We synthesized a series of enediyne containing Combretastatin A-4 analogues and evaluated the growth inhibitory activity of analogues against two human hepatoma cell lines (Hep G2, Hep 3B), two breast cancer cell lines (MDA-MB-231, MCF-7) and human lung cancer cell line (A-549). It was found that compounds 39a, 39d, 39e, 39f and 40a produced growth inhibitory activity for the five human cancer cell lines. Among them, compound 40a showed the best inhibitory activity (IC50 = 0.8 £gM for A-549 and IC50 = 8.53 £gM for Hep 3B) which were comparable to the Combretastatin A-4 (IC50 = 1.57 £gM for A-549 and IC50 = 14.32 £gM for Hep 3B).

A total of 27 new enediyne analogs of combretastatin A-4 (CA-4), with different substituents (Cl, Br, OCH3, SCH3 or N(CH3)2 ) of the aryl A-ring, were designed, synthesized, and evaluated for their growth inhibition activity against human tumor cell lines. Among them, compound 14a showed most significant inhibition activity against Hep3B (0.22 £gM), A-549 (0.49 £gM) and MCF-7 (0.22 £gM), and these activities were greater than CA-4. The enediyne analog 14a may be consider as a new drug candidate worthy of further investigation and development as a potential antitumor agent.

We designed a new series of enediyne and their derivatives, and evaluating for their growth inhibition activity against human tumor cell lines. This dissertation is composed of two parts. First, compounds 6c, 6e, 6g and 7g displayed good growth inhibition activity against A549 (non-small-cell lung cancer), AGS (human stomach adenocarcinoma), PC-3 (prostate cancer), BT483 (breast carcinomas), HeLa (human cervical epithelioid carcinoma), OVCA (ovarian cancer cell line), SKHep (hepatocellular carcinoma), H460 (human lung cancer cell line) and SW620 (Human Colorectal Cancer Cell Line), especially compound 7g is better, and its average IC50 is 8.79£gM. Second, compounds 25a and 26 displayed better growth inhibition activity against HeLa (human cervical epithelioid carcinoma), OVCA (ovarian cancer cell line), AGS (human stomach adenocarcinoma) and H460 (human lung cancer cell line), and their average IC50 are10.82 and 11.08 £gM.

A series of anilines and enediynes containing Combretastatin A-4 analogues were synthesized. The growth inhibitory activity of these analogues were evaluated against three human cancer cell lines including human hepatoma (Hep G2), breast cancer (MBA-MB231) and lung cancer (A-549) cell lines. The IC50 values of the synthesized compounds are between 10-5-10-7 £gM. We found that compounds 11e, 12e and 13e were observed good growth inhibitory activity for the three human cancer cell lines, especially for A549 (IC50 = 0.22, 0.40 and 0.16 £gM respectively).

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A fenstatina, quimicamente designada de 4-metoxifenil-3,4,5-trimetoxifenilmetanona, denominada RR07, Ã uma bisarilcetona de esqueleto estilbenÃide que pode ser obtida a partir da combretastatina A4, com reconhecida atividade citotÃxica, por meio da oxidaÃÃo de Jacobsen. Dentre as muitas atividades desencadeadas pelos estilbenos destacam-se atividade antiangiogÃnica, citotÃxica e antitumoral. Para avaliar o seu potencial antineoplÃsico, um estudo farmacolÃgico de suas propriedades anticÃncer foi realizado em vÃrios modelos biolÃgicos. Utilizando o ensaio do MTT foi feito um estudo comparativo da citotoxicidade de molÃculas estilbenÃides com estruturas relacionadas ao composto RR07, onde foi observado que a presenÃa do anel A (3,4,5-trimetoxifenil) Ã essencial para a atividade citotÃxica da molÃcula. Determinou-se inicialmente a atividade citotÃxica, frente a 13 linhagens tumorais pelo ensaio de reduÃÃo do MTT sendo testados 11 compostos, (RR01, RR02, RR03, RR04, RR05, RR06, RR07, RR08, RR09, RR10, RR11), onde o estilbenÃide RR07 destacou-se como um dos mais citotÃxicos apresentando valores de CI50 que variaram de 0,009 a 17,49 M. Posteriormente, os estudos de mecanismo de aÃÃo com o RR07 nas concentraÃÃes de 0,25; 0,50; 1,00; 2,00 e 4,00 M, revelaram reduÃÃo, de forma concentraÃÃo-dependente, na viabilidade celular pelos mÃtodos do azul de tripan e de BrdU (bromodeoxiuridina). As anÃlises morfolÃgicas feitas por hematoxilina/eosina mostraram nÃcleos metafÃsicos, onde no ensaio de incorporaÃÃo por brometo de etÃdio/laranja de acridina evidenciou-se morte celular por apoptose nas concentraÃÃes de 0,25 e 0,50 M, com cÃlulas em necrose nas concentraÃÃes de 1,00; 2,00 e 4,00 M, destacando-se alteraÃÃes como desintegraÃÃo membranar e picnose nuclear, nas concentraÃÃes de 0,25 M e 1,00 M, respectivamente. Nos ensaios por citometria de fluxo foi observado fragmentaÃÃo do DNA com parada de ciclo na fase G2/M a partir da concentraÃÃo 0,25 M. A anÃlise pelo ensaio do cometa, para o RR07 revelou atividade genotÃxica do tipo concentraÃÃo-dependente entre cÃlulas mononucleadas de sangue perifÃrico (PBMC), principalmente em 2,00 e 4,00 M. A avaliaÃÃo do RR07 no ensaio com tubulina isolada revelou inibiÃÃo na polimerizaÃÃo da mesma em uma concentraÃÃo de 10 M. A anÃlise antitumoral evidenciou inibiÃÃo de 30,9% e 48,19%, respectivamente, para as doses de 20 e 40 mg/kg/dia de RR07 por via intraperitoneal (ip), e 55,68% de inibiÃÃo para a associaÃÃo de 10 mg/kg/dia de 5-fluorouracil com 20 mg/kg/dia de RR07 (ip), em camundongos transplantados com Sarcoma 180, onde se verificou reduÃÃo no crescimento tumoral e alteraÃÃes renais iniciais e reversÃveis. Desta forma, a fenstatina, RR07, apresenta-se como uma proposta de ferramenta farmacolÃgica Ãtil para a pesquisa de novos derivados. / The phenstatin, chemically known as 4-methoxyphenyl-3,4,5-trimetoxiphenylmethane, was called in the present study as RR07. This compound is a bisarylketone with stilbenoid skeleton obtained from combretastatin A4, with recognized cytotoxic and antineoplasic activities. Then, a detailed study of RR07 anticancer properties was conducted in several biological models. The cytotoxic activity of eleven compounds (RR01, RR02, RR03, RR04, RR05, RR06, RR07, RR08, RR09, RR10, RR11) was determined against thirteen tumor cell lines by MTT assay. Structure-activity relationship pointed out that the presence of ring A (3,4,5-trimethoxiphenyl) is essential for the cytotoxic potential. The RR07 was one of the most cytotoxic compounds, showing IC50 values ranging from 0.009 to 17.49 ÂM. Investigations on mechanism of action (0.25, 0.50, 1.00, 2.00 and 4.00 ÂM) showed a concentration-dependent manner cell viability reduction (trypan blue dye exclusion test) and proliferation decreasing (BrdU assay). Morphological alterations determined by hematoxylin/eosin and acridine orange/ethidium bromide fluorescent double staining methods indicated an increased number of apoptotic cells at lowest concentrations (0.25 and 0.50 ÂM) and necrotic cells at higher concentrations (1.00, 2.00 and 4.00 ÂM). Flow cytometry analyses showed that RR07 induced DNA fragmentation and cell cycle arrest at G2/M starting at 0.25 ÂM. In the comet assay performed with human peripheral blood mononuclear cells (PBMC), RR07 caused DNA strand breaks only at higher concentrations (2.00 and 4.00 ÂM). Experiments with isolated tubulin, RR07 also induced tubulin polymerization inhibition in a concentration of 10 μM. In vivo antitumor experiments showed that Sarcoma 180 transplanted-mice treated with RR07 intraperitoneally (ip) presented a tumor growth inhibition ratios of 30.9% and 48.19% (20 mg/kg/day and 40 mg/kg/day, respectively) and inhibition of 55.68% in associated treatment (5-Fluoruracil 10 mg/kg/day + RR07 20 mg/kg/day). Histopathological analyses of kidneys, spleen and livers revealed incipient and reversible alterations. In summary, RR07 can be considered as a pharmacological useful tool as well as a lead molecule to obtain novel compounds with low toxicity and promising antitumor properties.

Lors de son développement, une tumeur ne peut survivre sans passer par une étape invasive afin de subvenir à ses besoins en nutriment et en oxygène. Cette étape, appelé angiogénèse tumorale, conduit à la formation de vaisseaux sanguins dits « tumoraux », différents des vaisseaux sanguins normaux. Afin de stopper la croissance de la tumeur, il est possible de détruire les vaisseaux sanguins tumoraux formés pendant l’angiogénèse tumorale grâce à des molécules antivasculaires. Ces molécules vont désorganiser la structure du vaisseau et diminuer le flux sanguin au sein de la tumeur pour mener à la nécrose de cette dernière. Parmi ces molécules antivasculaires,la prodrogue phosphate de la combrétastatine A-4 naturelle (CA-4) est le composé actuellement le plus efficace(en développement clinique de phase III contre le cancer de la thyroïde). L’isocombrétastatine A-4 (isoCA-4),possédant une structure de type 1,1-diaryléthylène, est un analogue très puissant développé au laboratoire. Cette molécule est isomère de la CA-4 et permet d’obtenir les mêmes activités biologiques que la CA-4. L’objectif de cette thèse est d’étudier ce motif 1,1-diaryléthylène dans le cadre de molécules cytotoxiques en synthétisant et en évaluant in vitro plusieurs séries de molécules de type « iso ». L’autre partie de cette thèse est dédiée à la synthèse d’analogues hétérocycliques de l’isoCA-4, qui permettront de réaliser les premières études de relation structure-activité sur l’isoCA-4, où son cycle B a été remplacé par un hétérocycle. Ce travail nous a permis de confirmer que la structure de l’isoCA-4 peut servir de base de travail pour développer d’autres agents antivasculaires. / For a tumor, the angiogenesis is a vital step for its development. The spread of the tumor is necessary characterized by an extension of the surrounding vasculature, in order to provide the nutriments and the oxygen required to the growth of the tumor. Resulting from the angiogenesis, the new tumorous blood vessels formed represent an excellent target to treat cancer by aiming specifically at the heart of the tumor. By means of vascular disrupting agents (VDA), it is possible to cut the tumor off the blood flow to trigger the necrosis within the tumor. Among the current VDA, the natural combretastatin A-4 (CA-4) is a strong compound that exhibits excellent antitumoral activities. An isomer of the CA-4, the isocombretastatin A-4 (isoCA-4), was developed inour laboratory to propose an alternative and a new family of VDA. The isoCA-4 is characterized by a 1,1-diarylethylene core, which we studied in this thesis, through 3 series of molecules related to this new structure inmedicinal chemistry. We also synthesized heterocyclic analogues of the isoCA-4, in order to explore the capacity of the isoCA-4 to serve as a basis for developing new antimitotic compounds.

En 2015, on estime le nombre de nouveaux cas de cancer à plus de 385000 et le nombre de décès à 149500. Ces chiffres, plus élevés chez l'homme que chez la femme, connaissent ces dernières années une nette recrudescence chez les patients de sexe féminin. Globalement, la principale difficulté pour lutter contre les cancers se situe dans la capacité à les détecter avant qu'ils ne métastasent et dans les nombreux phénomènes de résistance aux traitements chimiothérapeutiques.De par son implication dans la croissance des cellules cancéreuses, le réseau vasculaire tumorale représente une cible intéressante ainsi qu'une alternative prometteuse aux traitements actuels. Ainsi, la classe de composés qualifiés de VDAs (pour « Vascular Disrupting Agents ») visent les cellules endothéliales ainsi que les péricytes, provoquant ainsi des phénomènes d'ischémie et de nécrose cellulaire. Un exemple de ce type de composés n'est autre que la Combrétastatine A-4 (ou CA-4), qui est un composé naturel extrait d'un arbuste sud-africain, le Combretum caffrum. Cette molécule a été pour la première fois étudiée par G.R. Pettit en 1989 qui a alors démontré qu'elle pouvait inhiber très efficacement la polymérisation des dimères α et β de tubuline en microtubules, résultant alors en la non-formation du cytosquelette, et l'apoptose de la cellule. Le développement de nouveaux analogues de la CA-4 s'avère crucial car ce composé dispose non seulement d'une solubilité réduite mais peut aussi être instable lorsqu'il est administré. Le travail effectué lors de cette thèse a donc consisté dans un premier temps en la synthèse de nouveaux dérivés de la CA-4 en remplaçant le noyau B par différents hétérocycles et en effectuant plusieurs pharmacomodulations ; ces derniers étant alors évalués pour leurs propriétés inhibitrices de la polymérisation de la tubuline et antiprolifératives. Avec plus de 24 millions de personnes touchées dans le monde, les pathologies neurodégénératives représentent un problème majeur de santé publique. Il est également très important de considérer l'incidence future de ces pathologies, et on estime à plus de 42 millions le nombre de personnes qui seraient atteintes par ce fléau d'ici à 2020. La famille des protéines kinases DYRK a fait l'objet ces dernières années d'une attention toute particulière de par son implication dans de nombreux phénomènes physiologiques et notamment au niveau des phases primaires du développement du système nerveux central. Plus spécifiquement, les kinases DYRK1A et DYRK1B ont été étudiées de par leurs implications dans de nombreux cancers (notamment le glioblastome) et certaines pathologies neurocérébrales. Le dérèglement de l'expression de ces protéines pourrait être la cause de retards mentaux, du développement de la maladie d'Alzheimer, de la trisomie 21, ainsi que de phénomènes de résistance. Nous avons identifié au sein de notre laboratoire une molécule inhibant ces 2 kinases. Initialement développé dans le but d'inhiber la protéine kinase CK2 (caséine kinase 2), notre composé a montré une activité inhibitrice et une sélectivité sur DYRK1A et DYRK1B. De par son activité, cette molécule est aujourd'hui notre composé « hit » et nous visons, à travers les travaux de cette thèse, la synthèse de multiples analogues dans le but d'améliorer l'activité initiale mais aussi sa spécificité sur DYRK1A ou sur DYRKB. Dans cette optique, nous avons réalisé différentes pharmacomodulations de nos composés dits « indénobenzo[b]thiophènes » et étudier les effets de tels composés sur DYRK1A, DYRK1B, DYRK2 mais aussi sur CK2. Ceci nous a permis d'affiner nos connaissances vis-à-vis des kinases de type DYRK et de sélectionner les molécules les plus prometteuses afin de (i) réaliser une étude autour de leurs caractéristiques physico-chimiques (Log P, solubilités dans différents solvants), (ii) d'analyser leurs comportements au niveau de la barrière hémato-encéphalique (BHE) et (iii) de réaliser des nano-encapsulations / Up to now, cancer is the second deadliest pathology in the World and is still considered as one of the most challenging public health issue. Globally, it has been assessed to be the main pathologic cause of death by the World Health Organization. This bad prognosis is partly due to the ability of cancer cells to give metastases but also to resistances phenomenon impeding drastically the effect of chemotherapeutic and radiotherapeutic treatments. As a consequence, there is currently a critical lack of effective treatments which would completely eradicate tumor cells, with minimal side effects. In spite of some difficulties in this competitive research area, the discovery of cancer therapeutics remains stimulating and we aim to achieve the synthesis of novel anticancer agents.Given its pivotal role in tumor growth and survival, the tumor vasculature represents an attractive target for anticancer therapy. Apart from angiogenesis inhibitors that compromise the formation of new blood vessels, the class of vascular disrupting agents (VDAs) targets endothelial cells and pericytes of the already established tumor vasculature, resulting in tumor ischemia and necrosis. A striking example of VDA is the combretastatin A-4, also known as CA-4, which was originally isolated from the bark of the South African willow tree Combretum caffrum by the American scientist G.R. Pettit in 1989. These products demonstrated to be efficient against a wide array of cancers such as breast, colon, lung or ovarian cell lines. New CA-4 analogs containing different heterocycles instead of the hydroxymethoxy substituted pharmacomodulable B ring were prepared and evaluated for their in cellulo tubulin polymerization inhibition and antiproliferative activities. In the other hand, tumor cell survival is a complex process which remains poorly understood. As part of the survival machinery, chemoresistances and DNA repairs are central elements regulated by a prosurvival/proapoptotic signal balance. Protein kinases are known to be directly involved in this signal transmission through molecular interactions. As such, DYRK kinases and most specifically DYRK1A/1B, were part of numerous recent studies due to their involvement in cancer and other pathologies. DYRK1B (also called Mirk kinase) is an ubiquitous kinase which was proved to be over-expressed in many cancers such as pancreatic, ovarian or colon. Its involvement as a regulator of DNA repair and tumor cell survival was assessed, phosphorylating specifically serine, threonine and tyrosine residues. Also, another closely related isoform known as DYRK1A, was mapped in the Down syndrome critical region located itself on chromosome 21. Interestingly, this kinase was not only uncovered to play a fundamental role in glioblastomas survival but was also associated with abnormal brain development in early stages and mental retardations. Particularly, DYRK1A was found to hyperphosphorylate microtubule-associated tau protein, resulting into genesis of neurofibrillary tangles. As a consequence, DYRK1A has become one of the most targeted proteins in order to improve cognitive impairment of patients suffering from Down syndrome or Alzheimer’s disease. Initially designed to target protein kinase CK2, one of our molecules was also tested on DYRK kinases. This compound exhibited a strong activity against DYRK1A/DYRK1B whilst being inactive on other protein kinases. Consequently, it was considered as our hit compound and (i) we synthetized derivatives as dual or single inhibitors of DYRK1A/DYRK1B, (ii) evaluated their biological activities (with emphasis on the blood brain barrier), and (iii) finally synthesized nanoparticles loaded with our inhibitors

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-04-04T13:49:10Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Maria Ester Barros_CD_4.pdf: 6351675 bytes, checksum: 1634d753cabb911bd4c0cf8e58182237 (MD5) / Made available in DSpace on 2017-04-04T13:49:10Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Maria Ester Barros_CD_4.pdf: 6351675 bytes, checksum: 1634d753cabb911bd4c0cf8e58182237 (MD5) Previous issue date: 2015-05-01 / CNPQ / A primeira parte deste trabalho envolveu o estudo de docking molecular, a síntese, e a avaliação das atividades antitumorais e anti-inflamatórias de lactonas -insaturadas, análogos estruturais da (-)-Massoialactona, produto natural com conhecida atividade farmacológica. O alvo biológico selecionado para o estudo de docking foi a CRM-1, e tal estudo revelou o potencial biológico dos análogos propostos. Estes análogos foram sintetizados em rendimentos que variaram entre 65-75%, e as suas atividades antitumoral e anti-inflamatória foram avaliadas, sendo que um deles mostrou-se bastante promissor. Estudos posteriores de docking molecular da lactona mais promissora revelou que, apesar dos isômeros R e S assumirem diferentes encaixes no sítio ativo da CRM-1, não há diferença significativa na energia de ligação entre esses isômeros e o alvo molecular estudado. A segunda etapa deste trabalho envolveu o estudo de docking molecular, a síntese, e a avaliação da atividade antitumoral de Z-estilbenos, análogos da Combretastatina A-4 (CA-4). O alvo biológico selecionado para o estudo de docking molecular foi o domínio da colchicina na tubulina. Os cálculos teóricos dos análogos estruturais propostos revelaram que seis desses compostos apresentam diferenças significativas nos valores de energia de ligação quando comparados com o valor teórico calculado para a CA-4, ou seja, teoricamente seriam mais ativos que o produto natural. Dos análogos selecionados a partir do docking molecular, dois foram sintetizados em bons rendimentos empregando-se como precursores teluretos vinílicos e organotrifluoroboratos de potássio. A posterior avaliação da atividade antitumoral dos compostos sintetizados revelou que os resultados obtidos estavam em concordância com o previsto no estudo teórico. / The first part of this work involved the study of molecular docking, synthesis, and the evaluation of anti-tumor and anti-inflammatory activities of unsaturated-lactones, structural analogues of (-)-Massoialactone, a natural product with known pharmacological activity. The biological target selected for the docking study was the CRM-1 and this study revealed the biological potential of the proposed analogues. These analogues were synthesized in yields ranging from 65-75%, and its anti-tumor and anti-inflammatory activities were evaluated, being one of them very promising. Further studies of molecular docking using the most promising lactone revealed that despite the R and S isomers take different fittings in the active site of CRM-1, there is no significant difference in binding energy between these isomers and the molecular target studied. The second step of this work involved the study of molecular docking, synthesis, and evaluation of antitumor activity of Z-stilbenes, analogues of combretastatin A-4. The biological target selected for the study of molecular docking was the domain of colchicine in tubulin. Theoretical calculations of the proposed structural analogues revealed that six of these compounds shown significant differences in binding energy values compared to the theoretical values calculated for the CA-4, ie, theoretically, the proposed compounds would be more active than the natural product. From the selected analogs proposed by molecular docking, two were synthesized in good yields employing vinyl tellurides and potassium organotrifluoroborates as precursors. Further evaluation of the antitumor activity of the synthesized compounds showed that the results were in agreement with the theoretical study.

La combretastatine A-4 (CA-4), produit naturel isolé d’un arbuste d’Afrique du sud (Combretum caffrum K.), a montré des propriétés antitumorales intéressantes. Grâce à sa capacité à empêcher la polymérisation de la tubuline, ce stilbénoïde possède des propriétés cytostatiques sélectives à l’égard de différentes lignées cellulaires cancéreuses. Certains dérivés hydrosolubles de la CA-4 comme la CA-4P (fosbretabuline) et le composé AVE8062 (ombrabuline) sont actuellement en essais cliniques pour le traitement de différents cancers. Trois séries de dérivés de la CA-4 ont été synthétisées : les Z-stilbènes, les 1,2- diaryl-1,2-éthanediones et les 5,6-diaryl-2,3-dihydropyrazines. Dans ces composés, le cycle B est remplacé par divers hétérocycliques (indole, benzofurane, benzothiophène, thiophène) attachés à la position C2. Ces dérivés ont été évalués pour leur capacité à inhiber l'assemblage de la tubuline. Le produit Z-stilbènes portant un noyau benzo[b]thiophène a montré une activité antitubuline comparable à celle de la colchicine et de la deoxypodophyllotoxine. L’effet sur l'organisation intracellulaire des microtubules et les propriétés antimitotiques de ce composé ont été ensuite testés sur les lignées cellulaires de kératinocytes SKv-a et HaCaT. Enfin, des essais préliminaires de vectorisation de ce composé dans des nanoparticules lipidiques solides (SLN) ont été réalisés / Combretastatin A-4 (CA-4), a natural product first isolated from the South African bush willow tree (Combretum caffrum K.), possesses interesting antitumor properties. Due to its capacity to inhibit tubulin polymerization, this stilbenoid shows selective cytostatic activities against various cancer cell lines. Some water-soluble CA-4 derivatives such as CA-4P (fosbretabuline) and AVE8062 (ombrabuline) are currently undergoing clinical trials for the treatment of various cancers. Three series of CA-4 analogues, Z-stilbenes, 1,2-diaryl-1,2-ethanediones and 5,6-diaryl-2,3-dihydropyrazines, were synthesized. In these compounds, the B ring is replaced by various heterocycles (indole, benzofurane, benzothiophene or thiophene) attached at the C2 position. These derivatives were evaluated for their ability to inhibit tubulin assembly. Compound Z- stilbenes bearing a benzo[b]thiophene ring showed an antitubulin activity comparable to that of colchicine and deoxypodophyllotoxine. Its effect on the intracellular organization of microtubules and antimitotic properties were then tested on two keratinocyte cell lines HaCaT and SKV-a. Finally, preliminary essays to the vectorization of this compound in solid lipid nanoparticles (SLN) were carried out