Synthesis, spectral, crystal structural, antimicrobial, DNA interaction and thermal behavior of some new zinc halide complexes: 3D supramolecular structure of zinc bromide complex

2.1 Materials and methods

All solvents used in the synthesis and analysis, 3-phenyl-propenal, 2,2-dimethyl-propane-1,3-diamine, zinc salts and other chemicals were purchased from Aldrich and Merck, and used without further purification. The FT-IR spectra were recorded on the JASCO-680 model in the range of 400–4000 cm⁻¹ using KBr disks. Elemental analysis (C, N and H) was conducted on a CHNS-932(leco) elemental analyzer. UV–Vis spectra were recorded in the 200–800 nm range using a JASCO-V570 spectrometer at DMF. ¹H and ¹³C NMR spectra were recorded by a Brucker DPX FT-NMR spectrometer at 400 MHz in CDCl₃ using TMS as internal standard. Molar conductance of the Schiff base ligand and their zinc complexes were determined in DMF (1.0 × 10⁻³ M) at room temperature by a Metrohm 712 conductometer. The melting or decomposition temperatures (°C) of the complexes were recorded on the Kruss instrument. In biological tests, Nutrient agar (Merck, Germany) was used as the solid medium for preparing nutrient plates, while Mueller Hinton broth (Scharlab) was used as the liquid culture media. Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538 and Bacillus subtilis ATCC 6633 were selected for antibacterial studies and Candida albicans was a candidate for antifungal test. Thermal behaviors were investigated by Diamond TGA PerkinElmer.

2.2 Synthesis of Schiff base ligand (L)

The Schiff base ligand was synthesized according to the previous report via a condensation reaction between 2,2-dimethylpropane-1,3-diamine and 3-phenyl-2-propenal (Joohari, 2013). Ligand was filtered and washed twice with cooled ethanol and recrystallized from ethanol–dichloromethane solvent mixture.

2.3 Synthesis of complexes

Zinc complexes were prepared by addition of an ethanolic solution of the Schiff base (1 mmol, 0.3305 g) to the corresponding zinc salt solution (1 mmol in ethanol) under vigorous stirring at room temperature. The zinc complex precipitate was filtered, then washed with cooled ethanol and dried under vacuum. Crystals of ZnLBr₂ suitable for X-ray analysis were obtained by slow evaporation from acetonitrile as solvent at room temperature. Physical and spectral data of ligand and its zinc complexes have been collected in Tables 1–3.

2.4 Single-crystal X-ray diffraction

Suitable single crystal for structure determination was selected and mounted on glass fibers using a Bruker Kappa Apex II CCD diffractometer with graphite-monochromatized Mo-Kα radiation (λ = 0.71073 Å). Data were collected at room temperature by scanning ω and ϕ in set of blocks, with exposure times of 20 s. Data were corrected for Lorentz and polarization effects. Absorption corrections were based on fitting a function to the empirical transmission surface as sampled by multiple equivalent measurements using the SADABS part of the APEX II package since there were no clear faces for indexation. The structure was solved using direct methods and refined by the full-matrix least-squares method on F² data using SHELXTL. Tables 4–6 show the crystallographic data, selected bond distances and hydrogen bonds of ZnLBr₂ respectively.

2.5 Antimicrobial activity bioassay procedure

The ligand and its zinc complexes were tested for their in vitro antibacterial activities against Gram-negative bacteria of E. coli and P. aeruginosa; Gram-positive bacteria of S. aureus and B. subtilis and fungal strains of C. albicans and Aspergillus oryzae by disk diffusion method. For investigation into the effect of concentration, compounds were dissolved in DMSO at three different concentrations (100, 50 and 25 mg/mL). Enriched culture of the microorganisms was prepared by incubation of them in Muller–Hinton broth medium at 37 °C for 24 h. For all tests, 15 mL of sterilized Nutrient Agar as solid medium was solidified into sterilized petri plates and 0.1 mL of culture microorganism swabbed evenly on the surface of the solid medium and kept for 15 min for adsorption. Blank paper disks (6.00 mm in diameter) were saturated with a stock solution of test compounds and placed on the surface of the agar plates. All the plates were incubated at 37 °C. The growth inhibition zone around each disk as inhibitory effect was measured in millimeters after 24 h of incubation.

2.6 DNA cleavage experiments

DNA cleavage potential of the ligand and its zinc complexes has been studied by gel electrophoresis method. DNA of the E. coli cells was extracted using the DNA Mini Prep Extraction Kit (BIONEER, Korea) according to the manufacturer’s instructions and was kept at 40 °C for the following tests. The test samples for the study of DNA cleavage potential of the compounds were prepared at a concentration of 5 mg/mL solutions in DMSO. 4 μL of each titled samples was added to 4 μL of isolated DNA in the individual microtube and incubated for 2 h at 37 °C. Extracted DNA alone as positive control and DNA treated with 30% H₂O₂ (X) as negative control were also used for monitoring the probable DNA cleavages. Then DNA containing samples were mixed with bromophenol blue dye (6 mL glycerin; 0.9 g bromophenol blue/10 mL). Finally, pure DNA, mixture of DNA and H₂O₂, ladder (1 Kb) and mixture of DNA and compound test were loaded carefully into the wells. Electrophoresis was performed at a constant 100 V of electricity for around 30 min. Resulting bands of electrophoresis were visualized by UV light (Transilluminator, UVItec, England) and photographed.

3.1 IR and UV–Visible spectra

In order to clarify the mode of bonding and the effect of coordination of the ligand, IR spectra of the free ligand and its Zn(II) complexes were studied and for easy comparison have been listed in Table 2. In the free ligand spectrum, week absorptions at 3058, 3025, (2954, 2924) and (2866, 2830) cm⁻¹ are assigned to the vibration stretching of aromatic, olefinic, aliphatic and iminic groups C–H respectively that smoothly shift to the lower or higher frequencies after complexation. The observation of a strong absorption at 1635 cm⁻¹ in the ligand spectrum is attributed to the vibration frequency of the azomethine moieties (ν(C⚌N)). Coordination of the Schiff base to the zinc ion through the nitrogen atoms of the azomethine groups leads to the reduction of electron density in the azomethine bond and this occurrence causes a shift to lower frequencies of azomethine vibration in all the zinc complexes (Khalaji et al., 2011; Rahaman et al., 2005; Chakraborty et al., 2007). New weak absorptions in the region of 443–445 cm⁻¹ in the IR spectra of complexes, are attributed to the vibration frequencies Zn–N bonds that indicates the participation of the azomethine nitrogen in coordination to zinc ion (Shebl, 2009; Anitha et al., 2012).

The electronic absorption spectra of freshly prepared DMF solution of the Schiff base and its Zn(II) complexes were recorded at room temperature and their spectral data have been tabulated as Table 2. The free Schiff base ligand shows a broad band appeared at 278 nm and it is attributed to π–π^∗ internal electron transfer that may be due to aromatic rings, carbon–carbon and/or azomethine double bond. Coordination of ligand to metal ion via azomethine moieties leads to a shift to a longer wavenumber at 286 nm. For Zn(II) complexes with d¹⁰ electronic configuration, d–d electronic transition is not expected but observation of the band due to metal to ligand charge transfer (MLCT) is expectable but it is not observed in the titled compounds.

3.2 ¹H and ¹³C NMR spectral studies

As more evidence to confirm the structure and bonding pattern of the titled complexes, ¹H and ¹³C NMR spectra of the ligand and its Zn(II) complexes were recorded in CDCl₃ solution and the data have been presented in Table 3. ¹H and ¹³C NMR spectra data of the ligand and ZnLX₂ strongly support the structural formula. In the ¹H NMR spectrum of the free ligand, the doublet signal at 8.08 ppm is corresponded to the resonance of H_f (azomethine hydrogens) with J = 7.20 Hz due to coupling with H_e. Binding of the azomethine nitrogens to the zinc ion causes a shift to downfield regions at all zinc complex spectra (Amirnasr et al., 2002; Chohan, 2009). In the free ligand spectrum, the aromatic hydrogens of H_c, H_c′ and H_b, H_b′ and H_a appeared at 7.44 and 7.37 ppm as broad doublet and multiplet signals respectively. In the complex spectra, H_c and H_c′ appeared as a doublet of doublet signal for ZnLCl₂ and as a multiplet signal for ZnLBr₂ and ZnLI₂ at the region 7.59–7.70 ppm. Also in all complexes, the resonance of H_a overlapped with the signals of H_b and H_b′ and except for entry 4, in the other complexes shifted to the up field regions. In the ligand spectrum, two signals at the 7.31 and 6.93 ppm were safely assigned to ethylenic protons of H_e and H_d. After complexation, in all complexes, the resonance of H_d and H_e showed considerable shift to the upfields at 8.14–8.26 ppm as doublet of doublet signal for H_e and doublet signal for H_d. As seen in Table 3, the coupling constant for ethylenic protons is in the range of 15–16 Hz that strongly confirms trans-position of these protons around –C⚌C– as shown in Fig. 1. In the free ligand spectrum, H_g and H_h appeared as quartet and triplet peaks at 3.71 and 1.24 ppm due to coupling with each other. After coordination of the ligand, signals of these protons were up or down fielded.

In the ¹³C NMR spectrum of the ligand, resonance signal of azomethine carbons (C₇, C_7′) occurred at 173.01 ppm. Good coordination of the azomethine nitrogens to zinc ion led to the observation of this signal in the upfield region at the range of 170.65–170.93 ppm. The aromatic, olefinic and aliphatic carbons signals appeared at 163.72(C₆), 138.07(C₄), 128.82(C₁), 128.53(C₃), 127.26(C₂), 126.42(C₅), 58.22(C₈), 46.25(C₉) and 18.46(C₁₀) ppm shifted to upfield or downfield ranges of 150.04–150.22(C₆), 134.46–134.50(C₄), 131.03–131.16(C₁), 129.05–129.13(C₃), 128.69–128.73(C₂), 124.23–124.33(C₅), 69.97–71.84(C₈), 36.69–36.91(C₉) and 24.53–24.93(C₁₀) ppm. These signals and their shifts in complexes as compared with free ligand are in good agreement with the structure of zinc halide complexes.

3.3 Crystal structure

The ZnLBr₂ complex structure has been identified by single crystal X-ray analysis. The ORTEP view of the complex is shown in Fig. 2. Crystallographic data and refinement have been tabulated as Table 4. Some selected bond lengths and angles are given in Table 5. The complex crystallizes in a triclinic system with the space group of $\mathit{P}\overline{1}$ , with two crystallographically independent molecules noted as A and B per asymmetric unit cell (Fig. 3). The crystal structure of the complex reveals the presence of a structure consisting of a bidentate N₂-donor Schiff base, two bromide anions and a zinc(II) ion in the discrete monomeric species. Both Zn1A and Zn1B are almost in slightly distorted tetrahedral environments (τ₄ = 0.94 for Zn1A and τ₄ = 0.93 for Zn1B whereas τ₄ = 0 and τ₄ = 1 are attributed to square planar geometry and tetrahedral geometry respectively) (Yang et al., 2007).

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Figure 2

ORTEP diagram of complex 1 with the atom numbering scheme. Atoms are represented by 25% probability thermal ellipsoids. Hydrogen atoms are omitted for clarity.

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Figure 3

Unit cell structure of ZnLBr₂ complex indicating two molecules with different symmetry equivalence in the asymmetric unit.

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The average bond lengths of Zn–Br (2.37 Å in both molecules) and Zn–N (2.05 Å in both molecules) well agreed with the Zn–Br and Zn–N bond lengths in other reported tetrahedral zinc complexes, such as [Zn(nca₂en)Br₂] (2.35 Å for Zn–Br and 2.05 Å for Zn–N) and [Zn(Salen)Br₂] (2.35 Å for Zn–Br and 2.08 Å for Zn–N) (Dehghanpour and Mahmoudi, 2007; Taylor et al., 2006). It is to be noted that Zn–N bond distances in the current structure are considerably shorter than some Zn–N distances reported for some organo-zinc structures (2.156-2.410 Å) (Melnik et al., 1995; Jastrzebski et al., 2006). The angle values around the zinc center [N–Zn–Br: 106.19–113.76°; N–Zn–N: 96.69 and 98.67°; Br–Zn–Br: 113.94 and 114.48°] are in good agreement with other ZnN₂Br₂ tetrahedral complexes (Dehghanpour and Mahmoudi, 2007; Taylor et al., 2006; Khalaji et al., 2010). The N–Zn–N and Br–Zn–Br bond angles are respectively smaller and larger than the ideal tetrahedral values. Of course, the angle value of N–Zn–N (96.69 and 98.67°) in the current structure is smaller than that in [Zn(CH₂)₃NMe₂))₂] (109.7°) while is larger than that in [Zn(Me₄en)C₅H₇)Cl] (84.5°) that each two mentioned complexes contain non-Schiff base chelating ligands (Melnik et al., 1995). In the Zn1A molecule, the aromatic rings of the ligand (L) are almost coplanar with the dihedral angle of 6.63°, and the dihedral angle between the aromatic rings of the ligand (L) in Zn1B molecule is 21.04°. Detailed structural analysis shows that there are three non-classical hydrogen bonding C–H…Br (C13B–H13B…Br1A = 2.98 Å; C15B–H15B…Br2A = 2.95 Å; C17B–H17B…Br2A = 2.85 Å) between the discrete molecules in the structure (Table 6). These interactions, which are in the expected ranges (Horn et al., 2003; Steiner, 2002), connect the molecules together forming a three dimensional network (Fig. 4). Also the analysis of π–π and C–H…π interactions in this complex shows that the π–π interaction between the phenyl rings of the adjacent Zn1B molecules (Fig. 5) was 3.847 Å and an inter-planar atom–atom distance was 3.471 Å between the parallel stacking pairs with a dihedral angle of 0.0°. Several C–H…π interactions exist in the packing structure of the ZnLBr₂ complex (Fig. 5). The most significant interactions of these types are listed in Table 6. Distances and angles are well placed within the accepted range of C–H…π interactions (Nishio et al., 1998). These interactions play an important role for stabilizing the molecular structure and give rise to a 3D supramolecular structure.

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Figure 4

Aggregation of ZnLBr₂ compound via C–H...Br interactions.

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Figure 5

π–π and C–H…π interactions in ZnLBr₂ complex (green: Zn1A molecule; blue: Zn1B molecule).

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3.4 Thermal analysis (TG/DTG) of complexes

Investigation into thermal stability and existence of any water in the coordination sphere of the complexes has been performed at a heating rate of 10 °C/min under nitrogen atmosphere in the range from room temperature up to 1000 °C. As seen in Fig. 6, thermo-grams of all complexes did not show any weight loss up to 200 °C confirming the absence of any lattice or coordinated water molecules. Regarding TG/DTG plots of all complexes, it was found that ZnLCl₂ and ZnLI₂ complexes were decomposed via four thermal steps while the degradation of ZnLBr₂ occurred via about seven thermal steps. It is to be noted that the final steps are very near to each other so that they are distinguishable only based on DTG plot changes. Resulting data of these thermal steps including temperature range, mass loss (%) and thermo-kinetic activation parameters for each decomposition step have been listed in Table 7. The suggested lost segments at each decomposition step for all complexes have also been found in Table 7. As mentioned in Table 7, at the end of decomposition process, the complexes lose 88.22–98.40% of their total mass and finally leave out zinc metal as residue. As a typical model, the suggested decomposition pathway for the zinc chloride complex is illustrated in Fig. 7. This complex loses the segments of C₁₃H₁₁, C₃H₃, C₃H₅ and C₄H₇Cl₂N₂, via steps 1–4 respectively and some zinc metal residue remained in the final. In continuation of the thermal investigation, Arrhenius constant (A), activation energy (E^∗), enthalpy (ΔH^∗), entropy (ΔS^∗) and Gibbs free energy (ΔG^∗) of the decomposition process for each thermal step have been evaluated by using TG/DTG plots and Coats–Redfern relationship (Coats and Redfern, 1964). The positive values of activation energies (E^∗) evaluated in the range of 34.41–158.61 kJ mol⁻¹ for the thermal decomposition steps is an evidence of the thermal stability of the complexes. In all decomposition stages ΔH^∗ and ΔG^∗ values were found positive in the range of 28.69–153.73 kJ mol⁻¹ for ΔH^∗ and 161–256 kJ mol⁻¹ for ΔG^∗ reflecting endothermic character of decomposition processes. In most of the thermal decomposition steps, negative values in the range of −3.1 to −267 kJ mol⁻¹ K⁻¹ were obtained for entropy of activation (ΔS^∗). These negative values indicate that the processes are slower than the normal decomposition reactions or suggest that the thermally activated compounds have a more ordered or more rigid structure than that of the not-decomposed compounds/fragments (Vinodkumar et al., 2000; Ismail et al., 2012).

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Figure 6

TG/DTG plots of (A) ZnLCl₂, (B) ZnLBr₂ and (C) ZnLI₂.

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Figure 7

Proposed decomposition pathway of zinc chloride complex.

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3.5 Antibacterial and anti-fungal activity

Schiff base ligand and zinc(II) complexes were screened in vitro for their abilities as antibacterial and antifungal activities against E. coli and P. aeruginosa as Gram-negative; S. aureus and B. subtilis as Gram-positive bacteria and against C. albicans and A. oryzae as fungal strains. Antibacterial and antifungal activities as the zones of inhibition for all compounds are tabulated as Tables 8 and 9. Resulting data of these investigations revealed that the inhibitory effect is susceptible to the concentration of the compound used for inhibition so that the activity is greatly enhanced at the higher concentration of compound. In case of antibacterial activity, the ZnLI₂ complex was found highly effective against E. coli, S. aureus and B. subtilis in the following order B. subtilis > S. aureus > B. subtilis while against P. aeruginosa, ZnLCl₂ showed better antibacterial activity. Antifungal study of zinc complexes exhibited good activity against C. albicans and Aspergillus niger than the free ligand. The growth inhibitory effect of complexes against A. niger was found better than against C. albicans. The zinc bromide complex demonstrated the highest zone of inhibition in three concentrations against A. oryzae while zinc iodide complex showed the highest zone of inhibition against C. albicans.

In general, the activity of Schiff bases may be due to the chelating properties of the azomethine group used in metal transport across the bacterial membranes or in attaching to a specific site of the bacterial cells from which it can interfere with their growth (Rehman et al., 2008; Amin et al., 2010). Coordination of ligand to the metal ion makes the ligand a stronger antibacterial agent so that the zinc complexes show increased antibacterial and antifungal activity than the free ligand. This enhanced activity may be due to electron delocalization over the whole chelate ring upon complexation. Such chelation increases lipophilicity and enhances permeation through the lipid layer of the cell membrane (chelation theory) (Singh et al., 2009; Angelusiu et al., 2010).

3.6 DNA cleavage efficiency

Interactions between Zn (II) complexes and DNA were investigated by the cleavage assay of plasmid DNA (pMalC₂X DNA of E. Coli). The ability of compounds to cleavage plasmid DNA was analyzed by monitoring the conversion of supercoiled circular DNA (Form I) to nicked (Form II) and the linear (Form III) form of DNA. The amounts of strand scission were assessed by agarose gel electrophoresis. During electrophoresis, depending on the rate of migration, three forms of DNA are separated. The compact Form I migrates relatively fast while the nicked Form II migrates slowly, and the linearized form (Form III) migrates between Forms I and II. These changes are clearly observed in the gel electrophoresis by the change or disappearance of the intensity of the supercoiled band and appearance of new bands or smear in gel diagram (Fig. 8). In this figure L, X and C are attributed to the ladder, mixture of DNA and H₂O₂ and control DNA respectively. Lanes 1 to 3 are attributed to ZnLCl₂, ZnLBr₂ and ZnLI₂ mixed with DNA respectively. As illustrated in Fig. 8, a difference was observed in the bands of ZnLBr₂ and ZnLI₂ complexes (lanes 2 and 3) as compared to those of the control DNA showing the partial cleavage of DNA but for ZnLCl₂ (lane 1) any apparent cleavage of DNA is observed. The presence of a smear in the gel diagram indicates that the mechanism may involve radical cleavage (Kaczmarek et al., 2009; Refat et al., 2009; Zhang and Lippard, 2003). DNA cleavage potential of the compounds may suggest that the compound inhibits the growth of pathogenic organisms by cleavage of their genome.

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Figure 8

Agarose gel electrophoresis photograph for DNA cleavage study of Zn(II) complexes. Lane L = ladder, Lane X = DNA + H₂O₂, Lane C = control DNA. Lane 1 = DNA + ZnLCl₂, Lane 2 = DNA + ZnLBr₂ and Lane 3 = DNA + ZnLI₂.

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