Synthesis, Characterization, and Biological Studies of Organotin(IV) Derivatives with o- or p-hydroxybenzoic Acids

Organotin(IV) complexes with o- or p-hydroxybenzoic acids (o-H2BZA or p-H2BZA) of formulae [R2Sn(HL)2] (where H2L = o-H2BZA and R = Me- (1), n-Bu- (2)); [R3Sn(HL)] (where H2L = o-H2BZA and R = n-Bu- (3), Ph- (4) or H2L = p-H2BZA and R = n-Bu- (5), Ph- (6)) were synthesized by reacting a methanolic solution of di- and triorganotin(IV) compounds with an aqueous solution of the ligand (o-H2BZA or p-H2BZA) containing equimolar amounts of potassium hydroxide. The complexes were characterized by elemental analysis, FT-IR, Far-IR, TGA-DTA, FT-Raman, Mössbauer spectroscopy, 1H, 119Sn-NMR, UV/Vis spectroscopy, and Mass spectroscopy. The X-ray crystal structures of complexes 1 and 2 have also been determined. Finally, the influence of these complexes 1–6 upon the catalytic peroxidation of linoleic acid to hydroperoxylinoleic acid by the enzyme lipoxygenase (LOX) was kinetically studied and the results showed that triorganotin(IV) complex 6 has the lowest IC50 value. Also complexes 1–6 were studied for their in vitro cytotoxicity against sarcoma cancer cells (mesenchymal tissue) from the Wistar rat, and the results showed that the complexes have high activity against these cell lines with triphenyltin((IV) complex 4 to be the most active one.


Introduction
Organotin compounds have many important applications and uses [1,2]. Commercially, organotin compounds are used as industrial and agricultural biocides because they have high antifungal properties [3,4]. The in vitro fungicidal or antibacterial properties of organotins have been found to exhibit the general order of activity: RSnX 3 < R 2 SnX 2 < R 4 Sn R 3 SnX, with the anionic X group to exert little influence on activity [5,6]. The combination of two biologically active entities, however, in the same molecule could enhance their activity [7]. For example, triphenyltin(IV) derivatives of phthalic acid and salicaldehyde have significant activity toward a range of fungi [8,9]. Recently, interests in organotin(IV) carboxylates are increasing due to their possible medical uses as antitumor agents [10]. For example, the fluoro-substituted carboxylate ligands with di-and triorganotins produced several antitumor active compounds [11]. Hubert et al. concluded that antitumor active tin compounds possess available coordination positions around tin atom and also have relatively stable ligand-tin bonds with low hydrolytic decomposition [12]. Thioamides-organotin complexes, on the other hand, have shown high antitumor activity, which is rather related to the ligand type and not to the geometry of the compounds [13][14][15][16][17]. Given that the antitumor action of Sn(IV) compounds may not be due to their direct interaction with DNA constituents [18][19][20][21][22], their reaction with enzymes like lipoxygenase is always of interest in the attempt to elucidate their mechanism of action [13][14][15][16][17]. This antitumor activity of the organotin complexes follows the same order of lipoxygenase inhibition, an enzyme taking part in the inflammation mechanism and tumor genesis [13][14][15][16][17].

Thermal Analysis.
The TGA/DTA data curves for complexes 1-2 show that they decompose generally in one stage. Thus, thermal analysis in flowing nitrogen shows that complex 1 decomposes between 125 and 315 • C with 72% mass loss which corresponds to the methyl groups of the metal and the ligand molecules (the calculated mass loss is 72%), compound 2 decomposes between 35 and 490 • C with 74% mass loss due to the butyl groups of the metal and the ligand molecules (the calculated mass loss is 76.50%) (Scheme 2).
The TGA/DTA data in flowing nitrogen data curves for complexes 3-6 show that they decompose generally in two stages. The first stage of decomposition of compound 3 lies between 20 and 255 • C that corresponds to 13% mass loss of one of the metal butyl groups (the calculated mass loss is 13%), compound 4 decomposes between 35 and 215 • C with 16% mass loss of one of the metal phenyl groups (the calculated mass loss is 16%), compound 5 decomposes between 25 and 255 • C that corresponds to 13% mass loss of one of the metal butyl groups (the calculated mass loss is 13%), and compound 6 decomposes between 25 and 177 • C with 15% mass loss of one of the metal phenyl groups (the calculated mass loss is 16%). The second stage of decomposition is between 255 and 400 • C in case of 3 which corresponds to 44% mass loss of the ligand (o-hydroxybenzoic acid) and another metal butyl group (the calculated mass loss is 45%), between 215 and 500 • C (4) corresponding to 43% mass loss of the ligand (o-hydroxybenzoic acid) and another metal phenyl group (the calculated mass loss is 42%), compound 5 decomposes between 255 and 412 • C which corresponds to 45.60% mass loss of the ligand (p-hydroxybenzoic acid) and another metal butyl group (the calculated mass loss is 45%), and compound 6 decomposes between 177 and 435 • C with 42% mass loss which corresponds to the ligand (p-hydroxybenzoic acid) and another metal phenyl group (the calculated mass loss is 42%) (Scheme 2).    Table 1.
The v as (COO − ) vibration of the free ligand appears at 1656 cm −1 while the v s (COO − ) at 1324 cm −1 for o-H 2 BZA [25], and the v as (COO − ) vibration of p-H 2 BZA ligand appears at 1687 cm −1 while the v s (COO − ) at 1360 cm −1 [27].  [30], while when the ligand chelates, the Δv [v as (COO − )-v s (COO − )] is considerably smaller than that observed for its ionic compounds. For asymmetric bidentate coordination, the values are in the range of monodentate coordination [30]. When the -COO − group bridges metal ions, the Δv [v as (COO − )-v s (COO − )] value is higher than that of the chelated ions and nearly the same as that observed for ionic compounds [30]. In our case, the Δv [v as (COO − )-v s (COO − )] values of the ionic compounds of the o-NaHBZA and p-NaHBZA ligands (sodium salts) are 205 cm −1 and 131 cm −1 , respectively [26]. Therefore, in the diorganotin complexes 1 and 2, where higher Δv values were observed (243 1 and 209 2 cm −1 ), an asymmetric bidentate coordination of the ligand to the metal ion is expected (see crystal structure, Mössbauer spectra).The same is true in the case of triorganotin complexes 3-6 with significantly higher Δv values than the corresponding ones of the sodium salts of the ligands (281 3, 280 4, 217 5, and 270 6 cm −1 ), indicating clearly an asymmetric bidentate coordination mode of the ligands and suggesting trigonal bipyramidal geometry for complexes 3-6 in the solid state in agreement with the results of Mössbauer and 119 Sn-NMR spectra.
The isomer shifts values of complexes 1-6 are in the range of δ 1.22 to 1.56 mm s −1 ( Table 2), indicating that tin is in the (4+) oxidation state in all cases [2,32,33]. The spectra of Table 2: 119 Sn Mössbauer spectroscopic data for complexes 1-6 at 80 K. diorganotin(IV) complexes 1-2 consist of two symmetrical Lorentzian doublets which indicate the presence of two tin atoms in different chemical environments with the same ratio 1 : 1 (56 : 44% 1 and 29 : 71% 2, resp.). This may be due to the presence of two different isomers in the unit cells with variable bond distances. The quadrupole splitting values (Δ) of complexes 1-2 are 3.09 mm s −1 and 3.65 mm s −1 for 1 and 3.53 mm s −1 and 3.68 mm s −1 for 2, suggesting distorted trans-R 2 octahedral geometry (2.4-5.5 mm s −1 ) [2,32,33] in the solid state in agreement with X-ray structures. The calculated C-Sn-C angles ( • ) from Mössbauer spectra [31] for the compounds 1 and 2 ( Table 2) [34], suggesting that the OH groups are not involved in bonding with the tin atom. The two doublet signals at 7.83-7.79 and 6.82-6.79 1, 7.69-7.65 and 6.75-6.72 2, 7.72-7.69 and 6.73-6.70 3, 7.81-7.78 and 6.69-6.65 4, and 7.75-7.71 and 6.76-6.73 6 ppm are assigned to the protons of the phenyl group of the ligands a and b, respectively (Scheme 1) [34]. In case of complex 1, the single signal at 0.83 ppm is assigned to the protons of the methyl group of the metal.
In case of complexes 6 and 7, 119 Sn-NMR spectra were also recorded. 119 Sn-NMR spectra showed resonance signals at −20.0 (6) and −212.0 (7) ppm, respectively. Although the shift ranges are somewhat dependent on the nature of the substituents at the tin atom the δ( 119 Sn) values (in ppm) for R 3 Sn(IV) complexes with five-coordinated Sn vary from +25 to −329 ppm [33]. Thus, the tin(IV) atoms are five coordinated in case of complexes 5 and 6, and therefore the expected geometry arrangement around triorganotin(IV), in solution, is suggested as trigonal bipyramidal.

Crystal, Molecular Structures of [(CH 3 ) 2 Sn(OOCC 6 H 4 OH) 2 ]
(1) and [(n-C 4 H 9 ) 2 Sn(OOCC 6 H 4 OH) 2 ] (2). The crystal structures of complexes 1 and 2 had been determined previously, at 193 and 295 K with R% values of 6.4 and 3.8, respectively [23,24]. However, we redetermined the structures here of both 1 or 2 complexes at room temperature (293 K), with R% 2.18 and 4.83, respectively, for comparison and in order to investigate the influence of the temperature on their structures [33]. ORTEP diagrams of complexes 1 and 2 are shown in Figures 1 and 2.
Compounds 1 and 2 are covalent monomers in the solid state with a distorted octahedral geometry around the metal ion. Table 3 summarizes Sn-O and other selected bond distances and angles found for organotin complexes reported here and elsewhere [23,24]. They differ only slightly.

Study of the Peroxidation of Linoleic Acid by the Enzyme
Lipoxygenase in the Presence of Complexes 1-6. The influence of complexes 1-6 on the oxidation of linoleic acid by the enzyme (LOX) was studied in a wide concentration range. The degree of (LOX) activity (A, %) in the presence of these complexes was calculated according to the method described previously [13].     very strong cytotoxic activity against leiomyosarcoma cells. In accordance with the LOX inhibition activity, the five-coordinated triorganotin complexes 5, 3, and 6 showed stronger antiproliferative activity against sarcoma cell lines (see LOX inhibition activity). The order of activity of complexes 1-6 is found to be 4 > 6 > 5 3 = 2 1. It is noteworthy to mention that triorganotin(IV) compounds 4-6 were found to inhibit LOX activity and anti-proliferate sarcoma cells stronger than di-organotins. Between diorganotin(IV) derivatives of o-H 2 BZA 1 and 2, the dibutyltin complex exhibits significant stronger cytotoxic activity than the corresponding one of dimethyltin complex. This is also observed in the case of substituted salicylic acids reported previously [34], where diethyl derivatives were found to show approximately 10 times lower activity than the corresponding dibutyltin complexes against human mammary tumor cell lines (MCF-7) and human colon carcinoma cell line (WiDr) [34].    Table 4 summarizes the IC 50 (μm) values for LOX inhibition in comparison with cell activity of organotin(IV) complexes 1-6 against sarcoma cells.

Computational Methods-Docking Study.
In order to investigate further the complex-LOX interactions, we performed computational molecular docking studies for the complexes 1 and 2 were X-ray data are available. The binding energy (E) of the substrate (S: linoleic acid) to its binding site in the enzyme LOX (E) when ES is the complex formed was E: −7.89 kcal/mole [13]. The corresponding binding energies of inhibitors 1 and 2 (I), in ESI, are calculated to −8.48 (1) and −8. 23 (2) kcal/mol, respectively, while the binding energies of EI are estimated to −9.7 Kcal/mol (1) and −11.4 (2) kcal/mol. According to the binding energy (E) values of ES in contrast to those of EI or ESI, it is found that both ESI and EI complexes could be formed. Figures 3 and 4 show the binding site of compound 1 toward LOX in ESI and EI, respectively. Compounds 1 and 2 bind to both ESI and EI complexes at the same pocket where the strong inhibitors of LOX bind [15], supporting its strong inhibition activity, found experimentally. Since high inhibition activity of LOX has been detected for all cytotoxic organotin(IV)-thione compounds tested previously, [13][14][15][16][17] strong activity would also be expected for compounds 1 and 2, although weaker than the others, tested in this study.

Conclusion
Organotin(IV) complexes (1)-(7) are found to inhibit strongly the peroxidation of linoleic acid by the enzyme lipoxygenase. Five-coordinated organotin(IV) complexes 6, 4, and 5 (IC 50 = 11, 19, and 24 μm, resp.) showed strongest LOX inhibition activity than the corresponding sixcoordinated one (Table 4). Compounds 1-6, also, showed strong antitumor activity against sarcoma cells ( Table 4). The highest antiproliferate activity against sarcoma cell lines is also shown by the five-coordinated triphenyltin(IV) complexes 4 and 6 (IC 50 = 5-10 and 25-35 nm, resp.).  Between tri-and diorganotin complexes, five-coordinated organotin complexes which have one free coordination position were found to exhibit stronger antiproliferative and LOX inhibition activity. These findings are in accordance to Huber et al. [12] who suggested that the structures of organotin carboxylates antitumor active compounds are characterized by (i) the availability of coordination positions at Sn and (ii) the occurrence of relatively stable ligand-Sn bonds, for example, Sn-N and Sn-S and their slow hydrolytic decomposition. Therefore, the geometrical feature of this type of complexes (1-6) seems to play important role in their antitumor and LOX inhibition activity.

Materials and Instruments.
All solvents used were of reagent grade, while o-, p-hydroxybenzoic acid, and organotins chlorides (Alderich,USA), (Merck, Germany) were used with no further purification. Elemental analysis for C and H was carried out with a Carlo Erba EA MODEL 1108. Infrared spectra in the region of 4000-370 cm −1 were obtained in KBr discs. The 119 Sn Mössbauer spectra were collected at 80 K, with a constant acceleration spectrophotometer equipped with CaSnO 3 source kept at low temperature. A Jasco UV/Vis/NIR V570 series spectrophotometer . Complexes 1-2 were synthesized as follows: a suspension of the ligand (ohydroxybenzoic acid, 0.138 gr, 1 mmol 1-2) in 5 cm −3 distilled water was treated with a solution of KOH 1 N (1 cm −3 , 1 mmol 1-2) and a clear solution was immediately formed. 5 cm −3 methanolic solution of diorganotin(IV) chloride ((CH 3 ) 2 SnCl 2 , 0.109 gr, 0.5 mmol for 1 and (n-C 4 H 9 ) 2 SnCl 2 , 0.152 gr, 0.5 mmol for 2) was then added to the above solution. A white precipitate formed and the mixture was stirred for 4 hours. The precipitate was then filtered off, washed with 3 mL of distilled water and dried in vacuo over silica gel. Crystals of 1 and 2 complexes suitable for X-ray analysis were growth by slow evaporation of CH 3 OH/CH 3 CN solutions (1 : 1). Complexes 3-6 were synthesized as follows: a suspension of the ligand (o-, p-hydroxybenzoic acid, 0.069 gr, 0.5 mmol 3-6) in 5 cm −3 distilled water was treated with a solution of KOH 1 N (0.5 cm −3 , 0.5 mmol 3-6) and a clear solution was immediately formed. 5 cm −3 methanolic solution of triorganotin(IV) chloride ((n-C 4 H 9 ) 3 SnCl, 135.6 μL, 0.5 mmol for 3 and 5, (C 6 H 5 ) 3 SnCl, 0.193 gr, 0.5 mmol for 4 and 6) was then added to the above solution. A white precipitate formed and the mixture was stirred for 4 hours. The precipitate was then filtered off, washed with 3 mL of distilled water, and dried in vacuo over silica gel.

Study of the Lipoxygenase Inhibition and Biological
Tests. Experimental details for LOX inhibition activity of complexes 1-6 and their in vitro cells toxicity studies were described earlier [13][14][15].
The structure was solved with direct methods with SHELXS97 [40] and refined by full-matrix least-squares procedures on F 2 with SHELXL97 [41]. All nonhydrogen atoms were refined anisotropically, hydrogen atoms were located at calculated positions and refined as a "riding model" with isotropic thermal parameters fixed at 1.2 times the U eq of appropriate carrier atom. Crystallographic data (excluding structure factors) for the structures reported in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication nos. CCDC 705791 (1) and CCDC 705792 (2). Copies of the data can be obtained free of charge on application to CCDC (Cambridge, UK).