Syntheses , Characterization , and Antimicrobial Screening of N-( benzothiazol-2-yl ) benzenesulphonamide and its Cu ( I ) , Ni ( II ) , Mn ( II ) , Co ( II ) , and Zn ( II ) Complexes

Abstract: N-(benzothiazol-2-yl)benzenesulphonamide (BS2ABT) was synthesized by the condensation (by refluxing) of 2-aminobenzothiazole and benzenesulphonylchloride in acetone at 140°C. The resulting crude precipitates were recrystallized from dimethylformamide (DMF). Five metal complexes of copper(I), nickel(II), manganese(II), cobalt(II) and zinc(II) of the ligands were synthesized. The compounds were characterized using magnetic susceptibility measurements, UV/VIS spectrophotometry, infra red, proton and C nmr spectroscopies. The antimicrobial tests of the ligands and its metal complexes were carried out on both multi-resistant bacterial strains isolated under clinical conditions and cultured species using agar-well diffusion method. The multi-resistant bacterial strains used were Escherichia coli, Proteus species, Pseudomonas aeroginosa and Staphylococcus aureus which were isolated from dogs. The culture species were Pseudomonas aeruginosa (ATCC 27853), Escherichia Coli (ATCC 25922) Staphylococcus aureus (ATCC 25923), and the fungi, Candida krusei (ATCC 6258) and Candida albicans (ATCC 90028). The tests were both in vitro and in vivo. Thus the Inhibition Zone Diameter (IZD), the Minimum Inhibitory Concentration (MIC), and the Lethal and Effective Concentrations (LC50 and EC50) were determined. The antimicrobial activities of the compounds were compared with those of Ciprofloxacin and trimethoprim-sulphamethoxazole as antibacterial agents and Fluconazole as an antifungal drug. All the compounds showed varying activities against the cultured typed bacteria and fungi used. However, they were less active than the standard drugs used except Fluconazole which did not show any activity against Candida krusei (ATCC 6258) but most of the compounds synthesized were very active against it. The Lethal Concentration (LC50) ranged from 26.25±4.9-1833.88±186.92 ppm. These are within the permissible concentrations.


Introduction
Interest in the coordination chemistry of thiazole and its derivatives with metal ions has risen due to the important role they play in biological systems 1 .It is known that metal chelates of ligands with sulphur or nitrogen donor atoms have interesting physicochemical properties as well as physiological activities 2,3 .Mixed ligand complexes of Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) derived from 1,10-phenanthroline and o-vanillidene-2-aminobenzothiazole, and Schiff base metal complexes of o-vanillidene-2-amino-N-(2-pyridyl)-benzene sulfonamide have been studied.The metal complexes show more potent activities compared with Schiff base ligands 4 .Studies have shown that the metal complexes of sulfa drugs promote rapid healing of skin disorder, for instance, silver(I)sulfadiazine complex is used for human burnt treatment and zinc(II)sulfadiazine in preventing bacterial infections in burnt animals 5 .Mercury(II), and copper(II) complexes of 6-methyl-2-aminobezothiazole have equally been discovered to show high activity against Aspergillus niger, Alternaria alternate, Curvularia plunata, and Penicillium fumculorus 6 .Furthermore, mercury(II) complex of sulfathiazole has been discovered to show high antibacterial activity against Escherichia Coli 7 which causes sepsis and also diarrhea in humans.Obasi et al worked on some sulfonyl derivatives of 2-aminothiazole, and the results obtained showed that the compounds were significantly active against Staphylococcus aureaus and Escherichia coli 8 .Some novel N-(Benzothiazol-2-yl)ethanamides were also synthesized and characterized by Obasi et al., and were screened in vitro and in vivo for antibacterial activity.The compounds were very stable and showed high antibacterial activities against both gram-positive and gram-negative bacteria tested 9 .The present work is aimed at synthesizing new derivative of 2-aminobenzothiazole and its metal(II) complexes, characterizing them, and investigating how their structural differences affects antimicrobial activities when compared with conventional sulfonamides.

Reagents and Apparatus
The ligand, N-(benzothiazol-2-yl)benzenesulphonamide (BS2ABT) was prepared based on our modified method from that by Sprague et al 10 .All reagents were of analytical grade and were used as supplied except otherwise stated.UV-Visible spectra of the ligand and complexes were obtained on UV-2550 UV-VIS Spectrophotometer, (SHIMADZU).FTIR spectra of the compounds were run as Nujol mulls on FTIR-84005 FTIR Spectrophotometer, (SHIMADZU). 13C and 1 H NMR spectra were recorded on Bruker-BioSpin 500 MHz NMR Spectrometer (UK) using DMSO and CDCl 3 as solvents respectively.The proton NMR peaks were observed at 500 MHz whereas the carbon-13 spectra were observed at 125 MHz.The magnetic susceptibility of the complexes were determined using Sherwood Scientific Magnetic Susceptibility Balance, Mk1 Model (Cambridge, UK) at Department of Pure and Applied Chemistry, University of Strathclyde, Scotland, UK.

Antimicrobial properties
In The antibacterial and antifungal activities of the ligand, BS2ABT, and its complexes, Ni(II)BS2ABT, Mn(II)BS2ABT, Co(II)BS2ABT, Zn(II)BS2ABT, Cu(I)BS2ABT against these multi-resistant bacteria were determined using the agar well diffusion method as described by Chah et al. 11 .Mueller-Hinton agar plates were inoculated with 0.1 ml of 3 hr broth culture of the test bacteria.Using a cork borer, wells (7 mm in diameter and 2.5 mm deep) were bored into the inoculated agar.The test compounds were solubilised in 20 % v/v dimethyl sulfoxide (DMSO) and 0.05 ml of each compound at a concentration of 20 mg/ml were delivered into the wells.One of the wells contained 20% v/v DMSO and served as control.The plates for antibacterial screening were incubated at 37 o C for 24 hours while the fungi were incubated at 30 o C for 48 hrs and assessment of activity was based on the measurement of the diameter of inhibition zone (IZD) around the wells.The test was performed in triplicates, mean IZD was recorded to the nearest whole millimetre.
The minimum inhibitory concentrations (MICs) of the test compounds were determined using the agar dilution method as described by Ojo et al. 12 .Two-fold serial dilutions of test compounds were made in 20% v/v DMSO.One millilitre of each serial dilution was added to 19 ml of sterile Mueller-Hinton agar maintained at 45 o C, thoroughly mixed and poured into a sterile plate and the medium allowed to solidify.The final concentrations of the compounds ranged from 20 mg/ml to 1.25 mg/ml.Amended media were incubated overnight at 37 o C to check for sterility.Overnight nutrient broth cultures of the test bacteria were adjusted to contain approximately 10 8 cfu/ml and 0.025 ml of each of the test organisms was spot-inoculated on the amended culture media.Inoculated plates were incubated at 37 o C for 24 hours and observed for presence of visible growth.The minimum inhibition concentration was determined as the value of the lowest concentration that completely suppressed growth of the organisms.

In vivo Tests [Brine Shrimps Lethality Test (BSLT)]
The method of McLaughlin and coworkers was used to study the bioactivity of the synthesized compounds 13 .Artemia salina eggs obtained from a pet shop in Davis California was incubated in natural sea water (from Bar Beach, Lagos, Nigeria) in a dam-well under room condition.About ten (10) 48h-shrimp nauplii in 1mL of autoclaved sea water were put into Bijou bottles using a Pasteur pipette under a stereo-microscope with a light source.They were separated into 7 groups in triplicate.Increasing concentrations (10, 100, 1000 ppm) of the synthesized compounds were added into each of the triplicate and distilled water was added into the control group.The nauplii were incubated at room temperature (37 o C) for 24h after which the survivors in each well were counted.The results were analysed using Finney Probit Analysis (MS-DOS-Computer-Program) to determine the LC 50 at 95% confidence interval.Weak nauplii were noted as an indication of central nervous system depression.

Results and Discussion
The equations of reactions for the syntheses of the ligand, BS2ABT and its metal complexes are represented in Schemes 1-6.

Physical Properties of the Compounds
Table 1 shows some physical properties of both ligand and its complexes.The melting point of the ligand, BS2ABT is 130-132 o C while those of the complexes range from 181-271 o C. The ligand and its copper complex are dirty white, the nickel and zinc complexes are white, while the manganese and cobalt complexes are shiny white and shiny blue respectively.The ligand, BS2ABT is amorphous; its nickel, manganese and cobalt complexes are crystalline while the copper complex is powdery.

Electronic Spectra
The electronic transition result of the compounds synthesized are recorded in Table 2. Two bands were observed for the ligand at 201 nm and 264 nm.They are due to π→π* transitions.

Nickel Complexes of all the Ligand
For most octahedral and tetrahedral Ni(II) complexes, three bands are expected, however for some tetrahedral Ni(II) complexes like [NiI 4 ] 2-two broad bands may be found 14,15 .Thus three bands were observed for Ni(II) complex of BS2ABT (20,700 cm -1 , 31,750 cm -1 , and 33, 160 cm -1 ), and the transitions are assigned as follows; Manganese Complexes of all the Ligand Three bands were observed for the Mn(II) complex synthesized (13, 620 cm -1 , 20, 560 cm -1 , and 37, 740 cm -1 ), the transitions are assigned:

Cobalt Complexes of the Ligand
In a cubic field, three spin-allowed transitions are anticipated because of the splitting of the free-ion ground 4 F term and the accompanying 4 P term.Of course it is essentially a 2electron transition from t 2g 5 e g 2 to t 2g 3 e g 4 .Two bands were observed for the Cobalt complex (11,460 cm -1 and 13, 610 cm -1 ), it is assigned:

Zn(II) Complexes of the Ligand
Three bands were observed for the Zn(II) complex synthesized, they are probably due to Metal-Ligand Charge Transfer (MLCT) transition.We deduced a tetrahedral geometry for the Zn(II) complex.

Cu(I) Complexes of the Ligand
Two bands were observed for the Cu(I) complex of the ligand synthesized (11,150cm -1 and 13,620 cm -1 ).Based on the fact that the Cu(II) complex was reduced to Cu(I) in this synthesis, there are no dd transitions 16 .With this fact, coupled with the colour of the complex, we presumed that the bands observed are as a result of charge transfer transitions.
We therefore proposed tetrahedral geometry for the copper complex synthesized.The molar absorptivities of the ligand and its complexes are also shown in Table 2.

IR Spectra of the BS2ABT and of Its Complexes
Table 3 gives the essential peaks of the ligand and complexes and presents a scheme for determining the mode of ligation of the ligand.The broad peaks at 3464 cm -1 , 3380 cm -1 , and 3400cm -1 were assigned N-H Stretching vibrations of the ligand BS2ABT, and its Co(II), Ni(II), Mn(II), Zn(II) and Cu(I) complexes respectively.It was also observed that there was a reduction in the stretching vibration in (N-H) of the metal complexes compared to the ligand's values.This indicates that coordination may have taken place via the N-H of the ligand.The strong peaks around 2850-2985cm -1 are assigned the C-H stretching vibrations in both the ligand, BS2ABT and its metal complexes.The strong peaks around 1603 -1650 cm -1 were assigned the C=C stretching vibration of the aromatic ring.The two strong peaks observed in the ligand and metal complexes around 1450-1560 cm -1 were assigned C=N stretching vibration of benzothiazole ring.It was also observed that there was a reduction of the frequencies to about 55 cm -1 in the complexes when compared with the ligand.We therefore state that there was coordination though the C=N of the benzothiazole ring.The strong to medium peaks around 1305-1385 cm -1 were assigned to SO 2 stretching vibration.The two medium to weak peaks observed for the ligand and the metal complexes between 818-961 cm -1 were assigned C-H to bending vibration of substituted benzene ring.However the peaks around 576-657 cm -1 were assigned C-S-C stretching vibration of thiazole ring.The two peaks each observed in the complexes around 325-360 cm -1 were assigned the metal-coordinating atom, M-N, and M-Cl stretching vibrations, where N is the nitrogen atom in N-H and thiazole ring.
1 H and 13 C NMR Spectral Data 1 H and 13 C NMR Spectra data of the ligand and its complexes are shown on Tables 4  and 5 respectively.The peaks at 9.73 ppm (1H, s) for BS2ABT, 8.32 ppm (1H, s) for Zn(II)BS2ABT and Cu(I)BS2ABT are assigned to N-H protons.The peaks at 7.26 ppm (5H, m) for BS2ABT, 7.31 ppm (5H, m) for Zn(II)BS2ABT and Cu(I)BS2ABT are assigned to phenyl protons.The peaks at 7.88 ppm (4H, m) for BS2ABT, 7.80 ppm (4H, m) for Zn(II)BS2ABT and Cu(I)BS2ABT are assigned to benzothiazole protons.Proton nmr of the complexes of Mn(II)BS2ABT, Ni(II)BS2ABT, and Co(II)BS2ABT were non-definite and incomprehensible.This is indicative of paramagnetic nature of these complexes.
Zn(II)BS2ABT and Cu(I)BS2ABT showed the same peak patterns, and the peaks at 3.34 ppm (singlet) and 4.13 ppm (triplet) observed in their spectra may be due interference of magnetic field from the metal ions in the complexes.Peaks at 136.9 ppm in the ligand and 167.6 ppm in the metal complexes are assigned benzothiazole ring carbon (C1), peaks at the range of 132.9-136.8ppm in the complexes and 129.8 ppm in the ligand are assigned benzothiazole ring carbon (C2).Peaks at 133.1 ppm the ligand and at the range of 142.5-142.7 ppm in the metal complexes are assigned benzothiazole ring carbon (C3).Peaks at 128.4 ppm in the ligand and the range of 129.7-133.0ppm in the metal complexes are assigned benzothiazole ring carbon (C4).Peaks at 126.0 ppm in the ligand, and the range of 125.3-127.8ppm in the metal complexes are assigned benzothiazole ring carbon (C5).Peaks at 125.0 ppm in the ligand, and the range of 124.2-126.4ppm in the metal complexes are assigned benzothiazole ring carbon (C6).Peaks at the range of 126.4-129.7 ppm in the ligand and its metal complexes are assigned benzothiazole ring carbon (C7).Peaks at the range of 129.3-136.7 ppm in the ligand and its metal complexes are assigned phenyl ring carbon (C8).Peaks at the range of 125.3-129.7 ppm in the ligand and its metal complexes are assigned phenyl ring carbon (C9).Peaks at the range of 123.3-126.0ppm in the ligand and its metal complexes are assigned phenyl ring carbon (C10).Peaks at the range of 113.4-124.0ppm in the ligand and its metal complexes are assigned phenyl ring carbon (C11).

Magnetic Properties of the Complexes
The result of the magnetic properties of the complexes is shown in Table 1.The result gave an interesting data.It was generally observed that the metal complexes were of low spin.This is an indication that the ligand is a strong field and thus was able to cause pairing of the electrons.As expected the zinc complex investigated gave very small effective magnetic moment, µ eff (0.67 BM).Therefore the zinc complex is diamagnetic, and have sp 3 hybridized geometry, thus tetrahedral structure.Ni(II)BS2ABT complex showed effective magnetic moment of 2.88 BM.This showed high spin configuration and correspond to two unpaired electrons, indicating paramagnetism.It has sp 3 hybridized geometry, thus tetrahedral structure.Co(II)BS2ABT showed effective magnetic moment of 2.28 BM.This is an indication of low spin paramagnetism corresponding to one unpaired electron.We proposed sp 3 d 2 hybridization of octahedral geometry.The manganese complex investigated showed effective magnetic moment of 1.79 BM.This is indication of low spin paramagnetism corresponding to an unpaired electron.We proposed d 2 sp 3 hybridization of octahedral geometry.The Cu(I) complex investigated showed diamagnetism, indicating no unpaired electrons in the metal d-orbitals.Since there is no possibility of electron pairing in the dorbitals, we are presuming that the ligand may have induced reduction of the Cu 2+ to Cu + .This is also confirmed by the dirty white colour of the complex formed.We also proposed sp 3 hybridization of tetrahedral geometry for all the Cu(I) complex investigated.

Antimicrobial Activity of the Ligand and of Its Metal Complexes
The antimicrobial activities of the ligand and of its metal complexes are recorded in Tables 6a and 6b.Table 6a showed the activities against multi-resistant bacterial strains isolated under clinical conditions.The Inhibitory Zone Diameter (IZD) in mm and Minimum Inhibitory Concentration (MIC) in mg/ml of the compounds were determined.Two strains each of E.
Coli (E.Coli strain 1 and E. Coli strain 15), and Proteus species (Proteus spp strains 25 and Proteus spp strains 26), Pseudomonas aeroginosa strains 34 and multi-resistant Staphylococcus aureus (SR) strain, all isolated from dogs at clinical conditions were used.Ciprofloxacin and trimethoprim-sulphamethoxazole were used as the standard drugs.We determined the MIC on the concentration range 0.125-10 mg/ml.We discarded concentrations above 10 mg/ml.Based on this, only the ligand showed activity against the tested microbes-E.Coli Strain 15 and Pseudomonas aeroginosa strains 34 with MIC of 10 mg/ml and IZD of 10 mm.The complexes showed no detectable activity against the multiresistant bacteria tested.Ciprofloxacin, and trimethoprim-sulphamethoxazole were used as the antibacterial standard drugs while Fluconazole disk was used as antifungal standard drugs.
The MIC were determined majorly on the concentration range of 0.125-10 mg/ml.Based on this, all the compound synthesized showed activity against at least one of the tested microbes.We adjudged that as in the case of the activity against the multi-resistant bacteria, BS2ABT showed the highest of activity in that it was active against most of the typed strains used-both the bacteria and fungi with MIC of 2.5 mg/ml and IZD of 10 mm against Staphylococcus aureus (ATCC 25923), followed by the nickel complex with MIC of 5 mg/ml and IZD of 10 mm against Staphylococcus aureus (ATCC 25923).All the compounds showed activity against Staphylococcus aureus (ATCC 25923).None of the complexes of BS2ABT was active against Pseudomonas aeruginosa (ATCC 27853).All the compounds synthesized did not show activity against Escherichia Coli (ATCC 25922).
As with the case of the fungus, Candida krusei (ATCC 6258), the ligand synthesized was active against Candida albicans (ATCC 90028) with MIC 10 mg/ml and IZD of 11 mm.Like also the case with C. Krusei, none of the metal complexes showed activity against the C. albicans typed strain used, showing that the ligand has more active antifungal properties than the synthesized complexes.Fluconazole is primarily fungistatic but can be fungicidal against certain organisms in dose-dependent manner.Fluconazole was only active against the typed strain Candida albicans (ATCC 90028) but not against C. Krusei tested strains.This was confirmed from literature 17 .We can conclude that the compounds showed some degree of activity against the tested microorganisms which to a large extent can be compared with the standard drugs used.Since the standard antifungal drug used did not show activity against the Candida krusei (ATCC 6258), we can say that the ligand, BS2ABT wase more active that the fluconazole.

Lethal Concentration (LC 50 ) and Effective Concentration (EC 50 )
The result of the Cytotoxic tests viz; Lethal Concentration (LC 50 ) and Effective Concentration (EC 50 ) is recorded in Table 7.
The result showed that all the synthesized compounds showed high levels of bioactivity against 48 h-nauplii.BS2ABT showed the highest bioactivity (26.25±4.9ppm) with EC 50 of 26 ppm while Co(II)BS2ABT showed the lowest bioactivity (1833.88±186.92ppm) with EC 50 of 183.4 ppm.Comparing the complexes, the level of bioactivity is in the order Cu(I)BS2ABT > Ni(II)BS2ABT > Zn(II)BS2ABT > Mn(II)BS2ABT > Co(II)BS2ABT.BSLT is a rapid, inexpensive and single bioassay for testing bioactivity of natural and synthetic products, which in most cases correlates reasonably well with cytotoxicity and antitumor properties of the products.The results of Brine Shrimps Lethality Test (BSLT) established that the ligand and the complexes are very potent bioactive compounds.EC 50 value for general bioactivity is approximately one tenth of the value is the LC 50 in BSLT.The surviving nauphii were dull and inactive, which may be a sign of Central Nervous System (CNS) depression.

Conclusion
N-(benzothiazol-2-yl)benzenesulphonamide and its metal complexes were synthesized.The compounds were characterized using magnetic susceptibility, UV/VIS spectrophotometer, infra red, proton and 13 C nmr.The spectral analyses confirmed the structures of the compounds synthesized.The antimicrobial tests of the ligand and its metal complexes were carried out on both multi-resistant bacterial and fungal strains isolated under clinical conditions and cultured species using agar-well diffusion method.The tests were both in vitro and in vivo.The antimicrobial activities of the compounds were compared with those of ciprofloxacin and trimethoprim-sulphamethoxazole as antibacterial agents and Fluconazole as an antifungal drug.N-(benzothiazol-2-yl)benzenesulphonamide, showed activities against multi-resistant Escherichia coli and Pseudomonas aeroginosa.All the compounds showed varying activities against the cultured typed bacteria and fungi used.However, they were less active than the standard bacterial drugs used, and since the standard antifungal drug (fluconazole) used did not show activity against the Candida krusei (ATCC 6258), we can conclude that the compound, BS2ABT which showed activity against it was more active than the fluconazole and can be recommended for preclinical screening.The Lethal Concentrations (LC 50 ) were within the permissible concentrations.

Table 1 .Table 2 .
Physical properties of the ligand, BS2ABT and of its metal Complexes, and the Magnetic Properties of the Complexes.UV/Visible spectral Result of BS2ABT and its complexes.
Antimicrobial activity of the compounds against Typed Strains (ATCC Cultures) microorganisms.Typed strains (ATCC Cultures) vitro Tests Multi-resistant bacterial strains isolated under clinical conditions and Typed strains (ATCC Cultures) were used in the study.The bacterial strains used were Escherichia coli strains (E.

Table 3 .
IR Spectra of the BS2ABT and of its complexes in cm -1

Table 4 .
1 H NMR Spectra of the BS2ABT and of its complexes in ppm.

Table 5 .
13C NMR Spectra of the BS2ABT and of its complexes in ppm.

Table 6a .
Antimicrobial activity of the ligand and of their metal complexes against multi-resistant bacterial strains isolated under clinical
Table 6b showed activities of the compounds against Typed Strains (ATCC Cultures) microorganisms.The bacteria cultures used are Pseudomonas aeruginosa (ATCC 27853), Escherica coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923).The fungi, Candida krusei (ATCC 6258) and Candida albicans (ATCC 90028) were also used.As with the multi-resistant bacteria Strains, the Inhibitory Zone Diameter (IZD) in mm and Minimum Inhibitory Concentration (MIC) in mg/ml of the compounds were determined.