Synthesis and Evaluation of Antimicrobial Activity of Metal Complexes of 4-(2'-Hydroxy Phenyl Imino) Phenyl Sulphonamide

Document Type: Research Paper


Department of Pharmaceutical Sciences, Utkal University, Vanivihar, Bhubaneshwar, Orissa, India


       Keeping in view the promising potential of carbonic anhydrase inhibitor based antimicrobials and enhancement of carbonic anhydrase inhibitory activity by metal complexes of sulfonamides, with an aim to develop better antimicrobial agents we have attempted investigation of antimicrobial activity of metal complexes of 4-(2'-hydroxy phenyl imino) phenyl sulphonamide. Sulfanilamide was taken as the starting material to synthesize 4-(2'-hydroxy phenyl imino) phenyl sulphonamide. Cu (II), Zn (II), Co (II), Ni (II) and Pb (II) complexes were synthesized following reported methods. The in vitro screening was carried out using two gram positive bacteria (S. aureus, E. faecalis) and two gram-negative bacteria (E. coli, P. aeruginosa) by disc diffusion method. Metal complexes were found to enhance theantimicrobial potental of the ligand.


1. Introduction

       Development of carbonic anhydrase (CA) inhibitor based antimicrobials has shown promising results because of the presence of carbonic anhydrases in a multitude of bacteria [1-4]. Metal complexes of sulfonamide CA inhibitors generally act as 10-100 times more potent inhibitors of isozymes CA I, CA II and CA IV compared to the parent sulfonamide from which they were obtained [5, 6]. In this context we have undertaken the antimicrobial evaluation of Zn (II), Cu (II), Pb (II), Ni (II) and Co (II) complexes of 4-(2'-hydroxy phenylimino) phenyl sulphonamide. For this purpose the in vitro susceptibility of two gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis) and two gram-negativebacteria (Escherichia coli, Pseudomonas aeruginosa) to the synthesized compoundswas investigated.


2. Materials and methods

2.1. Chemistry

      The purity of the ligand was checked by TLC using chloroform-methanol-DMF (100:05:05, v/v/v) as developing solvent and iodine as visualizing agent. Melting points were determined in open capillary tubes with the help of melting point apparatus (Sisco) and were uncorrected. The physical constants of        compounds are summarized in Table 1. Infrared spectra were recorded on Shimadzu-8400 S spectrophotometer using KBr powder. The conductances of the compounds were determined in DMF with conductivity meter (CM-180, Elico). The electronic spectra were recorded with the help of UV-Visible double beam spectrophotometer (CECIL CE 7200). The complexes were analysed for their metal content by Aanalyst-200 atomic absorption spectrophotometer (Perkin Elmer). Elemental analysis was done with Euro-EA analyzer. The title compounds were synthesized according to the published method starting from sulfanilamide [7-9].


2.2. Synthesis of 4-(2'-hydroxy phenyl imino) phenyl sulphonamide

      Sulfanilamide (0.05 mol) and salicylalde-hyde (0.05 mol) were added into 20 ml of absolute ethanol containing a few drops of glacial acetic acid in a 250 ml round bottomed flask. The reaction mixture was refluxed for 2 h. It was then cooled and ice-cold water was added .The product so formed was filtered, washed, dried and recrystallised from alcohol.


2.3. Synthesis of [CuL2(AcO)2]

      To an ethanolic (20 ml) solution of 4 - (2' - hydroxy phenyl imino) phenyl sulphonamide (0.008 mol), an ethanolic solution of the corresponding metal (II) acetate (0.004 mol) was added. The mixture was refluxed for 3 h. The solution was reduced to half of its volume. It was left overnight at room temperature. The product so obtained was filtered, washed repeatedly with water and dried. The Zn (II) and Pb (II) complexes were prepared in a similar manner.


2.4. Synthesis of  [CoL2Cl2]

      An ethanolic (10 ml) solution of 4 - ( 2 ' - hydroxy phenyl imino) phenyl sulphonamide (0.008 mol) was mixed with metal (II) chloride (0.004 mol) in ethanol (10 ml) followed by few drops of acetic acid (pH=6). The mixture was then refluxed for 1 h on a water bath till the complex precipitated. The solid product obtained was filtered, washed with distilled water and dried. In a similar process Ni (II) complex was synthesized.


Scheme 1. Metal complexes of 4-(2'-hydroxy phenyl imino) phenyl sulphonamide.

Figure 1. Relative antimicrobial activity of the synthesized compounds.


2.5. Evaluation of antimicrobial activity

      The in vitro screening was carried out using two gram-positive bacteria (S. aureus, E. faecalis,) and two gram-negative bacteria(E. coli, P. aeruginosa). Strains were obtained from Post Graduate Department of Microbiolgy, Orissa University of Agricultural Technology, Bhubaneshwar. The organisms were identified following the standard micro-biological methods [10]. The compounds were screened for their antibacterial activity using disc diffusion method [11-13]. The compounds were dissolved in dimethyl formamide (6%), which was previously tested for antibacterial activity against all test bacteria and found to have no antibacterial activity. A solution with a concentration of 30 mg/ml was made for each test compounds and finally sterilized by filtration using 0.45 m millipore filters. The sterile discs (Hi-media, 6 mm in diameter) were impregnated with 10 μl of the test solutions (300 μg/disc) and placed in inoculated agar. The density of the bacterial suspension was standardized by using McFarland standard method [11-13]. Nitrofurantoin (300 μg/disc) and ciprofloxacin (25 μg/disc) were used as standard drugs. The control was prepared using dimethyl formamide. The inoculated plates were incubated at 37 °C for 24 h. The antibacterial activity of test compounds against the bacterial strains is given in Table 3 as zone of inhibition. The relative antibacterial activity of test compounds is illustrated in Figure 1.

       The results obtained are expressed in meanE standard deviation of three determinations.


 Table 1. Physicochemical data of the synthesized compounds.


3. Results and discussion

     The IR band at 1674.27 cm-1 (Table 2) due to C=O stretching of salicylaldehyde shifted to 1626 cm-1 (C=N) in the spectrum of the ligand suggesting formation of Schiff base of salicylaldehyde and sulfanilamide. Low molar conductivities (5-11 2.mol-1) in DMF solutions measured for 1:2 (M:L) complexes indicate the non-electrolyte nature. The band for C=N stretching for ligand was observed at lower frequency by 9-35 cm-1 in the metal complexes, indicating participation of the azomethine nitrogen in the complexation. The shifting of the OH band at 3394.83 cm-1 in the spectrum of free ligand to 3338-3346 cm-1 in the spectra of metal complexes also indicate coordination of the oxygen of OH group to the metal ions. These suggest the bidentate coordination of ligand to the metal that is further supported by the appearance of weak, low frequency new bands at 530-550 cm-1 (M-N) and 440-450 cm-1 (M-O). The electronic spectra of these complexes are also consistent with an octahedral environment around the Co (II) ion. The spectra displayed band at, 29455 cm-1 (339.5 nm) attributed to 4T1g_4T2g transitions, in a low-spin octahedral geometry. The electronic spectra of Cu (II) complex showed band at 28169 cm-1 (355 nm) assigned to charge transfer bond, supporting octahedral geometry of the complex. The quantitative assay of elements and metals generated values close to the theoretical values of the metals and elements in the proposed complex. The analytical data in the study are in agreement with previously reported metal complexes [7] and the proposed structure.


Table 2. IR (KBr; n , cm-1), Electronic (nm), conductance and elemental data.



Table 3. In vitro antibacterial activities of the synthesized compounds against pathogens by disc diffusion method.

"-" Indicates no zone of inhibition. All the values are meanESD of three determinations. Values showed significant difference from solvent
control at p < 0.001.


      The antimicrobial activity of the ligand and its metal complexes were significantly different from that of solvent control (pE. coli, P. aeruginosa and E. faecalis. The Co (II), Zn (II) and Pb (II)complexes were equally active against S. aureus (Table 3). The Cu (II) and Ni (II)complexes were more potent than NF but the zones of inhibition were less compared to that of other metal complexes. From the relative antimicrobial action (Figure 1), it is evident that complexation of the ligand with metals has enhanced their antimicrobial potential. The enhancement was more pronounced in case of Pb (II), Co (II) and Zn (II) complexes. Further study to elucidate the pharmacokinetic behavior of these complexes and their in vivo action are to be made to prove their therapeutic utility.



      The authors are thankful to HOD, Department of Chemistry, Utkal University for providing instrumental facility and HOD, Department of Microbiology, OUAT for providing the pure bacterial strains.

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