Correlation between the structure and biological activity studies of supramolecular coordination azodye compounds

2.1 Synthesis of ligands

(4-Alkylphenylazo)-5-sulfo-8-hydroxyquinolines (HL_n) were typically prepared by adding 25 mL of distilled water containing hydrochloric acid (12 M, 2.68 mL, 32.19 mmol) to aniline (10.73 mmol) or p-derivatives. To the resulting mixture, stirred and cooled to 0 °C, a solution of sodium nitrite (10.73 mmol, in 20 mL of water) was added dropwise. The so-formed diazonium chloride was consecutively coupled with an alkaline solution (sulfonate) (10.73 mmol) in 20 mL of ethanol containing 602 mg (10.73 mmol) of potassium hydroxide. Immediately, the formed red precipitate was filtered and washed several times with water. The obtained crude product was purified by crystallization from hot ethanol (yield ∼ 60–80%). The analytical data were confirmed by expected composition (Table 1). The ligands were also characterized by ¹H NMR and IR spectroscopy.

The synthesis of ligand is summarized in Fig. 1. However a detailed procedure is given in the following reactions:

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Figure 1 The formation mechanism of azodye ligands (HL_n). n = 1, R = OCH₃ (HL₁); n = 2, CH₃ (HL₂); n = 3, H (HL₃); n = 4, Cl (HL₄); and n = 5, NO₂ (HL₅).

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The resulting ligands are:

• (4-Methoxyphenylazo)-5-sulfo-8-hydroxyquinoline (HL₁).

• (4-Methylphenylazo)-5-sulfo-8-hydroxyquinoline (HL₂).

• (4-Phenylazo)-5-sulfo-8-hydroxyquinoline (HL₃).

• (4-Chlorophenylazo)-5-sulfo-8-hydroxyquinoline (HL₄).

• (4-Nitrophenylazo)-5-sulfo-8-hydroxyquinoline (HL₅).

2.2 Synthesis of metal complexes

The appropriate ligand (0.01 mol) dissolved in ethanol (20 cm³) was added dropwise into an ethanolic solution (20 cm³) of metal salt (0.01 mol) with stirring. After the complete addition, 0.50 g of sodium acetate was added to the solution and the reaction mixture was refluxed for 2 h. The solution, thus obtained was concentrated to half of its original volume by evaporation using a hot plate and allowed to cool to room temperature. During this, a microcrystalline solid was separated, which was isolated by filtration, washed with hot water followed by ethanol and ether and dried in air.

2.3 Microbiological investigation

For this investigation the agar well diffusion method was applied (Alghool et al., 2010). The antibacterial activities of the investigated compounds were tested against two local Gram positive bacteria (Bacillus cereus and Staphylococcus aureus) and two local Gram negative bacteria (Escherichia coli and Klebsiella pneumoniae) on the nutrient agar medium. Also, it was tested against four local fungal species (Aspergillus niger, Alternaria alternata, Penicillium italicum and Fusarium oxysporium) on the DOX agar medium. The concentrations of each solution were 50, 100 and 150 μg/ml. By using a sterile cork borer (10 mm diameter), wells were made in agar medium plates previously seeded with the test organism. 200 μl of each compound was applied to each well. The agar plates were kept at 4 °C for at least 30 min to allow the diffusion of the compound. The plates were then incubated at 37 °C or 30 °C for bacteria and fungi respectively. Penicillin and miconazole were used as antibacterial and antifungal substrates respectively. The diameters of inhibition zone were determined after 24 h and 7 days for bacteria and fungi, respectively.

2.4 Measurements

Elemental microanalyses of the separated ligands and solid chelates for C, H, S, and N were performed in the Microanalytical Center, Cairo University, Egypt. The analyses were repeated twice to check the accuracy of the analyzed data. The ¹H-NMR spectrum was obtained with a JEOL FX90 Fourier transform spectrometer with DMSO-d₆ as the solvent and TMS as an internal reference. Infrared spectra were recorded as KBr pellets using a Pye Unicam SP 2000 spectrophotometer. Ultraviolet–Visible (UV–Vis) spectra of the compounds were recorded in nuzol solution using a Unicom SP 8800 spectrophotometer.

3.1 Structure and stability

The presence of a sulfonate group in the quinoline ring confers special characteristics to the ligand, introducing changes in spectroscopic and structural properties of the metallic complexes.

It has been known that 8-hydroxyquinoline exists, in solution, in a monomer dimer equilibrium (El-Sonbati et al., 2002, 2010a, 2012b; Cook and Rotello, 2002; Albrecht et al., 1999). Our results of this paper suggest that in the monomeric form a strong intramolecular hydrogen bond is present. This is in agreement with previous results (Suzuki, 1967). The two such monomers lead to the dimer by forming an additional hydrogen bond yielding the bifurcated hydrogen bonds and H–N–H nitrogen bridges (Fig. 2A).

In addition to the two bifurcated intra/intermolecular OH … N hydrogen bonds (Figs. 1 and 3), two more intermolecular hydrogen bonding interactions are observed between nitrogen atom of the azo/azomethine group and hydrogen atom of the hydroxyl group. This additional H-bond does not influence the intramolecular distance which shows a band at a lower frequency than the intermolecular interaction. Reason for this behavior might be due to the additional H-bond which influences the hydrogen bonding ability of the sulfonyl group by electronic and/or steric factors. The overall structure of the dimer is close to planar with a slight shift of the two quinoline units from the plane. The two hydroxyquinoline units of the dimer (Fig. 2B′ and C₂) are in one plane. The intermolecular as well as intramolecular hydrogen bonding occur between the hydroxyl group and the quinoline nitrogen atom. The intermolecular hydrogen bond distance is shorter than the intramolecular one. This observation was also reported for other 8-hydroxyquinoline dimers and might be due to an unfavored small O–H–N angle for the intramolecular interaction (Jeffery et al., 1989; Cook and Rotello, 2002; Albrecht et al., 1999).

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Figure 3 Intermolecular hydrogen bond.

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Hydrogen bonding represents one of the most versatile interactions that could be used for molecular recognition. In view of the large differences in the substituent effects (e.g., the Hammett-type substituent constants for p-positions and sulfonyl group); it might be possible to tune the strength of the hydrogen bond effectively by linking the hydrogen-bonding site to a reaction center through a conjugated spacer, and by altering the charge state of the reaction center in the solution. At the hydrogen-bonding end, azo/azomethine is used as a proton acceptor to form a hydrogen bond with the OH group of ligand.

An electron-withdrawing bridge would be expected to increase the acidity of the proton donor and hence increase its binding ability. As the electron-withdrawing character of an azo group is relevant to the interesting signal-amplifying behavior (Suzuki, 1967) the results indicate that in the HL_n, the effects of the bridges are electron-withdrawing and electron-donating, respectively. Accordingly, the efficiency of sp²-hybridized bridges is N⚌N > C⚌N.

Coggeshall and El-Sonbati et al. (Coggeshell, 1947; El-Sonbati et al., 2002a, 2004a, 2007, 2010a, 2012b) found three kinds of bonded –OH structures on the basis of the frequencies: (i) only oxygen is in the bridge while hydrogen is free, (ii) a polymer chain is formed in which both hydrogen and oxygen atoms participate in the hydrogen bond, and (iii) dimer associates are formed.

Intramolecular hydrogen bond between nitrogen atom of C⚌N(CN_Py)/–N⚌N– (five/six-membered) system and hydrogen atom of phenolic hydroxyl hydrogen atom and hydrogen (C₈–OH) are illustrated in Fig. 2B, C and C′. Intermolecular hydrogen bonding can form a cyclic dimer through the O−H---OH type between C₈–OH/⚌N of one molecule and C₈–OH/N⚌N group of another one (Fig. 3H, G and F) and/or - - - -N type between C₈–OH of one molecule and CNpy/–N⚌N– of another (Fig. 3D).

In general, hydrogen bonds involving OH groups are proton donors and their O atoms are proton acceptors. Both intra and intermolecular OH–N may form a number of structures in a simultaneous equilibrium.

3.2 ¹H NMR spectra

The ¹H NMR spectra of all the ligands were recorded in DMSO-d₆ at room temperature. The signal due to methyl and methoxy proton appeared as a singlet at 1.55 and 3.84 ppm, respectively. In the aromatic region, a few doublets and in few cases some overlapping doublets/multiplets are observed in the range δ 6.78–8.40 ppm. These doublets/multiplets are due to aryl protons of three benzene rings. Another singlet corresponding to one proton for all compounds is observed in the range δ ∼ 9.2–10.40 ppm. This signal disappeared when a D₂O exchange experiment was carried out. It can be assigned either to OH or NH, in either case it is strongly deshielded because of hydrogen bonding with the other atom (N/O) (Figs. 2 and 3). It may be noted that the integration of this signal perfectly matches with one proton and there is no other fragment(s) of this signal, which suggest that only one tautomeric form of the ligand exists in the solution under the experimental conditions. Comparing with the solid state study, we prefer to assign this signal to OH, however, assignment of this peak to NH cannot be ruled out provided solid state structural evidence is not considered (Jadeja et al., 2004). As reported in previous studies (El-Sonbati et al., 2011; Shoair et al., 2001), this hydrogen bonding lead to a large deshielding of these protons. The shifts are in the sequence: p-NO₂ > p-Cl > H > p-OCH₃ > p-CH₃. The appearance of signals due to HC⚌N [∼8.94 ppm (1H)] protons of the same positions in the ligand and its diamagnetic complexes shows the non-involvement of this group in coordination (Shoair et al., 2001).

3.3 Electronic spectra

HL_n exhibited bands at 32,500–32,150 cm⁻¹ (CN) (π–π^∗), 33,450–33,340 cm⁻¹ (H-bonding and association), 40,038–39,460 cm⁻¹ (phenyl) (Ph–Ph^∗, π–π^∗) and 29,340–29,230 cm⁻¹ transition of phenyl rings overlapped by a composite broad (π–π^∗) of the azo structure. The band due to the n → π^∗ transition obtained in the visible region is associated mainly with the color of the respective compound (El-Sonbati et al., 2012b). The band due to π → π^∗ transition moves to lower energy. These shifts or the disappearance of the bands is indicative of coordination of the ligands to M(II). The position of their bands varied from one dye to the other which may be attributed to the p-phenylazo substituent’s variable donating power.

In general, most of the azo compounds give spectral localized bands in the wavelength range 46,620–34,480 cm⁻¹ and 31,250–270, 370 cm⁻¹. The first region is due to the absorption of the aromatic ring compared to ¹B_b and ¹L_b of mono substituted benzene and the second region is due to the conjugation between the azo group and the aromatic nuclei with intermolecular charge transfer resulting from π-electron migration to the diazo group from electron donating substituents. The p-substituents increase the conjugation with a shift to a longer wavelength. Most of the simple p-substituted compounds are in the azoid form in cyclohexane and alcohols. The substituted effect is related to the Hammett’s constant values (El-Sonbati et al., 2010c, 2012b). The position of the π–π^∗ transition of the azo groups remains one of the most interesting unanswerable questions of molecular spectroscopy. For azo benzenes, as the possibilities of the mesomerism became greater, the stabilization of the excited state is increased relative to that of the ground state and a bathochromic shift of the absorption bands follows (El-Sonbati et al., 2010c). Based on the MO theory the energy terms of the molecular orbital became more closely spaced as the size of the conjugated system increases (Jean, 2004). Therefore, with every additional conjugated double bond the energy difference between the highest occupied and the lowest vacant π-electron level became smaller and the wavelength of the first absorption band corresponding to this transition is increased. The azo group can act as a proton acceptor in hydrogen bonds (El-Sonbati et al., 2010c, 2012b). The role of hydrogen bonding in azo aggregation has been accepted for some time.

3.4 Microbiological investigation

The antimicrobial activity of HL_n ligand was tested against bacteria and fungi; we used more than one test organism to increase the chance of detecting their antimicrobial activities. The used organisms in the present investigations included two Gram positive bacteria (B. cereus and S. aureus) and two Gram negative bacteria (E. coli and K. pneumoniae) in addition to four species of fungi (i.e. A. niger, P. italicum, Alternaria alternata and F. oxysporium). The results of the antibacterial activities of the synthesized compounds are recorded in Table 2. All the used HL_n and some of their metal complexes were found to have antibacterial activity against Gram negative bacteria namely; E. coli (inhibition zone of HL₁ = 5 and 4 mm at concentration = 50 and 100 μg/ml) and inhibition zone of HL₄ = 2 mm and CuL₄ = 3 mm and NiCl₂ = 3 mm at concentration = 100 μg/ml). In addition to that K. pneumoniae was also affected by HL_n and their metal complexes i.e. (inhibition zone of HL₁ = 3 mm and CuL₁ = 4 mm at concentration = 50 μg/ml) and (inhibition zone of HL₃ = 2 mm and CuL₃ = 4 mm at concentration = 50 μg/ml. CuL₂, CuL₃ and CuL₅ were found to have high antibacterial activity than HL₂, HL₃ and HL₅ against K. pneumoniae, this means CuL_n complexes are more active than HL_n as shown in Figs. 4 and 5. Whereas the HL_n and their derivatives also have effect against Gram positive bacteria; namely B. cereus (inhibition zone of HL₃ = 2 mm and HL₄ = 4 mm at concentration = 50 μg/ml) and inhibition zone of CuL₃, CuL₄ and CuL₅ = 2, 2.5 and 3 mm, respectively at concentration = 50 μg/ml). But very low effects were recorded against S. aureus. HL₄, HL₅ and HL₁ are more active than penicillin against B. cereus, E. coli and K. pneumoniae as shown in Fig. 6.

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Figure 4 Effect of HL₂ and HL₅ on growth of Klebsiella pneumoniae on the nutrient agar medium using concentrations a = 50 μg/ml and b = 150 μg/ml.

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Figure 5 Effect of CuL₂ and CuL₅ on growth of Klebsiella pneumoniae on the nutrient agar medium using concentrations a = 50 μg/ml and b = 150 μg/ml.

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Figure 6 Antibacterial activity data of (4-alkylphenylazo)-5-sulfo-8-hydroxyquinolines (HL_n) and their metal complexes. Inhibition zones were recorded as mm.

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Zhandarev et al. (2006) found that dichlorotetrahydroquinolinol and dichloromethyloxyquinolinol caused the maximum antibacterial effect with respect to Gram negative bacterial species (i.e. E. coli). The same previous authors recorded that some quinolinols and their tetrahydro derivatives caused pronounced growth inhibition of S. aureus. The results (Table 2) reveal that the ligand is moderately toxic against bacteria, while all the chelates are more toxic than ligand. Among all the chelates the Cu²⁺ chelates are toxic against bacteria, also Cu²⁺ complexes are more active than Ni²⁺ complexes against B. cereus, E. coli and K. pneumoniae. The relation between the values of inhibition zone and ionic radius for complexes is shown in Figs. 7 and 8 and it is clear that the values of inhibition zone increase with increasing ionic radius. Our results of the antifungal activities of the synthesized compounds are recorded in Table 3. The results of the examination of antifungal activity of HL_n ligands (Table 3) revealed that the ligands have low toxicity than their complexes against fungi as shown in Fig. 9. Patel (2009) found that 5-(4-N,N-Diethylaminosulfonly phenylazo)-8-hydroxy quinolines and their metal chelates have moderate and high toxicity against some fungi including A. niger and Penicillium expansum. The same previous author found that Cu²⁺ chelates are more toxic against the tested fungi than other chelates.

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Figure 7 The relation between ionic radius and inhibition zone (mm) (In the case of using concentration = 150 μg/ml against Bacillu scereus).

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Figure 8 The relation between ionic radius and inhibition zone (mm) (In the case of using concentrations a = 50 μg/ml and b = 150 μg/ml against Escherichia coli).

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Figure 9 Antifungal activity data of (4-alkylphenylazo)-5-sulfo-8-hydroxyquinolines (HL_n) and their metal complexes. Inhibition zones were recorded as mm.

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As shown in Tables 2 and 3, the values of inhibition zone for ligands (HL_n) are related to the nature of the p-substituent as they increase according to the following order p-(NO₂ > Cl > H > CH₃ > OCH₃) (El-Sonbati et al., 2011, 2012a; El-Ghamaz et al., 2011, 2012; Abou-Dobara et al., 2013). This can be attributed to the fact that the effective charge experienced by the d-electrons increased due to the electron withdrawing p-substituent (HL₄ and HL₅) while it decreased by the electron donating character of HL₁ and HL₂. This is in accordance with that expected from Hammett’s constant σ^R as shown in Figs. 10 and 11 correlates the values of inhibition zone (mm) with σ^R, it is clear that these values increase with increasing σ^R. It is important to note that the existence of a methyl and/or methoxy group enhances the electron density on the coordination sites and simultaneously decreases the values of inhibition zone.

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Figure 10 The relation between Hammett’s substitution coefficient (σ^R) and inhibition zone (mm) (In the case of using concentrations a = 100 μg/ml (HL_n) and b = 50 μg/ml (CuL_n) against Bacillus cereus).

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Figure 11 The relation between Hammett’s substitution coefficient (σ^R) and inhibition zone (mm) (In the case of using concentrations a = 150 μg/ml (HL_n) and b = 100 μg/ml (CuL_n) against Fusarium oxysporium).

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