INTERACTION OF ORGANOTIN MOIETIES WITH DNA AND NUCLEIC ACID CONSTITUENTS

Abstract

Due to tremendous contribution in agricultural, industrial, nanotechnology and pharmaceutical fields, the significance of organometallic compounds escalated rapidly in recent years. Organotin(IV) compounds have extensive industrial applications viz. stabilizers for poly(vinyl chloride), homogeneous catalysts, disinfectants, antifouling paints, timber preservatives, anti-wear agents, recycling agents, flame retardants and corrosive inhibitors. They are renowned for their practical applications in agriculture as pesticides, fungicides, bactericides and biocides, and are also well known to exhibit potential biological properties such as antimicrobial, antibacterial, antifungal, antiviral, antiherpes, antituberculosis, anti-inflammatory, antitumor and antihypertensive activities. Amongst metal-based non-platinum chemotherapeutics, organotin(IV) compounds lie forefront for their potential application in cancer therapy. Several organotin(IV) derivatives with ligands having donor atoms viz. N, O or S, are reported to exhibit antitumor activity comparable to that of standard antitumor drug, cis-platin. The biological activity of these compounds may be due to the presence of vulnerable Sn–X (X = N, O, S or halogen) bonds (due to longer covalent bonds, high polarizability and less thermodynamic stability) yielding RnSn(4–n)+ (n = 2 or 3) species, which have high affinity for negatively charged cellular moieties such as amino acids, proteins, nucleic acids and DNA. These species also play key role in delivering the active species to the site(s) of action. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are the biopolymers of nucleic acids (nucleotides) which are essential to store, transmit and express the genetic information in living organisms. The nucleic acid constituents are the precursors of obligatory metabolites and main energy donors for all cellular processes. Purines are the most ubiquitous nitrogen-containing heterocycles, which are involved in many metabolic processes, such as cofactors associated with a great number of enzymes and receptors, notably ATP, GTP, GDP, cAMP, cGMP, AcCoA, NAD, NADP, FAD, PAPS and SAM; playing vital roles at various cell cycle phases, in cell signalling and many other fundamental biological processes. The antitumor platinum-based drug, cis-platin binds at N7 position of two successive guanines (purine base) of same strand or two different strands of DNA, thereby, hinders transcription and replication. Guanosine (a purine nucleoside) comprises of guanine attached with ribofuranose ring, whereas 5’-guanosine monophosphate (5’-GMP) (nucleotide) consists of a phosphate group attached at C’5 of ribose of guanosine. 5’-Inosine iii monophosphate (5’-IMP) or inosinic acid is a ribonucleotide of hypoxanthine. It has been reported that 1,10-phenanthroline, a bidentate chelate ligand, is effective for cancer treatment when used in combination with other antitumor drugs. Keeping in view of their extensive bioavailability in cellular fluids, important roles in biochemical processes and non-toxicity in low amounts, the selected ligands are ideal for the present studies. In order to maintain the clarity in the presentation, the work in the thesis is systematically divided into the following chapters. First chapter presents the general introduction and an overview of some important applications of organotin(IV) compounds, nucleic acid derivatives and DNA. A critical and comprehensive review on the antitumor activity and possible modes of action of organotin(IV) derivatives has also been presented. A brief literature survey on solution studies of organotin(IV) compounds with biologically important ligands and DNA binding studies of organotin(IV) complexes has also been presented. Second chapter features the details of make, purity and other specifications of the materials used in the present study. The specifications of the instruments used to carry out the potentiometric studies, elemental analyses, spectroscopic studies viz. IR, far-IR, multinuclear (1H, 13C, 119Sn) NMR, UV-Visible and fluorescence have been mentioned. The details of the procedures of various biological studies (gel electrophoresis, cytotoxicity (MTT assay) studies, enzyme (lipid peroxidase, lactate dehydrogenase) assays, acridine orange assay, comet assay of the synthesized compounds have also been described. Third chapter includes the solution studies of organotin(IV) compounds with 5’- guanosine monophosphate (5’-HGMP)2– and guanosine (HGUO) (H indicate the N1 site of nitrogen base is protonated; used only in chapter 3 for clarity purpose). The potentiometric studies of MenSn(4–n)+ (where n = 2 or 3) with (5’-HGMP)2– and guanosine in aqueous solution at 298.15 ± 0.1 K and ionic strength 0.1 M of KNO3 in 1:1 and 1:2 ratios have been performed. The protonation constants of ligands, hydrolysis constants of MenSn(IV)(4–n)+ (where n = 2 or 3) species and formation constants of complex species formed in aqueous solution at different pHs have been calculated by using computer program, SCOGS (Stability Constants of Generalized Species). The output files obtained from the SCOGS program for a given system have been employed in Origin 6.1 software to draw speciation curves. From speciation diagrams, the formation of various species at different pHs have been discussed. The results indicate that for both 1:1 Me2Sn(IV)-ligand and Me3Sn(IV)-ligand systems (where ligand = (5′-HGMP)2− and (HGUO)), M(HL) species is the major species (87–100%) existing between pH ~ 6.0–7.0 (physiological pH) along with traces of M(HL)H-1 (4–10%), iv M(HL)(OH) (0.2–3%) and M(OH)2 (0.4–0.5%) (M = MenSn(IV)(4–n)+; n = 2 or 3). Multinuclear (1H, 13C, 119Sn) NMR studies of the solutions of both 1:1 and 1:2 systems at different pHs were recorded. From the chemical shifts, coupling constants 2J(1H−117/119Sn) and 1J(13C−117/119Sn), and C–Sn–C bond angles, the possible geometries of various species have been proposed. M(HL) species adopt a distorted octahedral geometry in case of Me2Sn (IV)-ligand system and a distorted trigonal bipyramidal/distorted tetrahedral geometry in case of Me3Sn(IV)-ligand system. As the pH increases beyond 8.0, concentration of hydrolyzed species increases with the release of the ligand. Same results are obtained for 1:2 systems except the formation of hydrolyzed species starts before 5.0 pH. Fourth chapter deals with the synthesis and characterization of some new mixed diorganotin(IV) (R = Me, Ph, Bu and Oct) and triphenyltin(IV) derivatives of guanosine and 1,10-phenanthroline in 1:2:1 and 1:1:1 ratio, respectively. The probable structure for the synthesized derivatives on the basis of spectral studies viz. IR, far-IR and multinuclear (1H, 13C, 119Sn) NMR has been proposed to be a distorted octahedral geometry around tin. The in vitro cytotoxicity activities against HEK293 (human embryonic kidney) and a panel of four cancer cell lines of human origin, viz. MCF7 (mammary), HepG2 (liver), DU145 (prostrate) and HeLa (cervical) have been screened and the IC50 values indicate that the dimethyltin(IV) and dioctyltin(IV) derivatives are found to be inactive against all cell lines, where as the di-/triphenyltin(IV) derivatives show good activity (higher than cis-platin in few cases). Dibutyltin(IV) derivative exhibits the highest activity against all cell lines, except DU145. Enzyme assays viz. lipid peroxidase and lactate dehydrogenase assays, performed on two selected complexes (diphenyltin(IV) and triphenyltin(IV) derivatives) indicate that both the complexes cause apoptosis along with a minor necrosis. Acridine orange and comet assays on HepG2 and HeLa cell lines also support that apoptosis is the main cause for cell death. DNA binding studies have been performed by using UV-Visible and fluorescence spectroscopy, and the determined binding constants showed that the complexes have good binding affinity with CT DNA, suggesting that the synthesized derivatives binding to DNA led DNA damage, one of the causes for apoptosis. Further, the interaction of organotin(IV) moieties might takes place via electrostatical binding or groove insertion of DNA. Gel electrophoresis results of all the complexes indicate the promotion of the conversion of DNA from super coiled form I to nicked circular form II, implicating the role of organotin(IV) compounds in the process of DNA cleavage. v Fifth chapter deals with the synthesis and characterization of some new diorganotin(IV) (R = Me, Ph and Bu) derivatives of 5’-inosine monophosphate (5’-IMP)2– (1:1) and mixed diorganotin(IV) (R = Me, Ph and Bu) derivatives of (5’-IMP)2– and 1,10-phenanthroline (1:1:1). On the basis of spectral studies (IR, far-IR, and 1H, 13C, 119Sn NMR) of the synthesized derivatives, a distorted octahedral geometry around tin is proposed. Owing to the poor solubility, biological studies of dimethyltin(IV) derivatives could not be performed. The diphenyltin(IV) inosinate is slightly more active against MCF7 as compared to that of cis-platin but it shows very less activity in comparison to 5-fluorouracil. The compounds dibutyltin(IV) inosinate and the mixed diphenyltin(IV) and dibutyltin(IV) derivatives are found to be much more active against MCF7 cell lines. The dibutyltin(IV) derivatives are found to be very active against HepG2 cell lines. The IC50 values of the diphenyltin(IV) derivatives against HepG2 cell line suggests that the compounds are inactive for the reason unknown. The presence of phenanthroline moiety induces the cytotoxicity, hence, the mixed diphenyltin(IV) and dibutyltin(IV) derivatives a more active compared to that of respective diorganotin(IV) inosinates. However, the dibutyltin(IV) inosinate is found to be the most active complex against both MCF7 and HepG2 cell lines. Acridine orange and comet assays on MCF7 cell line support that the main cause for cell death is via apoptosis. UV-Visible spectral and fluorescence binding studies shows that the diphenyltin(IV) and dibutyltin(IV) derivatives have moderate binding affinity with DNA and the interaction might takes place via electrostatical binding or groove insertion of DNA, thus indicating that organotin(IV) binding with DNA is one of the causes for apoptosis. Gel electrophoresis results show the interaction of the complexes with DNA, but no cleavage has been observed. Chapter 6 concludes the results from the solution studies of organotin(IV) moieties with nucleic acid constituents and the studies of newly synthesized organotin(IV) derivatives. The solution studies and various biological studies have been performed in order to understand their mechanism of action. However, more detailed experimental studies are required to throw light upon their cytotoxic activities and possible modes of action. The future prospects of utility of organotin(IV) moieties in anticancer chemotherapy have also been discussed.