D. Tech. Pharmaceutical Sciences.

Among all the major infectious human diseases, gastro-intestinal infections caused by microbial pathogens are a major cause of morbidity and infant death in developing countries, largely due to inadequate sewage disposal and contaminated water. Traditional health practitioners in South Africa play a crucial role in providing health care to the majority of the population. Many plants are locally used by South African traditional healers to treat microbial infections related to gastro-intestinal tracts. Ethnopharmacological and ethnobotanical studies using traditional knowledge as a selection strategy has given priority to certain plants for isolation and identification of plant novel bioactive compounds. Pharmacological and phytochemical studies of the investigated twelve medicinal plant species (from 10 families) extensively used as antimicrobials against gastro-intestinal infections was necessary to validate the use of the plants. Furthermore, to provide sufficient preliminary information for the isolation and identification of active compounds that are present in the investigated plants. Plant parts were sequentially extracted using petroleum ether (PE), dichloromethane (DCM) and 70% ethanol (EtOH). Cold water and boiled (decoction) extracts of the plant materials were prepared non- sequentially. Among the extracts, EtOH yielded the highest amount of plant substances. A total number of 85 extracts were evaluated for antibacterial activity, 80 for antifungal activity, 64 for anti-inflammatory activity, and 27 biologically active extracts were tested for genotoxicity. The microdilution method was used to determine the minimum inhibitory concentration values in the antibacterial assay against two Gram-negative bacteria (Escherichia coli ATCC 11775 and Klebsiella pneumoniae ATCC 13883) and two Gram-positive bacteria (Bacillus subtilis ATCC 6051 and Staphylococcus aureus ATCC 12600). A modified microdilution method was used to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values in the antifungal assay against Candida albicans. Cyclooxygenase assay was used to evaluate the anti-inflammatory activity of the extracts against cyclooxygenase-1 and -2 (COX-1 and COX-2) enzymes. The plant extracts were screened first at a concentration of 250 ƒÊg/ml per test sample, and then further screened at concentrations of 125 and 62.5 ƒÊg/ml for extracts that inhibited the COX-2 enzyme. The Ames test was used to test for genotoxicity in extracts that showed interesting pharmacological activities using Salmonella typhimurium strain TA98. Among the screened extracts, 25 extracts showed good antibacterial activity with MIC values . 1.0 mg/ml. Dichloromethane extracts exhibited the greatest antibacterial activity, and Gram-positive bacteria were most susceptible. The best antibacterial activity was exhibited by Becium obovatum leaf EtOH extracts with an MIC value of 0.074 mg/ml. A broad spectrum antibacterial activity was observed by leaf extracts of Cucumis hirsutus (PE), Haworthia limifolia (PE), Protea simplex (PE and DCM) and Dissotis princeps (EtOH) against both Gram-negative and Gram-positive bacteria. No interesting antibacterial activity was exhibited by water extracts with the exception of Dissotis princeps water extract with a good antibacterial activity against Gram-positive and Gram-negative bacteria. In the antifungal assay, 6 extracts showed interesting antifungal activity. Protea simplex leaf PE extract showed the best fungicidal activity with an MFC value of 0.014 mg/ml. The best overall antifungal activity was observed in plant EtOH extracts. Some extracts from Agapanthus campanulatus (leaves and roots), Dissotis princeps (leaves), Gladiolus dalenii (corms) and Protea simplex (leaves) showed good activity against Candida albicans. Twenty one extracts inhibited the COX-1 enzyme, while fifteen extracts inhibited the COX-2 enzyme at the lowest screening concentration of 62.5 ƒÊg/ml. The highest COX-1 inhibition at a concentration of 62.5 ƒÊg/ml was exhibited by Diospyros lycioides leaf PE extract (89.1%) while Agapanthus campanulatus root DCM extract showed the highest COX-2 inhibitory activity (83.7%) at the same concentration. In the Ames test, no genotoxicity was observed in any of the extracts, however more tests need to be done to confirm these results. Thin layer chromatograms of the organic solvent plant extracts were developed. The fingerprints of the plant extracts showed colours of bands at different Rf values when viewed under UV254 and UV366 suggesting that the investigated plant species contained different compounds in the extracts. In the quest to understand the source of the plants pharmacological activities, total phenolic compounds including condensed tannins, gallotannins and flavonoids were quantitatively investigated in terms of their amounts in the aqueous methanol extracts of the plants materials using spectrophotometric methods. Alkaloids and saponins were qualitatively determined. The amounts of total phenolics were determined by the Folin Ciocalteu assay, condensed tannins were determined by the butanol-HCl assay, while rhodanine and vanillin assays were used to determine the amounts of gallotannins and flavonoids respectively. Dragendorff reagent was used to detect alkaloids in the plant extracts on thin layer chromatographic plates, while the froth test was employed to detect saponins. Secondary metabolites varied with plant parts and species with Cyperus textilis (leaf) having the highest amounts of total phenolics, condensed tannins and flavonoids. The highest amount of gallotannins was detected in Protea simplex leaf extracts. All the investigated plant materials with the exception of Haworthia limifolia leaf, Protea simplex leaf, Antidesma venosum leaf and Dissotis princeps leaf tested positively to saponins. Alkaloids were detected in Haworthia limifolia leaf (PE and EtOH), Cucumis hirsutus leaf (EtOH), Becium obovatum root (DCM), Protea simplex root and bark (EtOH), Agapanthus campanulatus root (DCM) and leaf (EtOH), Cyperus textilis root (DCM), Vernonia natalensis leaf (PE), Antidesma venosum leaf (PE), Diospyros lycioides leaf (PE) and Dissotis princeps leaf (DCM) extracts. The results obtained from the investigation of the pharmacology and phytochemistry of the plant species used to treat microbial infections related to gastro-intestinal tracts, provide sufficient preliminary information to validate the use of some of the plants in traditional medicine. The information provided might be considered sufficient for further studies aimed at isolating and identifying the active compounds in the plant species, and evaluating possible synergism amongst the isolated compounds. / Thesis (M.Sc)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Traditional medicine in South Africa is part of the culture of the people and has been in existence for a long-time. Although animal components form part of the ingredients used, plant material constitutes the major component. South Africa is endowed with vast resources of medicinal and aromatic plants which have been employed for treatment against various diseases for decades. A large number of South Africans still depend on traditional medicine for their healthcare needs due to its affordability, accessibility and cultural importance. Helminth infections are among the variety of diseases treated by traditional healers. These infections are regarded as neglected tropical diseases (NTDs) due to their high prevalence among the economically disadvantaged living in rural areas in different regions of the world. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

L’el•lipticina, un compost aïllat originàriament d’Ocrosia elliptica Labill. (família Apocinaceae), posseeix una elevada activitat antiproliferativa. La seva estructura química consisteix en un esquelet tetracíclic format de carbazole fusionat amb una piridina. L’el•lipticina i els seus derivats exerceixen els seus efectes citotòxics i antineoplàstics a través d’un mecanisme multimodal d’acció biològica (inhibició de l’ADN-topoisomerasa II, intercalació a l’ADN, unió covalent a l’ADN i generació de radicals lliures citotòxics mitjançant reaccions d’oxidació-reducció). Prèviament en el grup de treball, s’han estudiat les 1,4-benzodioxinoisoquinolines dissenyades i sintetitzades com a anàlegs de l’el•lipticina. Tenint en compte els precedents bibliogràfics i la recerca duta a terme pel nostre grup en relació amb la preparació de compostos amb activitat anticancerígena, els objectius d’aquest treball van adreçats a la preparació i avaluació biològica de compostos policíclics que continguin el nucli de carbazole unit a un anell D pentacíclic i incorporin diferents cadenes alquíliques laterals. Es tracta d’estructures dissenyades a partir de l’el•lipticina principalment per farmacomodulació modulativa. Els intermediaris clau per a la obtenció d’aquestes estructures tetracícliques són diens de tipus furo[3,4-b]indole. En un primer lloc s’estableix una ruta sintètica a partir de l’àcid indole-2-carboxílic (ruta A, de 8 etapes i un rendiment global del 8-22%). D’aquesta ruta, cal destacar-ne la utilització d’N,N-dimetilhidrazida com a grup ortodirigent de metal•lació, que s’oxida a àcid mitjançant MnO2 en una etapa posterior. També es proposen d’altres rutes a partir del propi nucli indòlic (ruta B, de 5 etapes i un rendiment global del 31-76%). De la ruta B en destaquen la utilització de la funció acetal com a grup orto-dirigent de metal•lació i la utilització de resina àcida d’intercanvi iònic com a reactiu sòlid. D’altres rutes a partir de l’àcid indole-3-carboxílic o de l’indole-3-acetonitril no han resultat satisfactòries. El tractament dels diens obtinguts amb els dienòfils adients condueix als compostos tetracíclics desitjats. De la sèrie de compostos sintetitzats, s’han realitzat proves de citotoxicitat en les línies cel•lulars de leucèmia K562, càncer de pulmó NCIH460 i càncer de còlon HT-29. Per tal d’avaluar també la selectivitat dels compostos respecte a les cèl•lules no canceroses, s’ha realitzat un control en cèl•lules no canceroses de tipus HuDe (fibroblasts). Tots els productes obtinguts i els principals intermediaris s’han caracteritzat per tècniques d’RMN, IR i p.f. Es posa especial èmfasi en l’estudi de la reactivitat i en l’estabilitat dels intermediaris implicats i aïllats de les diferents rutes sintètiques. Finalment, durant una estada a la Universitat de Huddersfield (Anglaterra), s’ha realitzat la síntesi en fase sòlida de derivats indòlics, així com la posta al punt d’un mètode analític basat en les tècniques d’HPLC-Masses (ESI-ToF) per a la quantificació del pèptid fosfo-pRbING procedent de la reacció de fosforilació del pèptid pRbING mediada per CDK4/6. Aquest nou mètode constitueix una alternativa a la determinació de l’activitat de compostos inhibidors de CDK4/6 amb l’avantatge que no necessita de derivats de fòsfor marcats radioactivament i, per tant, ofereix menys riscos i major seguretat en el treball del laboratori. / “Design and synthesis of polycyclic compounds that contain the indolyc nucleus. Evaluation of antitumor activity.” Ellipticine is a natural product isolated from Ochrosia elliptica with significant cytotoxic activity by several mechanisms of action. Its tetracyclic structure exhibits antineoplasic activity against several human cancer cell lines. Many syntheses have been reported by Gribble and col. and other authors, and its structure has been widely modified in order to obtain more efficacious, selective and less toxic new compounds. Our group had previously reported a series of ellipticine analogues including 1,4-benzodioxin nucleus. Herein, we present two new alternative pathways to prepare the intermediate isobenzofuroindole starting from either indole-2-carboxylic acid (8 steps, 8-22% overall yield) or 3-indolylalkanones (4 steps, 31-76% overall yield). Some interesting steps (involving protection of indole, directed ortho-metallation and cyclization) had been studied and developed in our laboratory. These methods lead us to the intermediate diene, which reacts with the corresponding dienophiles to obtain the desired compounds. These compounds were tested in cancer cell lines K-562 (leukaemia), NCI-H460 (lung) and HT-29 (colon), as well as in fibroblasts cell line HuDe to evaluate toxicity. All compounds were characterized by NMR, IR and m.p. techniques. Finally, over a PhD stage at the University of Huddersfield (England), some indole derivatives were synthesised by solid phase and a new HPLC-MS CDK4/6 inhibition test was designed. It was developed a 22-min HPLC-MS method to elute and quantify the phospho- pRbING product and the pRbING substrate from reaction samples containing buffer solution, ATP and CDK4/6. Peptide pRbING was efficiently quantified by HPLC-MS (ESI-ToF), within a range between 1 – 100 ppm and a detection limit as low as 3 pmol. This method should allow the evaluation of new CDK4/6 inhibitors.

Thesis Univ. College London, 2006. / Includes bibliographical references and indexes.

En l’espace d’une quarantaine d’année, les connaissances sur l’angiogenèse tumorale ont littéralement explosé. Dans les années 1970, Judah Folkman démontre que les tumeurs ont besoin d’être vascularisées pour continuer à proliférer. Peu de temps après, les protagonistes principaux de l’angiogenèse tumorale ont été découverts, de même que les mécanismes dans lesquels ils sont impliqués. La décennie suivante marque le début des recherches sur les molécules à visée anti-angiogénique et c’est en 2004 que le bevacizumab (Avastin, Roche), premier médicament anti-angiogénique utilisé en oncologie, voit le jour. Parallèlement à cela, l’essor de la modélisation à effets-mixtes couplée aux progrès des outils informatiques ont permis de développer des méthodes d’analyses de données de plus en plus performantes. Dès 2009 L’agence de régulation FDA (Food and Drug Administration) aux Etats-Unis a identifié le rôle central de la modélisation numérique pour mieux analyser les données d’efficacité et de toxicité, préclinique et clinique en cancérologie. Le but de ce projet est d’étudier les effets de différents inhibiteurs de l’angiogenèse sur la dynamique tumorale, en se basant sur une approche populationnelle. Les modèles développés seront des modèles à base d’équations différentielles ordinaires intègrant des données et des informations issues de la littérature. L’objectif de ces modèles est de caractériser la dynamique des tailles tumorales chez les animaux et chez les patients, afin de comprendre les effets des traitements anti-angiogéniques et apporter un soutien pour le développement de ces molécules ou pour la prise de décisions thérapeutiques par les cliniciens. / Within the last 40 years, knowledge of tumor angiogenesis has literally exploded. In the seventies, Judah Folkman demonstrated that tumors need to be vascularized to continue to proliferate. Shortly after, the main protagonists of tumor angiogenesis have been discovered, as well as the mechanisms in which they are involved. The next decade is the beginning of the research on molecules with anti-angiogenic effects and in 2004 bevacizumab (Avastin, Roche), the first antiangiogenic drug used in oncology, was available for treating solid cancer patients. Along with this, the increasing interest of mixed-effects modeling coupled with advances in computer tools allowed developing more efficient methods of data analysis. In 2009, the regulatory agency FDA (Food and Drug Administration) in the United States has identified the central role of numerical modeling to better analyze the efficacy and toxicity preclinical and clinical oncology data. The aim of this project is to study the effects of different angiogenesis inhibitors on tumor dynamics, based on a population approach. The developed models are models based on ordinary differential equations and that integrate data and information from the literature. The objective of these models is to characterize the dynamics of tumor sizes in animals and patients in order to understand the effects of anti-angiogenic treatments and provide support for the development of these molecules, or to help clinicians for therapeutic decision making.

O constante crescimento mundial da Pesquisa Clínica no desenvolvimento de novas drogas foi responsável pelo aumento do interesse em traçar as atividades desenvolvidas pelas Autoridades Regulatórias. Os dados foram obtidos através de revisão bibliográfica sistemática, destacando o tempo de aprovação dos protocolos clínicos e as normatizações vigentes: no Brasil, Estados Unidos da América, União Europeia, Canadá e Japão. Além disso, observou-se a atuação de profissionais experientes na realização de atividades no Comitê de Ética para Análise de Projetos de Pesquisa (CAPPesq) do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP). Com isso, foi possível identificar as diferenças significantes em relação à legislação e ao sistema regulatório dos países em questão. Deficiências no sistema regulatório brasileiro responsáveis pela demora no tempo de aprovação foram constatadas. Com a identificação destes fatores, foram elaboradas sugestões relacionadas à qualificação dos profissionais atuantes, organização das atividades desempenhadas pelas Autoridades Regulatórias e possíveis alterações administrativas. A diferente atuação das autoridades analisadas pode servir como fonte de aprimoramento do sistema regulatório nacional e, consequentemente, aprimorar o processo para aprovação e realização de protocolos clínicos com medicamentos testados no Brasil. / The world-wide Clinical Research for new drug development growth was responsible for the increase of interest in following the regulatory authorities activities. Data were collected through a systematic literature review.The main facts observed were clinical protocols time approval and guidelines in Brazil, United States of America, European Union, Canada and Japan. In addition, it was observed the activities realized by experienced professionals of the IRB of the University of São Paulo School of Medicine. Significant legislation and regulatory system differences between the countries were identified. Some deficiencies at Brazilian regulatory system, suggestions regarding the acting professionals qualification, organization of the regulatory authorities activities and possible administrative changes were discussed. The different performance of the regulatory authorities can serve as a source to upgrade the national regulatory system and consequently lead to improvements in process of approval and realization of clinical protocol with drugs tested in Brazil.

In type II diabetes, systemic metabolism is perturbed and on a cellular level the balance of fuel use is upset. More specifically, increased fatty acid use is seen alongside decreased glucose metabolism. This altered fuel use is mediated by changes in the activity and expression of multiple enzymes. One such enzyme within the glucose breakdown pathway is pyruvate dehydrogenase, whose activity is known to be reduced in the diabetic state. The field of real-time metabolic investigation has rapidly expanded over the past few years due to the invention of technology that has enabled the production of ¹³C labelled hyperpolarised compounds, which can generate high signal levels in magnetic resonance spectroscopy. This has provided the opportunity to measure real-time metabolism of injected hyperpolarised tracers both ex vivo and in vivo. This thesis aimed to develop the use of hyperpolarised compounds in vivo, to investigate the cardiac and hepatic metabolism of a diabetic rat model. We initially addressed the systemic nature of the disease by establishing a two-slice acquisition for obtaining cardiac and hepatic data during a single injection of hyperpolarised pyruvate. This was tested in the fed and fasted states before being used in the studies described in the subsequent chapters of this thesis. The value of hyperpolarised compounds in following metabolic modulation by drug treatment was explored in the next chapter. The effect on metabolism of two drugs targeted at pyruvate dehydrogenase, which differed in their isoform specificity, was investigated first in the perfused heart and subsequently in vivo, both in control and diabetic animals. Hyperpolarised magnetic resonance spectroscopy was combined with other established techniques to help both our understanding of the systemic changes that had occurred following treatment, and provide links between cardiac metabolism and function. The final chapter of this thesis explored the use of hyperpolarised ¹³C pyruvate to understand the effect of hypoxia on pyruvate dehydrogenase, firstly in healthy animals and subsequently in the diabetic, metabolically altered state. Understanding the combination of diabetes and hypoxia was interesting given the existence of several opposing metabolic effects seen in the two states. Overall this thesis has demonstrated developments in the use of hyperpolarised pyruvate that, when appropriately combined with other techniques, can yield valuable metabolic information, in terms of following disease progression, drug development, and understanding basic metabolism.

Metal-nucleic acid interaction studies have been gaining attention due to their biological and chemical importance. Nucleic acids are negatively charged bio-polymers and neutralization of their negative charge is essential for the stability and function. In the cells, organic positive ions (positively charged amino acids and polyamines) and some of the metal ions (e.g. Na+, K+, Mg2+...etc) neutralize the charge of nucleic acids. Whereas, interactions of some metal ions (e.g. Cd2+, Hg2+…etc) with nucleic acids destabilize the structure. The stability and conformation of nucleic acids alter due to metal interactions. Further, metal interactions with nucleic acids can bring changes in conformation of ribose, H-bonding and π-π stacking interactions. To understand the metal interactions with nucleic acids, various spectroscopic techniques are being used. However, X-ray crystallographic technique is advantageous over all other spectroscopic techniques since it gives thorough detail of coordination mode and structure. However, crystallization of large molecules like nucleic acids with metals is associated with great difficulty. In order to simplify the problem, nucleic acid constituents and derivatives have been used as model systems for metal-nucleic acid interactions. Nucleic acid constituents and derivatives are multidentate ligands. Moreover, binding mode of metal with nucleic acid constituents and derivatives depends on various factors include pH, temperature, type of metal…etc. Further, understanding of metal nucleic acid interactions can aid to develop new anticancer drugs targeting nucleic acids. For example, cisplatin is a platinum based anticancer drug, which coordinates to N(7) of guanine in DNA brings cell death. There have been several reports in literature on the complexes of metal nucleic acid constituents. However, much more research is warranted for thorough understanding of metal-nucleic acid interactions. On the other hand, nucleic acid constituents and derivatives are used extensively in anticancer drug development. Some of nucleic acid constituent derivatives, 5-Fluro uracil and 6-Mercaptopurine, are currently in use for the treatment of colorectal cancer and leukemia, respectively. Moreover, cisplatin is a platinum based anticancer drug used in the treatment of various types of cancers. However, use of these drugs for long time poses severe side effects and drug resistance. Most of the side effects are due to non bio-compatibility of drugs. To overcome problems associated with present anticancer drugs, bio-compatible metal based anticancer drug development could be an attractive and alternative strategy. To address this, in this study, we report synthesis of a number of new metal complexes of nucleic acid constituents and their derivatives and characterization by various spectroscopic techniques. Also, the interactions of Ni, Cu and Zn ions with various nucleic acid constituents and their derivatives have been elucidated by single crystal X-ray crystallography. Interestingly, Ni, Cu and Zn ions showed various coordination modes to nucleic acid constituents and their derivatives. Further, anticancer studies were carried out for all these complexes in various cancer cell lines. Several complexes showed better cytotoxicity than the well-known drug cisplatin. My thesis work is divided into five parts based on the nature of molecules. I. Synthesis, X-ray crystallographic and anticancer studies on metal (Zn/Ni) complexes of guanine (G) based nucleic acid constituents In order to understand (Zn/Ni) interactions with guanine based nucleic acid constituents and their anticancer activity, several (Zn/Ni) complexes of 5′-GMP, 5′-IMP and hypoxanthine complexes were prepared. The synthesized complexes are (1) [Zn (5′-GMP)]n.11H2O, (2) [Ni (5′-GMP)2 Na2 (μ-OH2)3 (H2O)8].2H2O, (3) [Ni (5′-IMP)2Na2 (H2O)12]n.5H2O and (4) [Ni (hx)2 (H2O)4] Cl2 [Here 5′-GMP = 5′-Guanosine Mono Phosphate, 5′-IMP = 5′-Inosine Mono Phosphate and hx = Hypoxanthine). These complexes were characterized by various spectroscopic and X-ray crystallography techniques. Complex 1: The X-ray structure revealed that zinc is coordinated to 5′-GMP through N(7) position of purine and phosphate moieties, the uncoordinated water molecules are making interesting complicated network of hydrogen bonds in the unit cell. The geometry of zinc coordination centre is distorted tetrahedral. Fascinatingly, zinc exhibited two different coordination environments. In one case, all phosphate oxygens participated in coordination with zinc. In second case, N(7) position of purine and phosphate oxygens participated in coordination with zinc. Moreover, zinc formed a coordination polymer with 5′-GMP. The conformation of ribose changed upon zinc interaction with 5′-GMP from C(3′)-endo to C(2′)-endo, these results suggest that zinc interaction with nucleic acids may change their conformation. Complex 1 is stabilized in solid state by H-bonding and π-π stacking interactions. Complex 2: In complex 2, 5′-GMP is coordinated to nickel through N(7) position of purine but phosphate moiety did not take place in coordination. Two molecules of 5′-GMP and four water molecules coordinated to nickel and formed distorted octahedral geometry. The charge of complex 2 is balanced by sodium coordination to sugar hydroxyl groups and nickel coordinated water molecules. The geometry of sodium coordination centre is distorted octahedral. The conformation of 5′-GMP is altered due to nickel interaction. Moreover, complex 2 is stabilized in solid state by H-bonding and π-π stacking interactions. Complex 3: Nucleotide 5′-IMP also showed similar coordination modes like 5′-GMP towards nickel, where N(7) position of purine participated in coordination with nickel and phosphate moieties did not coordinate to nickel. Two molecules of 5′-IMP and four water molecules participated in coordination with nickel and formed distorted octahedral geometry. Interestingly, the charge of complex 3 is balanced by sodium coordination to sugar hydroxyl moieties. The geometry of sodium coordination centre is distorted octahedral. Moreover, nickel is forming coordination polymer with 5′-IMP. Further, nickel interactions with 5′-IMP brought changes in the conformation of ribose moiety. These results suggest that nickel interactions with nucleic acids may bring changes in their conformation. Interestingly, right hand helical structure formation is observed for complex 3 in crystal structure. Further, the chirality of complex 3 was confirmed by circular dichroism studies. Complex 3 is stabilized by both H-bonding and π-π stacking interactions in solid state. Complex 4: Surprisingly, nickel is coordinated to hypoxanthine through N(9) position of purine in acidic conditions and not through N(7) or N(3). The coordination mode of nickel with hypoxanthine is different from complexes 2 and 3. Two hypoxanthine moieties are coordinated to nickel in axial manner. The geometry of nickel coordination centre is distorted octahedral. Further, complex 4 is stabilized by H-bonding and π-π stacking interactions in solid state. Cytotoxicity studies of complexes 1-4 on various cancer cell lines revealed that complex 1 is better cytotoxic than complexes 2-4. Moreover, complex 1 exhibited comparable cytotoxicity with cisplatin on various cells lines and induced apoptotic cell death. II. Synthesis, structure elucidation and anticancer activity of copper-adeninyl complexes In order to understand copper-adenine interactions and anticancer activity, several copper complexes of adenine derivatives were prepared. Here, most of adenine derivatives used in complex preparation is known as cycline dependent kinase inhibitors. Prepared copper complexes are 1) [Cu (N6-benzyl adenineH)2Cl3 ].Cl.2H2O, 2) [Cu (2-amino-N6-benzyladenineH)2Cl3].(2-amino-N6-benzyl adenineH)2.3Cl.5H2O, 3) [Cu (α-(Purin-6-ylamino)-p-toluenesulfonamide H)2Cl4], 4) [Cu (kinetinH)2 Cl3].Cl.2H2O, 5) [Cu (N-1H-purine-6-yl-alanineH) (H2O) Cl3].H2O, 6) [(Cu (N-1H-purine-6-yl-alanineH)2Cl3).(Cu(N-1H-purine-6-yl-alanineH)Cl)2(μ-Cl)2].Cl.4H2O. All these complexes were characterized by X-ray crystallography and various spectroscopic techniques. Complex 1: Synthesis and X-ray structures of complex 1 were reported in literature. However, anticancer activity of complex 1 is not known. Therefore, it was prepared based on the reported lines to assess the anticancer activity. The anticancer activity of complex 1 was studied on various cell lines. Interestingly, complex 1 exhibited better cytotoxicity than cisplatin in MCF-7 and MDA-MB-231 cell lines. Complex 2: Ligand 2-amino-N6-benzyl adenine is coordinated to copper through N(9) of purine. In addition, two uncoordinated 2-amino-N6-benzyl adenine, three chloride and five water molecules are making it as a co-complex with uncoordinated ligands. The copper coordination centre adopted distorted trigonal bipyramidal geometry [3+2] with τ = 0.671 (α-β/60, where α, β are two greatest valence angles of coordination centre). Further, complex 2 is stabilized in solid state by both H-bonding and π-π stacking interactions. H-bonding is observed between N-H···Cl. Uncoordinated water molecules formed six-member rings with H-bonding network. The π-π stacking interactions are observed between phenyl and purine moieties. Complex 2 exhibited better cytotoxicity than 2-amino-N6-benzyl adenine and copper salt. Complex 3: Ligand α-(2-Amino purin-6-ylamino)-p-toluene sulfonamide is coordinated to copper through N(9) position and protonation is observed at N(3) position. Two molecules of α-(2-Amino purin-6-ylamino)-p-toluene sulfonamide and four chloride ions are forming a distorted octahedral geometry with copper. Complex 3 is stabilized by N-H···Cl and N-H···O H-bonding. Further, complex 3 exhibited better cytotoxicity than cisplatin in U251 cells. Complex 4: Kinetin is coordinated to copper through N(9) position of purine. Protonation is observed on N(3) position and balanced the charge of complex 4. Two molecules of kinetin and three chloride moieties are coordinated to copper and forming distorted trigonal bipyramidal geometry [3+2] with τ = 0.431. Moreover, complex 4 is stabilized by both H-bonding interactions and π-π stacking interactions. The H-bonding of complex 4 is observed between N-H···Cl and C-H···Cl. The π-π stacking interactions are observed between furanyl aromatic ring and imidazole ring of purine. Complex 4 exhibited better cytotoxicity than kinetin and copper salt. Complex 5: The N-1H-purine-6-yl-alanine is coordinated to copper through N(9) position of purine. Complex 5 crystallizes in the monoclinic space group P21 with Z=4. One molecule of N-1H-purine-6-yl-alanine, two chloride ions and one water molecule coordinated to copper. The geometry of copper coordination centre is distorted trigonal bipyramidal [3+2] with Cu(1) τ1 = 0.613 and Cu(2) τ2= 0.671. Protonation is observed on N(3) position. Complex 5 is stabilized by both H-bonding and π-π stacking interactions. The H-bonding of complex 5 is observed between N-H···Cl and C-H···Cl. The π-π stacking interactions are observed between imidazole moieties. Moreover, complex 5 exhibited better cytotoxicity than N-1H-purine-6-yl-alanine and copper salt. Complex 6: Complex 6 is a co-complex, where two different complexes are co-crystallized. The crystal structure of complex 6 indicate that geometry of Cu(1) and Cu(2) coordination centre are distorted trigonal bipyramidal [3+2] with τ1 = 0.3261 and τ2 = 0.8, respectively. Two molecules of N-1H-purine-6-yl-alanineH are coordinated to Cu(2) through N(9) position of purine. The N-1H-purine-6-yl-alanineH ligands are arranged in geometry in trans manner with respect to axis passing through the N(9) atom and copper. Whereas, in second co-complex two N-1H-purine-6-yl-alanineH are coordinated to Cu(1) through N(9) and N(3) position of purine. Both Cl(1) and Cl(3) coordinated to copper are forming a bridge between copper. In addition, one uncoordinated chloride and two water molecules are present in the unit cell. Complex 6 is stabilized in crystalline state by both H-bonding and π-π stacking interactions. Complex 6 exhibited better cytotoxicity than complex 5, N-1H-purine-6-yl-alanine and copper salt on various cell lines. III. Synthesis, structure and anticancer activity of zinc complexes of adenine derivatives In order to understand zinc interaction with adenine and their anticancer activity, several zinc complexes of adenine derivatives were prepared. The prepared complexes are (1) [Zn (N6-benzyladenineH).Cl3].2H2O, (2) [Zn2 (μ -N6-benzyladenine)2( μ-H2O)2(H2O)4].(OTf)4.H2O, (3) (N6-benzyl adenineH2) [ZnCl4].2H2O, (4) [Zn (2-Amino-N6-Benzylpurine)Cl3).2-Amino-N6-BenzylpurineH).EtOH, (5) (2-Amino-N6-(3-picoyl)purineH2)[ZnCl4].H2O, (6)(2-Amino-N6-(3-picoyl)purineH2)[ZnCl4].HCl, (7) (2-Chloro-N6-(3-picoyl) purineH2) [ZnCl4].H2O, (8) ((α-Purine-6-ylamino)-p-toluene sulfonamide H)2[ZnCl4].2HCl.2H2O. Complex 1: The N6-benzyl adenine is coordinated to zinc through nitrogen atom N(7) of purine. One molecule of N6-benzyl adenine and three chloride ions are coordinated to zinc and forming distorted tetrahedral geometry. Interestingly, the nitrogen atom N(1) of purine is protonated. Complex 1 exhibited strong H-bonding interactions between N-H···O, N-H···Cl and N-H···N. The complex 1 showed better cytotoxicity than N6-benzyl adenine and ZnCl2. Complex 2: The N6-benzyl adenine formed a dimeric complex with zinc at neutral pH. Complex 2 crystallizes in the triclinic space group P-1with Z=1. Two Zn metal centres are bridged by two molecules of N6-benzyl adenine through nitrogen atoms N(3) and N(9) of purine forming a di-nuclear complex, further two zinc centres is bridged by two water molecules and other two water molecules on the other side completing the octahedral coordination for the Zn. Complex 2 is stabilized in crystalline state by H-bonding interactions. The H-bonding of complex 2 is observed between O-H···O and N-H···O. Complex 2 exhibited better cytotoxicity than N6-benzyl adenine and ZnCl2 on various cell lines. Complex 3: The N6-benzyladenine is not coordinated to the Zn metal at acidic pH and forms an ion-pair complex. Ion-pair complex 3 crystallizes in the monoclinic space group Cc with Z=4. The protonation is observed at N(1) and N(9) atoms of N6-benzyl adenine. The positive charges on N6-benzyl adenine is neutralized by the presence of two chloride ions in [ZnCl4]2-. Alternative arrangement of cation and anion arrangement is observed in complex 3. Water channel formation is observed between cation and anion arrangement. Moreover, complex 3 is stabilized by H-bonding and π-π stacking interactions. H-bonding is observed in complex 3 between N-H···Cl, O-H···Cl and N-H···O. The π-π stacking interactions in complex 3 are observed between benzyl six-membered aromatic ring and purine six-membered rings. Complex 3 exhibited better cytotoxicity than N6-benzyl adenine and ZnCl2 in various cell lines. Complex 4: Ligand 2-amino-N6-benzyl adenine resulted in a different structure from N6-benzyl adenine with zinc. One molecule of 2-amino-N6-benzyl purine is coordinated to zinc through nitrogen atom N(7) of purine. Surprisingly, one uncoordinated positively charged 2-amino-N6-benzyl purineH is present in the asymmetric unit, which is balancing the charge of zinc complex 4. Protonation is observed on N(3A) atom. Interestingly, tautomeric proton is located on coordinated purine of N(9) atom and uncoordinated purine of N(7A) atom. Geometry of ‘Zn coordination centre’ is distorted tetrahedral. Complex 4 is stabilized by H-bonding and π-π stacking interactions. The H-bonding interaction in complex 4 is observed between N-H···O and N-H···Cl. The π-π stacking interactions are observed between five-member aromatic rings and six-membered aromatic rings. Complex 4 exhibited better cytotoxicity than 2-amino-N6-benzyl purine and ZnCl2 in various cell lines. Complex 5: 2-Amino-N6-(3-picoyl) purine forms an ion-paired complex with zinc at acidic pH. The protonation in 2-Amino-N6-(3-picoyl) purine is observed at N(3) of the purine and picolyl N(14). The positive charge of 2-Amino-N6-(3-picoyl) purine is neutralized by the presence of two chloride ions in [ZnCl4]2-. Moreover, complex 5 exhibited both H-bonding interactions and π-π stacking interactions. The H-bonding interactions are observed between N-H···Cl, N-H···N, O-H···Cl, N-H···O and C-H···N. One uncoordinated water molecule is present in unit cell, which is involved in H-bonding with both ions. The π-π stacking interactions are observed between purine five-membered rings and purine six-membered ring. Complex 5 exhibited better cytotoxicity than cisplatin in HeLa and MDA-MD-231 cells. Complex 6: 2-Amino-N6-(3-picoyl) purine formed similar structure of complex 5 in strong acidic conditions. Complex 6 exhibited both H-bonding and π-π stacking interactions. The H-bonding in complex 6 is observed between N-H···Cl and N-H···N. In complex 6, the π-π stacking interactions are observed between pyridyl six-membered rings and purine six-membered rings. Purine-Purine stacking interactions are observed between purine six-membered ring and five-membered rings. Complex 6 exhibited better cytotoxicity than cisplatin in HeLa, MCF-7, MDA-MB-231 and HeLa-Dox cells. Interestingly, complex 6 arrested (G2/M phase) cell cycle in HeLa and MCF-7 at higher concentration and induced apoptosis. Complex 7: 2-chloro-N6-(3-picoyl) purine formed ion-pair complex with zinc. The protonation in 2-chloro-N6-(3-picoyl) purine is observed on N(9) of purine and N(14) of picolyl atoms. The positive charge of 2-chloro-N6-(3-picoyl) purine is neutralized by the presence of two chloride ions in [ZnCl4]2-. Complex 7 is stabilized by both H-bonding and π-π stacking interactions. The H-bonding is observed between N-H···Cl, O-H···Cl and N-H···O in complex 7. The π-π stacking interactions are observed between pyridyl six-membered ring and six-membered ring of purine. Complex 7 exhibited better cytotoxicity than cisplatin in HeLa, MCF-7, U251 and HeLa-Dox cells. Complex 8: (α-Purine-6-ylamino)-p-toluene sulphonamide formed ion-pair complex with zinc. Ion-pair complex 8, crystallizes in the triclinic space group P-1 with Z=4. The protonation on (α-Purine-6-ylamino)-p-toluene sulfonamide is observed at N(9) and N(1) atoms of purine. The positive charge of the ligand is neutralized by two chloride ions present in [ZnCl4]2 -. The H-bonding is observed between N-H···Cl, O-H···N, N-H···O and O-H···Cl. The π-π stacking interactions are observed between benzyl rings of benzene sulfonamide moieties. Complex 8 exhibited better cytotoxicity than cisplatin in HeLa, MCF-7 and HeLa-Dox cells. Moreover, these complexes induced apoptotic cell death as revealed by Annexin V/PI assay, FACS and microscopy analysis. IV. Synthesis, structure and cytotoxicity studies of zinc complexes of uracil-1-acetic acid and N6-adeninebutyric acid To understand the zinc interactions with nucleic acid constituent derivatives and their anticancer activity, zinc complexes of uracil-1-acetic acid and N6-adeninebutyric acids were prepared. (1) [Zn (uracil-1-acetato)2 (H2O)4] and complex (2) [Zn (N6-adeninebutyric acid)2 (H2O)2]) were characterized by X-ray crystallography and various spectroscopic techniques. The X-ray structures showed acetate moiety coordination to zinc rather than purine and pyrinidine moities. The geometry of zinc coordination centre is distorted octahedral. Complexes 1 and 2 are stabilized by non-covalent interactions. Anticancer studies of these complexes showed better cytotoxicity than cisplatin in MDA-MB-231cells. V. Copper (II) complexes of 6-mercaptopurine, hypoxanthine and uracil-1-acetic acid: Synthesis, structures, antioxidant and potent anticancer activity To delineate copper interactions with purine and pyrimidine derivatives and anticancer activity, several copper complexes of 6-mercaptopurine, hypoxanthine and uracil-1-acetic acid were prepared. The prepared complexes are (1) [Cu (6-MP) (bpy) Cl2], (2) [Cu (hx) (phen) Cl2].H2O and (3) [Cu (bpy)2 (uracil-1-acetato)].6H2O)] (bpy = 2, 2′-bipyridine, phen = 1, 10-phenanthroline, 6-MP = 6-Mercapto Purine and hx = hypoxanthine). All these complexes were chracterized by various spectroscopic and X-ray diffraction techniques. Complexes 1 and 2 crystallize in the monoclinic space groups Cc and C2/c, respectively with eight molecules in the unit cell. All the complexes 1-3 adopt distorted trigonal bipyramidal geometry. Surprisingly, most potent coordination sites of sulfur in 6-MP and acetato in uracil-1-acetato did not participate in coordination with copper. In complexes 1 and 2, the N(7) position of purine and the N(3) position of pyrimidine in complex 3 are coordinated with copper. All these complexes 1-3 are stabilized by non-covalent interactions in solidstate. Anticancer studies showed better cytotoxicity for copper complexes than cisplatin, 6-meracptopurine and temozolomide in various cell lines. Interestingly, copper complexes of 6-MP and hypoxanthine showed antioxidant activity and reduced ROS level in cells. In contrast, copper complex of uracil-1-acetic acid produced ROS in cells. In contrast, copper hypoxanthine showed better cytotoxicity than cisplatin in HeLa-Dox cells. All these complexes induced apoptotic cell death. In summary, we studied the interaction of metal-nucleic acid constituents and derivatives by X-ray crystallography. We found new coordination modes for Ni, Cu and Zn towards various nucleic acid constituents and derivatives. Some of these complexes showed better cytotoxicity than well known anticncer drugs cisplatin, 6-meracptopurine and temozolomide. Complex [Cu (hx) (phen) Cl2].H2O showed better cytotoxicity than cisplatin in doxorubicin resistant (HeLa-Dox) cells. These complexes induced apoptotic cell death in various cancer cells. All in all, the results of present studies/findings could form a potential lead for the development of newer anticancer therapeutics.

Nous proposons, dans cette thèse, d'utiliser les techniques de modélisation en pharmacométrie selon l'approche de population afin de décrire les cinétiques de plusieurs marqueurs tumoraux sériques, et d'analyser leurs potentielles applications. Dans un premier temps, nous avons construit un modèle reliant les cinétiques de taille tumorale et de CA-125 dans le cancer de l'ovaire. Nous avons ensuite évalué son application pour : i) la prévision de la réponse tumorale au niveau individuel ; ii) la prédiction précoce de la survie au niveau d'une population dans le développement du médicament. Dans un second temps, nous avons réalisé un travail plus méthodologique sur la modélisation des cinétiques conjointes de PSA et d'un nouveau marqueur, le nombre de cellules tumorales circulantes (CTCs), dans le cancer de la prostate. Un modèle atypique combinant plusieurs innovations en pharmacométrie a été développé. En perspective, un lien va être établi avec la survie. En conclusion, la modélisation mathématique est un outil efficace pour l'évaluation précoce de l'efficacité des traitements / Our thesis project aimed at building mathematical models, using population approach, for different serum tumor markers, in order to describe their kinetics and to assess their potential applications. In a first intent, we built a semi-mechanistic model linking tumor size changes and CA-125 kinetics induced by chemotherapy in ovarian cancer patients. This model allowed assessment of CA-125 as: i) a biomarker for tumor size dynamics and treatment efficacy for clinical purposes; ii) an early predictor of clinical benefit during drug development. Then, we realized a more fundamental work by developing a semi-mechanistic model for characterizing the relationships between PSA kinetics and circulating tumor cell count dynamics during treatment in metastatic prostate cancer patients. This is an atypical model combining several advanced features in pharmacometrics. We have planned to assess a link with survival. In conclusion, mathematical modeling could be an efficient tool for the early prediction of treatment efficacy