SN-Donor Methylthioanilines and Copper ( II ) Complexes : Synthesis , Spectral Properties , and In Vitro Antimicrobial Activity

Methylthioanilines, a series of sulfur-nitrogen donor ligands substituted with OCH3, CH3, Cl, and Br, and their copper(II) complexes have been synthesized and characterized by H and CNMR, elemental analysis, FTIR, UV-Vis and EPR spectra, molar conductance, and magnetic susceptibility measurements.The NMR spectra of the ligands revealed that the para/ortho protons and para carbon were sensitive to the electronic effect of substituents. The CHNS analysis presented CuLCl2 (L = OCH3, CH3, Cl) and CuL2Cl2 (L = Br) stoichiometries for the copper complexes. FTIR spectra showed that the bidentate ligands were coordinated to the copper ion through their nitrogen and sulfur atoms.The electronic spectra have suggested square planar and octahedral geometries for these complexes. The EPR spectra demonstrated that the solid state copper(II) complexes possess dx 2 –y 2 orbital ground state and g‖ > g⊥ > 2.0023 in a tetragonal environment. The compounds were evaluated for in vitro antimicrobial activity against S. aureus, B. subtilis, E. coli, and C. albicans. The copper complexes showed higher activity than the parent ligands against S. aureus and B. subtilis; the electron-donating OCH3 and CH3 derivatives were more active than the withdrawing Brand Cl-substituted compounds.


Introduction
Thiomethylated anilines belong to a class of nitrogensulfur (SN) donor groups.They find application in preparation of sulfoxides [1] which are desulfurized to generate methylated anilines [2] and as starting materials [3] for deriving aminobenzaldehydes which are also useful precursors to many important heterocyclics.By coupling 2-(methylthioaniline) with another suitable aromatic polymer, a suitable chelating resin [4] has been derived for use in preconcentration of metal ions such as Cd, Hg, Ni, Co, Cu, and Zn for analytical purposes.Substituted 2-(methylthio)anilines were synthesized from the reaction of corresponding anilines with aliphatic disulfides in the presence of Lewis acid catalysts, particularly aluminum chloride and copper iodide at high temperatures of >100 ∘ C; mixtures of ortho-and para-substituted methylthiolated products resulted [5].A two-pot synthetic route can also be used to generate substituted 2-(methylthio)anilines.By alkaline hydrolysis of the appropriate 2-aminobenzothiazoles at a high temperature and subsequent methylation with methyl iodide, the crude substituted 2-(methylthio)anilines were derived [6].The biological relevance of Cu(I)/Cu(II) in living systems includes their presence as cuproproteins to transport molecular oxygen and acts as good catalysts in related oxidationreduction processes.aniline ligands are potential SN bidentate ligands of which bioactivity has not been investigated.The synthesis, NMR, FTIR, UV/Vis, EPR, molar conductance, magnetic measurements, and in vitro antimicrobial studies of ortho-substituted-6-(methylthio)anilines and their copper(II) complexes are reported in this study.

Materials and Physical Measurements.
All the reagents and solvents were of analytical grade and used as obtained from commercial suppliers (Sigma Aldrich and Merck).CHNS analyses were determined on Elementar Analysensysteme varioMICRO V1.6.2.One-and two-dimensional NMR spectra ( 1 H, 13 C, DEPT135, COSY, HMBC, and HSQC) were obtained in CDCl 3 on a Bruker Avance 400 MHz NMR spectrometer.Chemical shifts were recorded in ppm with reference to the residual solvent proton relative to tetramethylsilane.Infrared spectra of the compounds were determined in the region 4000-400 cm -1 on PerkinElmer Spectrum 100 ATR-FTIR spectrometer.Far-infrared spectra for the complexes in the region 700-30 cm -1 were obtained as mulls held between polyethylene discs and recorded on Perkin Elmer Spectrum 400 FTIR/FIR spectrometer.The UV/Vis electronic spectra (250-1100 nm) were determined in DMF on a PerkinElmer Lambda 25 UV/VIS Spectrometer.The molar conductance was measured in DMF at room temperature on AZ5 86555 p H /mV/Cond./TDS/Temp at 10 -3 M. Magnetic susceptibility measurements were taken on a Sherwood magnetic susceptibility balance Mark 1.A Galenkemp melting point apparatus was used to determine the melting points (uncorrected).The powder electron paramagnetic resonance (EPR) spectra were recorded on a Bruker ESP 300E X-band EPR spectrometer with 100 kHz field modulation.Other experimental parameters: 9.762 GHz, 16.05 G modulation amplitude, 20 mW power, 1.25 ms time constant, and 100 s sweep time.anilines.Orthosubstituted-2-(methylthio)anilines were prepared in a twopot reaction involving the conversion of o-anisidine, otoluidine, o-chloroaniline, and o-bromoaniline to the corresponding aminobenzothiazoles [7,8] which were hydrolyzed and methylated to yield the crude products (Scheme 1) [6].

2-Methoxy-6-(methylthio)aniline (L1)
. o-Anisidine (1.00 g, 8.11 mmol) and potassium thiocyanate (3.16 g, 32.50 mmol) were rapidly stirred in glacial acetic acid (16 mL) and bromine liquid (1.30 g, 8.11 mmol) was added dropwise.The mixture was stirred for 10 h keeping the temperature below 35 ∘ C, during which a precipitate was formed.This mixture was filtered and the precipitate washed with water.The combined filtrate was neutralized to pH 7 with aqueous ammonia solution during which a shiny brown precipitate formed and the mixture was filtered and dried (2.00 g).The crude 6-methoxy-2-aminobenzothiazole (0.54 g, 3.00 mmol) was slowly added to potassium hydroxide (1.54 g, 27.40 mmol) in 2 mL water.The mixture was slowly heated to 135 ∘ C allowing the water to evaporate.The temperature was then increased to 165 ∘ C and held there for 2 h.The reaction mixture was allowed to cool to room temperature and quenched with 2 mL of water.The mixture was filtered to remove the unreacted 2aminobenzothiazole.The filtrate was collected and the water removed under vacuum.Iodomethane (0.19 mL, 3.00 mmol) and 4 mL ethanol were each added to the residue and the slurry was stirred for 16 h after which ethanol was removed in vacuo.The residue was dissolved in water, neutralized to pH 7 with concentrated HCl and the product extracted with dichloromethane.Removal of the solvent under pressure yielded crude 2-methoxy-2-(methylthio)aniline.This was purified by column chromatography on silica gel using hexane/ether (6:1 vol/vol) as eluent to afford the pure oil.Yield: 55%, mp 68-70 ∘ C. Colour: Brown.Anal.Calc.for C 8 H 11 NS (M r 169.2):C, 62.70; H, 7.24; N, 9.14; S, 20.92.Found: C, 61.97; H, 7.44; N, 9.02; S, 20.64%.FTIR (cm −1 ): 3383, 3293 V asy/sym (NH 2 ), 1620 (NH 2 ), 1269 v(C-N).UV  max (DMF, nm (, M -1 cm -1 ): 284 sh , 308 (12240).

Antimicrobial Studies.
The microorganism strains, growth media, and sterile assay disks (diameter 6 mm) were purchased from Microbiologics, Merck, Becton Dickinson and Company in South Africa.Ampicillin powder was obtained from Roche Diagnostics, Germany.Doubledistilled water was collected from the Pharmaceutics Unit of Faculty of Pharmacy, Rhodes University.Sterile saline was prepared by dissolving 0.85 g saline in double-distilled water and making up to 100 mL.McFarland (0.5) solution was prepared by adding 0.5 mL of 1.175 % BaCl 2 .2H 2 O to 99.5 mL of 1 % H 2 SO 4 [9].Agar disc diffusion method [10,11] was employed to determine the susceptibility of Staphylococcus aureus ATCC 6538, Bacillus subtilis (subsp.spizizenii) ATCC 6633, Escherichia coli ATCC 8739, and for antifungal activity against Candida albicans ATCC 2091 to the synthesized compounds.Ampicillin (AMP) and ketoconazole (KTZ) were used as positive controls for the antibacterial and antifungal tests, respectively.The preparation of the growth media, reference drugs, agar plates, the culture of microbial strains, and inoculation of agar plates followed standard procedures [12,13].Each microbial inoculum was standardized with reference to 0.5 McFarland solution [14].250 g of each test compounds dissolved in DMF was delivered on to sterile assay discs.Ampicillin and ketoconazole (125 g) were measured onto separate discs and allowed to dry under the laminar flow.Six discs were placed on each inoculated agar plate containing the appropriate growth medium and incubated for 24 h (bacteria) and 60 h (fungus) at 35 ∘ C. The diameter of zone of inhibition of the microbial growth by each compound was measured.The tests were carried out in triplicate and the mean values were recorded in Table 3.

Synthesis and Properties.
The ligands are soluble in common organic solvents and their copper(II) complexes are soluble in DMSO and DMF.The compounds were obtained in low to moderate yields and are stable in air.The experimental CHNS analyses corresponded with the calculated values; the complexes showed CuLCl 2 stoichiometry except the Brsubstituted Cu(L4) 2 Cl 2 .The molar conductance measurements (DMF) were in the range 28.3-33.4Ω -1 cm 2 mol -1 ; thus the complexes behave as nonelectrolytes with the chlorine species covalently bound to the Cu(II) ions.

NMR Spectra of the Ligands.
The atom labeling for 1 H and 13 C NMR shifts of the ligands have been recorded in Figure 1.The NMR resonances (Table 1) were assigned with the aid of DEPT135, COSY, HMBC, and HSQC spectra.The thiomethyl protons (-SCH 3 ) appear as singlet peaks absorbing between 2.40 and 2.43 ppm.The broad singlet peaks in the range 3.57-4.02ppm were assigned to amine (-NH 2 ) protons.The aromatic protons appear as doublets and triplets in the downfield region 6.61-7.42ppm.Additional single peaks at 3.83 and 2.15 ppm were observed in L1 and L2 due to -OCH 3 and -CH 3 proton resonances respectively.The methyl carbon (C7) resonated in the range 18.42-18.80ppm and the aromatic carbon atoms have higher absorptions between 112.29 and 147.17 ppm.The methyl and methoxy carbon atoms of L1 and L2 resonated at 17.04 and 55.36 ppm, respectively.The electronic effect of substituents on the NMR shifts of amine protons as well as the ortho (H3) and para (H5) protons was observed.These protons were more deshielded in the derivatives with electron-withdrawing substituents (L3 and L4), resonating at higher frequencies compared to those of substituents L1 and L2.A similar shift was observed at the para carbon atom (C3) (Table 1).

IR Spectra.
In the vibrational spectra of the ligands, the strong asymmetric and symmetric stretches due to vN-H of the primary amine group were observed within the ranges 3459-3383 and 3358-3293 cm -1 , respectively [7,14,15].The frequencies of both bands were lowered in the complexes to 3340-3251 and 3199-3171 cm -1 , respectively.The reduced frequency of these stretches upon chelation has been interpreted to be the result of the electron density of the nitrogen being directed to the metal ion, leaving the amino protons less tightly bound to the nitrogen [16].N-H scissor and vC-N which appeared as medium bands in the ligands in the ranges 1620-1611 and 1292-1269 cm -1 in the spectra of the ligands were shifted to 1585-1576 and 1273-1245 cm -1 , respectively, in the Cu(II) complexes.A shift to lower frequency of vC-N could be due to the decrease in the C-N double bond character.These reductions in frequencies upon chelation suggested the coordination of the ligands to the copper ion through the nitrogen lone pair [16].The bands due to vC-S-C (∼1100 cm -1 ) and vC-S (∼780-650 cm -1 ) of the thioether group were not observed as they were too weak [17].In the far IR region, new medium bands in the spectra of the complexes were observed and assigned as vCu-N (414-406 cm -1 ), vCu-Cl (314-290 cm -1 ), and vCu-S (280-275 cm -1 ) [18].

Antimicrobial Susceptibility Testing.
The agar disc diffusion technique was used to assess the antimicrobial activity of the synthesized compounds using the sterile assay discs of diameter 6 mm.The results have been recorded in Table 3.The gram-positive S. aureus and B. subtilis were susceptible to the compounds and were inhibited by the measured diameters of 8-20 mm while the gram-negative E coli and the fungus C. albicans were resistant.The copper complexes with methoxy and methyl substituents demonstrated better activity than the compounds with the electron-withdrawing substituents, with the diameters of inhibition in the range 19-20 mm.

Conclusion
6-(Methylthio)aniline derivatives and their Cu(II) complexes were prepared.The compounds were characterized by elemental analysis and spectroscopic means.The CHNS analysis showed the metal complexes stoichiometry as CuLCl 2 and CuL 2 Cl 2 .The electronic nature of substituents affected the NMR shifts of some protons in the ligands.The infrared spectral bands were consistent with primary amine groups, of which frequencies reduced in the copper complexes upon chelation.The substituted 6-(methylthioanilines) behaved as bidentate ligands binding with SN-donor atoms to the copper(II) ions.Molar conductance values were indicative of nonelectrolytic complexes.The electronic and powder EPR Scheme 1: Synthesis scheme for substituted 6-(methylthio)anilines.

Figure 1 :
Figure 1: Atom labeling for 1 H and 13 C NMR chemical shifts.

Table 2 :
EPR parameters for copper(II) complexes.→  * of the nitrogen lone pairs to the aromatic ring.In the spectra of the copper(II) complexes,  →  * transitions were bathochromically shifted to 273-281 nm.Two intense bands around 300-364 nm were assigned to n →  * transitions in the complexes.In the spectra of [Cu(L1)Cl 2 ], [Cu(L2)Cl 2 ] and [Cu(L3)Cl 2 ], moderately intense bands at 413, 428, and 454 nm, respectively, were assigned to 2 B 1g → 2 A 1g in a square planar geometry around the copper ion.The band at 436 nm in the spectrum of [Cu(L4) 2 Cl 2 ] was assigned to 2 E g → 2 T 2g transition in an octahedral geometry.The magnetic moments in the range 1.8-1.9BM indicate the availability of an unpaired electron in the copper(II) complexes which are magnetically dilute.

Table 3 :
Diameter of inhibition zones (mm) at 250 g disc -1 of samples.