La cycloaddition 1,3-dipolaire catalysée par le cuivre(I) entre un azoture et un alcyne (CuAAC), réaction de « chimie click » par excellence, suscite un grand intérêt en raison de son efficacité et de sa versatilité. L'objectif premier de cette thèse est d'appliquer cette réaction pour l'élaboration de structures 1,2,3-triazoles fonctionnalisées, en vue d'obtenir des ligands jouant le rôle de « pince à platine ». Les complexes de platine biologiquement actifs rapportés sont de type mono- ou bi-nucléaire et comportent un ou deux cycles triazole. Les complexes obtenus ont fait l'objet d'une étude in vitro d'interaction avec des nucléosides et de l'ADN soit sous forme d'hairpin (épingle à cheveux) soit sous forme plasmidique. Nos complexes ont montrés une réactivité similaire à celle du cisplatine, qui est la métallodrogue de référence. En parallèle, nous présentons les propriétés catalytiques et biologiques de complexes cuivre(I)-NHC (Carbène N-Hétérocyclique), dérivés du [CuCl(SIMes)], mis au point par Nolan et al. Dans un premier temps, un criblage d'activités catalytiques a été réalisé avec divers additifs aromatiques azotés afin d'améliorer l'efficacité de la CuAAC. Dans un deuxième temps, la cytotoxicité et l'activité antitumorale du complexe [CuCl(SIMes)] ont été considérées sur plusieurs lignées cellulaires. Nous rapportons ici, le premier exemple de cuivre(I)-NHC biologiquement actif, présentant une activité largement supérieure à celle du cisplatine. Enfin, la réactivité de ce complexe avec de l‟ADN plasmidique a été évaluée in vitro et nous rapportons sa capacité à couper l‟ADN. / The copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC), a “click chemistry” reaction, is of a great interest thanks to its efficiency and versatility. The present work aims to use this reaction for the elaboration of new functionalised 1,2,3-triazole, as “pincer ligand” to platinum. We have synthesized mono- or binuclear platinum anticancer complexes that contain one or two triazole cycles. These complexes were subject to in vitro tests, in order to investigate the interactions they can establish with both nucleosides and DNA. The results reveal a reactivity similar to cisplatin, the reference metallodrug. In parallel, we present the catalytic and biological properties of copper(I)-NHC complexes, like [CuCl(SIMes)] developed by Nolan et al. At first, a screening of the catalytic activity was realized with diverse nitrogen aromatics additives in order to improve the CuAAC efficiency. Secondly, the cytotoxicity and antitumoral activity of [CuCl(SIMes)] were considered on various cancer cell lines. We report here, the first example of a biologically active copper(I)-NHC, this complexe exhibiting a superior activity than cisplatin. Finally, the reactivity of this copper(I)-NHC with DNA was evaluated in vitro and highlights its capacity to cleave DNA.

The aim of this thesis was to find optimal therapy based on combination of membrane-anchored phagocytic ligands (mannose-(G)5-(K)10-STE, mannan-BAM, mannan-SMCC) with LPS (ligand of signal receptor)for treatment of murine melanoma B16-F10. Mixture of mannan-BAM with LPS applied in pulse regime proved to be the most effective, resulting in heigh reduction of tumor growth and significant prolongation of survival.

A descoberta de fármacos a partir de produtos isolados de organismos marinhos tem apresentado um grande crescimento nos últimos anos, principalmente devido aos avanços tecnológicos analíticos, síntese química e biotecnologia. Dentre estes, as esponjas representam uma das principais fontes de metabólitos protótipos para diversas atividades, destacando-se os efeitos antitumorais. Neste contexto, este trabalho teve como objetivo a investigação química e biológica de três esponjas coletadas na costa sul-brasileira: Haliclona tubifera, Polymastia janeirensis e Scopalina ruetzleri. Considerando a correlação entre câncer, distúrbios da coagulação e desbalanço de espécies reativas de oxigênio (EROs), foram realizados ensaios visando a aquisição destas atividades e a identificação de substâncias bioativas. Para a esponja H. tubifera foram observados interessantes efeitos antitumorais em células de glioma e neuroblastoma humano (IC50 < 20 μg/ml), além das atividades antioxidante e anticoagulante para a fração acetato de etila. O composto majoritário desta fração foi isolado como um derivado N-Boc e sua configuração foi estabelecida utilizando um novo protocolo de dicroísmo circular e semissíntese de derivados. Assim, este esfingosídeo de cadeia longa isolado (2R,3R,6R,7Z)-2-amino-7-octadecene-1,3,6-triol, foi denominado halisphingosine A. Um novo composto minoritário, halisphingosine B foi obtido usando técnicas de isolamento em escala nanomolar. Sua configuração absoluta foi estabelecida por comparação com o composto A. Da mesma forma, para a esponja S. ruetzleri, a fração acetato de etila demonstrou os resultados mais promissores. Um potencial efeito anticâncer e de inibição dos radicais peroxila foi observado. Além disso, um efeito modulador da peroxidação lipídica foi evidenciado em ensaio ex vivo de dienos conjugados. Através da análise por RMN de 1H, verificou-se que a fração era majoritariamente constituída por ácidos graxos, os quais foram derivatizados para caracterização por Cromatografia Gasosa (GC/FID). Foram identificados 32 ácidos graxos principalmente poli-insaturados (53%). Ácidos graxos minoritários não usuais para o ambiente marinho também foram caracterizados. A esponja P. janeirensis apresentou os efeitos citotóxicos mais promissores em células de glioma e neuroblastoma humano, com um IC50 < 1,0 μg/ml para o extrato aquoso (pH 6,8), sendo este efeito pH-dependente, uma vez que o extrato (pH 5,8) não alterou a viabilidade celular. Para P. janeirensis, foi também investigado seu potencial de defesa química em modelo de Zebrafish. Foi observado que o extrato aquoso desencadeia um efeito de fuga, alterando significativamente o comportamento espaço-temporal de peixes Danio rerio. Analisando em conjunto, os dados do presente trabalho representam uma nova contribuição para o estudo químico e biológico de espécies de esponjas marinhas da costa sul-brasileira e apontam as potencialidades destas esponjas na busca de moléculas protótipos para fármacos, especialmente relacionados à terapia do câncer. / Drug discovery from marine natural products has increased in the past few years, mainly due to technological advances in spectroscopy, chemical synthesis and biotechnology. Among all marine animals, sponges represent one of the major sources of prototype metabolites for several biological activities, highlighting the antitumor effects. In this context, this study carried out chemical and biological investigation of three sponges collected on the South Brazilian coastline: Haliclona tubifera, Polymastia janeirensis and Scopalina ruetzleri. Considering the correlation between cancer, clotting disorders and imbalance of reactive oxygen species (ROS), experiments were conducted for acquisition of these activities and identification of bioactive compounds. H. tubifera showed an interesting cytotoxic effect in human neuroblastoma and glioma cell lines (IC50 <20 μg/ml), antioxidant and anticoagulant effect for ethyl acetate (EtOAc) fraction. The major compound of EtOAc fraction was isolated as an N-Boc derivative and its configuration was established using a new circular dichroism protocol with the production of semi-synthetic derivatives. This long chain sphingoid base (2R,3R,6R,Z)-2-aminooctadec-7-ene-1,3,6-triol was named as halisphingosine A. A new minor compound, halisphingosine B was obtained using nanomol scale techniques and their absolute configuration was established by comparison with compound A. Likewise, for the sponge S. ruetzleri, the EtOAc fraction showed the most promising results. A potential anticancer effect, inhibition of peroxyl radicals and modulation effect of lipid peroxidation was observed. Fingerprint 1H NMR analysis showed that this fraction is mainly constituted of fatty acids. Through Fatty Acid Methyl Ester (FAMEs) analysis by GC/FID, it was possible to identify 32 fatty acids, of which around 50% were Polyunsaturated Fatty Acids (PUFAs). In addition, some minor unusual fatty acids for the marine biosphere were identified. It was observed for P. janeirensis the most promising cytotoxic effects on human glioma and neuroblastoma cells, with an IC50 <1.0 μg/ml to aqueous extract (pH 6.8), being this effect pH-dependent, since the extract (pH 5.8) did not affect the cell viability. Moreover, P. janeirensis was investigated along their potential chemical defense in Zebrafish model. Aqueous extract trigged an escape effect, significantly altering the spatio-temporal swimming activity of animals. Taken together, the data presented from this study represent a new contribution to chemical and biological research of marine sponge species from South Brazilian coastline, and point the potentialities of sponges to search chemical prototypes for drugs, especially related to cancer therapy.

L’évaluation de l’effet cytotoxique de différents jus de fruits naturellement riches en polyphénols vis-à-vis de quatre lignées de cancer colorectal a montré que le jus de la canneberge est particulièrement actif. En effet, les polyphénols de la canneberge induisent l’apoptose associée à une surexpression des deux facteurs de transcription pro-apoptotiques p73 et FOXO3a. Cette mort programmée est aussi associée à une diminution de l’expression de SIRT1 le déacétylateur de protéines non histone telles que p73, KU70, ou FOXO. D’autres événements précoces comme la production de ROS et les dommages à l’ADN connus pour réguler l’expression de SIRT1 ont été confirmés. Une deuxième étude avait pour objectif de valider le potentiel anticancéreux in vivo chez la souris Balb/C injectée de cellules d’adénocarcinome colique murin. Pour cela nous avons choisi le jus d’aronie noire qui a montré in vitro un profil de cytotoxicité intéressant. L’analyse des tumeurs a montré que l’administration de jus d’aronie entraine une réduction de la prolifération des cellules tumorales. Enfin, l’augmentation significative de la mobilité de LC3 suggère l’activation d’une mort cellulaire autophagique. Afin d’évaluer l’utilisation clinique des polyphénols, nous avons évalué les effets cytotoxiques des polyphénols de myrtille sur des lymphocytes, de patients atteints de LLC. Nos résultats montrent que l’extrait polyphénolique induit une apoptose dépendante du stress oxydant et impliquant aussi des protéines pro-apoptotiques dans des cellules de patients atteints de LLC mais pas dans les cellules de sujets sains. / The evaluation of the cytotoxic effect of different fruit juices, naturally rich in polyphenols, on four different colorectal cancer cell lines proved that cranberry juice was the most active. Indeed, cranberry polyphenols induce apoptosis associated with the overexpression of two important proapoptotic transcription factors, p73 and FOXO3a on one hand. Furthermore, it has been also correlated with a decrease in the expression of SIRT1, the deacetylase of several non-histone proteins such as p73, KU70 and FOXO. Other early events such as ROS production and DNA damage, which are known to regulate the expression of SIRT1 were confirmed. The second study aims at validating the potential anticancer effects in an in vivo model of colorectal cancer in BALB/c mice injected subcutaneously of murine colon adenocarcinoma cells. Accordingly, we chose the black chokeberry juice, which showed an interesting cytotoxic profile in vitro. The analysis of tumors demonstrated that the administration of chokeberry juice leads to a reduction in tumor cell proliferation. Finally, the significant increase in the mobility of LC3 suggests the activation of autophagic cell death. To validate the clinical use of polyphenols, we evaluated the cytotoxic effects of blueberry polyphenols on lymphocytes of CLL patients. Our results show that the polyphenolic extract induces an oxidative stress-dependent apoptosis that involve various pro-apoptotic proteins in cells of patients with CLL but not in healthy subjects.

Orientadores: João Ernesto de Carvalho, Mary Ann Foglio / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T00:15:44Z (GMT). No. of bitstreams: 1 Longato_GiovannaBarbarini_D.pdf: 11931836 bytes, checksum: 6a9508b3296a68bdd39eb4630f1e89de (MD5) Previous issue date: 2014 / Resumo: O câncer é a segunda causa de morte da população nos países desenvolvidos e em desenvolvimento. Caracteriza-se pelo desenvolvimento de células instáveis que tendem a não responder aos estímulos internos e externos que controlam a proliferação, diferenciação e morte celular. O crescente entendimento da biologia tumoral tem fornecido alvos moleculares para a triagem orientada de quimioterápicos e de agentes quimiopreventivos, geralmente de origem natural ou sintetizados com base em produtos naturais. A partir do extrato bruto diclorometânico das folhas de Piper regnellii foram isolados dois compostos, o eupomatenóide-5 e o apiol. O eupomatenóide-5 é uma neolignana cuja potencial atividade anticâncer foi demonstrada in vitro (cultura de células tumorais humanas) e in vivo (tumor sólido de Ehrlich) em trabalhos anteriores publicados pelo nosso grupo. Dentre as linhagens mais sensíveis, MCF-7 (mama) e 786-0 (rim) foram selecionadas para dar continuidade aos estudos de mecanismo de ação deste composto. Os resultados obtidos no presente estudo sugerem que o eupomatenóide-5 possui ação multialvo, uma vez que demonstraram claramente que existe um mecanismo de ação diferente para cada linhagem celular. Sugere-se que o efeito antiproliferativo do eupomatenóide-5 esteja associado com indução de apoptose em linhagem MCF-7 e necrose programada em linhagem 786-0. O apiol, outro composto isolado, é um fenilpropanóide com conhecida ação bloqueadora dos canais de cálcio. Sabe-se que a inibição de canais de cálcio tem relação com a reversão da resistência a múltiplas drogas (MDR) e, por este motivo, o efeito do apiol sobre a glicoproteína-P, uma das principais proteínas moduladoras dessa resistência, foi avaliado; entretanto o apiol não inibiu sua ação. Por outro lado, neste estudo foi possível evidenciar que embora tenha apresentado atividade citostática baixa para as linhagens tumorais, ele potencializou a atividade específica dos quimioterápicos doxorrubicina e vincristina quando associado a estes em linhagens tumorais com e sem fenótipo de resistência, sugerindo um efeito sinérgico. Além disso, o apiol potencializou a atividade antiproliferativa do eupomatenóide-5 e essa associação reduziu a quantidade de células tumorais de Ehrlich em modelo in vivo, sem sinais de toxicidade evidentes. Os resultados encorajam o aprofundamento desses estudos para a determinação dos mecanismos de ação envolvidos nas atividades anticâncer do eupomatenóide-5 e de reversão da MDR do apiol / Abstract: Cancer is the second leading cause of mortality worldwide. The loss of normal cell growth control is the main event in the development of cancer and includes specific steps known as cell proliferation, differentiation and apoptosis. Natural compounds are good source of new anticancer agents, being the investigation into cell death mechanisms involved in this activity a significant endeavor. From the crude dichloromethane extract of Piper regnellii leaves two compounds were obtained: eupomatenoid-5 and apiole. Eupomatenoid-5 is a neolignan whose in vitro (tumor cell line culture) and in vivo (Ehrlich solid tumor) anticancer activity was previously reported by our group. Among the cancer cell lines most sensible to eupomatenoid-5 treatment, MCF-7 (breast) and 786-0 (kidney) were chosen to continue the cell death mechanism of action studies. The results obtained suggest that eupomatenoid-5 is a multiple-target compound. Our findings support the possibility that eupomatenoid-5 might be acting in both cell death via: apoptosis in MCF-7 cell line and programmed necroptosis in 786-0 cell line. Apiole, the other compound isolated, is a phenylpropanoid and a calcium channel blocker. Recent pharmacological studies have reported the ability of several calcium channel blockers to reverse the multidrug resistance (MDR) and, therefore, the effect of apiole on P-glycoprotein (P-gp), the main protein involved in MDR, was evaluated. Apiole did not inhibit P-gp and, moreover, presented low cytotoxic activity for the most tumor cell lines. Nonetheless, the combined therapy with chemotherapies (doxorubicin and vincristine) was efficient in potentializing their antiproliferative action in tumor cell lines with or without resistance phenotype, suggesting synergic effect. Furthermore, apiole potentiated the antiproliferative activity of eupomatenoid-5 and this association reduced the in vivo tumor cell quantity with no side effects. The results encourage further studies to be developed with the aim to determine the mechanisms of action involved in the anticancer activity of eupomatenoid-5 and in the MDR reversion by apiole / Doutorado

The development of reliable and cost-saving methods to select pre-clinically new potential drugs with unknown and original mechanisms of action for cancer therapy has become crucial. Previous investigations demonstrated that infrared spectra of cancer cells exposed to well-known anticancer drugs used in the clinics provide a global fingerprint of all the metabolic modifications induced. Nowadays several natural products have been recognized for their medicinal values. Polyphenolic compounds constitute one of the largest groups of plant metabolites and many studies have demonstrated their anticancer properties at multiple steps of carcinogenesis. Taking into account the large diversity of polyphenolic structures in nature and their numerous targets against carcinogenesis, the step of selection becomes essential as it is virtually impossible to classify them using traditional classification techniques. While FTIR spectroscopy appears to have a definite potential to sort anticancer drugs on the basis of the metabolic modifications they induced, the present challenge in this thesis is to evaluate the drug-induced spectral changes in cancer cells on a larger scale. The coupling of FTIR spectroscopy with a high throughput screening extension could become a useful method to generate drug classifications based on the “modes of action”. In a first step, the IC50 was evaluated for each polyphenol to normalize all spectral experiments that will be carried out on breast cancer cells. The first experiments revealed dispersive artifacts (wide variations of absorbance distribution and Mie scattering effects) contributing to spectral measurements. To minimize them, the best option was covering uniformly the surface of spectral measurement with cells (detailed in chapter III). Once the protocol refined, it was applied to the study of MDA-MB-231 breast cancer cells exposed for 24 hrs to 15 polyphenols. Through unsupervised and supervised statistical analyses, a distinction between polyphenol treatments could be well established. Complex effects of polyphenols on cancer cells were revealed, suggesting that mechanisms specific to each polyphenol were evidenced by the whole infrared spectrum. Clustering of polyphenol-induced spectral signatures by Hierarchical Cluster Analysis indicates that some of the polyphenols share similar effects on MDA-MB-231 (detailed in chapter IV). This experiment was then extended to another breast cancer cell line, MCF-7. It demonstrated a cell line dependency to polyphenolic treatments. Finally a subcellular investigation of treated MCF-7 breast cancer cells in their live state was done using Raman imaging. A distinction between nucleus and cytoplasm of treated cells brought supplementary information regarding the effect of polyphenols, leading to subcellular biological assumptions on polyphenol effects. These results paves the way for a new classification based on infrared spectral signatures that reflect the overall chemical modifications experienced by the cells when exposed to drugs. / Le développement de méthodes fiables et peu cher pour sélectionner de potentiels nouveaux médicaments présentant un mécanisme d’action original et inconnu avant toute étape clinique devient crucial. De précédentes études ont pu démontrer que les spectres infrarouges de cellules cancéreuses exposées à des agents anticancéreux connus et utilisé dans le monde clinique fournissent une empreinte globale de toutes les modifications métaboliques induites. La spectroscopie infrarouge est un outil innovant qui semble prometteur pour offrir un aperçu global des processus biologiques et physiologiques qui sont menés par un médicament dans des cellules cancéreuses. De nos jours, de nombreux produits naturels ont été reconnus pour leurs propriétés médicinales. Les polyphénols constituent l’un des plus vastes groupes de métabolites végétaux et de nombreuses études ont démontré leurs propriétés anticancéreuses à de multiples étapes de la carcinogénèse. En prenant en compte la très grande diversité de structures polyphénoliques existantes dans la nature et leurs nombreuses cibles anti-tumorales, l’étape de sélection est devenue essentielle comme il est virtuellement impossible de les classifier grâce à des techniques de classification traditionnelles telles que les études –omiques. Dès lors, le défi de cette thèse est d’évaluer les variations spectrales induites par un polyphénol dans des cellules cancéreuses à une plus grande échelle. Le couplage de la spectroscopie IRTF avec une extension de criblage de haut débit pourrait devenir une méthode utile pour générer des classifications de molécules sur base de leur « modes d’action ». Dans un premier temps, la concentration qui inhibe 50% de la croissance des cellules cancéreuses fut déterminée pour chaque polyphénol et chaque lignée de cellules cancéreuses. Le traitement des cellules à cette concentration permet une normalisation interne des expériences réalisées ultérieurement en spectroscopie infrarouge. Une fois le protocole établi, la lignée MDA-MB-231 fut exposée durant 24 heures à 15 polyphénols différents. Au moyen d’analyses statistiques multivariées supervisées et non supervisées, une distinction parmi les polyphénols a pu être établie et des effets complexes des polyphénols sur les cellules cancéreuses ont pu être révélés, suggérant des mécanismes d’action spécifiques à chaque polyphénol mis en évidence par spectroscopie infrarouge. Finalement, une étude subcellulaire sur cellules vivantes fut réalisée par imagerie Raman sur une seconde lignée de cellules cancéreuses mammaires appelées MCF-7. Cela permis de compléter en partie l’information macroscopique offerte par la spectroscopie infrarouge par une information microscopique sur l’effet de certains polyphénols. Cette thèse a ouvert la voie pour de nouvelles techniques de classification d’agents anticancéreux basées sur la spectroscopie infrarouge, technique sensible à l’ensemble des modifications chimiques subies par des cellules. / Option Chimie du Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Nectandra membranacea (Lauraceae) est une plante très commune aux Petites Antilles. Aucune utilisation traditionnelle de cette plante n’est connue mais le genre Nectandra, qui représente le deuxième plus important de la famille des Lauraceae avec 114 espèces reconnues, présente des propriétés analgésiques, anti-inflammatoires et énergétiques (Le Quesne et al 1980). Nectandra a également démontré une activité antitumorale (Silva-Filho, 2004). Les nombreuses propriétés du genre et le peu de connaissance chimique sur cette espèce nous ont incités à réaliser une étude phytochimique et à évaluer le potentiel biologique des différentes parties de cette plante. L’analyse des lipides a été effectuée, et les principales classes de métabolites secondaires (terpènes, flavonoïdes, alcaloïdes) ont été recherchées dans les feuilles, les écorces et les fruits de Nectandra membranacea. Afin de suivre les variations de la composition chimique en fonction du cycle de vie du végétal, l’étude a été réalisée sur des échantillons (feuilles, écorces et fruits), récoltés à deux stades du cycle de la plante : la germination (stade A) et la fructification (stade B). N. membranacea, grâce à la présence de nombreuses familles chimiques, possède des activités biologiques intéressantes. Les huiles essentielles ont montré des activités anticancéreuses, anti-inflammatoires, antioxydantes et antiherpétiques. Les extraits polyphénoliques issus des différentes parties de la plante ont montré une très grande activité antioxydante (ORAC et DPPH) et in vitro (cellules saines) ainsi que des propriétés anti-inflammatoires notables. Les extraits d’alcaloïdes totaux issus des trois parties ont été évalués pour la maladie d’Alzheimer et ont révélé de très bons effets neuroprotecteurs, plus particulièrement pour les extraits de feuilles récoltées lors de la germination. Ce travail a permis d’élargir la connaissance des huiles essentielles de l’espèce (composition pour plusieurs parties et activité biologique). Des flavonoïdes ont été identifiés, mais la plante s’est révélée particulièrement riche en alcaloïdes (une quinzaine) de type isoquinoliques et indoliques. N. membranacea se révèle être une plante aux nombreuses propriétés, au regard de ses activités biologiques intéressantes. Des extraits pourraient être utilisés comme principes actifs dans des médicaments mais également en industrie cosmétique. / Nectandra membranacea (Lauraceae) is a very common plant in the French West Indies. No traditional use of this plant is known, but the genus Nectandra, which represents the second most important of Lauraceae family with 114 recognized species, has got analgesic, anti-inflammatory and energizing activities (Le Quesne et al 1980). Nectandra was also identified as a potential antitumor agent (Silva-Filho, 2004). Many properties of the genus, and some chemical knowledge of this species, have prompted us to perform phytochemical and biological study of different parts of the plant. Lipids and the main classes of secondary metabolites (terpenes, flavonoids, alkaloids) were investigated in leaves, bark and fruits of Nectandra membranacea. To determine the monitoring changes in the chemical composition, according to the life cycle of the plant, the study was carried out on samples (leaves, bark and fruits) harvested at two stages of the cycle plant : germination (stage A) and fruit stage (stage B). N. membranacea, thanks to the presence of many chemical families, has interesting biological activities. Essential oils have shown anticancer, anti-inflammatory, antioxidant activities. They revealed antiviral activity on the herpes virus. The polyphenolic extracts from different parts of the plant showed a high antioxidant activity (ORAC and DPPH tests) and in vitro (healthy cells) and significant anti-inflammatory properties. The total alkaloids extracted from the three parts were evaluated for Alzheimer’s disease and were found to have very good protective effects, especially the leaves’ extracts collected during germination. This work has expanded knowledge of the essential oil of the species (composition of several parts and biological activity). Flavonoids have been identified, but the plant was shown to be particularly rich in alkaloids (fifteen were identified) of indolic and isoquinolic type. N. membranacea presents many properties. In view of its interesting biological activities, it could be used for many drugs but also in cosmetic area.

Les cibles épigénétiques telles que les histones désacétylases (HDAC) sont étudiées comme nouveaux traitements anticancéreux, leur fonctionnement anormal étant associée aux processus cancéreux. De nombreux inhibiteurs de ces enzymes sont apparus tel que le SAHA ou le CI-994. Notre laboratoire a développé un nouvel analogue de la trichostatine A appelé NODH. Malgré leur activité anticancéreuse in vitro prometteuse, ces inhibiteurs souffrent de leur manque de solubilité, d'effets toxiques et de leur rapide métabolisation par l'organisme. Afin de pallier ces limitations, ces iHDAC peuvent être conjugués à des vecteurs capables de les transporter dans l'organisme et de les libérer une fois la cible tumorale atteinte. Ce travail de thèse a consisté dans un premier temps à développer des prodrogues acido-sensibles capable de libérer la substance active après endocytose par les cellules. Ces systèmes ont été appliqués aux trois iHDAC SAHA, CI-994 et NODH. Les prodrogues développées ont ensuite été accrochées sur des nanoparticules polymériques connues pour leur biocompatibilité et leur accumulation dans les tumeurs grâce à l'effet EPR. L'activité anticancéreuse de certaines nanoparticules a été validée par des tests in vitro et in vivo. Un deuxième travail a été entrepris concernant la préparation de prodrogues fluorescentes pH labiles capables de libérer la substance active et de moduler la fluorescence par la variation du pH. La dernière partie de ce travail a été consacrée à la mise au point d'une nouvelle méthode de synthèse par voie superacide potentiellement stéréosélective de benzofuranones nécessaires à la préparation d'analogues de NODH. / Epigenetic targets like histone deacetylases (HDAC) are studied as new anticancer treatments, their dysfunctioning being associated to cancer mechanisms. Several epigenetic target inhibitors have been developed like SAHA or CI-994. Our group has developed a new trichostatin A analogue called NODH. Despite their promising in vitro anticancer effect, these inhibitors suffer from lack of solubility, toxic effects and rapid clearance. These constrains make the HDACi good candidates for ligation to vectors able to carry them in the body and to release them when the tumor is reached. The first part of this thesis work has focused on the development of acid sensitive prodrugs able to release an active substance after cellular internalization by endocytosis. These systems were applied to three HDACi SAHA, CI-994 and NODH. These prodrugs were then conjugated to polymeric nanoparticles known for their biocompatibility and their accumulation in tumors exploiting the EPR effect. The anticancer activity of some nanoparticles has been validated by in vitro and in vivo tests. The second part of this work was carried out in order to prepare pH sensitive fluorescent prodrugs able to release the active molecule and to modulate the fluorescent effect by pH changes. The last part of this work was dedicated for the development of a new synthesis in suparacid potentially stereoselective of several benzofuranones required for the preparation of NODH analogs.

There is an urgent need to develop reliable and cost-saving methods to select pre-clinically new drug candidates with original mechanism for cancer therapy. Previous results have shown that IR spectra of cancer cells exposed to various drugs provided a global signature of all the metabolic changes induced by the treatments. In this thesis, we attempted to develop a selection criterion – based on FTIR spectroscopy – for potential antitumor compounds according to their mechanism of action. <p>In chapter III, it was demonstrated that spectral variations in IR spectra of cancer cells induced by a treatment can be correlated to the mechanism of the drug. Human prostate cancer PC-3 cells were exposed to 7 well-described anticancer drugs belonging to 3 distinct classes. Each class is characterized by a unique mode of action. Drugs known to induce similar types of metabolic disturbances appear to cluster when spectrum shapes are analyzed. Chapter IV generalized the results obtained on PC-3 cells with six other cell lines. We showed that the spectral signatures of drug effects are mainly independent of the cell line. Chapter V indicated that, while the cell cycle phase influence IR spectra of cells, the drug spectral signature was dominated by global metabolic modifications and not much by the cell cycle perturbations due to this drug. <p>Chapter VI and VII focused on lipids. While the precise identification of particular molecules is particularly complex with IR spectroscopy, we attempted to extract more precise information and to assign spectral variations to specific changes in lipids. IR spectra of lipids contain very interesting details on their nature and structure. We achieved to build a tool which quantifies five major lipid classes in complex mixtures such as total lipid cell extracts. However, based on this tool, the treatments used do not induce any variation in the lipid cell composition (for five classes).<p>Finally, in chapter VIII, we applied the method developed previously on a new potential class of anticancer molecules: the polyphenols. A global method was particularly interesting as the development of therapy using these compounds is hampered by the complexity of the multiple anticarcinogenic mechanisms of these molecules. We have noticed the similarities and discrepancies among 3 very close synthetic molecules and the observations were coherent with previous biological data. We also compared them with 3 natural molecules already in clinical phase for treatment of various cancers.<p>In conclusion, we developed an objective classifier for potential anticancer drugs based on their global effects on cancer cells. Applied to a larger scale, this method could constitute a first step in the screening method to select drugs with original mode of action.<p>There is an urgent need to develop reliable and cost-saving methods to select pre-clinically new drug candidates with original mechanism for cancer therapy. Previous results have shown that IR spectra of cancer cells exposed to various drugs provided a global signature of all the metabolic changes induced by the treatments. In this thesis, we attempted to develop a selection criterion – based on FTIR spectroscopy – for potential antitumor compounds according to their mechanism of action. <p>In chapter III, it was demonstrated that spectral variations in IR spectra of cancer cells induced by a treatment can be correlated to the mechanism of the drug. Human prostate cancer PC-3 cells were exposed to 7 well-described anticancer drugs belonging to 3 distinct classes. Each class is characterized by a unique mode of action. Drugs known to induce similar types of metabolic disturbances appear to cluster when spectrum shapes are analyzed. Chapter IV generalized the results obtained on PC-3 cells with six other cell lines. We showed that the spectral signatures of drug effects are mainly independent of the cell line. Chapter V indicated that, while the cell cycle phase influence IR spectra of cells, the drug spectral signature was dominated by global metabolic modifications and not much by the cell cycle perturbations due to this drug. <p>Chapter VI and VII focused on lipids. While the precise identification of particular molecules is particularly complex with IR spectroscopy, we attempted to extract more precise information and to assign spectral variations to specific changes in lipids. IR spectra of lipids contain very interesting details on their nature and structure. We achieved to build a tool which quantifies five major lipid classes in complex mixtures such as total lipid cell extracts. However, based on this tool, the treatments used do not induce any variation in the lipid cell composition (for five classes).<p>Finally, in chapter VIII, we applied the method developed previously on a new potential class of anticancer molecules: the polyphenols. A global method was particularly interesting as the development of therapy using these compounds is hampered by the complexity of the multiple anticarcinogenic mechanisms of these molecules. We have noticed the similarities and discrepancies among 3 very close synthetic molecules and the observations were coherent with previous biological data. We also compared them with 3 natural molecules already in clinical phase for treatment of various cancers.<p>In conclusion, we developed an objective classifier for potential anticancer drugs based on their global effects on cancer cells. Applied to a larger scale, this method could constitute a first step in the screening method to select drugs with original mode of action.<p><p>Afin d’améliorer les thérapies contre le cancer, il devient actuellement cruciale de développer une méthode pour améliorer la sélection préclinique de nouvelles molécules, potentiellement anticancéreuses. Des publications précédentes ont mis en évidence que les spectres infrarouges de cellules cancéreuses exposées à différents agents thérapeutiques fournissent une empreinte globale de l’ensemble des changements métaboliques induit par ce médicament. Dans cette thèse, nous proposons d’utiliser la spectroscopie infrarouge pour mettre au point un critère de sélection basé sur le mode d’action des agents anticancéreux. Plusieurs aspects de la technique ont été investigués. Nous avons d’abord démontré la possibilité d’utiliser les spectres infrarouges de cellules cancéreuses de prostate PC-3 traitées avec 7 drogues pour classer ces molécules selon leur mode d’action. Nous avons ensuite reproduit les résultats obtenus sur PC-3 avec 6 autres lignées cellulaires et montré que la signature spectrale obtenue était largement indépendante de la lignée. Par la suite, nous avons étudié si l’effet sur le cycle cellulaire induit par de nombreuses molécules anticancéreuses, pouvait expliquer certains changements spectraux observés suite au traitement. Nous avons pu montrer que la majorité des variations spectrales n’étaient pas liées à une perturbation du cycle cellulaire. Nous nous sommes ensuite concentrés sur une classe de molécules en particulier: les lipides. Après avoir mis en évidence l’ensemble des informations contenues dans un spectre infrarouge de lipides, nous avons mis au point un modèle permettant de quantifier 5 classes de lipides dans des mélanges complexes tels que des extraits lipidiques provenant de cellules. Néanmoins, aucune variation du contenu en ces 5 classes de lipides n’a été observée pour les traitements utilisés dans cette étude. Enfin, nous avons appliqué la méthode mise au point dans cette thèse à une classe de molécules prometteuses :les polyphénols. Une approche globale s’avère particulièrement intéressante pour ces composés étant donné qu’ils présentent des mécanismes anticancéreux multiples et complexes. Nous avons comparé 3 molécules naturelles en phase clinique pour le traitement de certains cancers et 3 molécules synthétiques présentant une structure très proche. Par notre méthode, nous avons mis en évidence certaines similarités et différences de ces 6 molécules en termes d’effets globaux sur les cellules. En conclusions, nous avons développé un outil objectif de classification pour les molécules anticancéreuses potentielles, basée sur le mécanisme global des composés. Appliquée à plus large échelle, cette méthode pourrait constituer une première étape permettant de sélectionner les molécules avec un mode d’action original. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

Nous avons synthétisé des composés d'étain avec des acides undécylénique, ricinoléique et caprylique. Ils ont une activité importante contre certaines souches de microrganismes, puisque ils agissent pour certains d’entre eux à des concentrations nanomolaires. Staphylococcus aureus semble être 4000 fois plus sensible à leur toxicité que les cellules de mammifères. Nous avons obtenu des composés du ruthénium qui présentent cytotoxicité contre des cellules cancéreuses suivant un mécanisme d'action différent de ceux observés pour le Cisplatine ou d'autres composés de Ru, grâce à leur grande stabilité dans les réactions de substitution. Enfin, pendant la vectorisation des composés dérivés du Ru avec une Affitine nous avons pu acquérir des connaissances importantes sur un éventuel mécanisme d'action de ce type de molécules dont le potentiel redox très abaissé par rapport aux composés correspondants pourrait être responsable de la polymérisation de protéines cibles par transfert d’électron. / Related to antimicrobial research, we synthesized tin compounds derived from undecylenic, ricinoleic and caprylic acids and we found that they show very high activity against some strains of bacteria and yeast, even in nM range, being up to four thousand times more potent against Staphylococcus aureus than against mammalian cells. For ruthenium compounds, in turn, we have confirmed that the mode of action of some compounds that were synthesized recently is undoubtedly different from Cisplatin or other ruthenium compounds, because of their high stability toward substitution reactions. Finally, during the vectorization of compounds derived from Ru with Affitin we have gained important knowledge about a possible mechanism of action of this type of molecule: it could indeed be possible that these compounds which have a very reduced redox potential compared to corresponding compounds can cause polymerization of proteins by electron transfer.