Antimicrobial metal-based thiophene derived compounds

Abstract A novel series of thiophene derived Schiff bases and their transition metal- [Co(II), Cu(II), Zn(II), Ni(II)] based compounds are reported. The Schiff bases act as tridentate ligands toward metal ions via azomethine-N, deprotonated-N of ammine substituents and S-atom of thienyl moiety. The synthesized ligands along with their metal complexes were screened for their in vitro antibacterial activity against six bacterial pathogens (Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus and Bacillus subtilis) and for antifungal activity against six fungal pathogens (Trichophytonlongifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glabrata). The results of antimicrobial studies revealed the free ligands to possess potential activity which significantly increased upon chelation.


Materials and methods
All the chemicals used were of analytical grades and purchased from Sigma Aldrich and were used without further purification. All ligand synthetic reactions were carried out in solvents that were purified and dried before use, using standard literature methods. Gallenkamp melting point apparatus was used for recording melting points. The infrared absorption spectra were recorded as KBr pellets on Excalibur FTR-IR 4800 MX spectrophotometer (Bio-Rad Laboratories, Cambridge, MA) in the frequency range 200-4000 cm À1 . Elemental analysis was carried out on Perkin Elmer. 1 H and 13 C NMR spectra were recorded on a Bruker SpectrospinAvance400 DPX spectrometer using TMS as internal standard and d 6 -DMSO as a solvent. Electron impact mass spectra (EIMS) were recorded on JEOL MS Route instrument. UV-Vis spectra were recorded on ultraviolet spectrometer-1700 in the frequency range of 250-800 nm. Conductivity meter Jen way model 70 was used for measuring the conductivity of metal complexes using 0.001 molar solutions in DMSO at room temperature. A Stanton SM12/S Gouy balance was used to measure the magnetic susceptibility of the metal complexes at room temperature using mercury acetate ligand as a standard. In vitro antibacterial and antifungal properties were studied at HEJ Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Pakistan.

Synthesis of ligands
1-(Thiophene-2-yl)ethanone (0.002 M, 0.25 g) dissolved in ethanol (20 mL) was added to a stirred solution of ethane-1,2-diamine/propane-1,3-diamine/butane-1,4-diamine respectively in ethanol (15 mL) in an equimolar ratio followed by 2-3 drops of acetic acid. The reaction mixture was stirred for 8 h with continuous monitoring through TLC and then kept overnight at room temperature. The solid crystallized product was obtained which was filtered, washed with ethanol and dried. It was recrystallized in a mixture of hot ethanol:acetone (1:1).

Synthesis of metal(II) complexes
All metal complexes were prepared according to the standard procedure in which ethanol solution (15 mL) of the respective metal(II) salt (5 mmol) was added respectively into a refluxed ethanol solution (30 mL) of the Schiff base ligand (10 mmol). The mixture was refluxed for 3 h, during which a precipitated product was formed. It was then cooled to room temperature, filtered, washed with hot ethanol and finally with diethyl ether. The precipitated product thus obtained was dried and crystallized in a mixture of hot aqueous methanol (1:2) to obtain TLC pure product.

Antibacterial activity (in vitro)
Agar-well diffusion method 17 was used to test antimicrobial activity and toxicity of all the newly synthesized acetyl thiophene derived Schiff bases (L 1 )-(L 3 ) and their resulting metal (II) complexes (1)-(12) against four gram-negative (E. coli, S. flexneri, P. aeruginosa and S. typhi) and two gram-positive (S. aureus, B. subtilis) bacterial strains ( Figure 1). The wells (6 mm in diameter) were dug in the media with the help of a sterile metallic borer with centers at least 24 mm apart. Bacterial inoculums after 6-8 h of growth approximately having104-106 colony forming units (CFU)/ mL were used. The recommended concentration of the test sample (1 mg/mL in DMSO) was introduced in the respective wells. Other wells supplemented with DMSO and reference antibacterial drug (imipenem) served as a negative and positive control, respectively. The plates were incubated at 37 C for 24 h. The activity was determined by the measurement of the diameter of zones which showed complete inhibition (mm). Separate studies of DMSO were carried out to rule out any role of DMSO in antibacterial activities but expectedly it showed no activity against any of the bacterial strains used in this study.

Antifungal activity (in vitro)
Antifungal activities of all compounds were studied 17 against six fungal strains (T. longifusus, C. albicans, A. flavus, M. canis, F. solani and C. glabrata). Sabouraud dextrose agar (Oxoid, Hampshire, England) was seeded with 10 5 (cfu) mL À1 fungal spore suspensions and transferred to petri plates. Discs soaked in 20 mL (200 lg/mL in DMSO) of the compounds were placed at different positions on the agar surface. The plates were incubated at 32 C for 7 d. The results were recorded as a percentage of inhibition and compared with standard drugs miconazole and amphotericin B.

Minimum inhibitory concentration (MIC)
Compounds showing promising antibacterial (>80%) activity were selected for minimum inhibitory concentration (MIC) studies 18 . Disc diffusion method was applied to determine minimum inhibitory concentration by preparing discs containing 10, 25, 50 and 100 lg mL À1 concentrations of the compounds along with standards at the same concentrations.
All the synthesized metal(II) complexes were microcrystalline in nature and were intensely colored except the Zn(II) complexes, which were off-white in color. The metal(II) complexes decomposed without melting and were soluble in ethanol, methanol, DMSO and DMF. The analytical data of the complexes indicated a 1:2 (metal: ligand) stoichiometry. The spectral data as well as elemental analysis of the synthesized ligands and their metal(II) complexes were in good agreement with their structure, indicating the high purity of all the compounds.

IR spectra
The IR spectra of Schiff bases reveal presence of the active donor sites like azomethine-N (-C¼N), aliphatic ammine (-NH 2 ) and C-S moieties which are responsible for chelation with the transition metal atoms. The Schiff bases (L 1 )-(L 3 ) possessed typical azomethine (-C¼N) stretching 19 at 1655-1683 cm À1 , hence confirming production of the required product. One of each amino group remained unchanged during condensation reaction and their bands were also observed at 3348-3387 cm À1 , respectively, as they did not take part in the reaction. All the ligands showed C-N and C-S stretching 20 at 1062-1065 and 882-889 cm À1 , respectively. The IR spectral comparison of the Schiff bases (L 1 )-(L 3 ) with their metal(II) complexes (1)- (12) indicated that the Schiff bases were principally coordinated to the metal(II) ions in a tridentate manner. The vibrations of azomethine group in all the metal complexes (1)- (12) shifted to lower frequency (10-20 cm À1 ) at 1638-1668 cm À1 demonstrating the coordination of the azomethine nitrogen 21 with the metal(II) atoms. Shifting of (-NH 2 ), C-N and C-S vibrations at 3348-3387, 1062-1065 and 882-889 cm À1 to 3330-3375, 1040-1045 and 868-877 cm À1 , respectively, supported the coordination of ligands with metal atoms. Appearance of the new bands at 543-580 and 460-471 cm À1 could be assigned 22 to v(M-N) and v(M-S) vibrations in their metal complexes, which were actually absent in their ligands also confirmed the mode of chelation. Therefore, these clues supported the evidence of the participation of azomethine-N, thienyl-S in the coordination. This data provides good evidence for the coordination of the metal(II) ions to the synthesized Schiff base ligands. 1 HNMR spectra 1 H NMR spectra of the Schiff bases (L 1 )-(L 3 ) and their diamagnetic Zn(II) complexes were recorded in DMSO-d 6 and spectral data is provided in the experimental section. The 1 H NMR spectra of Schiff basesdisplayed 23 characteristic amino (NH 2 ) protons at 5.70-5.75 ppm as a singlet which provided evidence for the condensation of only one amino group of diammines with acetyl thiophene. All of the ligands (L 1 )-(L 3 ) showed methyl (CH 3 ) protons at 2.31-2.35 ppm as a singlet and thienyl protons at 7.02-7.60 ppm as a doublet and double of doublet. The methylene (CH 2 ) protons of ligand (L 1 ) were observed at 3.06-3.71 ppm as a triplet, propylene protons of ligand (L 2 ) were found at 2.55-3.65 ppm as a triplet and multiplet. Similarly, butylene protons of ligand (L 3 ) appeared at 2.46-3.70 ppm as a triplet and multiplet. The complexation of the azomethine (C¼N) nitrogen was proven by the downfield shifting of the methyl protons in their Zn(II) complexes, which could be attributed to the discharging of electronic cloud toward the Zn(II) ion. All protons underwent downfield shift by 0.06-0.18 ppm due to the increased conjugation on coordination with the zinc metal atom. Largely, the number of protons calculated from the integration curves 24 and obtained values of the CHN analysis agreed were found to be in agreement.  24 . Furthermore, the conclusions drawn from these studies present further support to the modes of bonding discussed in their IR and 1 H NMR spectra.

Mass spectra
The mass fragmentation pattern of the ligands (L 1 )-(L 3 ) followed the cleavage of C¼ N, C-N, N-H, C-C, C¼ C and C-S bonds. The mass spectral data and the most stable fragmentation values of the ligands were depicted in the experimental section. All the ligands showed pronounced molecular ion peaks. The mass spectra of ligand (L 1 ) displayed a molecular ion peak at m/z 168.1 (calc. 168.26) of [C 8 H 12 N 2 S] þ , which loses a methyl group (CH 3 ) as a radical to give the most stable fragment at m/z 153 of [C 7 H 9 N 2 S] þ . Similarly, the molecular ion peak of ligand (L 2 ) was observed at m/z 182.2 (calc. 182.29) of fragment [C 9 H 14 N 2 S] þ and most stable fragment [C 8 H 11 N 2 S] þ was found at m/z 167. In the same way, the molecular ion peak [C 10 H 16 N 2 S] þ of ligand (L 3 ) appeared at 196.2 (calc. 196.31) and its base peak [C 9 H 13 N 2 S] þ was observed at m/z 181. The mass spectral data of the Schiff bases strongly confirmed the best possible structure of ligands and also their bonding pattern.

Molar conductances and magnetic measurements
The molar conductance of the metal complexes was studied when dissolved in DMSO. The data of molar conductance was (86.8-65.2 ohm À1 cm 2 mol À1 ) found to be in the range which indicated the electrolytic 25 (Table 1).

Electronic spectra
The electronic spectral values of the metal(II) complexes are recorded in Table 2. The electronic spectra of Co(II) complexes displayed well resolved absorption bands in the region 7590-7645, 17 490-17 642 and 23 134-23 252 cm À1 which may be assigned to 4 T 1 g ! 4 T 2 g(F), 4 T 1 g ! 4 A 2 g(F) and 4 T 1 g ! 4 Tg(P) transitions, respectively 29 . A high-energy band at 29 858-30 030 cm À1 was due to metal ! ligand charge transfer. These bands suggest  Table 2. Conductivity, magnetic and spectral data of metal(II) complexes (1)- (12). No.
88. presence of a high-spin octahedral geometry around the Co(II) ion. The electronic spectral data of Ni (II) complexes exhibited 30 the  d-d bands in the region of 9875-9939, 15 263-15 379 and 25 743-25 865 cm À1 , which could be assigned to the spin allowed transitions of 3 A 2 g(F) ! 3 T 2 g(F), 3 A 2 g(F) ! 3 T 1 g(F) and 3 A 2 g(F) ! 3 T 1 g(P), respectively, consistent with the octahedral geometry. A high-intensity band was also observed at 29 585-29 690 cm À1 due to metal ! ligand charge transfer. The spectra of Cu(II) complexes demonstrated two weaker bandsat 15 681-15 802 and 19 415-19 617 cm À1 , which represent the transitions 2 B 1g ! 2 A 1g and 2 B 1g ! 2 E g , respectively, indicating their octahedral geometry. The highenergy band was also observed at 29 892-29 967 cm À1 assigned to the metal ! ligand charge transfer. On the basis of electronic spectra, octahedral geometry around the Cu(II) ion was suggested 31

[Zone of inhibition (mm)]
Gram-negative Gram-positive against C. glabrata. It is obvious from the data reported in Table 4 that ligand (L 1 ) displayed overall good fungal activity as compared to the other three ligands. The Zn(II) complex (4) of (L 1 ) was found to be the most active complex. The metal(II) complexes showed enhanced 34 activity results compared to their non-coordinated Schiff bases upon complexation ( Figure 2).

Minimum inhibitory concentration (MIC)
The transition metal(II) complexes which displayed promising antibacterial activity (more than 80%) were selected for MIC studies and results are presented in Table 5. MIC values of the metal complexes fell in the range of 18.42-39.91 lg/mL. Amongst these, the complex (1) was found to be the most active possessing maximum inhibition 18.42 lg/mL against bacterial strain E. coli.

Conclusions
The newly synthesized Schiff bases acted as tridentate ligands for chelation with the Co(II), Cu(II), Ni(II) and Zn(II) metal atoms. Physical (magnetic and molar conductance), spectral (IR, NMR and electronic) and analytical (C, H, N, Co, Cu, Ni and Zn%) data established that the Schiff base ligands are coordinated with the metal ions through azomethine-N, thienyl-S and ammine-N presenting an octahedral geometry. The obtained results of antibacterial and antifungal activities revealed that the metal complexes are found to be more biological active against one or more bacterial and/or fungal strains as compared to their parent non-coordinated Schiff base ligands. Usually, it is claimed that the functional groups like azomethine-N, thienyl-S and ammine-N present in the compounds are responsible for the enhancement of bacterial and fungal activities. These findings propose that the synthesized metal-based compounds have the potential to be developed therapeutic agents.