological processes either by involvement in essential biological processes, as toxic constituents, or as remarkable diagnostic and therapeutic agents [1].Metals can readily lose electrons and transform into positively charged ions, making them soluble in biological fluids [2].Most biological components including proteins and DNA are electron-rich, in contrast to metal ions, which are electron-deficient.Metal ions have a general affinity to bind and interact with biological molecules as a result of the attraction of these opposing charges [3].Among the metal ions that are present in nature are the biologically relevant transition metals, which can exist in more than one oxidation state and play important roles in chemical activity.Transition metals are involved in oxygen transport and storage, electron transfer, and enzymatic conversion of small molecules [4].Transition metals participate in the functioning of various enzymes and proteins as catalytic cofactors including cobalamin, ureases, hydrogenases, superoxide dismutases, acetyl-coenzyme A synthases, carbon monoxide dehydrogenases, methyl-coenzyme M reductases, ceruloplasmin, metallothionein, Cu-Zn superoxide dismutase, cytochrome c oxidase, and in few metalloproteins [5].
The first-row transition metal consists of ten elements with electronic configurations of [Ar] 3d 1-10 4s 1-2 .The involvement of 3d electrons has a substantial impact on the properties of first-row transition metal complexes including their magnetic nature, variable oxidation states, and catalytic activity [6].Metal ions can be used in organic synthetic reactions as catalysts for N-alkylation and C-alkylation [7].The ability to participate in both one-and two-electron elementary reactions makes first-row transition metals different from second-and third-row transition metals, with reactions taking place via two-electron processes [6].Apart from catalytic activity, they are also popular choices for biological activity.Transition metal complexes exhibit a wide variety of biological applications including antibacterial, antifungal, antitumor, and antiviral [8][9][10].
Transition metal complexes containing naphthalimide cores are gaining more interest in the development of DNA-targeting agents and chemotherapeutics [39][40][41][42].The metal complexes with naphthalimide ligands show enhanced cellular uptake and accumulation within the tumor cells when compared to naphthalimide ligands alone [43].The well-defined features of naphthalimide such as DNA binding ability and enhanced cytotoxicity against a variety of cell lines encouraged us to synthesize naphthalimidebased ligands and their metal complexes.The present study emphasizes the antibacterial activity of affordable and accessible first-row transition metal coordination compounds.

Materials and methods
All chemicals were purchased commercially and used without purification.Cobalt(II) nitrate hexahydrate, nickel(II) nitrate hexahydrate, copper(II) nitrate trihydrate, zinc(II) nitrate hexahydrate, and ethylenediamine were purchased from S.D.Fine Chemicals (India); ortho-vanillin was purchased from Spectrochem (India).

Synthesis of ligand (L)
The precursor 2-(2-aminoethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (A) was synthesized according to the procedure described in the literature [44].The methanolic solution of ortho-vanillin (0.63 g, 4.2 mmol) is added dropwise to the suspension of A (1.0 g, 4.2 mmol) in the methanol solution.At room temperature, the reaction mixture is stirred for 24 h.The yellow precipitate obtained is filtered, washed with methanol, and dried to obtain the ligand L (yield: 0.63 g, 66%).IR (cm

Methods and instrumentation
IR spectra of L and transition metal complexes were recorded in KBr disks from 4000 to 400 cm −1 on a Nicolet 6700 spectrometer. 1H and 13 C NMR of L and 1-4 were recorded on a JEOL 400 MHz spectrometer using DMSO-d 6 solvent.The EI/ESI-MS of all the compounds were measured in DMF solvent on a Shimadzu QP210S, Waters Xevo G2-XS and Waters SYNAPT G2.UV-Vis absorption spectra were measured on a JASCO UV-visible spectrophotometer.The emission spectra were recorded on a Spectrofluorimeter model F-7000 (Hitachi Japan) having a Xenon lamp.The EPR spectrum of the Cu(II) complex was recorded on a JES-FA200 ESR spectrometer with an X band at 298 K.

X-ray crystallography
The single crystal X-ray data of 3 was collected on a Rigaku diffractometer with a CCD detector having a graphite monochromatic Mo-Ka radiation source (0.71073 Å).The unit cells were determined using APEX 2 software.Unit cell refinement and integrated intensities determination were performed using SAINT [45].SADABS is used for absorption correction effects [45].To process the data SAINT PLUS has been used.The structure was solved on Olex2 [46] and SHELXT [47] programs and the data were refined using the SHELXL [48] program.CCDC 2241514 contains the supplementary crystallographic data for 3.

Antibacterial activity
All the bacterial cultures were maintained using BHI agar plates at 37 � C in aerobic conditions.The colonies isolated were diluted in fresh BHI broth to achieve the turbidity of 0.5 McFarland standards.The aliquots of this bacterial suspension were used for further experiments.Each test sample was prepared to the highest concentration of 1000 mM in DMSO and serially diluted to a working concentration ranging from 100 to 0.02 mM.The final concentration of DMSO was kept at less than 2.5% v/v in all the experiments.

Minimum inhibitory concentration (MIC)
Minimum inhibitory concentration (MIC) was determined following guidelines as described by the Clinical and Laboratory Standards Institute (CLSI).Briefly, overnightgrown bacterial cultures were diluted in fresh BHI to achieve the turbidity of the 0.5 McFarland standards.Two-fold serial dilutions of test samples in culture medium were mixed into the wells of a 96-well microplate.The microplate was incubated for 24 h at 37 � C in aerobic conditions.Then the incubation bacterial growth was recorded visually.These appropriate negative controls and positive controls were used in successive experiments.

Minimum bactericidal concentrations (MBC)
MIC dilution tubes, the first three or five tubes were plated (which was sensitive in MIC) and incubated for 24 h, then the next day the colony count was taken.MBC was done to see whether there was the bacteriostatic or bactericidal effect of the extract (drug) against the organism; if there was no growth then it was the bactericidal effect, if there was growth then it was the bacteriostatic effect.

Results and discussion
The synthetic route for the synthesis of L and the metal complexes (1-4) has been described in Schemes 1 and 2, respectively.L was synthesized via a condensation reaction between 2-(2-aminoethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (A) and ortho-vanillin in methanol at room temperature (Scheme 1).The ligand was characterized with FT-IR, NMR, LCMS, and elemental analysis techniques.The IR bands at 1694 and 1659 cm −1 correspond to the C ¼ O group.The proton NMR peak at 13.25 ppm (s, 1H) corresponds to OH, the peaks at 4.37 ppm (t, 2H) and 3.89 ppm (t, 2H) correspond to methylene protons and the peak at 3.69 ppm (s, 3H) corresponds to methyl protons of L. The 13 C NMR of L shows peaks at 167.76 ppm and 164.07 ppm corresponding to carbonyl carbon (C ¼ O) and the peaks around 56 ppm correspond to methylene carbon.Complexes 1-4 have been synthesized by reacting L and metal salts (Co(NO 3 ) 2 �6H 2 O, Ni(NO 3 ) 2 �6H 2 O, Cu(NO 3 ) 2 �3H 2 O, Zn(NO 3 ) 2 �6H 2 O) in the stoichiometric ratio of 2:1 in chloroform and methanol mixture (Scheme 2).The 1 H NMR spectra of nickel and zinc complexes (i.e. 2 and 4) show proton signals in the diamagnetic region i.e. at 0-10 ppm.The OH protons that were observed in L at 13.25 ppm disappeared after complexation in both 2 and 4.This result indicates that coordination occurs via deprotonation.

Crystallographic analysis of 3
The ORTEP diagram of 3 is given in Figure 1.Complex 3 crystallizes in a monoclinic crystal system with the space group of P2 1 /c.The asymmetric unit of the unit cell consists of half of the molecule and the other half of the molecule is generated by a center of inversion (Figure 2).Two equivalents of ligand are coordinated to Cu(II) in a bidentate manner with NO donor sites.Crystallographic refinement data of 3 are given in Table 1.The bond lengths and angles of 3 are listed in Tables S1 and S2, respectively.The coordination geometry around Cu(II) is defined as an almost regular square planar, as the coordination bond angles are close to an ideal square planar geometry (Figure 3); (O1-Cu1-O1 i ¼ 180.0 � , O1 i -Cu1-N1 i ¼ 91.98 � , O1-Cu1-N1 ¼ 91.98 � , O1-Cu1-N1 i ¼ 88.02 � , O1 i -Cu1-N1 ¼ 88.02 � , N1 i -Cu1-N1 ¼ 180.0 � ).The average distance of Cu1-O1 is 1.8628 Å and Cu1-N1 is 2.023 Å. Complex 3 shows intermolecular p … p stacking interaction between 1,8-naphthalimide rings with a distance of 4.024 Å (Figure 4).The complex also shows short contact between the oxygen of 1,8-naphthalimide moiety and the hydrogen of ortho-vanillin with a distance of 2.490 Å (Figures S7 and S8).Further, the s 4 value has been calculated for 3 which quantitatively measures the geometry around Cu(II) center.The value is found to be s 4 ¼ 0, indicating the perfect square planar geometry for 3.The proposed structures of 1, 2, and 4 are shown in Figure 5.

EPR spectroscopy
The X-band EPR spectrum of 3 was measured in the solid state at 298 K and is given in Figure 6.The spectrum showed two g values, g | (g z ) and g ?(g x ¼ g y ), indicating axial geometry with equivalent x and y-axes.For 3 the relation g | (2.14) > g ?(2.08) > g e (2.0023) is observed, which suggests square planar geometry [49] with the ground state of d x2-y2 .The exchange parameter (G) has been calculated using Eq.(1).(1) There will be an exchange interaction in the polycrystalline complexes if the G value is less than 4 (G < 4), and there will not be much of an exchange interaction if the G value is larger than 4 (G > 4) [50,51].The G value for 3 is 1.74 which is less than 4, indicating the presence of exchange interaction between the copper centers.

Photophysical properties
The photophysical properties of L and 1-4 were examined by absorption and emission spectroscopic techniques.The absorption and emission spectra were measured using various solvents such as phosphate buffer (pH 7.2, 0.1 M), water, ethanol, acetonitrile, and DMF (Figures S10 and S11).The band at 340 nm corresponds to internal charge transition (ICT) and the bands at 220-280 nm correspond to p!p � transitions for L (Figure 7a).There is no variation observed for the ligand transition in all the complexes (1-4).The low energy band observed at 700 nm for 3 is due to metal-to-ligand charge transfer transitions (MLCT) (Figure S10c).No prominent peaks were observed for 1, 2, and 4 in the low-energy region.
The emission spectra for the ligand and complexes were recorded with excitation of 340 nm and the corresponding emission is observed around 400 nm for ligand and complexes.After complexation fluorescence intensity increased in comparison to the ligand (Figure 7b).Complexes are highly fluorescent in aqueous solution compared to organic solvent (Figure S11).The photophysical data of ligands and complexes are tabulated in Tables 2 and 3, respectively.

Antibacterial activity
The in vitro antibacterial activity of L and 1-4 were studied against gram-positive (Streptococcus mutans (SM), Staphylococcus aureus (SA) and gram-negative [Escherichia coli (EC), Klebsiella pneumoniae (KL)] bacterial strains.The study focuses on the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration of all the synthesized compounds.The MIC value determines the minimum concentration required to prevent the visible growth of the bacteria as the MBC determines the minimum concentration required to kill 99.9% of the bacteria [52].Among the tested compounds (Table 4) 2 is effective against gram-negative strain i.e.K. pneumoniae with a MIC value of 6.25 mM which is similar to the MIC value of ciprofloxacin.Complex 3 is more effective against gram-positive strain i.e.Staphylococcus aureus with a MIC value of 3.125 mM which is less than the MIC value of ciprofloxacin (Figure 8).Similarly, the MBC values have been shown to kill the bacterial strain at 12.5 mM for 2 and 6.25 mM for 3 (Figure 9).MBC determination is a common approach for estimating bacterial eradication and is a recognized measure for evaluating new antimicrobial medicines [53].MBC was done to see whether there was a bacteriostatic or bactericidal effect of the drug against the bacteria.If there is no growth then it is a bactericidal effect and if there is growth then it is a bacteriostatic effect [54] (Table 5).The metal complexes containing 1,8-naphthalimide are more active against bacteria than free 1,8-naphthalimide under identical experimental conditions [55,56].According to Overtone's concept, lipophilicity plays an important role in determining

Conclusion
We synthesized 1,8-naphthalimide-based ligand (L) and their Co(II) (1), Ni(II) (2), Cu(II) (3), and Zn(II) (4) complexes.The synthesized compounds were characterized with FT-IR, 1 H and 13 C NMR, UV-Vis, and fluorescence spectroscopic techniques.The structure of 3 was confirmed by a single crystal X-ray diffraction.Further, the EPR spectra of the Cu(II) complex are in accord with the obtained crystal structure.The metal-to-ligand stoichiometry is 1:2 in all complexes.The ligand environment forms square planar geometry around Cu(II) and Ni(II), whereas it is tetrahedral around Co(II) and Zn(II).
The fluorescence study showed that the complexes are highly fluorescent compared to the free ligand.The antibacterial assay revealed that the first-row transition metal complexes derived from 1,8-naphthalimide ligand are effective against both gramnegative and gram-positive bacterial strains.The complexes are more effective than  free ligands.In particular, Ni(II) complex 2 is active against gram-negative bacteria (Klebsiella pneumoniae) with similar activity as ciprofloxacin with MIC of 6.25 mM.Cu(II) complex 3 is active against gram-positive bacteria (Staphylococcus aureus), two times better than the standard ciprofloxacin with a MIC value of 3.125 mM.

Scheme 1 .
Scheme 1. Synthetic route for the synthesis of L.

Figure 1 .
Figure 1.ORTEP diagram of 3 with the atom numbering scheme and with thermal ellipsoids at the 50% probability level.

Figure 5 .
Figure 5. Proposed structures of 1, 2 and 4 based on spectral characterization data.

Table 2 .
Photophysical data of L.

Table 5 .
Minimum bactericidal concentration (MBC) for different concentrations of L, 2 and 3 after 24 h.