ORGANOTIN (IV) COMPLEXES OF DRUGS: DNA INTERACTION AND IN VITRO CYTOTOXIC STUDIES

Abstract

Organometallic compounds served as potential antitumour, antimicrobial and antimalarial agents, which revealed the metal-specific modes of action. A variety of medicinal and biological applications of organometallics due to their structural diversity and chemical tunability have emerged as a new field of research called bioorganometallic chemistry. Few interesting features of organometallic compounds which make them fit to use in biological system compared to organic compounds include; (1) a variety of coordination numbers of metal in metal complexes, provide them high structural diversity, (2) the prediction of ligand exchange reactions in the metal complexes makes them important candidates for combinatorial synthetic strategies and high biological activities, (3) the characteristic properties of metals viz. catalytic properties, Lewis acidities, redox activities, ability to access radical, radioactivity, magnetic and spectroscopic properties, allow the tailored functions in metal complexes. All the features described above are very important to design the new pharmacophores, which could not be acquired by mere organic synthesis. Organotin compounds found widespread applications in the field of industrial, agricultural, synthetic and biological fields. The biocidal uses of organotin compounds as acaricide, fungicide and in agriculture, as preservatives of wood and for the protection of textiles, cordage fibers, paper, leather and electrical equipment etc. revealed their high toxicity towards living cells. A significant research has been carried out to investigate the biological or pharmaceutical role of organotin compounds, which prove them potential apoptotic inducer, antiviral, anticancer, antibacterial, antifungal, antineoplastic and anti-tuberculosis agent. During last few decades organotin compounds occupy an important place in cancer chemotherapy reports, which place them among the most reliable compounds to replace the platinum based antitumour drugs in order to avoid the serious side effects and to overcome the drug resistance related to platinum based drugs. In order to understand the distribution of the species of a particular compound in a system under study, it is must to account the data related to various equilibria established and stability constant calculated for the particular species involved. Inside the living systems organotins can interact with proteins, nucleic acids, carbohydrates and lipids biomolecules through heteroatoms like N, P, O, and S, thus the discussion of their equilibria and formation of the different species of ii biologically active ligands with organotins is of considerable importance. However, the study in this regard with commercially available drugs are quite rare, thus we carried out the potentiometric study of MESNA (sodium 2-mercaptoethanesulphonate) with organotin(IV) chloride. Many researchers adopted the idea of syntheses of the organotin complexes with commercially available drugs in order to achieve the desired features. In this way the metal can stabilize the drug which in turn may help the metal complex in transportation in order to reach their potential target and avoid needless side reactions. In this view a number of organotin(IV) complexes with commercially available drugs have been synthesized and many of them exhibited improved biological activities. Further, the non-steroidal anti-inflammatory drugs (NSAIDs) have been found quite effective against various types of cancers viz. breast, esophageal, stomach, prostate, bladder, ovarian, lung and colorectal cancers and successfully used in combination therapies with antitumour drugs. Thus, we chose to synthesize the organotin-drug complexes, study their various possible mode of binding with DNA, and further investigate their biological activities. In order to achieve the desired objective the thesis has been divided into seven chapters which are described in detail herein. First Chapter highlights the importance of organotin(IV) complexes in various fields including commercial, agricultural, synthetic and biological uses. The critical discussion on the mode of action of organotin(IV) complexes inside the biological system and their DNA binding properties has been presented. A huge literature survey based on organotin complexes having antimicrobial and antitumour activities has been exemplified. Further, the importance of potentiometric study of these complexes has been highlighted with various examples. Second Chapter describes the purity and make of different chemicals used in the current study. This chapter also includes the specification of various instruments used during the experimental work. The details of the methods and procedures which were used in common in all Chapters have also been discussed in this section. Third Chapter includes the potentiometric study of interaction of [Me2Sn(IV)]2+ and [Me3Sn(IV)]+ with a prodrug, sodium 2-mercaptoethane sulfonate (MESNA) abbreviated as (HL) in aqueous solution (I = 0.1 M KNO3, 298 K). The concentration distribution of various species formed in the solution was studied with a change in pH (~3–11). A strong coordination of MESNA with metal through S atom of thiol group has been elucidated. In Me2Sn(IV) – HL system, the iii species [Me2Sn(L)]+ (53.1–75.6%) is predominating at acidic pH (3.73 ± 0.02), and the species [Me2Sn(L)2OH]– (29.4–38.5%) is predominating at basic pH (10.32 ± 0.08). Whereas for [Me3Sn(IV)]+ system, [Me3SnL] (37.0–57.4%) is the major species at pH 7.65 ± 0.03 and [Me3Sn(OH)] (49.9–67.2%) and [Me3Sn(L)(OH)]– (30.2–46.5%) are the major species at pH 11.05 ± 0.01. However, at physiological pH (7.01 ± 0.32), in both (1:1) and (1:2) Me2Sn(IV) – HL systems, the species [Me2Sn(L)(OH)] (67.2–89.9%) is predominating whereas for Me3Sn(IV) – HL (1:1) and (1:2) systems, [Me3Sn(OH)] (53.5%) and [Me3SnL] (56.8%) are the respective predominated species. In order to characterize the possible geometry of the proposed complex species, multinuclear (1H, 13C and 119Sn) NMR studies were carried out at different pHs. No polymeric species were detected in the experimental pH range. Fourth Chapter discussed the synthesis of the diorganotin(IV) complexes (1-4) of MESNA (sodium 2-mercaptoethanesulfonate HSCH2CH2SO3Na) and a mixed ligand complex of dibutyltin(IV), 1,10-phenanthroline and MESNA (5). All the complexes were characterized thoroughly with the help of analytical and various spectroscopic techniques viz. FTIR, NMR (1H, 13C, and 119Sn NMR) spectroscopy and ESI MS spectrometery. Various spectrophotometric studies were carried out to decipher the binding mode of MESNA and its diorganotin complexes 1-5 with calf thymus DNA (CT DNA) and thus, to calculate the binding constant (Kb). Absorption spectrophotometric study confirmed the interaction is through partial intercalation of all the complexes including MESNA, inside the DNA helix and calculated binding constant (Kb) is in the order of 103 M-1. A series of emission spectrophotometric experiments support the results obtained through the absorption spectrophotometric studies. Circular dichroic (CD) spectroscopic analysis and viscosity measurement of CT DNA further complemented the fact that the partial intercalation plays a major role in the interaction of the studied complexes with CT DNA. All the studies corroborated that complex 2 bound to CT DNA with maximum affinity followed by complex 5 among all the complexes. Involvement of hydroxyl radicals as an active species in the cleavage activity of pBR322 plasmid DNA is proved by carrying out agarose gel electrophoresis. The cytotoxicity of the complexes was evaluated with MTT assay against DU145 (prostate cancer), HCT-15 (colon adenocarcinoma) and HeLa (cervical cancer) cell lines. Complexes 4 and 5 demonstrated remarkable cytotoxicity in vitro, even more than 5-fluorouracil, against prostate cancer cell lines, which possesses intrinsic and acquired resistance to cis-platin. Complex 5 displayed highest cytotoxicity against all the cell lines viz. DU145, HCT-15 and HeLa with IC50 iv values 7.15±0.7 μM, 0.83±0.1 μM and 1.85±0.4 μM, respectively. The cause of cell death was further investigated through acridine orange/ethidium bromide staining of cell lines and DNA fragmentation assay which revealed that the other processes along with the apoptosis plays role in cell death. Fifth Chapter deals with the syntheses of tri- and diorganotin(IV) derivatives of non-steroidal anti-inflammatory drug (NSAID), sulindac (Sul), 1-7 viz. [Me3Sn(Sul)], [Ph3Sn(Sul)], [Bu3Sn(Sul)], [Ph2Sn(Sul)2], [Me2SnCl(Sul)]2, [Bu2Sn(Sul)2] and [Oct2Sn(Sul)2]. The complexes have been characterized by analytical and spectroscopic (FTIR, 1H, 13C, 119Sn NMR and ESI MS) techniques. Crystal structure of 3 indicates trigonal bipyramidal geometry around tin atom in solid state, where axial positions were occupied by O atoms of carboxylate and sulphoxide groups. Carboxylate oxygen is coordinating to Sn atom in a monodentate fashion. Dissociation of the coordinate bond between Sn and O (sulphoxide group) resulted in tetrahedral structure of the complex as witnessed from solution study in NMR. Similar structures were proposed for complexes 1 and 2. Optimized geometry and electronic structures of complexes obtained from DFT calculations reveals that Sn atom in complexes 4 and 6 are hexa-coordinated with highly distorted octahedral geometry, whereas 5 is penta-coordinated with distorted trigonal bipyramidal geometry and 7 is tetra-coordinated with monodentate carboxylates. Probable mode of DNA binding with ligand (Sul) and complexes 1–7 has been revealed via various biophysical techniques (UV–visible spectroscopy, fluorometry and circular dichroism). Intrinsic binding constants (Kb) obtained from UV–visible spectroscopy for Sul and complexes 1 – 7 are 3.69 × 104 M-1, and 7.3 × 103 M-1, 1.14 × 104 M-1, 1.47 × 104 M-1, 1.55 × 104 M-1, 1.49 × 104 M-1, 2.02 × 104 M-1, 1.17 × 104 M-1, respectively. The quenching constants (Ksv) using flourometric titrations, calculated from competitive binding of ethidium bromide (EB) vs Sul/complexes (1–7) with CT DNA, also corresponds to the above results. Circular-dichroism (CD) spectroscopic patterns of CT DNA with Sul and complexes 1–7 have been investigated. All the above results revealed that the complexes bind with DNA through partial intercalative mode. pBR322 Plasmid fragmentation has also been studied by gel electrophoresis, which shows the fragmentation of circular DNA by increase in nicked form and also with the appearance of linear form with increasing concentration of drug or complexes. Further, in vitro cytotoxicity of Sul and synthesized complexes against HCT-15 (colon adenocarcinoma), MCF-7 (mammary cancer), HeLa (cervical cancer), and LNCaP (androgen-sensitive prostate adenocarcinoma) cancer cell lines have been determined, which indicated a low v to moderate cytotoxicity of these complexes toward MCF-7, HeLa and LNCaP cancer cell lines. However, all the complexes demonstrated good in vitro antitumour activity towards the HCT-15. Sixth Chapter encompasses the synthesis and characterization of organotin(IV) derivatives of non steroidal anti-inflammatory drug Ibuprofen (IBF) viz. [(Me3Sn)(IBF)] (1), [(Bu3Sn)(IBF)] (2), [Ph3Sn(IBF)] (3), [(Me2Sn(IBF))2O]2 (4) and [Bu2Sn(IBF)2] (5). The crystal structure of complexes 3, [Ph3Sn(IBF)], gives the clearity about the structure of triorganotin complexes. Triorganotin complexes are characterized with highly distorted tetrahedral (td) geometry with anisobidentate mode of co-ordination of the carboxylate group with tin atom. Moreover, the DFT calculation and other studies varified a dimer distannoxane type of structure for complex 4, [(Me2Sn(IBF))2O]2. Complex 5 was found to exhibit a highly distorted octahedral geometry around tin atom. To investigate the DNA binding profile of the synthesized complexes, UV-visible and fluorescence titrations were performed, which revealed an intercalative type of binding with DNA for IBF and complex 5 and external binding in case of the complexes 1 and 2. Plasmid DNA fragmentation studies indicate that the complexes and IBF cleaved the pBR322 plasmid potentially. Further, the IBF and complexes 1-5 were screened in vitro for cytotoxicity against human cancer cell lines viz. DU145 (prostate cancer), HCT-15 (colon adenocarcinoma) and Caco-2 (colorectal adenocarcinoma) through MTT reduction assay and the cause of cell death was investigated through acridine orange/ethidium bromide staining of cells and DNA fragmentation assay. Complex 4 displayed the maximum cytotoxicity against prostate and colon cancer cell lines, with IC50 values 3.97±0.81 μM and 2.188±0.67 μM, respectively, while complex 3 was found highly regressive against colorectal cancer (IC50 = 1.21±0.84 μM) cell lines. Seventh Chapter encloses the synthesis and characterization of trimethyltin(IV) (1) and tributyltin(IV) (2) complexes of an anticoagulant drug warfarin (WR). The full geometry optimization and simple harmonic frequency calculations in gaseous phase by DFT indicated that both the complexes have distorted tetrahedral geometry around tin centre. The synthesized complexes have been investigated for their DNA binding profile (mode and extent of binding with CT DNA) through UV-visible and fluorescence spectrophotometric titrations, which revealed a partial intercalation of complexes inside the base pairs of DNA, and the binding constant (Kb) calculated through UV-visible titration study was found to be in the order of 104 M-1. The partial intercalation of the complexes inside the DNA has also been confirmed through a decrease in the viscosity of CT DNA with the increasing complex concentration. Both the complexes were found vi to be potential plasmid cleaving agent which has been confirmed though gel electrophoresis of pBR322 plasmid with increasing the complexes concentrations. Both the organotin(IV) complexes have been found to exhibit a greater potential towards DNA binding and fragmentation in comparison to that of WR. Complexes and WR were screened in vitro for anti-tumor activity against HCT-15 (colon adenocarcinoma), MCF-7 (mammary cancer), HeLa (cervical cancer), and LNCaP (androgen-sensitive prostate adenocarcinoma) cell lines of human origin, which exhibited a good activity of complexes against prostate cancer cell line.