Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Leishmanioses são um grupo de doenças com um largo espectro de manifestações clínicas, as quais variam desde lesões cutâneas até o envolvimento visceral severo, podendo levar ao óbito. A leishmaniose é, ainda hoje, uma doença negligenciada, estando entre os agravos prioritários do programa de pesquisa sobre doenças da pobreza da Organização Mundial da Saúde (OMS). Além de não haver vacinas disponíveis, a terapia é baseada em medicamentos injetáveis que causam sérios efeitos colaterais, tornando o tratamento inviável para muitos países endêmicos. Drogas derivadas de metal representam um novo arsenal terapêutico antimicrobiano e anti-câncer. Os inibidores de peptidase/agentes quelantes tais como 1,10-fenantrolina e seus derivados, no estado livre de metal ou como ligantes com metais de transição, interferem com a função de vários sistemas biológicos. Em trabalhos anteriores, nosso grupo descreveu que o parasito L. braziliensis produziu moléculas gp63 sensíveis a 1,10-fenantrolina. No presente trabalho, demonstramos a distribuição celular da molécula gp63 em uma cepa virulenta de L. braziliensis por meio de análises bioquímicas e imuno-histoquímica. Depois disso, relatamos os efeitos inibitórios de três compostos derivados da 1,10-fenantrolina, 1,10-fenantrolina-5,6-diona (phendio), [Cu(phendio)2] e [Ag(phendio)2], nas atividades metalopeptidases celulares e extracelulares produzidas por promastigotas de L. braziliensis, bem como as suas ações sobre a viabilidade do parasita e na interação com as células de macrófagos murinos. As moléculas gp63 foram detectadas em compartimentos de parasitos, incluindo membrana citoplasmatica e bolsa flagelar. O tratamento de promastigotas de L. braziliensis durante 1 hora com 1,10-fenantrolina e seus derivados resultou numa inibição significativa da viabilidade celular e mostrou um mecanismo de ação irreversível. Estes inibidores de metalopeptidases induziram apoptose em promastigotas de L. braziliensis, demonstrada através da marcação com anexina/iodeto de propídio e ensaio TUNEL. O pré-tratamento de promastigotas com os inibidores de metalopeptidases induziram uma diminuição na expressão de moléculas de superfície gp63, assim como uma redução significativa no índice de associação com macrófagos. Em paralelo, macrófagos infectados com L. braziliensis e tratados com 1,10-fenantrolina e seus derivados promoveram uma potente redução sobre o número de amastigotas intracelulares. O tratamento de macrófagos com 1,10-fenantrolina e seus derivados não induziram o aumento de óxido nítrico. A ação combinatória sobre a capacidade de crescimento entre os compostos derivados da 1,10-fenantrolina e Glucantime, quando ambos foram utilizados em concentracões sub-inibidoras, também foi observada. In vivo os compostos derivados da 1,10-fenantrolina e seus drivados foram capazes de controlar o tamanho das lesões a partir da terceira semana de tratamento em relação ao controle não tratado em hamsters infectados quando administrado por via intraperitoneal. Os animais tratados com os compostos apresentaram maior resposta intradérmica (DTH) aos antígenos de L. braziliensis. Coletivamente, a 1,10-fenantrolina e seus derivados metálicos apresentam uma nova perspectiva de estudos para o desenvolvimento de novos fármacos anti-L. braziliensis / Leishmaniasis is a group of diseases with a wide spectrum of clinical manifestations, which range from self-limited skin lesions to severe visceral involvement that can lead to death. Leishmaniasis is still a neglected disease, and it is among the priorities of the research program on diseases of poverty of World Health Organization (TDR/WHO). There is no available vaccine and the treatment is based on drugs that cause serious side effects, and are unaffordable in several endemic countries. Metal-based drugs represent a novel antimicrobial and anti-cancer therapeutics arsenal. Peptidase inhibitors/chelating agents such as 1,10-phenanthroline and its substituted derivatives, either the metal-free state or as ligands coordinated to transition metals, interfere with crucial functions of several biological systems. In previous works, our group described that L. braziliensis produced gp63 molecules sensible to 1,10-phenanthroline. Herein, we initially studied the cellular distribution of gp63 in a virulent strain of L. braziliensis by biochemical and immunocytochemical analyses. After that, we reported the inhibitory effects of three 1,10-phenanthroline derivative compounds, 1,10-phenanthroline-5,6-dione (phendio), [Cu(phendio)2]and [Ag(phendio)2], on both cellular and extracellular metallopeptidase activities produced by L. braziliensis promastigotes as well as their actions on the parasite viability and on the interaction with murine macrophage cells. The gp63 molecules were detected in several parasite compartments, including cytoplasm, membrane lining the cell body and flagellum, and flagellar pocket. The treatment of L. braziliensis promastigotes for 1 hour with 1,10-phenanthroline and its derivatives resulted in a significant inhibition of cell viability and showed an irreversible mechanism of action. These metallopeptidase inhibitors induced apoptosis in L. braziliensis promastigotes as judged by annexin/propidium iodide staining and TUNEL assays. The pre-treatment of promastigotes with metallopeptidase inhibitors induced a reduction on the expression of surface gp63 as well as a significant reduction on the association index with macrophages. In parallel, the treatment of L. braziliensis-infected macrophages with the 1,10-phenanthroline and its derivatives promoted a powerful reduction on the number of intracellular amastigotes. The treatment of macrophage cells with 1,10-phenantroline and its derivatives did not induce nitric oxide production. A synergistic inhibition on the growth ability was observed when 1,10-phenanthroline derivative compounds and Glucantime were used at sub-inhibitory concentrations. In vivos the compounds 1,10-phenanthroline derivatives and its were able to control the lesions size from the third week of treatment on, when compared to untreated control infected hamsters. The animals treated with the compounds showed greater response to the intradermal antigens of L. braziliensis. Collectively, the 1,10-phenanthroline and its metal-based drugs present a good perspective for prospective studies to the development of new anti-L. braziliensis drugs

Photodynamic therapy(PDT) has recently emerged as a promising new non-invasive treatment modality for a large number of neoplastic and non-neoplastic lesions. Photoexcitation of a photosensitizing drug in the tumor tissue causes generation of reactive oxygen species which results in cell death. The current porphyrinic photosensitizers suffer a wide range of drawbacks leading to the development of the chemistry of alternative photosensitizing agents in PDT. Among them, the 4d and 5d transition metal-based photosensitizers have been explored extensively with the exception of the 3d metal complexes. The objective of this thesis work is to design and synthesize photoactive iron(III) abd cobalt(III) complexes and evalutate their photonuclease and photocytotoxic potential. Bioessential 3d metal ions provide an excellent platform for metal-based PDT drug designing as because of its varied spectral, magnetic and redox properties, with its complexes possessing rich photochemical behavior in aqueous and non-aqueous media. We have synthesized binary iron(III) complexes as netropsin mimics using amino acid Schiff bases derived from salicylaldehyde/napthaldehyde and arginine/lysine. The complexes were found to be good AT selective DNA binders and exhibited significant DNA photocleavage activity. To enhance the photodynamic potential, we further synthesized iron(III) complexes of phenolate-based ligand and planar phenanthroline bases. The DNA photocleavage activity of these complexes and their photocytotoxic potential in cancer models were studied. ROS generated by these complexes were found to induce apoptotic cell death. Ternary cobalt(III) complexes were synthesized to study the effect of the central metal atom. The diamagnetic cobalt(III) complexes were structurally dissimilar to their iron(III) analogues. Although the Co(III)/Co(II) redox couple is chemically and photochemically accessible but the Co(III)-dppz complex, unlike its iron(III)-dppz analogue, exhibited selective damage to hTSHR expressing cells but not in HeLa cells. A structure-activity relationship study on iron(III) phenolates having modified dppz ligands was carried out and it was found that electron donating group on the phenazine unit and an increase of the aromatic surface area largely improved the PDT efficiency. Finally, SMVT targeted iron(III) complexes with biotin as targeting moiety were synthesized and the in vitro efficacy of the complexes was tested in HepG2 cells over-expressing SMVTs and compared to HeLa amd HEK293 cells. The complexes exhibited higher phytocytotoxicity in HepG2 than in HeLa and cells and HEK293 cells. An endocytotic mode of uptake took place in HepG2 cells whereas in HEK293 cells, uptake is purely by diffusion. This is expected to reduce the side-effects and have less effect on cells with relatively less SMVTs. In summary, the present research work opens up novel strategies for the design and development of primarily iron-based photosensitizers for their potential applications in PDT with various targeting moieties.

Photodynamic therapy (PDT) is a non-invasive treatment of cancer with an advantage of having localized photo-activation of the drug at the targeted tumor cells leaving the healthy cells unaffected by the photo-toxicity of the PDT agent. Organic molecules and 4d/5d metal complexes have been extensively studied for their DNA cleavage activity and photo-cytotoxicity in UV and/or visible light. The photoactivity of the current PDT drugs is due to reactive singlet oxygen species. To address the hypoxic nature within neoplasia and to get a realistic scenario to build model and potent PDT agents, attempts have been made in this thesis work to design and synthesize new cobalt and copper complexes having a variety of ancillary ligands and planar phenanthroline bases showing efficient visible light-induced anaerobic plasmid DNA cleavage activity. The disulfide and thiol compounds are known to generate thyil radical in anaerobic medium in presence of some electron donating solvent. To exploit this chemistry of the sulfur anion radical as a reactive species damaging DNA under light irradiation, we have prepared copper(II) complexes of bis(2-hydroxybenzylamino-ethyl)disulfide and D-penicillaminedisulfide and characterized. The complexes are moderate binders to calf thymus DNA and exhibit plasmid DNA cleavage activity in red light. Near-IR light-induced double-strand DNA cleavage activity is observed for the complexes having 3,3' -dithiodipropionic acid and phenanthroline bases. These complexes show lethal double strand breaks in SC DNA responsible for the inhibition in DNA repair mechanism in the cells thus becoming potent candidates as transcription inhibitors. The work has been extended to achieve better visible light-induced plasmid DNA cleavage activity and UV light-induced photocytotoxicity using a more bio-compatible metal ion, viz. cobalt(II) with the same ligand system and enhancement in the photocytotoxicity is observed. To investigate the role of the disulfide ancillary ligands, complexes of salicylideneaminothiophenol bound to the copper(II) are prepared and the complexes show significant plasmid DNA cleavage activity in red light. Finally, ternary cobalt(III) phenanthroline base complexes are prepared to study their DNA cleavage activity in red light and photo-cytotoxicity in UV light. The complexes show efficient plasmid DNA cleavage activity in red light, significant cytotoxicity in UV light, low dark cytotoxicity, and protein (BSA, lysozyme) cleavage activity in UV light. The mechanistic aspects of the photo-induced DNA and protein cleavage activity of the complexes have been studied. A dual involvement of the charge transfer and d-d band is observed in the photosensitization process leading to generation of reactive oxygen species. In summary, the thesis work presents cobalt and copper complexes having thiolate and disulfide moieties that are designed and synthesized as new photodynamic therapeutic agents showing anaerobic DNA cleavage activity in red light and photocytotoxicity. The present study opens up new strategies for designing and developing cobalt and copper based photosensitizers for their potential photochemotherapeutic applications under hypoxic reaction conditions. References: Lahiri, D. et al., J Chern. Sci, 2010, 122, 321-333; Inorg. Chern., 2009, 48, 339-349; Dalton Trans. 2010,39,1807-1816; Polyhedron, 2010, 29, 2417-2425.

Photo sensitizers showing visible light induced DNA cleavage activity are of current importance for medicinal applications related to photodynamic therapy (PTD) considering greater skin penetration of light near 700 nm. While organic molecules and complexes of 4d-5d metal ions are extensively studied for their DNA photo-damage properties in UV and visible light, the chemistry of 3D metal complexes showing visible light-induced DNA cleavage activity is relatively unexplored efforts have been made in this thesis work to design new ternary copper (II) complexes having a-amino acids Such copper (II) complexes with tunable coordination geometry could find potential applications in PDT. Ternary Copper (II) complexes containing L-methionine, S-methy1-L-cysteine and phenanthroline bases are prepared and characterized. They display DNA binding and visible light induced DNA cleavage activity. An enhancement of the DNA cleavage activity is observed for analogous ternary copper (II) complexes contained L-lysine with a pendant cationic amine moiety as a photo-induced DNA Cleavage activity using binary and ternary copper (II) complexes of L-arginine and phenanthroline bases. We have observed AT selective DNA binding and visible light –induced DNA cleavage activity. The crescent-shaped bis-arginine Copper (II) complex mimics the natural antiviral antibiotic netropsin. T o investigate the role of the pendant groups of the amino acids, we have explored the DNA binding and DNA cleavage activity of analogues L-glutamine and L-asparagine complexes. We have prepared ternary copper (II) complexes containing two photosensitizers, viz., L-tryptophan (L-trp) and dipyridoquininoxaline/dipyridophenazine to achieve double strand breaks forming linear DNA. Complex [Cu(L-trp)(dppz)(H2O)+ shows a stacking arrangement of the indole and dppz rings giving a separation that fits with the base pair separation of ds-DNA. Photosensitizes in these complexes approach two different complementary stands of the ds-DNA, leading to double strand breaks and formation of linear DNA. Mechanistic studies on the DNA photocleavage reactions reveal the formation of singlet oxygen(1O2)species by a type-II pathway in preference to the hydroxyl radical generation. A process leading to an efficient DNA cleavage activity on visible light irradiation. The observation of sequence selectivity and double strand DNA cleavage on red light exposure by national design of the complexes is significant considering importance of the results in the chemistry of photodynamic therapy of cancer. The results of this dissertation open up new avenues for designing and developing 3d metal-based photosensitizers with potential utility in nulcleic acid chemistry.

La synthèse et l'étude de rotaxanes et de porphyrines sont deux domaines particulièrement actifs de la recherche en chimie. Cependant, les composés combinant les propriétés intéressantes de ces deux types de structures sont plus rares. De nouveaux multi-rotaxanes à base de porphyrines, dont la préparation représente un défi synthétique, sont décrits dans cette thèse.Des porphyrines liées à deux ou quatre anneaux coordinants ont été synthétisées. Des rails moléculaires à deux chélates ont été enfilés dans les anneaux grâce à l'effet template du cuivre(I); l'introduction de bouchons a mené à la formation de rotaxanes. Dans le cas du bis-macrocycle porphyrinique, un [4]rotaxane a été obtenu. Des études de complexation hôte/invité avec des ligands azotés rigides ont montré que ce rotaxane est un récepteur moléculaire qui peut s'adapter à la taille du substrat invité en se "gonflant" ou en se "dégonflant".Dans le cas du tétra-macrocycle porphyrinique, la formation d'un [3]rotaxane d'architecture originale a étéobservée.La synthèse d'un nouveau bis-macrocycle plus rigide est en cours. Ce composé sera utilisé pour la construction d'un [4]rotaxane, qui pourrait montrer un caractère de presse moléculaire capable de modifier la conformation d'un substrat invité en le comprimant. / Rotaxanes and porphyrins are two particularly active fields of research in chemistry. However,molecules that combine the interesting properties of these types of structures are not so common. In this thesis we describe new porphyrin-based multi-rotaxanes, whose syntheses constitute interesting challenges.Porphyrins linked to two or four coordinating macrocycles were synthesised. The "gathering-andthreading" effect of copper(I) was used to thread molecular rods through the rings; the subsequent introduction of stoppers led to the formation of rotaxanes. In the case of the porphyrinic bis-macrocycle a [4]rotaxane was obtained. Host/guest complexation studies with rigid nitrogen ligands showed that the rotaxane behaves as a distensible molecular receptor that can adopt an "inflated" or "deflated" conformation and adjust its shape to the size of the guest. In the case of the porphyrinic tetra-macrocycle the formation of a [3]rotaxane of novel architecture was observed.The synthesis of a new, more rigid bis-macrocycle is in progress. This compound will be used for the construction of a [4]rotaxane that could act as a molecular press able to change the conformation of a guest substrate by compression.

Ferrocene is an important molecule in the field of chemical biology due to its stability, unique redox property and significant lipophilicity for better cellular delivery. The medicinal importance of ferrocene is well recognized after its successful incorporation into breast cancer drug tamoxifen and antimalarial drug chloroquin. Designing ferrocene conjugated transition metal complexes is an interesting area of research in the field of photodynamic therapy, a new modality of light activated cancer treatment. The objective of the present thesis work is to develop photoactive ferrocene conjugates showing DNA photocleavage and photocytotoxic activity. We have synthesized the ferrocene conjugated imidazophenanthroline derivative which exhibits visible light induced DNA photocleavage activity and photocytotoxicity in HeLa cell line. The corresponding phenyl analogue is found to be inactive. Polypyridyl platinum(II) complexes of ferrocenyl as well as phenyl moiety are prepared and studied their interactions with calf thymus DNA. The cytotoxicity of the complexes enhance significantly upon irradiation of UV-A light of 365 nm. To enhance the photodynamic potential and to understand the role of organometallic ferrocenyl moiety, ferrocene conjugated terpyridyl copper(II) complexes having planar phenanthroline bases are prepared. The interaction of these complexes with duplex DNA and their photo-induced DNA cleavage and anticancer activity in HeLa cancer cells are studied. The complexes are able to generate ROS in the presence of visible light which causes DNA damage as well as cell death. The importance of ferrocenyl moiety is evidenced from the less activity of the corresponding phenyl analogues complex. We have prepared copper(II) complexes of ferrocenyl methyl dipicolylamine ligand to understand the role of terpyridyl moiety. These complexes lacking any conjugation between the copper(II) and the ferrocenyl moiety are found to be less active compared to the terpyridyl conjugated system. The copper(II) complexes are found to show undesirable dark cytotoxicity in the presence of cellular thiols like GSH. To overcome the dark toxicity problem and to understand the mechanistic aspects of DNA photocleavage and photocytotoxicity, a series of binary ferrocene conjugated terpyridyl complexes of Fe(II), Co(II), Cu(II) and Zn(II) are prepared and their DNA photocleavage and anticancer activity studied. The zinc(II) complex having redox-active ferrocenyl moiety and redox-inactive zinc(II) center exhibits significant PDT effect with low dark toxicity compared to its copper(II) analogue. The ferrocenyl moiety plays an important role towards showing photocytotoxic activity since its phenyl analogue is inactive in nature. Finally, the present thesis work opens up a new strategy for designing and developing new ferrocene based metal complexes as novel photosensitizers for PDT applications.

The present thesis deals with different aspects of the chemistry and photo-biology of various ferrocene-conjugated metal complexes, their interaction with double helical DNA, DNA photocleavage and photo-enhanced cytotoxicity in visible light. Phenyl analogues of the active complexes have been synthesized and used for comparison in biological assays. Chapter I provides an introduction to the potential of metal complexes as photochemotherapeutic agents with special reference to organometallic compounds. A brief overview of Photodynamic Therapy (PDT) as a new modality of cancer treatment has been given. Various modes of non-covalent interactions of small molecules with duplex DNA are mentioned. Recent reports on the metal-based photocytotoxic and DNA cleaving agents including photoactivatable organometallic compounds are discussed. The objective of the present investigation is also presented in this chapter. Chapter II presents the synthesis, characterization, structure, DNA binding, DNA photocleavage, photocytotoxicity, mechanism of cell death and cellular localization of ferrocene-conjugated L-methionine reduced Schiff base Cu(II) complexes of phenanthroline bases. To explore the role of the ferrocenyl moiety the phenyl analogues of the ferrocenyl complexes are synthesized and used as controls for comparison purpose. Chapter III deals with the photo-induced DNA cleavage and photo-enhanced cytotoxicity of ferrocene-appended L-tryptophan Cu(II) complexes of heterocyclic bases. The synthesis, characterization, structural comparisons, DNA binding, DNA photocleavage, photocytotoxic activity and cell death mechanism in visible light are discussed in detail. Chapter IV describes the synthesis, characterization and structure of ferrocenylmethyl-L-tyrosine Cu(II) complexes of phenanthroline bases. The complexes are evaluated for DNA binding, DNA photocleavage and photocytotoxic activity in visible light. The cellular localization of the complexes and the mechanism of cell death induced by the complexes are also discussed. Chapter V presents the photocytotoxic effect of ferrocene-conjugated L-amino acid reduced Schiff base Cu(II) complexes of anthracenyl/pyrenyl imidazophenanthroline. The ability of the complexes to bind to double helical DNA and cleave it under photo-illumination conditions is described. Evaluation of the complexes as photochemotherapeutic agents and comparison with currently clinically available drug Photofrin are presented. The mechanism of cancer cell death and cellular localization of the complexes are studied by fluorescence microscopy. Chapter VI describes the synthesis, characterization and photochemotherapeutic efficacy of Cu(II) complexes having ferrocene-appended L-amino acid reduced Schiff base ligands and the naturally occurring polyphenol curcumin. Stabilization of curcumin by complexation to metal for improved photodynamic effect in cancer cells is described with comparison to the parent dye and clinically used drug Photofrin. The mechanism of cell death induced by the copper complexes and their localization in cancer cells are also presented. Finally, the summary of the dissertation and conclusions drawn from the present investigations are presented. The references in the text have been indicated as superscript numbers and compiled at the end of each chapter. The complexes presented in this thesis are represented by bold-faced numbers. Crystallographic data of the structurally characterized complexes are given in CIF format in the enclosed CD (Appendix-I). Due acknowledgements have been made wherever the work described is based on the findings of other investigators. Any unintentional omission that might have happened due to oversight or mistake is regretted.