Potential bioactive mononuclear diorganotin(IV) phenoxyacetohydroxamate complexes: synthesis, characterization and antimicrobial evaluation

IR spectra

A comparison of the infrared (IR) spectra of diorganotin(IV) phenoxyacetohydroxamates with that of free ligand PhOAHK scanned in the 4500–450 cm⁻¹ region has supported their formation. The PhOAHK exhibited characteristic bands at 3217, 1643, 1365 and 985 cm⁻¹ attributed to ν(N-H), ν(C=O), ν(C-N) and ν(N-O) modes respectively. The complexes of composition Me₂Sn(PhOAH)₂ and n-Bu₂Sn(PhOAH)₂ displayed these bands at 3292, 3292; 1598, 1598; 1374, 1373 and 982, 989 cm⁻¹ due to respective modes. The retention of bands due to the ν(N-H) mode in complexes has suggested the exclusion of coordination through nitrogen. The observed trend of shifts in the ν(C=O) mode to lower wavenumbers by 45 cm⁻¹ and those of the ν(C-N) and ν(N-O) modes to higher wavenumbers have suggested bonding through carbonyl and hydroxamic oxygen atoms [O,O coordination], establishing the bidentate nature of the ligand. The appearance of new bands in the 530–499 cm⁻¹ region (not present in the free ligand) assigned to the ν(Sn-O) mode has substantiated their formation.

¹H NMR spectra

A comparison of the ¹H nuclear magnetic resonance (NMR) spectra of di-organotin(IV) hydroxamates with that of free ligand has further supported their formation. The free PhOAHK exhibited proton resonances due to NH, aromatic and aliphatic –CH₂– groups at δ 8.08; δ 6.79–7.31 and δ 4.24–4.90 ppm, respectively. Complexes of composition [Me₂Sn(PhOAH)₂] and [n-Bu₂Sn(PhOAH)₂] have displayed these diagnostic resonances at δ 9.12–10.92, δ 9.16–10.88 ppm; δ 6.85–7.30, δ 6.84–7.29 ppm and δ 3.61–4.72, δ 3.45–4.71 ppm respectively. The signals due to methyl and n-butyl groups attached to tin appeared at δ 0.76 ppm and at δ 0.84–0.88 (t; CH₃), 1.26–1.35 (m; γCH₂), 1.39–1.48 (m; βCH₂) and 1.55–1.63 (m; αCH₂) ppm range, respectively.

Mass spectra

The electrospray ionization mass spectra (ESI-MS) of [Me₂Sn(PhOAH)₂] (I) and [n-Bu₂Sn(PhOAH)₂] (II) have been recorded. Complex (I) has displayed a low intense molecular ion peak at m/z (%) 480 (2.8). Complexes (I) and (II) have exhibited the most abundant peak at m/z (%) 315 (100) and 400 (100) corresponding to [Me₂Sn¹¹⁹(PhOAH)]⁺ and [n-Bu₂Sn¹²⁰(PhOAH)]⁺, respectively. The fragment ions observed at higher than molecular mass at m/z (%) 630 (3.5) and 799 (6.4) in (I) and (II) have occurred due to the association of Me₂Sn- and n-Bu₂Sn- moiety with the respective complexes (Table 1). Most of the significant peaks have been rationalized in terms of fragment ions of specific composition and fit in well with a successive fragmentation pattern of mononuclear complexes. Based on physicochemical and spectral studies, six-coordinate distorted octahedral geometry around tin in complexes may tentatively be proposed as shown in Figures 2 and 3.

Figure 2:

Proposed structure of [Me₂Sn(PhOAH)₂].

Figure 3:

Proposed structure of [n-Bu₂Sn(PhOAH)₂].

Cyclic voltammetry

Complex of composition [Me₂Sn(C₆H₅OCH₂CONHO)₂] exhibited two reduction waves at 0.573 and +0.727 V and one feeble oxidative peak at −0.559 V as a counterpart of a former reductive peak. [n-Bu₂Sn(C₆H₅OCH₂CONHO)₂] displayed two reduction waves at +0.725 and −0.554 V and one oxidation wave at −0.520V. No anodic counterpart of reductive wave at +0.725 V appeared in regions different from those observed for free ligand (one oxidative and one reductive peak), suggesting these to be metal centered rather than ligand centered. The Ipa/Ipc ratio (<1) has suggested quasi-irreversible reduction. The electrochemical data reveal that the tin (IV) state is easily reduced to tin (II) by a two-step reduction process.

Thermogravimetric study

The thermal behavior of (I) and (II) studied by the thermogravimetric analysis (TGA) technique in the N₂ atmosphere has shown these to be thermally stable up to 88.30°C and 109.17°C, respectively. Mass loss of 71.87 and 76.06% in a single step in the 88.30°C–360.25°C and 109.17°C–435.25°C range accounted for the formation of SnO as the gray-black residue and methyl phenyl ether and acetamide/pentanamide as the tentative volatile organic decomposition products in accordance with the equations in Scheme 2.

Scheme 2:

Thermal decomposition of complexes.

Antimicrobial activity

The in vitro antimicrobial activity of the ligand (PhOAHK) and di-organotin(IV) hydroxamates has been evaluated against selected Gram-positive bacteria Bacillus cereus and Staphylococcus aureus and Gram-negative bacteria Salmonella typhimurium and Escherichia coli and pathogenic fungi Aspergillus niger and Alternaria alternata at different concentrations in dimethyl sulfoxide (DMSO) using the standard minimum inhibitory concentration (MIC) method recommended by the National Committee for Clinical Laboratory Standards. The commercial antibiotics chloramphenicol and nystatin have been used as standards for the comparison of results.

Antibacterial activity

PhOAHK showed inhibitory effect against test bacteria in the MIC 12.5–25 μg/mL range. Both complexes I and II have shown remarkably enhanced inhibitory activity at the MIC 1.56–3.125 μg/mL range (Figure 4) relative to that of chloramphenicol at the MIC 3.91–7.80 μg/mL range against test bacteria.

Figure 4:

In vitro antibacterial spectrum.

Antifungal activity

The ligand PhOAHK has been observed to exhibit high inhibitory effect against A. niger and A. alternata at MIC 1.56 and 3.125 μg/mL, respectively. Complexes of composition [Me₂Sn(PhOAH)₂] and [n-Bu₂Sn(PhOAH)₂] have shown inhibitory activity similar to that of the ligand against A. alternata at MIC 3.125 μg/mL and a lower activity than the ligand against A. niger. The antifungal activity of complexes has been found to be quite promising against A. alternata compared to standard nystatin, which shows MIC 3.90 μg/mL against test fungi (Figure 5).

Figure 5:

In vitro antifungal spectrum.

The enhanced antimicrobial activity of complexes relative to free ligand may be explained on the basis of chelation theory, whereby the polarity of central metal ion is reduced because of partial sharing of its positive charge with the ligand on complexation, which favors permeation of the complex through the lipid cell membrane (Chaudhary et al., 2002). The observed promising antimicrobial activity of complexes can also be attributed to the biological significance associated with organotin moiety and phenoxyacetohydroxamate ligand.

Materials and instrumentation

The purity of dimethyltin dichloride (Merck, Hohenbrunn, Germany) and di-butyltin dichloride (Merck, Hohenbrunn, Germany) was checked by their melting point (105°C and 42°C), respectively. Phenoxyacetic acid (Merck, Hohenbrunn, Germany) and hydroxylamine hydrochloride (Sisco, Bombay, India) were used as such without further purification. The purity was checked by their melting point at 98°C–102°C and 151°C, respectively. The reagent-grade solvents were dried and distilled prior to use. Potassium phenoxyacetohydroxamate was synthesized by a reported method (Hauser and Renfrow, 1967). The carbon, hydrogen and nitrogen analyses were obtained on a Carlo-Erba 1106 Elemental Analyzer. Tin was estimated as tin dioxide by treating the complex with conc. H₂SO₄ (2 volume) and conc. HNO₃ (3 volume) and heating to 800°C. The molar conductance (10⁻³ M solution in methanol) of complexes was measured at 18±1°C using CON 510; Bench conductivity/TDS meter (cell constant K=1.0). The molecular weights of the complexes were determined by the Rast’s camphor method. The IR spectra were recorded in KBr pellets on a Nicolet-5700 FTIR Spectrophotometer (Thermo Electron Corporation, Netherlands). ¹H NMR spectra were recorded on a BRUKER AVANCE II 400 Spectrometer (Billerica, MA, USA) using TMS as an internal standard and DMSO-d6 as the solvent. The ESI-MS were recorded on a Waters QTOF-MICROMASS mass spectrometer (Water Corporation, Milford, USA). Cyclic voltammetric measurements were carried out on an Autolab Potentiostat 128N electrochemical analyzer (Metrohm Autolab B.V., Kanaalweg, Netherlands) in methanol in single compartmental cell of volume 10–15 mL containing a three-electrode system comprising a Pt-disk working electrode, Pt-wire as auxiliary electrode and Ag/AgCl electrode as reference electrode. The supporting electrolyte was 10⁻²m KCl in 10⁻⁴m methanol. Thermograms (TGA curves) were recorded on a NETZSCH STA 449F1 thermal analyzer (NETZSCH Technologies India Pvt. Ltd., Chennai, India) in N₂ atmosphere. A heating rate of 10°C/min was employed. The thermocouple used was Pt/Pt-Rh 10% with a temperature range of 10°C–800°C.

Antimicrobial activity

The in vitro antibacterial and antifungal activity of di-organotin(IV) hydroxamate complexes was studied against test bacteria and fungi by the MIC method in a 96-well microtiter plate (tissue culture grade) by the twofold serial dilution method using oxidation-reduction colorimetric indicator Resazurin dye blue in its oxidized state, which turned pink when reduced by viable cells according to Clinical and Laboratory Standards Institute (CLSI M07-A9). A stock solution of test ligand and complexes was prepared in DMSO (100 μg/mL) for the twofold serial dilution. The test material (complex) (100 μL) was then added in the first row of wells in the microtiter plate. After proper mixing (broth and test complex), 100 μL was withdrawn from the first well (containing test complex) with a sterile tip and same was added to 100 μL of the broth in the second well. Then, 100 μL was withdrawn from the second well and added to the third well. This way a range of twofold serial dilutions were prepared from the initial 100 μg/mL to 50, 25, 12.5, 6.25, 3.125 and 1.56 μg/mL. The resazurin indicator (0.18%) prepared in distilled water, sterilized by filtration and stored at 4°C for a week was added to each well. The wells were inoculated with 10 μL of the bacterial culture/fungi culture, and the contents were mixed properly on a flat surface. The plates thereafter were wrapped with aluminium foil and incubated at 37°C for 24 h in the case of bacteria and at 28°C for 72 h for fungi. A change in color from purple to pink indicates the growth of bacteria and fungi. The lowest concentration of test complex that prevented this color change was considered as MIC. The results were compared with a standard antibacterial drug for S. typhi, E. coli, B. cereus and S. aureus (chloramphenicol) and an antifungal drug for A. niger and A. alternata (nystatin). All the experiments were carried out in triplicate (Greenwood et al., 1997).

Syntheses of [Me₂Sn(PhOAH)₂] and [n-Bu₂Sn(PhOAH)₂

To a solution of Me₂SnCl₂ (0.80 g, 0.0036 mol) and n-Bu₂SnCl₂ (1.11 g, 0.0036 mol) in distilled benzene (10 mL), an equimolar amount of potassium phenoxyacetohydroxamate (1.5 g, 0.0073 mol) in methanol (10 mL) was added in separate experiments and refluxed for 24 h. The white solid formed during the course of the reaction was filtered and identified as KCl. The addition of petroleum ether to the filtrate gave light brown solids (yield=1.52 g=87% and 1.73 g=84%, respectively). For C₁₈H₂₂O₆N₂Sn/480 (%) Calc. C 45.00; H 4.58; N 5.83; Sn 25.00. Found: C 44.87; H 4.33; N 5.42; Sn 24.87. ∧m: 8.23 S cm² mol⁻¹. M.pt: 85°C–87°C. For C₂₄H₃₄O₆N₂Sn/564 (%) Calc. C 51.06; H 6.02; N 4.95; Sn 21.27. Found: C 51.43; H 6.41; N 4.69; Sn 22.48. ∧m: 8.61 S cm² mol⁻¹. M.pt: 101°C–103°C.