Synthesis , Characterization , and Biological Activity of Some TransitionMetal Complexes of N-Benzoyl-N ′-2-thiophenethiocarbohydrazide

In the present study, Mn(II), Fe(II), Ni(II), and Cu(II) complexes of N-benzoyl -N′-2-thiophenethiocarbohydrazide (H2 BTTH) have been synthesized and characterized by elemental analysis, magnetic susceptibility measurements, infrared, NMR, electronic, and ESR spectral studies. The complexes were found to have compositions [Mn(H BTTH)2], [Ni(BTTH)(H2O)2], [Cu(BTTH)], and [Fe(H BTTH)2EtOH]. The antibacterial and antifungal properties of H2 BTTH and its metal complexes have been screened against several bacteria and fungi.


Introduction
The expansion of research in the coordination chemistry of nitrogen-sulphur donor ligands such as substituted thiosemicarbazides [1], thiosemicarbazones [2][3][4], and dithiocarbazates [5], during the recent years has been due to their remarkable antineoplastic activity against a variety of tumors [6] in addition to their applicable antifungal [7] and antibacterial [8] activities.Sulphur and nitrogen containing ligands and their transition metal complexes were also used as corrosion inhibitors [9,10] and extreme pressure lubricant additives [11].Keeping in view the above biological activity of the sulfur and nitrogen containing ligands, we planned to undertake the synthesis, characterization, antibacterial, and antifungal activity of N-benzoyl-N -2-thiophenethiocarbohydrazide (H 2 BTTH) (Figure 1) and its Mn(II), Fe(II), Ni(II), and Cu(II) complexes.This ligand is expected to form addition complexes without loss of protons, and deprotonated complexes by loss of one or both the hydrazinic protons.

Starting Materials.
All the chemicals used were of analytical grade.Ammonium polysulphide [12] and carboxymethyl-2-thiophenedithioate [13] were prepared by literature methods.

Preparation of N-benzoyl-N -2-thiophenethiocarbohydrazide (H 2 BTTH)
. N-benzoyl-N -2-thiophenethiocarbohydrazide (H 2 BTTH) was prepared by mixing solutions of benzoic acid hydrazide (20 mmol) and carboxymethyl-2thiophenedithioate (20 mmol) each dissolved separately in 50 mL of 0.5 N NaOH and allowing the mixture to stand at room temperature for 2 hrs.The product precipitated by adding dilute AcOH dropwise to the above ice-cold mixture, was filtered off, washed with H 2 O, dried, and recrystallized from EtOH.

Instrumentation.
Complexes were analyzed for their metal content, following a standard procedure [14] by decomposing the complexes with a mixture of HNO 3 and HCl followed by H 2 SO 4 .Sulfur and chloride were determined as BaSO 4 and AgCl, respectively.Carbon, hydrogen, and nitrogen were estimated on EA 1108 CHN Elemental Analyzer.Magnetic susceptibility measurements were made at room temperature on a Cahn-Faraday balance using Hg [Co (NCS) 4 ] as calibrant.Electronic spectra were recorded on a CARY-2390 UV-Visible Spectrophotometer as Nujol mulls [15].IR Spectra were recorded in the 4000-400 cm −1 region (KBr disc) on a JASCO FT/FR-5300 spectrophotometer.The 1 H and 13 C NMR spectra were obtained in DMSO-D 6 on a JEOL FX-300 Q FT/NMR spectrometer using TMS as internal reference.ESR spectra were recorded on a Xband spectrometer model EPR-112 using DPPH as a g marker.The electrical conductivity of the pressed pellets of the complexes was obtained by a conventional two-probe method in the 303-383 K range with contact made on the pellet surfaces using graphite paint.

Bactericidal Screening.
The antibacterial activity of the ligand and the complexes was evaluated using the disc diffusion technique [16].A stock solution of 2000 μg cm −3 was made by dissolving 2 mg cm −3 of each compound in DMSO and it was serially doubled diluted up to five dilutions, giving the concentrations of 1000, 250, 125, and 62.5 μg cm −3 .Filter paper (Whattman no.42) discs (6 mm dia) were soaked in these solutions of different concentrations and placed on nutrient agar plates.The plates were then incubated for 24 hrs at 37 • C. The inhibition zones around the discs were measured after 24 hrs.Co-trimoxazole was used as a standard drug in the form of disc, containing trimethoprim = 1.25 μg and sulfamethoxazole = 23.75 μg per disc.The zones of inhibition were found to be 20, 32, and 18 mm against Staph aureus, Escherichia coli, and Pseudomonas aeruginosa, respectively, in agreement with the sensitive zone reported in the literature.
2.8.Fungicidal Screening.The antifungal activity was evaluated by a drug dilution technique.The solution of the test compounds were prepared as described earlier to which sabouraud's dextrose broth and slightly turbid suspension of fungus in normal saline (10 μL) were added and placed in an incubator for 48-72 hrs.A turbidity in the solution indicated the growth of fungus, which is represented as -sign; however, a clear solution showed that there was no growth of fungus and is represented as + sign.The cases where compounds showed antifungal activity, no growth was observed in the solution.Amphotericin B was used as a standard drug.

Results and Discussion
All the complexes are insoluble in water, methanol, and ethanol but are soluble in polar organic solvents such as DMSO and DMF.It was determined by Job's method that the complexes having 1 : 1 metal-ligand stoichiometry (Table 1) are formed by loss of two protons from the ligand, generating a conjugated system.Because of steric considerations, all the four potential sites cannot be attached to a single metal and, therefore, the ligand binds in a polymeric fashion.The following equations represent the formation of the ligand and the complexes: 3.1.Magnetic Moments and Electronic Spectra.The magnetic moments and electronic spectral data of the complexes are given in Tables 1 and 2, respectively.[Mn(H BTTH) 2 ] shows a magnetic moment, 5.76 B.M. and exhibits a band   peak at 3125 cm −1 due to ν(NH), suggesting loss of one hydrazinic proton via enolisation/thioenolisation.A strong band at 1640 cm −1 in the ligand due to ν(C=O) is found to be absent in these complexes, and in place of this a new band due to ν(N=C) of NCO appears, suggesting that enolic oxygen is involved in bonding.[Mn(HBTTH) 2 ] shows a negative shift of 15 cm −1 in ν(C=S), suggesting an additional bonding through thione sulfur.Furthermore, the spectra of these complexes show a positive shift of 20 cm −1 in ν(N-N) Table 3: Important IR spectral bands (cm −1 ) and their assignments.complexes [19] suggesting the involvement of these groups as bonding sites.These observations show the involvement of thiolato sulfur and both the hydrazinic nitrogens, in addition to the enolic oxygen in bonding.Thus, H 2 BTTH acts as a binegative tetradentate ligand in the 1 : 1 complexes.

NMR Spectra.
The 1 H NMR spectrum of H 2 BTTH (Figure 4, Table 4) in DMSO-d 6 shows a signal at δ 9.88 and 9.24 ppm due to the presence of -NH-NH-protons which are lost on D 2 O exchange.The protons due to the thiophene ring appear at δ 7.12 (s,1H), 7.32 (d,1H), and 7.56 (s,1H) ppm and the benzene ring protons appear as a multiplet at at δ 6.64-6.92(m,5H) ppm [20].
The NH signals are absent in the 1 H NMR spectrum of [Ni(BTTH)(H 2 O) 2 ], (Figure 4, Table 4) suggesting loss of both NH protons via enolization and thioenolization.The thiophene ring protons show three separate signals at δ 7.08 (q, 1H), 7.28 (d, 1H), and 7.52 (s, 1H) ppm and the benzene ring protons are observed at δ 6.61-6.88(m,5H) ppm.4).The latter is formed due to the thioenolization of the ligand.All the thiophene and benzene ring protons are observed nearly at the same position in the complex as compared to those of the H 2 BTTH, suggesting noninvolvement of the ring sulphur in bonding.value of 2.02 characteristic of square planar geometry around Cu(II) (Figure 6).6) and its complexes has been tested against S. aureus, E. coli, and P. aeruginosa.H 2 BTTH, [Mn (H NTTH) 2 ] and [Cu(BTTH)] show antibacterial activity starting from 62.5 to 2000 μg cm −3 against S. aureus and the activity increases with an increase in the concentration.The ligand is found to be active at a concentration of 1000 μg cm −3 against E. coli.[Mn (H NTTH) 2 ] and [Cu(BTTH)] also show activity against E. coli at 500 and 250 μg cm −3 , respectively.Only [Cu(BTTH)] has been found active against P. aeruginosa at 250 μg cm −3 .
On the basis of physicochemical studies and the foregoing discussion, the proposed structures of the complexes is shown in Figure 7.

Conclusion
The magnetic and electronic spectral studies suggest square planar geometry for [Cu(BTTH)] and octahedral geometry for rest of the complexes.The infrared spectral studies of the 1 : 1 deprotonated complexes suggest bonding through enolic oxygen, thiolato sulfur, and both the hydrazinic nitrogens.
The 13 C NMR spectrum of [H 2 BTTH] (Figure 5, Table 5) shows eleven signals, of which two signals at δ 181 and 163 ppm are due to the C=S and C=O carbons, respectively.The chemical shifts for the benzene and thiophene ring carbons in H 2 BTTH are (δ, ppm) C(2, 6) 151, 146, C(4) 136,

Figure 7 :
Figure 7: Proposed structure of the complexes.

Table 1 :
Analytical data and physical properties of the complexes of N-benzoyl-N -2-thiophenethiocarbohydrazide.

Table 2 :
Electronic spectral bands and their assignments.

Table 6 :
Antibacterial activity of H 2 BTTH and its complexes.Pseudomonas aeruginosa.Control: Co-trimoxazole.+ and −: over the parentheses indicate the zone of inhibition and no zone of inhibition, respectively.

Table 7 :
Antifungal activity of H 2 BTTH and its complexes.Candida albicans.Control: Amphotericin B. Growth and no growth of fungi are represented by − and + sign, respectively.Thus, H 2 BTTH acts as a binegative tetradentate ligand.The ESR spectrum of [Cu(BTTH)] shows that the unpaired electron is present in the d x 2 −y 2 orbital of Cu(II).