Synthesis , Characterization , and Thermal and Antimicrobial Activities of Some Novel Organotin ( IV ) : Purine Base Complexes

A new series of organotin(IV) complexes with purine bases theophylline (HL) and theobromine (L) of the types R 3 Sn(L), R 2 Sn(L)Cl, R 3 Sn(L)Cl, and R 2 Sn(L)Cl 2 (R = C 6 H 5 CH 2 –; p-ClC 6 H 4 CH 2 –) have been synthesized in anhydrous THF. The complexes were characterized by elemental analysis, conductancemeasurements, molecular weight determinations, UV-vis, IR, H, CNMR, andmass spectral studies. Various kinetic and thermodynamic parameters of these complexes have also been determined using TG/DTA technique. The thermal decomposition techniques indicate the formation of SnO 2 as a residue. The results show that the ligands act as bidentate, forming a five-member chelate ring. All the complexes are 1 : 1 metal-ligand complexes. In order to assess their antimicrobial activity, the ligands and their corresponding complexes have also been tested in vitro against bacteria (E. coli, S. aureus, and P. pyocyanea) and fungi (Rhizopus oryzae and Aspergillus flavus). All the complexes exhibit remarkable activity, and the results provide evidence that the studied complexes might indeed be a potential source of antimicrobial agents.

Compounds containing purine play a significant role in many biological systems [20], where they exist in nucleic acids, several vitamins, coenzymes, and antibiotics.These provide potential binding sites for metal ions, and any information on their coordinating properties is important as a means of understanding the role of the metal ion in biological systems.Theophylline (1, 3-dimethylxanthine) and theobromine (3, 7-dimethylxanthine) are referred to as purine bases and constitute an important class of antiinflammatory agents [21].Theophylline has biological importance as it is structurally related to nucleic acids components [22] and thus can be used as a drug for the treatment of asthmatic bronchitis and chronic obstructive bronchitis under a variety of brand names and as anticancer drugs.
The interest in metal complexes of theophylline and theobromine is stimulated by the fact that certain purine-metal complexes have been found to have therapeutic value as diuretics [23].It is also important to study the mode of binding of the ligand with the metal [24].Moreover, the study of the behavior of theophylline as a ligand can be useful for elucidating the metal and interligand interactions involving the purine bases of nucleic acids or their nucleosides, as the theophylline molecule can be considered as a model for the nucleoside guanosine.In continuation of our previous work, here we present the synthesis, mode of binding, thermal studies, and antimicrobial properties of some organotin(IV) complexes with purine bases theophylline (Figure 1(a)) and theobromine (Figure 1(b)).

Experimental
2.1.Physical Measurements.All reagents used were AR grade, and the solvents were purified and dried by standard methods, and moisture was excluded from glass apparatus using CaCl 2 drying tubes.The elemental analysis was carried out on an Elementar Analysensysteme GmbH Varion EL III, Germany.IR and far IR spectra were recorded on KBr and polyethylene discs, respectively, using a Perkin Elmer Spectrum 2000 FTIR spectrometer.The 13 C and 1 H NMR spectra were recorded in CDCl 3 , respectively, on a Bruker Spectrospin Advance 300 spectrometer.Tetramethylsilane was used as an internal reference for 1 H NMR. Varian Cary 100 UV-VIS spectrophotometer was used to record the electronic spectra of the compounds.Conductance measurements were carried out on Elico Conductivity Bridge (Type CM82T), and mass spectra were obtained using Jeol SX-102(FAB) mass spectrometer.Thermal studies (TG/DTA curves) were recorded on a Rigaku Corporation Thermoflex PTC-10A, in static air at a heating rate of 10 ∘ C/min.The Pt crucible was used with alumina as the reference material.Chlorine was determined gravimetrically as silver chloride [25,26].Tin was also estimated gravimetrically as SnO 2 [27].The antibacterial activities were evaluated by the paper-disc plate method [28] and antifungal by agar plate technique [29].Molecular masses of the complexes were determined by Cottrell's method of boiling point elevation [30].

Antibacterial Activity.
For the evaluation of degree of inhibitory effects on the growth of a wide spectrum of microorganisms, antibacterial activity was performed against one Gram-positive (Staphylococcus aureus) and three Gram-negative (Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae) bacteria.In order to compare the results obtained the Imipenem was used as standard drug.
Determination of the antibacterial activity was carried out by the paper-disc plate method [28].The compounds were dissolved in DMF at 500 and 1000 ppm concentrations.The Whatman no. 1 papers with a diameter 5 mm were soaked in these solutions.These discs were placed on the appropriate nutrient medium (0.5% peptone, 0.15% yeast, 0.15% beef extract, 0.35% sodium chloride, and 0.13% KH 2 PO 4 in 1000 cm 3 distilled water which was autoclaved for 20 min at 15 psi before inoculation), previously seeded with organisms, in Petri dishes and stored in an incubator at 28 ± 2 ∘ C. The inhibition zone thus formed around each disc was measured (in mm) after 96 h.

Antifungal Studies.
Bioefficacies of the synthesized compounds were checked in vitro.The in vitro antifungal activities of the ligands and their complexes were evaluated against two pathogenic fungi, Rhizopus oryzae (causes zygomycosis) and Aspergillus flavus (causing aspergillosis of lungs) by the agar plate technique [29].In order to compare the results obtained, the Bavistin was used as standard drug.The compounds were directly mixed with the medium in 0.01 and 0.1% (in methanol) concentrations.The medium was then poured into Petri plates, and a small disc (0.7 cm) of the fungus culture was cut with a sterile cork borer and transferred aseptically into the centre of a Petri dish containing the medium, with a certain amount of the compound.Suitable checks were kept where the culture discs were grown under the same conditions on PDA without the compound.These Petri dishes were wrapped in polythene bags containing a few drops of alcohol and were placed in an incubator at 25 ± 2 ∘ C. Controls were also run, and three replicates were used in each case.The linear growth of the fungus was obtained by measuring the diameter of the fungal colony after four days.The amount of growth inhibition in each of the replicate was calculated by percentage inhibition = ( − ) × 100/, where  is diameter of the fungus colony in the control plate after 96 h and  is diameter of the fungus colony in tested plates after the same period.

Preparation of Complexes
were synthesized by the method given by Sisido et al. [31].The organotin(IV) complexes of theophylline (HL 1 ) and theobromine (L 2 ) were synthesized according to the procedure reported by Bhatia et al. [32].
A solution of theophylline (HL 1 , 1.80 g, 0.01 mol) in 25 mL THF and 5 g NaOH was stirred for about 12 hrs at room temperature.A solution of R 3 SnCl or R 2 SnCl 2 (0.01 mol) in 25 mL THF was added to it and stirred for 4H at room temperature.The contents were then filtered, and the filtrate was reduced to one-fourth of its original volume.The R 3 SnL 1 or R 2 Sn(L 1 )Cl complexes precipitated out on adding petroleum ether and were recrystallised from acetone.Theobromine complexes were synthesized by simply adding a solution of R 3 SnCl or R 2 SnCl 2 (0.01 mol) in 25 mL THF to a suspension of theobromine (L 2 , 1.80 g, 0.01 mol) in 25 mL THF and stirred for 3 hours at room temperature.The contents were filtered, and volume was reduced to one-fourth of its original volume.The R 3 SnL 2 (Cl) or R 2 Sn(L 2 )Cl 2 complexes precipitated out on addition of petroleum ether and were recrystallised from acetone.

Results and Discussion
Elemental analyses reveal that the complexes are of good purity and are yellow in colour.The complexes were found to be soluble in acetonitrile, THF, DMSO, acetone, and CHCl 3 .The complexes decomposed before melting.The analytical data of the complexes along with their decomposition temperature is given in Table 1.From the analytical data, it is clear that organotin(IV) derivatives react with these ligands in 1 : 1 molar proportions.Conductance measurements for these complexes in 10 −2 M nitrobenzene solution are in the range of 0.12-0.40ohm −1 cm 2 mol −1 , indicating that the complexes are nonelectrolytes.

Infrared Studies.
In case of theophylline complexes, the ligand can chelate with the metal ion either through C(6) carbonyl and N (7) or through N( 7) alone [24,32,33].In the present case, the ](C=O) stretching frequency for free ligand was observed at 1720 and 1660 cm −1 .On complexation, the peaks merged and appeared at ca. 1700 cm −1 .However, this shift cannot be attributed to the interaction of carbonyl at C (6) with Sn(IV), since the electronic spectra of these complexes rules out this possibility.Further, the disappearance of the band at 3200 cm −1 due to ](N-H) stretching in the complexes indicate that the ligand is linked to Sn(IV) through the deprotonation of N (7).
In case of free theobromine, the ](C=O) stretching frequency appears at 1700 and 1665 cm −1 and remains unaltered on complexation.However, the ](C=N) + ](C=C) stretching frequency is shifted from 1595 and 1550 cm −1 in free theobromine to ca. 1580 and 1530 cm −1 in complexes, indicating that the theobromine is linked to Sn(IV) through N( 9) [34].

Electronic Spectra.
In the electronic spectra of theophylline and theobromine, the band corresponding to  →  * transition of the carbonyl group at 270 nm (log 3.45) and 272 nm (log 3.5), respectively, almost remained unperturbed on complexation, confirming the noninvolvement of the carbonyl group of these ligands in complexation [32].Additional peaks at ∼210 nm and ∼250 nm corresponding to the phenyl ring of R moiety are observed in case of the UV spectrum of metal complexes.In addition to this, the ligand → metal charge transfer band is observed in the region 320-340 nm (log  ∼ 4.2) in the complexes. 13C NMR Studies.The 1 H NMR data of the complexes are tabulated in Tables 2 and 3.The peaks undergo a slight downfield shift on complexation, due to the change in the electronic environment of the ligand.In theophylline, the broad peak due to -N-H ( 10.4) disappears on complexation, confirming that the complexation involves the replacement of hydrogen atom in -N-H-group.In theobromine, the peak corresponding to H-8 ( 7.1) undergoes significant downfield shift on complexation, confirming the involvement of N-9 in complexation [35]. 13C NMR data have been recorded for both the ligands and their corresponding organotin(IV) complexes, and these spectra also support the authenticity of the proposed structures.(C 6 H 5 CH 2 ) 3 Sn(L

Mass Studies.
The fragmentation pattern of the complexes has been analysed on the basis of mass spectra.The RSn +. , R + , and Sn + ions dominate the mass spectra.The carbonium ion R + constitutes the base peak in the mass spectra of all the complexes.Schemes 1 and 2 represent the fragmentation pattern for theophylline and theobromine moieties, respectively, in the organotin(IV) complexes.

Thermal Studies. Thermal studies have been carried
out for all the complexes.The thermogravimetric (TG) studies reveal that only two of the prepared complexes (C 6 H 5 CH 2 ) 3 Sn(L 1 ) and (p-ClC 6 H 4 CH 2 ) 3 Sn(L 1 ) undergo decomposition in two steps, whereas the rest undergo decomposition in three steps.The first step (423-673 K) involves the loss of R groups, the second step corresponds to the loss of chlorine atom(s), and the final step involves the formation of SnO 2 .The mass loss data is given in Table 4.The DTA curves reveal that all the decomposition steps, except the step corresponding to the formation of SnO 2 , are exothermic in nature.The last step corresponding to the formation of SnO 2 is endothermic in nature.
The order () and activation energy (  ) have been elucidated for the various steps using Coats-Redfern [36] as well as Horowitz-Metzger [37] methods.The values of activation energy and entropy of the reaction have been calculated using both methods and found to be in close agreement.The order of reaction in each case is one.ΔH (enthalpy of reaction) have been calculated, using the TG [38] and DTA curves, respectively [39,40].The kinetic parameters m/e: 179.06 m/e: 179.05 m/e: 179.05 Scheme 1: Fragmentation pattern for theophylline complexes.
m/e: 180.06 m/e: 180.06 m/e: 180 m/e: 180  for the thermal decomposition of the complexes are given in Table 5.
On the basis of the above data, it can be concluded that the complexes with R = p-ClC 6 H 4 CH 2 -have lower   value for step 1 as compared to the complexes with the same number of R groups, but R = C 6 H 5 CH 2 -because the electron withdrawing effect of chlorine makes the R-Sn bond weaker and facilitates its thermal degradation.Moreover, tri(pchlorobenzyl)tin derivative has the least activation energy, for the first step, due to the greater steric hindrance.

3.6.
In Vitro Antibacterial Studies.The metal complexes were screened versus E.coli, S. aureus, and P. pyocyanea bacterial strains, using the respective ligands as the standard for comparing the activities.The samples were screened at three different concentrations (25,50, and 100 g cm −3 ) in DMF.The inhibitory power of the metal complexes was observed to be greater than that of the control.The general order of activity versus the three microorganisms is S.aureus > P. pyocyanea > E.coli.The data is presented in Table 6.7 reveal that the organotin complexes with these ligands are much more active than the parent ligand against the same microorganisms.With increase in concentration of the compounds, there occurs increase in percentage of inhibition.Higher concentration proves inhibitory for fungal growth.

Sn
On the basis of spectral evidence, Figure 2 representing coordination has been prepared for one of the complexes synthesized.

Table 1 :
Physical characteristics and analytical data of theophylline and theobromine complexes.

Table 4 :
Phenomenological data for the thermal decomposition of the complexes.

Table 5 :
Kinetic parameters for the thermal decomposition of theophylline and theobromine complexes.

Table 7 :
Antifungal activity of theophylline and theobromine complexes.