Abstract
A novel series of complexes of the type [M(C24H16N4)X2]; where M = Co(II), Ni(II), Cu(II), Zn(II) and Cd(II); X = Cl− 1, 
, CH3COO− 1 has been synthesized by template condensation of 1,8-diaminonaphthalene and glyoxal in the presence of divalent metal salts in methanolic medium. The complexes have been characterized with the help of elemental analyses, conductance measurements, molecular weight determination, magnetic measurements, electronic, NMR, infrared and far infrared spectral studies. The low value of molar conductance indicates them to be non-electrolytes. On the basis of various studies a distorted octahedral geometry may be proposed for all of these complexes. These metal complexes were also tested for their in vitro antibacterial and antifungal activities to assess their inhibiting potential.
| Abbreviations | ||
| B.M., | = | Bohr Magneton |
| CFU, | = | Colony Forming Unit |
| DMF, | = | N,N dimethylformamide |
| DMSO, | = | Dimethylsulphoxide |
| IR, | = | Infra red |
| MIC, | = | Minimum Inhibitory Concentration |
| MRI, | = | Magnetic Resonance Imaging |
| MTCC, | = | Microbial Type Culture Collection |
| NMR, | = | Nuclear Magnetic Resonance |
| PDA, | = | Potato Dextrose Agar |
Introduction
Macrocyclic compounds and their derivatives are interesting ligand-system because they are good hosts for metal anions, neutral molecules and organic cation guests [Citation1]. The metal-ion and host-guest chemistry of macrocyclic compounds are very useful in fundamental studies e.g. in phase transfer catalysis and biological studies [Citation2]. The family of complexes with aza-macrocyclic ligands has been remained a focus of scientific attention for many decades [Citation3]. In-situ one pot template condensation reaction lie at the heart of the macrocyclic chemistry [Citation4]. Therefore, template reactions have been widely used for synthesis of macrocyclic complexes where generally the transition metal ion is used as templating agent [Citation5]. There is continued interest in synthesizing macrocyclic complexes because of their potential application in fundamental and applied sciences [Citation6,Citation7]. Macrocyclic complexes act as a model, mimicking naturally occurring metalloproteins and metalloenzymes [Citation8]. Macrocyclic Nickel complexes find use in DNA recognition and oxidation [Citation9]. Macrocyclic copper complexes find use in DNA binding and cleavage [Citation10] and copper containg proteins have been identified [Citation11]. Macrocyclic metal complexes of lanthanides e.g. Gd3 + are used as MRI contrast agents [Citation12]. Macrocyclic metal chelating agent (DOTA) is useful for detecting tumour lesions [Citation13]. The chemistry of macrocyclic complexes is also important due to their use as dyes and pigments [Citation14] as well as NMR shift reagents [Citation15]. Some macrocyclic complexes have received special attention because of their mixed soft- hard donor character and versatile coordination behaviour [Citation16] and of their pharmacological properties i.e. toxicity against bacterial and fungal growth [Citation17]. Prompted by these, in the present paper, synthesis and characterization of cobalt(II), nickel(II), copper(II), zinc(II), and cadmium(II) macrocyclic complexes derived from 1,8-diaminonaphthalene and glyoxal have been discussed. Besides the characterization of complexes by physicochemical technique like IR, NMR, elemental analyses, magnetic susceptibility and conductance measurements, the biological activities of the synthesized complexes have been examined against some bacterial strains viz. Bacillus subtilis (MTCC 8509), Bacillus stearothermophilus (MTCC 8508), Escherichia coli (MTCC 51), Pseudomonas putida and some fungal strains viz. Aspergillus flavus and Aspergillus niger. The results obtained were compared with standard antibiotics: Chloroamphenicol, Streptomycin and the antifungal drug Cyclohexamide.
Experimental
All the chemicals used were of AnalaR grade. 1,8-diaminonaphthalene and glyoxal procured from Acros, metal salts were purchased from s.d.-fine, Merck, Ranbaxy and were used as received.
Chemistry
Isolation of complexes
All the complexes were synthesized by template method i.e. by condensation of 1,8-diaminonaphthalene and glyoxal in the presence of the respective divalent metal salts. To a hot stirring methanolic solution (∼50 cm3) of 1,8-diaminonaphthalene (10 mmol) was added divalent cobalt, nickel, copper, zinc and cadmium salt (5 mmol) dissolved in the minimum quantity of MeOH (∼20 cm3). The resulting solution was refluxed for 0.5 h. After that, glyoxal (10 mmol) was added in the refluxing mixture and refluxing was continued for 8-10 h. The mixture was concentrated to half of its volume and kept in a desiccator overnight. On overnight cooling dark coloured precipitates formed which was filtered, washed with methanol, acetone, diethylether and dried in vacuo. Yield obtained ∼50-65%. The complexes are soluble in DMF and DMSO, but are insoluble in common organic solvents and H2O. They were found thermally stable up to ∼280°C and then decomposed.
The template synthesis of complexes may be represented by the following scheme:
Analytical and physical measurements
The microanalysis of C, H, and N were carried out at SAIF, Punjab University, Chandigarh. The magnetic susceptibility measurements were carried out at SAIF, IIT Roorkee. The metal contents in the complexes were determined by literature method [Citation18]. The IR spectra were recorded on a FT-IR spectrophotometer (Perkin Elmer) in the range 4000-200 cm− 1 using KBr pellets. The 1H NMR spectra (at room temperature) (in DMSO d6) were recorded on a Bruker AVANCE II 400 NMR spectrometer (400 MHz) at SAIF, Punjab University, Chandigarh. Electronic spectra (in DMSO) were recorded on Cary 14 spectrophotometer at room temperature. The FAB mass spectra (at room temperature) were recorded on TOF MS ES+ mass spectrometer at SAIF, Punjab University, Chandigarh. The conductivity was measured on digital conductivity meter (HPG system, G-3001). Melting points were determined by using capillaries in electrical melting point apparatus. The molecular weights were determined by Rast camphor method [Citation19].
Biological assay
Test microorganisms
Two Gram-positive bacteria Bacillus subtilis (MTCC 8509) and Bacillus stearothermophilus (MTCC 8508), two Gram-negative bacteria Escherichia coli (MTCC 51) and Pseudomonas putida (MTCC 121) and two fungal strains Aspergillus flavus, Aspergillus niger were used for biological assay.
In-vitro antibacterial activity
Primary screening
The antibacterial activities of the newly synthesized complexes were evaluated by Agar Well Diffusion Assay technique [Citation20] against two Gram- positive bacteria: B. subtilis (MTCC 8509) and B. stearothermophilus (MTCC 8508) and two Gram-negative bacteria: E. coli (MTCC 51) and P. putida (MTCC 121). The bacterial cultures were maintained on the nutrient agar media by sub-culturing them on the fresh slants after every 4-6 weeks and incubating them at the appropriate temperature for 24 h. All stock cultures were stored at 4°C. For the evaluation of antimicrobial activities of the synthetic compounds, suspension of each test microorganism was prepared. Turbidity of each suspension was adjusted to 0.5 McFarland units by suspending the cultures in sterile distilled water. The size of final inoculum was adjusted to 5 × 107 cfu/mL. A volume of 20 mL of agar media was poured into each petri plate and plates were swabbed with broth cultures of the respective micro-organisms and kept for 15 min for adsorption to take place. Wells of ≈ 8 mm diameter were punched in the seeded agar plates and a 100 μL volume of each test compound reconstituted in DMSO was added into the wells. DMSO was used as control for all the test compounds. To allow diffusion of the compounds in to the agar, the plates were hold at room temperature for 2 h. After that the palates were incubated at 37°C for 24 h. Antibacterial activities were determined by measuring the inhibition zone diameter. The entire tests were made in triplicates and mean of the diameter of inhibition was calculated.
Determination of minimum inhibitory concentration (MIC)
The minimum inhibitory concentration (MIC) is the lowest concentration of the antimicrobial agent that prevents the development of viable growth after overnight incubation [Citation21]. Nutrient broth was adjusted to pH 7.0 used for the determination of MIC [Citation22]. The inoculum of the test microorganisms were prepared by using 16 h old cultures adjusted by reference to the 0.5 McFarland standards (108cells/mL) [Citation23]. These cultures were further diluted up to 10 fold with nutrient broth to get the inoculum size of 1.2 × 107 cfu/mL. A positive control (containing inoculum but no compound) and a negative control (containing compound but no inoculum) were also prepared. A stock solution of 4 mg/mL of each compound was prepared in DMSO and further appropriately diluted to get final concentration ranging from 250 to 0.03 μg/mL [Citation21]. Separate flasks were taken for each test dilution. To each flask was added the 100 μL of inoculum. Then appropriately diluted test sample was added to each flask having broth and microbial inoculum. The contents of the flask were mixed and incubated for 24 to 48 h at 37°C. The test bacterial culture were spotted in a predefined pattern by aseptically transferring 5 μL of each bacterial culture on the surface of solidified agar plates and incubated at 37°C for 24 h for determining the MIC values.
In-vitro antifungal activity
Antifungal activities of the synthesized compounds were carried out against two fungal strains i.e. A. flavus and A. niger by agar plate technique [Citation20]. Further the antifungal (Percentage Inhibition) activities of these compounds were compared with standard drug cyclohexamide [Citation24]. Potato Dextrose Agar (PDA) medium was prepared in flask and sterilized. To check the growth of bacterial culture in the medium, requisite quantity of the standard antibiotic (Ampicilline) was added, so as to get their desirable final concentration of 100 μg/mL of the medium. Test samples were prepared in different concentrations (10 μg, 50 μg, 100 μg per mL) in DMSO and 200 μL of each sample was spread on PDA media containing sterilized petri plates. Mycelial discs taken from the standard cultures (A. flavus and A. niger) of fungi were grown on PDA medium for 5-7 days. These cultures were used for the purpose of inoculation in sterilized petri dish, aseptically. Standard cultures inoculated at 28 ± 1°C, were also used as the control. The efficiency of each sample was determined by measuring radial mycelial growth. The radial growth of the colony was measured in two directions at right angle to each other, and the average of two replicates was recorded in each case. Data were expressed as percent inhibition over control from the size of colonies. The percent inhibition was given in the was calculated using the formula:
C = diameter of fungus colony in the control plate after 96 h incubation;
T = diameter of the fungus colony in tested plate after same incubation period.
Result and discussion
Chemistry
The analytical data show the formula for macrocyclic complexes as: [M(C24H16N4)X2]; where M = Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and X = Cl− 1 
and CH3COO− 1. The tests for anions are positive only after decomposing the complexes with conc. HNO3, indicating their presence inside the coordination sphere. The low molar conductance values (10-17 ohm− 1 cm2 mol− 1) of complexes determined in DMSO indicates their non-electrolyte nature [Citation25]. All complexes give satisfactory elemental analyses results as shown in .
Table I. Analytical data of divalent cobalt, nickel, copper, zinc and cadmium complexes derived from glyoxal and, 8-diaminonaphthalene.
IR spectra
It was noted that a pair of bands are present in the spectrum of 1,8-diaminonaphthalene at 3,350 & 3,390 cm− 1 corresponding to ν(NH2) group are absent in the infrared spectra of all the complexes. Further, no strong absorption band was observed near 1,715 cm− 1 indicating the absence of > C = O group of glyoxal moiety. The disappearance of these bands and appearance of a new strong absorption band near 1,590-1,629 cm− 1 confirms the condensation of carbonyl group of glyoxal and amino group of diaminonaphthalene and formation of macrocyclic Schiff's base [Citation26] as these bands may be assigned due to ν(C = N) stretching vibrations [Citation27,Citation28]. The lower value of ν(C = N) may be explained on the basis of a drift of lone pair density of azomethine nitrogen towards the metal atom [Citation29,Citation30] indicating that coordination takes place through nitrogen of C = N groups. The bands present in the range 3,010-3,050 cm− 1 may be assigned due to ν(C–H) stretching vibrations of glyoxal and naphthalene moiety [Citation31]. The various absorption bands in the region 1,400-1,588 cm− 1 may be assigned due to ν(C = C) aromatic stretching vibrations of the naphthalene ring [Citation32,Citation33]. The bands in the region 740-785 cm− 1 may be assigned to ν(C–H) out of plane bending of aromatic ring [Citation34,Citation35]. The bands present in the region 1,000-1,300 cm− 1 in all complexes are assigned due to ν(C–N) stretching frequency [Citation31].
Far IR spectra
The far infrared spectra show bands in the region 420-450 cm− 1 corresponding to ν(M–N) vibrations Citation36,Citation37,Citation38. The presence of bands in all complexes in the region 420-450 cm− 1 originate from (M–N) azomethine vibrational modes and identify coordination of azomethine nitrogen [Citation39]. The bands present in the range 300-320 cm− 1 may be assigned due to ν(M–Cl) vibration Citation36,Citation37,Citation38. The bands present in the region 220-250 cm− 1 in all nitrato complexes are assignable to ν(M–O) stretching vibration [Citation36,Citation37].
NMR spectra
The 1H NMR spectrum of the zinc(II) complex shows peaks in the region 6.62-7.32 p.p.m. corresponding to aromatic ring protons [peak B—8H (b), peak C—4H (a)] [Citation40]. The singlet at 7.98 p.p.m. (peak A) may be assigned due to the azomethine (HC = N) protons (4H) [Citation41]
Figure 1. 1H NMR spectrum of [Zn(C24H16N4)(OAc)2] complex.
![Figure 1. 1H NMR spectrum of [Zn(C24H16N4)(OAc)2] complex.](/cms/asset/ee192ef0-cd9c-429d-be34-29d1d0622c67/ienz_a_339884_f0001_b.gif)
Mass spectra
The mass spectra of [Co(C24H16N4)(NO3)2] complex have been recorded and shown in . The complex shows the peak at m/z = 544 corresponding to the molecular ion peak (M+). This confirms the formation of the macrocyclic frame with molecular formula [M(C24H16N4)X2]. Another peaks at m/z = 353, 381, 413, 507 etc. may be corresponding to various fragments. The peak intensity gives an idea of the stability of the fragment.
Figure 2. FAB mass spectra of [Co(C24H16N4)(NO3)2] complex.
![Figure 2. FAB mass spectra of [Co(C24H16N4)(NO3)2] complex.](/cms/asset/dafebee0-d8de-4671-8151-9ca02851bf0c/ienz_a_339884_f0002_b.gif)
Magnetic measurements and electronic spectra
Cobalt complexes
The magnetic moment of the cobalt complexes was measured at room temperature and lay in the range 4.90-4.99 B.M. which corresponds to three unpaired electrons. The electronic spectra of the cobalt(II) complexes recorded in DMSO exhibits three absorption peaks in the region 8,150-9,280 cm− 1 (ν1), 13,500-15,550 cm− 1 (ν2) and 18,500-20,500 cm− 1 (ν3) respectively. The spectra resemble to those complexes reported to be octahedral [Citation42]. Thus, assuming the effective symmetry to be D4h, the various bands may be assigned to the transitions: 4T1g → 4T2g (F), (ν1) 8,150-9,280 cm− 1; 4T1g → 4A2g (F), (ν2) 13,500-15,550 cm− 1; 4T1g → 4T1g (P), (ν3) 18,500-20,500 cm− 1; respectively. It appears that the symmetry of these complexes is not idealized Oh, but is D4h. The assignment of the first spin-allowed band seems plausible since the first band appears approximately at half the energy of the visible band [Citation43].
Nickel complexes
The magnetic moment of the nickel complexes at room temperature lay in the range 2.94-3.10 B.M. These values are in tune with a high spin configuration and show the presence of an octahedral environment around the Ni(II) ion in all complexes. The spectra of Ni(II) complexes recorded in DMSO solution exhibit a well discerned band with a shoulder on the low energy side. The other two bands generally observed in the region 16,500-17,200 cm− 1 (ν2), and 27,800-28,300 cm− 1 (ν3), are assigned to 3A2g → 3T1g (F) (ν2), and 3A2g → 3T1g (P) (ν3), respectively. The first two bands result from the splitting of one band, ν1 and are in the range ∼ 9,650-10,200 and 11,850-12,350 cm− 1, which can be assigned to 3B1g → 3Eg and 3B1g → 3B2g, assuming the effective symmetry to be D4h (component of 3T2g in Oh symmetry) [Citation43]. The intense higher energy bands at 34,540 cm− 1 may be due to a π-π* transition of the (C = N) group. Various bands do not follow any regular pattern and seem to be anion independent. The spectra are consistent with distorted octahedral nature of these complexes.
Copper complexes
The magnetic moment of copper complexes lay in the range 1.76-1.80 B.M. The electronic spectra of the Cu(II) complexes exhibit bands in the region 17,750-19,500 cm− 1, with a shoulder on the low energy side at ∼14,500-16,000 cm− 1, and show that these complexes are distorted octahedral [Citation42,Citation43]. Assuming tetragonal distortion in the molecule, the d-orbital energy level sequence for these complexes may be: x2-y2 > z2 > xy > xz > yz and the shoulder can be assigned to: z2 → x2-y2 (2B1g → 2B2g) and the broad band contains both the xy → x2-y2 (2B1g → 2Eg) and xy, yz → x2-y2 (2B1g → 2A2g) transition [Citation44]. The band separation of the spectra of the complexes is of the order 2,500 cm− 1, which is consistent with proposed geometry of the complexes [Citation44]. Therefore it may be concluded that all the complexes formed by macrocycles with Cu(II) metals are distorted octahedral.
Proposed structure of complexes
Based on various studies like elemental analyses, conductance measurements, magnetic susceptibilities, infrared, NMR and electronic spectral studies the structure shown in may be proposed for all of the complexes.
Figure 3. Structure of the complexes. Where M = Co(II), Ni(II), Cu(II), Zn(II), Cd(II); X = Cl− 1, NO3− 1, CH3COO− 1.
Biological result and discussion
The newly synthesized compounds (1-11) were tested in the present investigation for their in-vitro antibacterial as well as antifungal activities. The antibacterial activities were studied against the two Gram-positive bacteria i.e. B. subtilis (MTCC 8509), B. stearothermophilus (MTCC 8508) and two Gram-negative bacteria i.e. E. coli (MTCC 51), P. putida (MTCC 121). The minimum inhibitory concentration (MIC) of all the complexes against Gram-positive and Gram-negative bacteria were determined by the method of NCCLS [Citation21] and given in the . In the whole series, compound (7) showed highest MIC 1 μg/mL against P. putida and E. coli (). Compound (6) possessed MIC of 2 μg/mL against B. subtilis, P. putida and E. coli. Compound (3) showed MIC of 2 μg/mL against the bacteria P. putida and MIC of 4 μg/mL against B. subtilis and E. coli. Compound (5) and (6) showed MIC of 4 μg/mL against B. subtilis and B. stearothermophilus, respectively. Further the antibacterial activities of these complexes were compared with standard antibiotics viz. Chloramphenicol and Streptomycin (). In general all the synthesized compounds showed the antibacterial activities, but they were found more potent inhibitors against Gram-negative bacteria as compared to the Gram-positive ones (). Some compounds were found to be more potent than the standard antibiotics against some species of bacteria. Compounds (3), (6) and (7) were found to be more potent than the commercial antibiotics by showing MIC of (1-2 μg/mL) (, ).
Table II. Minimum inhibitory concentration (MIC) of the complexes against test bacteria by using agar dilution assay.
Figure 4. Comparison of MIC of compounds with standard antibiotics up to the concentration 16 μg/mL. Bs—Bacillus subtilis (MTCC 8509); Bst—Bacillus stearothermophilus (MTCC 8508); Pp—Pseudomonas putida (MTCC 121); Ec—Escherichia coli (MTCC 51); Chloramphenicol, Streptomycin—standard antibiotics.
The antifungal activities of complexes were carried out against two fungal strains i.e. A. flavus and A. niger and then compared with standard drug Cyclohexamide (, ). The antifungal activities (percentage inhibition) are given in . In the whole series, compound (4) showed the highest percentage inhibition, whereas none of the tested compounds restrict the fungal growth excellently (). However, the tested compounds (1), (5), (7), (8) and (9) showed a moderate capability to check the growth of these fungal species ().
Table III. Antifungal (percentage inhibition) activities of the complexes against the fungal strains. (for concentration—100 μg/mL).
Figure 5. Comparison of the percentage inhibition of compounds with standard drug Cyclohexamide against fungal strains. AF—Aspergillus flavus; AN—Aspergillus niger; Cyclohexamide—standard drug.
Conclusions
It has been suggested that chelation/coordination reduces the polarity of the metal ion mainly because of partial sharing of its positive charge with donor group within the whole chelate ring system [Citation45,Citation46]. This process of chelation thus increases the lipophilic nature of the central metal atom, which in turn, favours its permeation through the lipoid layer of the membrane thus causing the metal complex to cross the bacterial membrane more effectively thus increasing the activity of the complexes. Besides from this many other factors such as solubility, dipole moment, conductivity influenced by metal ion may be possible reasons for remarkable antibacterial activities of these complexes [Citation47]. It also has been observed that some moieties such as azomethine linkage or heteroaromatic nucleus introduced into such compounds exhibit extensive biological activities that may be responsible for the increase in hydrophobic character and liposolubility of the molecules in crossing the cell membrane of the microorganism and enhance biological utilization ratio and activity of complexes [Citation48].
Acknowledgements
D.P. Singh thanks the University Grants Commission, New Delhi for financial support in the form of Major Research Project [MRP-F.No. 32-196/2006(SR)] and Krishan Kumar for the award of Project Fellowship under the above project. Thanks are also due to authorities of N.I.T., Kurukshetra for providing necessary research facilities.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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