Neste trabalho sintetizaram-se novos complexos imínicos e diimínicos de Cu(II) derivados da isatina, um indol endógeno, que foram então extensivamente caracterizados por análise elementar, medidas de condutividade molar, técnicas espectroscópicas: IV, UV/Vis e EPR, e por espectrometria de massa, ESI-MS. Estes compostos apresentaram equilíbrio ceto-enólico em solução, variando a geometria ao redor do íon Cu(II), ao se alterar o pH do meio, indo de tetraédrico em meio ácido à tetragonal em meio básico. Para a elucidação do papel do cobre no mecanismo de oxidação de carboidratos pelo oxigênio molecular, realizaram-se estudos cinéticos tendo como catalisadores estes complexos de cobre. Determinou-se a uma lei cinética global mais abrangente para estas reações, incluindo uma etapa dependente do cobre (a concentrações bem baixas), seguida de outra independente do metal. As etapas no mecanismo proposto, sob condições de pseudo-primeira ordem, combinam transferência eletrônica intramolecular, com provável redução do íon de cobre pelo substrato, levando a espécies muito reativas (OH•-, O2•-, H2O2, CO2•-), responsáveis pelo processo de iniciação e propagação, e a formação de produtos carbonílicos de cadeia curta (glicolato, glicerato e formiato). Para o estudo da interação metal-carboidrato, importante para se entender melhor o papel do cobre frente a este ligante biológico, complexos de Cu(II) com monossacarídeos foram sintetizados e caracterizados por análise elementar e termogravimétrica, medidas de condutividade molar, espectroscopias UV/Vis, IV e EPR, além da espectroscopia Raman, através da qual investigou-se o modo de ligação do carboidrato ao metal em cada um destes complexos. Foram ainda preparados e caracterizados por análise elementar, medidas de condutividade molar, espectroscopias UV/Vis, IV, EPR, ESI-MS e CD dois novos complexos imínicos quirais de Cu(II), com ligantes do tipo aminocarboidrato, e que também foram utilizados para estudos biológicos. Estudos de atividade biológica foram feitos in vitro, usando as linhagens celulares tumorais promonocítica sanguínea U937 e neuroblastoma SH-SY5Y, com os complexos imínicos de cobre, principalmente aqueles derivados da isatina. As células tratadas com os complexos mais ativos sofreram apoptose, verificada por ensaios citofluorimétricos, em que os complexos agiram em diferentes fases do ciclo celular de cada linhagem (fase G1, S ou G2/M). Através da técnica citofluorimétrica foi observada também a geração de radicais livres na célula e, através de ensaios imunológicos, determinou-se a quantidade de proteínas citoplasmáticas carboniladas e glicosiladas, geradas após os tratamentos com os compostos, em diferentes tempos de incubação. De uma maneira geral, estes estudos indicaram modulação da atividade biológica, com um comportamento antiproliferativo muito diferente, indo de baixa eficácia até alta eficiência. Dentre os compostos mais ativos, pode ser observada uma especificidade diferente, tanto com relação ao tipo de célula quanto ao seu modo de ação, evidenciando sua potencial aplicação como agentes antitumorais. / In this work, novel imine and diimine copper(II) complexes with ligands derived from isatin, an endogenous indol, were synthesized, and extensively characterized by elemental analysis, conductivity measurements, spectroscopic techniques (IR, UV/Vis, and EPR), and electrospray mass spectrometry (ESI-MS/MS). These compounds showed a keto-enolic equilibrium in solution, with variations in the geometry around the copper ion with increasing pH, varying from a more tetrahedral configuration in acidic medium to a tetragonal one in basic solution. In order to elucidate the role of copper in the carbohydrate oxidation by molecular oxygen, kinetic studies were performed using these complexes as catalysts. A more comprehensive global rate law was determined for this process, including a copper-dependent pathway (at very low concentrations), in addition to an independent one, both influenced by alkaline medium. The proposed mechanism, under pseudo-first order conditions, combine intramolecular electronic transfer with reduction of the copper ion by the substrate, leading to the formation of intermediary reactive species (OH•-, O2•-, H2O2, CO2•-), responsible for initiation and propagation steps, and of short chain carbonylic products (glycolate, glycerate and formiate ions). To better understanding metal-carbohydrate interactions, copper(II) complexes with simple monosacharides were isolated, and characterized by elemental and thermogravimetric analyses, and spectroscopic techniques (IR, UV/Vis, and EPR), besides Raman spectroscopy, used to investigate the binding mode of the carbohydrate moiety to the copper ion, in each one of these complexes. Additionally, two novel chiral imine copper(II) complexes, derived from aminocarbohydrate ligands, were prepared and characterized by elemental analysis, conductivity measurements, and spectroscopic techniques (IR, UV/Vis, EPR, ESI-MS and CD), and one of them was also used in biological studies. Biological activity studies were carried out with the imine and diimine copper(II) complexes derived from isatin, verifying antiproliferative effect toward some tumor cell lines (promonocite U937 and neuroblastoma SH-SY5Y). Cells treated with the most active complexes were committed by the apoptotic program, as verified by citofluorimetric assays, with the complexes interfering the cell cycle in different ways (G1, G2/M or S phase. Formation of free radicals was detected, and citoplasmatic carbonylated and glycosilated proteins inside the treated cells were determined by imunologic assays. In conclusion, these studies indicated modulation of the biological activity by the imine ligand in the copper(II) complexes, with very different antiproliferative behavior, going from undetectable activity to high efficacy. Among the most active compounds, a different specificity and action mode in both cell type could be observed, evidencing their potential application as antitumoral agents.

Os fármacos antiinflamatórios não-esteróides (FAINEs) são amplamente utilizados no combate a processos inflamatórios e dores, mas apresentam restrição de uso em razão de sérios efeitos colaterais sobre o trato gastrointestinal. A atividade biológica de complexos metálicos tem sido objeto de pesquisa de grande interesse na área de metalofármacos e compostos do tipo Cu(II)-FAINEs apresentam boa atividade antiinflamatória e efeitos colaterais reduzidos em relação aos fármacos orgânicos. Nesse trabalho, com o objetivo de contribuir para ampliar os estudos sobre o desenvolvimento de alternativas aos FAINEs, foram preparados e caracterizados complexos cobre(lI) com ibuprofeno, indometacina, naproxeno, sulindaco, meloxicam. Os compostos foram imobilizados em Hidrotalcita, um hidróxido duplo lamelar (HDL) de magnésio e alumínio (Mg/AI = 3) que é biocompatível e tem uso como antiácido estomacal. As interações dos metalofármacos com o HDL geraram materiais híbridos bioinorgânicos do tipo Cu-FAINElHDL, nos quais os complexos podem estar presentes de duas maneiras: intercalados entre as Ia meias ou adsorvidos nas superfícies externas do hidróxido duplo lamelar, dependendo do solvente utilizado. A intercalação de complexos neutros é favorecida em solvente misto álcooVamida no qual a Hidrotalcita sofre esfoliação promovida por um processo de hidrólise da amida. A estabilidade alguns dos complexos e dos materiais híbridos em condições gástricas simuladas (pH e temperatura) e as atividades antiinflamatória, analgésica e ulcerogênica in vivo dos sistemas contendo indometacina (Indo) foram investigadas. A intercalação do complexo Cu-Indo no HDL favorece sua estabilização, contribuindo para potencializar a sua atividade farmacolágica. Os materiais híbridos bioinorgânicos obtidos neste trabalho apresentam propriedades interessantes com vistas a potencial aplicação como sistemas de liberação controlada de fármacos. / Non-steroidal anti-inflammatory drugs are widely consumed to treat inflammatory diseases and pain but their clinical use are limited due to serious side-effects on the gastrointestinal tract. The bioactivity of metal complexes exhibits great interest in metal-based drug research. Cu(II)-NSAID compounds show good anti-inflammatory property and decreased side-effects compared to their organic parent drugs. This work aimed to contribute for development of alternative NSAIDs. Cu-NSAlD compounds containing the drugs Ibuprofen, Indomethacin, Naproxen, Sulindac and Meloxicam were synthesized and characterized. The compounds were also immobilized on Hydrotalcite, a layered double hydroxide (LDH) of magnesium and aluminum (Mg/Al = 3), that is biocompatible and used as stomach antacid. The interactions of the copper drugs with LDH led to Cu-NSAID/LDH- bioinorganic hybrid materiais. Two kinds of complex-LDH interactions were observed by changing the solvent: intercalation between LDH layers or adsorption on the LDH external surfaces. The intercalation of neutral complexes is favored in an alcohollamide solvent mixture where Hydrotalcite undergoes exfoliation process promoted by the amide hydrolysis. The stability of some complexes and their correspondent hybrid materials under gastric conditions (pH and temperature) and also in vivo anti-inflammatory, analgesic and ulcerogenic activities for Indomethacin (Indo)-containing systems were investigated. The stabilization of the Cu-Indo structure is promoted by intercalation of the complex into the LDH layers, contributing to increase its pharmacological activity. The bioinorganic hybrid materials here investigated also exhibit interesting properties for applications as controlled drug delivery systems.

Anticancer active metal complexes such as cisplatin are routinely used for treating various cancers since 1978. However, the side effects of cisplatin overwhelm its therapeutic potential, especially in the latter stages of treatment. The nonspecific cytotoxicity of drugs could be avoided if targeted delivery to cancer cells is achieved using two different methodologies namely, enhanced permeability and retention in solid tumors (EPR) and receptor mediated endocytosis using a homing agent (RME). Ru(II)-arene complexes which are delivered specifically into cancer cells by the transferrin enzyme are less toxic compared to other metal complexes. The thesis describes the synthesis and use of Ru(II)-η6cymene complexes with different ancillary ligands which modulates the anticancer activity and the utility of two macromolecular vehicles in directed drug delivery. Ru(II)-η6cymene complexes with different heterocyclic ancillary ligands are synthesized and their anticancer activity tested against various cancer cell lines. Ruthenium complexes with mercaptobenzothiazoles are found to be quite active against the H460 cell lines that overexpress transferrin receptors and non-cytotoxic to the normal cell line, HEL299. Biophysical studies show that complexes (H1 and H8) can unwind the pBR322 DNA and inhibit the Topo IIα enzyme. A unique biphasic melting curve of CT DNA is observed in the presence of H1 which is attributed to formation of a dinuclear species (H20). Half-sandwich complexes of 6-thioguanine (6-TG) have also been prepared to improve the delivery and efficacy of 6-TG which is used in spite of a deleterious photoreaction. The Ru complexes cytotoxic to several leukemia cell lines. As they are photostable and anticancer active, they are better than 6-TG. Anticancer activity exhibiting piazselenols are used as ancillary ligands to make Ru(II)-arene complexes. Unfortunately, 1H NMR spectra suggests that piazselenol complexes dissociate in solution. However, the nitro substituted piazselenol and its Ru complex show the greatest cytotoxicity (<0.1 µM) against the A2780 cell line. The utility of PAMAM dendrimers and hyper branched polymers (hybramers) conjugated with a homing agent to target cancer cells by EPR and RME is probed. A cytotoxic copper complex (CuATSM) is covalently attached to the macromolecules through a disulfide linker, cleaved in the presence of GSH. Targeting efficacy of the folic acid-dendrimer conjugates is checked against two glioma cell lines. The folic acid-dendrimer conjugate is more active compared to dendrimer conjugate without folic acid against folate-receptor-overexpressing LN18 cell line. Biotin conjugated dendrimer shows better accumulation in HeLa cells, which require high amounts of biotin for growth. In vivo studies demonstrate that the conjugate can cross the blood-brain barrier. These studies suggest that PAMAM dendrimer can be used as a targeted delivery vehicle for cytotoxic metal complexes. Hyperbranched polymers decorated with propargyl groups and hydrophilic OH terminated TEG groups are attached to biotin and a cytotoxic Cu complex. (CuATSM-SS-CONH-N3) through ‘click’ reactions and tested against the HeLa cell line. On the basis of the studies conducted, it is concluded that targeted delivery of cytotoxic metal complexes are possible in the case of Ru(II) half-sandwich complexes and macromolecular vehicles like dendrimers are suitable for specifically delivering copper complexes into cancer cells.

Many previous studies have demonstrated that gold compounds possess successful results in catalysis and in medicinal chemistry. The central aim of this dissertation is the design and synthesis of novel gold (I) acyclic diamino carbene complexes as a chemotherapeutic agent for triple-negative breast cancer (TNBC) and for catalysis. In this study, a series of chiral neutral and cationic gold (I) acyclic diamino carbene (ADC) complexes and neutral gold (I) bis- ADC complexes have been synthesized. As the chiral neutral gold (I) ADCs, four diastereomers of S binaphthyl L proline tertiary butyl ester gold (I) chloride, S binaphthyl D proline tertiary butyl ester gold (I) chloride, R binaphthyl L proline tertiary butyl ester gold (I) chloride, and R binaphthyl D proline tertiary butyl ester gold (I) chloride have been synthesized and characterized. Different chiral gold (I) ADC complexes with bulky chiral binaphthyl group and with different amine groups of morpholine, chiral proline methyl ester, and benzyl ester have been synthesized and characterized. After that four diastereomers of the nitrile adduct of cationic binaphthyl proline tertiary butyl ester nitrile and four diastereomers of the isonitrile versions of it have been synthesized and characterized. A series of gold (I) cationic bis ADC complexes have been synthesized and characterized. All these novel gold ADC complexes were tested for biological activity against TNBC cell line MDA-MB-231 and cationic S binaphthyl D proline ester isonitrile adduct, S binaphthyl D proline ester isonitrile adduct and R binaphthyl D proline ester isonitrile adduct gave promising inhibition rates. According to Lipinski's rule, lipophilicity determines the effectiveness of the drug absorption to the body through the lipid membrane. To determine the drug-likeness of the gold ADC complexes, log P values were calculated for some of the synthesized complexes using a modified shake flask method. Gold (I) ADC complexes have been renowned for their ability in catalysis, but enantioselective catalysis is not that well studied. A3 coupling reaction is a well-known reaction for the synthesis of propargyl amines. Here, A3 coupling reaction with a chiral amine has been performed using the previously synthesized four diastereomers of binaphthyl proline tertial butyl ester gold (I) ADCs (SL, RD, RL, SD) as the catalyst expecting four different diastereomers of the product. The reaction exhibited reasonable yields but with a low enantiomeric excess (ee%). However, it gave proof of the principle that asymmetric induction is possible with the synthesized novel chiral gold (I) ADC complexes.

Transition metal bis(thiosemicarbazone) complexes have been of great interest in the last five decades. One of the most striking features of these complexes is that they possess a wide range of biological properties including antimalarial, antibacterial and anticancer activity. Zinc and copper bis(thiosemicarbazone) complexes have recently attracted attention due to their intracellular fluorescence and anticancer activity, respectively. The present work “Targeting Cancer Cells and Live Cell Imaging Using Bis(thiosemicarbazone) Complexes of Copper and Zinc” is an effort to target cancer cells using folic acid or biotin linked anticancer active copper bis(thiosemicarbazone) complexes. Interestingly, bis(thiosemicarbazone) ligands form zinc complexes that could be used to image cancer cells and one of the ligands could be used for imaging zinc in the cells. Chapter 1, provides a brief introduction to metal complexes in medicine. Different classes of metallodrugs and their mechanism of action are listed. A short discussion on different types of diagnostic drugs and transition metal complexes possessing anticancer activity is presented. An overview of the strategies available to target cancer cells is included. Furthermore, the use of thiosemicarbazone compounds for anticancer activity is reviewed in detail. Recent examples of bis(thiosemicarbazone) compounds in medicinal studies is briefly mentioned. This section ends with the scope of the present work which involves bis(thiosemicarbazone) complexes of zinc and copper. Chapter 2, “Zinc bis(thiosemicarbazone) complexes for live cell imaging and anticancer activity” deals with the synthesis and characterization of a series of mononuclear and binuclear zinc bis(thiosemicarbazone) complexes by varying substituents at the diketone moiety or at the thiosemicarbazide fragment of the ligand. The crystal structures of mononuclear ligand benzil-bis(4-pyrrolidine-3-thiosemicarbazone) (BTSCH2), zinc glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarbazone) [Zn(GTSC)]3 and [Zn(BTSC)(DMSO)] complexes were determined using single-crystal X-ray crystallography. Here, the mononuclear zinc complexes were utilized as live cell imaging agents whereas binuclear zinc complexes proved to be anticancer agents. Among the many mononuclear complexes prepared, the trimeric zinc complex derived from glyoxal- bis(4-methyl-4-phenyl-3¬thiosemicarbazone) was found to be the most fluorescent complex owing to its unique structure. This permitted live cell imaging in a number of cancer cell lines. In comparison with the well studied zinc biacetyl-bis(4-methyl-3-thiosemicarbazone) Zn(ATSM) complex, which was used as a reference, [Zn(GTSC)]3 had a 2.5 fold higher fluorescence quantum yield in DMSO. The cellular fluorescence was measured in collaboration with Prof. K.Somasundaram’s laboratory at MCBL using flow cytometry. It was observed that [Zn(GTSC)]3 had 3 to 12 fold higher fluorescence than Zn(ATSM) in various cell lines (n = 9) of different tissue origin. Confocal fluorescence microscopy studies established that [Zn(GTSC)]3 localizes in the nucleus of human breast cancer MCF-7 and MDA-MB-231 cells within 30 minutes of addition. Moreover, [Zn(GTSC)]3 showed relatively less cytotoxicity compared to the Zn(ATSM) complex in all the cancer cell lines tested. DNA interaction studies such as binding and cleavage showed that [Zn(GTSC)]3 was less harmful to DNA as well. All these features make [Zn(GTSC)]3 a good fluorescent imaging agent for live cells. Binuclear zinc bis(thiosemicarbazone) complexes were also synthesized and their cytotoxicity was evaluated in different cancer cells. One of the ligands, 1,3-bis{biacetyl-2′-"-N-pyrrolidinethiosemicarbazide)-3′-(4"-N-thiosemicarbazide)} propane (ProBATpyrH4), and its zinc complex were found to show excellent anticancer activity against human hepatocellular cancer (HepG2) cell line. However, the cellular uptake studies as followed by flow cytometry revealed that these compounds do not fluoresce inside the cells. However, the DNA interaction studies using ethidium bromide displacement assay revealed that these complexes have better binding ability to DNA than mononuclear zinc complexes and the viscometric titrations suggested the binding mode to DNA is through partial intercalation. Apparently, these complexes do not induce DNA cleavage as evident from the cleavage experiments performed on pBR322 DNA. It is likely that their anticancer activity is due to unique DNA binding properties. Imaging zinc is important in the field of metallomics as alteration of zinc concentration in cells is associated with, or attributed to various diseases. In this regard, bis(thiosemicarbazone) ligands are useful. Chapter 3, “Imaging intracellular zinc using glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarbazone) ligand” deals with imaging zinc in live cells using the bis(thiosemicarbazone) ligand, GTSCH2. Since the trimeric zinc complex is fluorescent, the corresponding ligand, GTSCH2, was utilized to visualize the zinc present within cells. The ligand GTSCH2 is found to be a selective fluorescence “turn-on” sensor for zinc. This sensor exhibited excellent sensitivity and selectivity towards zinc over other physiologically relevant cations. The binding affinity of GTSCH2 to zinc was estimated to be 0.59 nM in an aqueous MOPS (50 mM, NaCl; 100 mM; pH 7.3) buffer containing 30% DMSO, from competitive binding experiments carried out with ethylene glycol tetraacetic acid (EGTA). The sensor displayed maximal fluorescence response to zinc ion when present in the ratio of 1:1 and displayed stable fluoresence in the pH range 5.0 to 7.8, which suggests that the probe may be suitable for imaging zinc in both normal and cancer cells. The potential of GTSCH2 to image zinc inside the cell has been demonstrated in two human breast cancer cell lines using confocal fluorescence microscopy. Unlike mononuclear zinc complexes, the mononuclear copper bis(thiosemicarbazone) complexes are cytotoxic. Chapter 4, “Anticancer activity of copper bis(thiosemicarbazone) complexes” deals with the synthesis, characterization and anticancer activity of mononuclear copper bis(thiosemicarbazone) complexes. All of them were characterized by spectroscopic methods and in three cases by single crystal X-ray diffraction. The redox properties, studied by cyclic voltammetry, showed reversible one electron- reduction process that varied from –0.53 V to –0.18 V vsSCE. Anticancer activity for the synthesized complexes and their ligands were tested against many human cancer cell lines where the complexes Cu(GTSC) and Cu(GTSCHCl) derived from glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarbazone) are found to be most cytotoxic (GI50 <0.1 µM to 2.1 µM) in five cancer cell lines tested. Moreover, the cytotoxicity is similar to that of adriamycin, a known anticancer drug, in all cell lines. However, it is less potent than a copper bis(thiosemicarbazone) analog, copper biacetyl-bis(4-methyl-3-thiosemicarbazone) Cu(ATSM), a well studied anticancer agent in many cell lines. Cellular studies were carried out for the selected complexes Cu(GTSC) and Cu(GTSCHCl) along with Cu(ATSM) on HCT116 colon cancer cells. The order of lipophilicity and cellular uptake as studied by ICP-OES are correlated with their cytotoxicity. Based on the interaction of these complexes with DNA using the ethidium bromide displacement assay, DNA -melting, -viscosity and -cleavage studies, it is suggested that these complexes intercalate partially with DNA. DNA cleavage studies using pBR322 DNA revealed that only Cu(GTSCHCl) complex cleaves DNA. Mechanistic discrimination studies suggest that the complex cleaves DNA through the hydrolytic pathway. Since the topoisomerase IIα (Topo IIα), a nuclear enzyme resolving topological problems of DNA, is considered as one of the possible molecular targets for a number of anticancer drugs including some of the copper thiosemicarbazone complexes, Topo IIαinhibition studies were carried out in human Topo IIα. Interestingly, many copper bis(thiosemicarbazone) complexes are able to inhibit Topo IIα activity by acting as Topo IIα poison. Cu(GTSCHCl) complex was found to be the most active in this series of complexes (90 % inhibition at 100 µM) and it inhibits the enzyme in a dose dependant manner. Based on the results, it was concluded that the cell death may be mediated, at least in part, through DNA cleavage and Topo IIαinhibition. Severe side effects, poor distribution profiles and or organ specific toxicity make the conventional chemotherapy of limited value with metal based drugs. Therefore, developing cancer-specific drug delivery systems is an urgent need in cancer therapy. Among the many strategies available to target cancer, targeting the receptors that are overexpressed in the cancer cell membrane is a novel strategy being used in recent studies. Therefore the last part my work, “Copper bis(thiosemicarbazone) complexes linked to poly(ethylene glycol) and multiwalled carbon nanotubes for targeted delivery to cancer cells ” was designed to target cancer cells. Here, copper complexes (therapeutic molecule) were attached with PEG and MWCNT (carrier) along with folic acid or biotin (targeting molecule). First, CuATSM–A was functionalized with a disulfide linker and connected with folic acid via a poly(ethylene glycol) (PEG600) linker. This was synthesized to target KB (human nasopharyngeal carcinoma) cells, a cell line that overexpresses the folate receptor on the cell surface. In order to investigate the targeting efficacy, the corresponding fluorescent labeled analogs and non-targeted PEG conjugates were synthesized. Flow cytometry studies with fluorescent marker (fluorescein isothiocyanate) labeled PEG analogs showed the targeting efficacy on KB cells. The copper complex, CuATSM–A, attached with biotin–PEG2000 also was synthesized to target high-biotin-using HeLa (human cervical carcinoma) cells. Multiwalled carbon nanotubes (MWCNT) were also used as nanocarriers. Here, the MWCNT was decorated with PEG600 diamine and then functionalized with the copper complex (therapeutic molecule), folic acid (targeting molecule), and FITC (fluorescent molecule). The conjugation of all the molecules with MWCNT is characterized by various spectroscopic techniques.