Copper(II) Complexes of 2-(Methylthiomethyl)anilines: Spectral and Structural Properties and In Vitro Antimicrobial Activity

Copper(II) complexes of 2-(methylthiomethyl)anilines (1a–1f) have been obtained and characterized by elemental analyses, IR, electronic spectra, conductivity, and X-ray crystallography. The complexes (2a–2f) have the structural formula [CuCl 2 L] with the bidentate ligand coordinating through sulfur and nitrogen.The single crystal X-ray diffraction data reveal that the copper complex (2f) has a tetragonally distorted octahedral structure with long Cu–Cl equatorial bonds. Magnetic susceptibility measurements indicate the availability of one unpaired electron in the complexes and the conductivitymeasurements inDMF show their behaviour as nonelectrolytes.The solid reflectance spectra and the electronic spectra of the complexes in DMSOwere determined.The ligands and their copper complexes were screened for in vitro antimicrobial activity against S. aureus, B. subtilis, E. coli, and C. albicans. The methoxysubstituted complex (2c) showed more promising antibacterial activity against S. aureus and B. subtilis than other compounds at the concentration tested.


Introduction
The alkylthioalkylated anilines have found application as intermediates in production of many organic compounds [1][2][3] including dyes, rubber, and herbicides [4].They act as coordinating ligands due to the presence of the aniline nitrogen and the thioether sulfur in their moiety.The hardborderline and soft nature of the nitrogen and sulfur, respectively, in alkylthioalkylated anilines permits the formation of stable complexes between them and metal ions under mild nonextreme reacting conditions.Donor groups commonly found in many known biologically active compounds and ligands used in pharmaceutical synthesis include the nitrogen, oxygen, sulfur, and chlorine atoms.Such biopotent organic compounds with their metal complexes are being explored for their activity against a wide range of microorganisms.Sulfur-containing ligands and complexes have been explored for biological activity and practical application [5][6][7].Some metal complexes of SN ligands were investigated and reported.Copper(II) complexes CuX 2 (N-SMe) (X = Cl, Br) obtained from alcohol solution at 0 ∘ C were not very stable [8].Ni(II) complexes of 2-methylthiomethylaniline [8] and 8-methylthioquinoline [9] have the composition NiX 2 (N-SMe) 2 (X = Cl, Br [8]; X = Cl, Br, I, NCS [9]).The Pd(II) and Pt(II) complexes of these ligands, on being heated in dimethylformamide were S-demethylated to yield the thiolo-bridged complexes M 2 Cl 2 (N-S) 2 .Complexes MX 2 (N-SMe) and [M(N-SMe) 2 ](ClO 4 ) 2 (M = Pd, Pt, Cu, Hg) were derived with 2-(2-methylthioethyl)pyridine [10] and 2-methylthiomethylpyridine [11].The structural, spectroscopic, and biological studies of alkylthioalkylated anilines and their copper complexes are less investigated in comparison to their sulfonamide analogues.Copper ions are biologically relevant in living systems as Cu(I)/Cu(II) cuproproteins which transport molecular oxygen and act as good catalysts in related oxidation-reduction processes.Here, the spectral, structural, and antimicrobial properties of copper(II) complexes of 2-(methylthiomethyl)anilines are reported with the spectral property and antimicrobial activity of the complexes compared to their corresponding ligands.

Materials and Physical Measurements.
The reagents and solvents used in the experimental procedures were of analytical grade and used without further purification.The elemental analysis was carried out on Elementar Analysensysteme varioMICRO V1.6.2GmbH. 1 H and 13 C NMR spectra of the ligands were obtained in CDCl 3 relative to the residual proton in the solvent on Bruker Avance 400 MHz NMR spectrometer.The midinfrared spectra (400-4000 cm −1 ) were determined as solids on PerkinElmer Spectrum 100 ATR-FTIR spectrometer.Far-infrared spectra (30-700 cm −1 ) were obtained in nujol mulls held between polyethene discs and recorded on Perkin Elmer Spectrum 400 FTIR/FIR spectrometer.The electronic spectra (250-1100 nm) of ligands and complexes were measured in DMF using PerkinElmer Lambda 25 UV/VIS Spectrometer.The solid reflectance spectra of the copper complexes (300-1500 nm) were obtained on Shimadzu UV-3100 UV-VIS-NIR Spectrometer.Conductivity measurements of the complexes were taken at room temperature on AZ 86555 conductivity instrument.A Gouy balance was used to determine the room temperature magnetic moments of the powdered samples employing Hg(II) tetrathiocyanatocobaltate(II) as a calibrant and diamagnetic corrections were made from Pascal's constants.

Crystallographic Measurements.
Crystallography data were collected at −73 ∘ C using a Bruker KAPPA APEX II diffractometer equipped with a graphite monochromator and a molybdenum fine focus sealed X-ray tube as source of X-ray (Mo- radiation,  = 0.71073 Å) and an Oxford Cryostream 700 system for sample temperature control.Bruker APEX II software was used for instrument control.The structures of the compounds were solved and refined using SHELXL-97 software package [13][14][15].Numerical absorption corrections were done and all nonhydrogen atoms were refined anisotropically.The positions and temperature parameters of the hydrogen atoms were fixed to the adjacent atoms.Diagrams and publication materials were generated using ORTEP [16]

Antimicrobial Susceptibility
Procedure.The ligands (1af) and copper complexes (2a-f) were screened in vitro for their antibacterial activity against Staphylococcus aureus ATCC 6538, Bacillus subtilis (subsp.spizizenii) ATCC 6633, and Escherichia coli ATCC 8739 and for antifungal activity against Candida albicans ATCC 2091.Ampicillin (AMP) and ketoconazole (KTZ) were, respectively, used as positive controls for the antibacterial and antifungal tests.All the growth media (Mueller Hinton agar (MHA), agar bacteriological, potato dextrose agar (PDA), and nutrient broth) were prepared in double-distilled water according to standard procedure.Sterile saline was prepared by dissolving 0.85 g saline in double-distilled water and making up to 100 mL.
McFarland solution (0.5 turbidity standard) was prepared by adding 0.5 mL of 1% barium chloride to 99.5 mL of 1% sulphuric acid [17].Agar disc diffusion method was employed to determine the susceptibility of the microorganisms to the test compounds [18,19].The preparation of the agar plates, culturing of the microbial strains, and the inoculation of the plates followed described procedure [20,21].Each microbial inoculum was standardized by reference to 0.5 McFarland turbidity standard [17].Stock solutions (100 mg/mL) of ampicillin and ketoconazole were also prepared and diluted to lower concentrations [21].

Agar Disc Diffusion Method.
The sterile assay disks were kept in sealed containers at 5 ∘ C and allowed to equilibrate to room temperature before use.The test compounds, namely, ligands (1a-2f) and complexes (2a-2f) were dissolved in DMF.Known concentrations of test solutions were delivered on to sterile assay disks of 6 mm diameter each using a micropipette; the quantity taken was 250 g per disc.125 g of Ampicillin and ketoconazole was measured on separate disks and allowed to dry under the laminar flow.Six disks were placed on each inoculated agar plate containing the appropriate growth medium and incubated for 24 h (bacteria) and 60 h (fungus) at 37 ∘ C. The diameter of zone of inhibition of the microbial growth by each compound was thereafter measured.The tests were carried out in triplicate and the mean values are recorded in Table 5.

Synthesis of Ligands and Complexes.
The ligands, 4-R-2-(methylthiomethyl)anilines (1a-1f), were prepared according to reported procedure [2].Appropriate aniline (10.7 mmol) and dimethyl sulfide (15.00 mmol) in dichloromethane were vigorously stirred at room temperature.-chlorosuccinimide (15.0 mmol) was added in small portions.The mixture was stirred for 10 min; triethylamine (15.0 mmol) was added and the mixture was heated at reflux for 12 h.The organic layer was extracted with 10% NaOH (25 mL) and dried over anhydrous magnesium sulfate.Solvent was removed in vacuo to give the crude which was purified by column chromatography on silica gel 60 (0.040-0.063 mm) using hexane: ether (4 : 1 vol/vol) as the eluent.Fractions were collected in test tubes in 30 mL portions and   value of each fraction was determined on TLC plate (silica gel 60 F 254 ).Fractions with similar   values were combined and dried in vacuo to remove the solvent and the NMR spectra obtained to identify the desired product (Scheme 1).
The copper(II) complexes (2a-2f) were prepared by adding equimolar amounts of cupric chloride dihydrate    (0.65 mmol) in ethanol (2 mL) to a stirred solution of the ligand (0.65 mmol) in ethanol or a mixture of ethanol/dichloromethane (2 mL).The mixture was further stirred for 1 h and the resulting solid precipitates were filtered off, washed with cold ethanol, and dried under vacuum (Scheme 1).

Results and Discussion
The synthesis route for the copper complexes is shown in Scheme 1.The complexes are stable solids in air, with varying shades of green colouration, and their structures were established from their elemental analyses, infrared and electronic spectra and X-ray crystallography.The results of the elemental analysis are in good agreement with the calculated values of 1 : 1 metal to ligand combination for the copper complexes.The complexes are completely soluble in DMF and DMSO, partially soluble in other polar solvents such as water, acetonitrile, and methanol but are completely insoluble in nonpolar organic solvents.Low molar conductance values between 27.2 and 38.3 Ω −1 cm 2 mol −1 obtained for the complexes in DMF indicate they are nonelectrolytes [22] and the nature of chlorine to metal bonds can be described as coordinative.The summary of the analytical data and other physical properties of the complexes are recorded in Table 1.

NMR Spectra.
The NMR shifts for the protons and carbon atoms of the respective ligands are shown in (Scheme 2, Table 2).The proton NMR spectra of the ligands can be classified into three distinct classes; the thiomethyl (-CH 3 ) and methylene (-CH 2 ) protons appear as singlet peaks and resonate in the ranges 1.97-2.02 and 3.59-3.70, respectively.The broad singlet peaks found between 3.81 and 4.76  are due to amine (-NH 2 ) protons and the peaks downfield in the region 6.58-7.96 which appear as multiplets are due to the aromatic protons.The ligands with the methyl or methoxy group show additional singlet peak due to methyl (-CH 3 ) protons at 2.25  or methoxy (-OCH 3 ) protons at 3.73 .

Infrared Spectra.
Selected infrared bands of the ligands and copper complexes are recorded in Table 2.The vibrational frequencies in the 2MT ligands (1a-1f) were Scheme 1: Synthesis of ligands (1a-1f) and copper complexes (2a-2f).characterized by those observed in primary amines [23].The N-H symmetric and asymmetric stretches were found between 3320 and 3400 cm −1 , respectively; NH 2 scissor was in the range 1590-1600 cm −1 and C-N stretching frequency was seen around 1280 cm −1 .The band expected from the thioether group due to C-S-C bend (around 1100 cm −1 ) and that due to C-S stretch between 650 and 780 cm −1 was not observed as they are weak bands and were masked by vibrations associated with the benzene ring [24].There was no deprotonation of the amine hydrogen atoms upon complexation as two N-H stretches were observed, shifted to lower energies by 100-200 cm −1 .The N-H bends were similarly shifted to lower frequencies (cm −1 ) in the complexes.The shift to lower frequency of these vibrational modes after chelation is a result of the electron density of the nitrogen being directed to the metal ion, leaving the amino protons less tightly bound to the nitrogen [25].Copper to ligand vibrations were seen in the far infrared region; VCu-N was observed in the range 425-450 cm −1 [26] and the vibrations due to Cu-Cl stretches consist of a mixture of medium and intense bands in the complexes between 268 and 365 cm −1 [27,28].In the crystal structure of complex (2f) below, the arrangement of the ligand atoms around the Cu 2+ center includes two chloride ions, one of them terminally bonded, while the other is linked to two other adjacent copper centres in a bridging mode.Frequencies between 268 and 303 cm −1 are assigned as VCu-Cl for equatorial bonds [29].Bands close to 320 cm −1 were assigned to Cu-S stretches [30].

Magnetic
Moment and Electronic Spectra.The magnetic moments of copper(II) complexes (2a-2f) are recorded in Table 1.The magnetic moments between 1.76 and 2.30 B. M. obtained for the complexes suggest the presence of one electron in the d 9 copper(II) configuration.The increase from the spin-only value of 1.73 B. M could be due to spin orbit coupling or orbital contribution from the unpaired electron in the ground state [44].The electronic spectra of the ligands and copper(II) complexes in DMSO are recorded in Table 2.The spectra of the ligands (1a-1f) consist of two high energy bands found in the range 250-320 nm arising from  →  * transitions of the phenyl ring; the ligands (1c) and (1f) show an additional band close to 360 and 390 nm, respectively, due to intraligand charge transitions of their methoxy and nitro groups.The electronic spectra of the copper(II) complexes in DMSO similarly show the  →  * transitions which are slightly shifted to shorter wavelengths as a result of decrease in conjugation of the system after complexation.Ligand to metal charge transfer transitions are observed; the band in the region 320-390 nm is assigned as N → Cu, while that between 400 and 450 nm is associated with S() → Cu [25].In the solid reflectance spectra of the complexes in Figure 3(a), two high energy bands due to charge transfer transitions are found near 350 and 400 nm, while the broad band in the range 700-800 nm is assigned to d → d transition [25].The description of the d → d band of the complexes changes in DMSO (Figure 3(b)) and a broad low-energy band is observed in the near-infrared between 880 and 920 nm.The shift to lower energies, by approximately 100 nm, is indicative of geometry change in the complexes as a result of probable coordination of DMSO to copper(II).From the crystal structure, the Cu-Cl distance in the bridging bonds is long and could imply a possible replacement of the axial binding site through the bridging chlorido ligand by the high coordinating DMSO molecule.Previous studies on electronic spectra of similar copper(II) complexes in DMF suggested the coordination of the solvent molecule to the metal ion resulting in distorted octahedral or tetragonal structures [19].The large bandwidth in the electronic spectra can be attributed to Jahn-Teller distortion which is commonly observed in octahedral Cu(II) complexes.4 shows the inhibitory activity of each ligand was improved upon chelation to copper ion.The higher activity of the complexes could be due to the increased lipophilicity conferred on the complex by the copper ion.It was also observed that the pure metal salt solution has an inhibitory effect on the microbial growth and it shows a measure of biological activity.In this study, the gram-positive bacteria were more susceptible to the test compounds than the gram-negative E. coli and the fungus C. albicans.Among the ligands and complexes screened, those with electron donating groups are seen to inhibit the microbial growth better than the electron withdrawing groups.The compounds with the methoxy moiety (1c) and (2c) demonstrate more inhibitory activity than other compounds (2b) with a methyl group showing a similar though less pronounced activity.

Conclusion
The copper(II) complexes (2a-2f) formed in a 1 : 1 ligand to metal reaction stoichiometry and were characterized by the elemental analysis, IR and X-ray crystallography.A change in the structure of the complexes in the solid state is suspected as a result of the coordination of DMSO to the copper(II).Screening of the ligands and their copper complexes for in vitro antimicrobial activity against S. aureus, B. subtilis, E. coli, and C. albicans was carried out using agar disk diffusion as well as microbroth dilution techniques.The methoxy complex (2c) showed promising antibacterial activityagainst S. aureus and B. subtilis, while E. coli was not susceptible to any of the compounds at the concentration tested.

Table 2 :
1 H and 13 C chemical shifts (, ppm) of the ligands.

Table 3 :
Selected IR bands and the electronic spectra of the ligands and complexes.
a In DMSO.b CT: charge transfer.

Table 5 :
Agar disk diffusion test of compounds against microbial strains.
a 250 g disc −1 sample concentration, disc diameter 6 mm.b NI: No inhibition.