The war between human and malaria has never stopped, and the development and application of antimalarial drugs has not eradicated this terrible disease. To fight drug-resistant malaria, many leads have been studied over the years. (1S,3R)-MMV008138 and MMV1803522 are two compounds that have been studied in the Carlier Group. My research focused on the structural variation of each of these compounds, in the hope that greater potency could be realized. Chapter 2 describes my work on (1S,3R)-MMV008138, which inhibits the enzyme PfIspD in the methylerythritol phosphate (MEP) pathway. This compound shows good in vitro potency against the drug resistant Dd2 strain of Plasmodium falciparum. However, this lead showed no activity in mouse models. This lack of activity may be due to poor metabolic stability of the compound. However, a significant increase in in vitro potency could also improve in vivo activity. Towards that end, I focused on further variation of the D-ring and A-rings. With the regard to the D-ring, we made five analogs of MMV008138 that replaced the 2,4-dichlorophenyl ring with dihalogenated thiophen-3-yl and thiophen-2-yl rings. We also explored the effect of installing a cyano group on the A-ring of MMV008138. Unfortunately, none of these new compounds were potent growth inhibitors of Dd2 strain P. falciparum. We conclude that the lead goes into a well-defined pocket within the PfIspD enzyme that only accommodates 2,4-dihalogenated phenyl D-rings. This pocket also cannot accept any substitution larger than F on the A-ring. Interestingly, the crystal structure of 5-cyano-substituted MMV008138 was obtained ((±)-2-50c). This is the first compound out of more than 100 analogs of MMV008138 family to be amenable to crystallization. The solid state conformation of the (±)-2-50c revealed that the C3-carboxyl group was in a pseudoequatorial orientation, and the C1-aryl group was thus in a pseudoaxial orientation. 1H NMR spectroscopic studies in CD3OD-D2O were carried out to determine the solution conformation. As expected from previous studies of ester derivatives of MMV008138, these studies indicated that in solution, 2-5 would adopt both the C3-carboxyl pseudoequatorial and pseudoaxial conformations. In Chapter 3, I describe the synthesis of analogs of the antimalarial drug candidate MMV1803522. This β-carboline-3-carboxamide shows good in vitro growth inhibition potency of Dd2 strain P. falciparum, operating by a still unknown mechanism. To investigate the pharmacophore of this lead, I first sought to determine whether the pyridine N (i.e. N2) of the β-carboline was important for in vitro potency. I prepared series of carbazole analogs of MMV1803522, which replace N2 with a CH. These compounds potently inhibited the growth of Dd2 strain P. falciparum. These results suggest that N2 of MMV1803522 is not involved in any energetically significant interactions with its target protein. To further identify the pharmacophore, we prepared truncated analogs lacking the A- and B- rings (biphenyl analogs), and tricyclic analogs that feature a reversed indole moiety. Unfortunately, the biphenyl analogs and reversed indole analogs show no growth inhibition at 10,000 nM the highest concentration tested. Lastly, I describe analogs of MMV1803522 in which the 3,4-dichlorophenyl ring of MMV1803522 was replaced with halogenated thiophene. This substitution was tolerated, but compounds were roughly half as potent as MMV1803522. / Doctor of Philosophy / Malaria, mainly caused by the infection of P. falciparum, is a serious worldwide disease. In 2020, there were 241 million cases of malaria infections and over 600,000 deaths from malaria. Combinations of commercially available antimalarial compounds, such as chloroquine, mefloquine and artemisinin, are commonly used as combination therapies to treat malaria. Since different antimalarial compounds have different mechanisms of action, this combination strategy can greatly slow down the spread of drug-resistant parasites. However, multiple drug-resistant strains of P. falciparum have been reported. Therefore, there is an urgent need for new antimalarial compounds with novel mechanisms of action. This dissertation involves my research on the investigation and optimization of two novel antimalarial compounds, MMV008138 and MMV1803522. MMV008138 is an inhibitor of the MEP pathway, which is an essential metabolic pathway and attractive target for antimalarial therapies, in malaria parasites. The parasites cannot survive, with the MEP pathway inhibited. Since the MEP pathway is not present in human, the MMV008138 molecule is unlikely to have toxicity to human. The MMV008138 molecule has been demonstrated to have great in vitro performance of inhibiting the MEP pathway in several studies, however, the in vivo performance in mouse models is yet to improve. This may be due to the poor metabolic stability of this compound. The compound decomposes in the mouse body before it takes effect. To enhance the metabolic stability and potency, I performed chemical modifications on the A- and D-rings of the MMV008138 compound. An X-ray crystal structure was obtained to help elucidate the conformer distribution of MMV008138. This crystal structure can be used to guide our understanding of the docking of this compound to the target enzyme in the future. MMV1803522 is another compound that shows great potency in vitro and in vivo. This compound is fully oxidized and contains four aromatic rings. However, the target enzyme and the mechanism of action of MMV1803522 is yet to be discovered, and the structure-activity relationship between the chemical structure and the biological activity of this molecule is still unknown. Therefore, I have developed synthetic methods to synthesize a series of compounds that are structurally similar to the MMV1803522 and found that potency of this molecule is not due to the nitrogen on the C-ring. Also, the number and size of the ring structures in the MMV1803522 may be crucial for this molecule to exhibit great potency in vitro and in vivo.

Made available in DSpace on 2015-05-14T12:59:52Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 3215575 bytes, checksum: 8de9cf3f200ed4dc0ee4d1b0b672ce4f (MD5) Previous issue date: 2012-08-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The number of alkaloids containing the 1-substituted tetrahydroisoquinoline skeleton is extensive and impressive versatility of this pharmacological class arouses interest in experimental pharmacologists. Applying the consecrated Pictet-Spengler reaction of four phenyltetrahydroisoquinoline alkaloids was obtained, three of which are unpublished. Two of them obtained with excellent yield (93.45%) in one step. Starting from allylbenzene that initially passed through an isomerization followed by oxidation by applying the Limieux-Johnson reaction were obtained from two other alkaloids with an overall yield of 50%. In experimental models of acute inflammation, the 1-(3-methoxy-4-hydroxyphenyl)-7-methoxy-1,2,3,4,tetrahydroisoquinoline (MTHP) significantly reduced (p <0.05) cell migration into the abdominal cavity of mice and the release of pro-inflammatory mediators (TNF-α, IL-1 and IL-6) in a dose one hundred thirty-eight times lower dosage than the dose of aspirin administered (200 mg/Kg).The MTHP causes hypotension in non-anesthetized normotensive rats, which can be attributed to the participation of endothelium-derived factors, including NO and metabolites COX. These data suggest that MTHP has anti-inflammatory and hypotensive effect related to different mechanisms, and further studies are needed to explore its potential. / O número de alcaloides que contém o esqueleto tetrahidroisoquinolínico 1-substituído é extenso e a impressionante versatilidade farmacológica desta classe desperta o interesse nos farmacologistas experimentais. Aplicando a consagrada reação de Pictet-Spengler obtivemos quatro alcaloides fenil tetrahidroisoquinolínicos dos quais três são inéditos. Dois deles obtidos com excelente rendimento (93,45%) em uma única etapa. Partindo-se de alilbenzenos que inicialmente passaram por uma isomerização seguida de oxidação via reação de Limieux-Johnson, foram obtidos os outros dois alcaloides com rendimento global de 50%. Em modelo experimental de inflamação aguda, a 1-(3-metoxi-4-hidroxifenil)-7-metoxi-1,2,3,4,-tetrahidroisoquinolina (MTHP) reduziu significativamente (p<0,05) a migração celular para a cavidade abdominal de camundongos bem como a liberação dos mediadores pró-inflamatórios (TNF-, IL-1 e IL-6) em uma dose cento e trinta e oito vezes menor que a dose de AAS administrada (200mg/Kg i.p.). MTHP provoca hipotensão em ratos normotensos não anestesiados, que pode ser atribuída à participação de derivados do endotélio, incluindo fatores de NO e metabolitos COX. Estes dados sugerem que MTHP tem efeitos anti-inflamatórios e hipotensores relacionados a diferentes mecanismos, e novos estudos são necessários para explorar seu potencial.

La synthèse de la 5-azagalanthamine, un analogue azoté de galanthamine, utilisée dans le traitement palliatif de la maladie d’Alzheimer, a été envisagée dans le cadre d’une étude de relations structure-activité. Durant cette thèse nous avons examiné quatre voies de synthèse afin d’accéder à la 5-azagalanthamine. La première voie est basée sur la réaction de Pictet-Spengler afin de fermer le dernier cycle de l’azagalanthamine. De nombreuses tentatives ont été effectuées sur différents types de substrats mais cette stratégie s’est révélée inefficace. La deuxième approche consiste en une oxydation d’anilide ortho-substitué par un groupement méthoxy, avec un réactif à base d'iode hypervalent pour accéder à une spirodiènone, un intermédiaire clé de la synthèse. En effectuant cette réaction nous n’avons pas obtenu le produit attendu, mais une 1,2-dispirodiénone, un motif inhabituel, et très rare. Après avoir optimisé les conditions réactionnelles, nous avons étudié la généralité de la réaction avec d'autres substrats. La modélisation moléculaire ainsi que des études de voltammétrie cyclique ont été réalisées (Chabaud, L.; Hromjakova, T.; Rambla, M.; Retailleau, P.; Guillou, C. Chem. Commun. 2013, 49, 11542-11544. Hromjakova, T.; Retailleau, P.; Grimaud, L.; Gandon, V.; Chabaud, L. et Guillou, C. accepté EurJOC, 2015, DOI 10.1002/ejoc.201501160).Ensuite nous avons examiné l’approche basée sur le couplage intramoléculaire pallado-catalysé. Après les premiers résultats encourageant avec le substrat modèle nous avons réalisé une optimisation des conditions réactionnelles. Une étude de généralité de la réaction avec d'autres substrats a été effectuée. Malheureusement, il s’est avéré que la substitution sur le cycle aromatique n’était pas bien tolérée conduisant à de faibles rendements. Par conséquent cette méthodologie n’a pas pu être appliquée à la synthèse de l’azagalanthamine. Dans la dernière voie de synthèse examinée nous avons repris les travaux antérieurs entrepris dans notre laboratoire concernant la réaction de Heck intramoléculaire. La diminution de la longueur de la chaîne liant les deux cycles a permis d’obtenir des résultats très prometteurs. / The synthesis of 5-azagalanthamine, the analogue of galanthamine that is used in Alzheimer treatment, was investigated for structure - activity relationship studies.During this thesis I explored four synthetic approaches with the aim of preparing the 5-azagalanthamine. The first one is based on a Pictet-Spengler reaction used for ring closure of the last cycle of azagalanthamine. We carried out many tests on various types of substrate but this strategy has proved to be ineffective. The second approach consists of an oxidation of ortho-methoxy substituted anilide by hypervalent iodine reagent to access a spirodienone, a key intermediate of the synthesis. Interestingly this reaction did not result in the expected compound but we observed the formation of an unusual motif, the 1,2-dispirodienone. After conditions optimisation we studied the scope and limitations with others substrates. Molecular modelling and cyclic voltammetry studies were also carried out (Chabaud, L.; Hromjakova, T.; Rambla, M.; Retailleau, P.; Guillou, C. Chem. Commun. 2013, 49, 11542-11544. Hromjakova, T.; Retailleau, P.; Grimaud, L.; Gandon, V.; Chabaud, L. and Guillou, C. accepté EurJOC, 2015, DOI 10.1002/ejoc.201501160).Then we investigated an approach based on palladium-catalysed intramolecular coupling. After the first encouraging results with the model substrate, we did optimization of the reaction conditions and the study of substrate scope. Unfortunately we discovered that the substitution was not well tolerated and decreased the yield. Therefore this methodology could not be applied to the synthesis of the azagalanthamine. In the last approach we used previous work of our laboratory on the intramolecular Heck reaction. The reduction of the length of the linker between both cycles showed to be beneficial. We obtained promising results with this approach.

Malaria is a serious mosquito-borne disease affecting the majority of Earth's southern hemisphere. While consistent efforts to curb malaria spread throughout 20th and early 21st century were largely successful, the recent rise in resistance to antimalarial treatments resulted in an increasing incidence rate and stalling mortality rate. This trend clearly signifies the need for the development of novel antimalarial agents able to circumvent current drug-resistance mechanisms. In 2014, in collaboration with Prof. Maria Belen Cassera from the University of Georgia, our group found that compound 1a (1R,3S-MMV008138), discovered from the publicly available Malaria Box, targets an essential biosynthetic pathway (MEP pathway) of malaria-causing parasite Plasmodium falciparum. Analogs of 1a synthesized in our laboratory were found effective against multi-resistant Dd2 strain of P. falciparum which, together with an absence of MEP pathway in humans, suggests that potent analogs of 1a may be safe and efficient antimalarial drug candidates. The initial bioassay studies determined that only one of four possible MMV008138 stereoisomers satisfactorily inhibits the target PfIspD enzyme. Thus a secure determination of stereochemistry in 1a analogs was of utmost importance to the structure-activity relationship studies performed in our group. The second chapter of this work discusses the validation of the previously known empirical stereoassignment method based on analysis of relative shift of 13C NMR resonances between cis and trans diastereomers and compares it to a new method based on 3JHH coupling constants developed in our laboratory. We demonstrate that the new method relying on the analysis of 1H-1H coupling is reliable over large samples of experimental data and suitable even when only a single diastereomer is produced in the synthetic process. Importantly, the origin of 3JHH coupling constants is well understood, unlike the source of relative differences in 13C NMR shifts observed in the older method. The empirical observations for both stereoassignment methods are supported by extensive density-functional theory calculations, which validate the new 1H-1H coupling-based assignment but do not provide a conclusive explanation for the origin of the 13C NMR-based method. In the third chapter, we discuss the replacement of the carboxylic acid moiety in 1a by alternative functional groups promising improved toxicity and bioavailability profile. The total synthesis of tetrazole (trans-23a) and phosphonic acid ((±)-62a) derivatives of 1a is discussed in detail. The tetrazole analog 23a was previously synthesized in the Carlier group as a diastereomeric mixture of cis and trans isomers (dr = 3:7), and it was tested for growth inhibition of multi-resistant P. falciparum with promising results. Later, the synthesis was revisited to obtain a stereochemically pure sample of trans-23a, which was expected to show improved potency compared to the original sample. Furthermore, the synthesis of pure trans-23a confirmed the accuracy of the previous assignment of cis and trans diastereomers in the mixture. Unfortunately, neither analog showed an improvement in potency relative to 1a. / Doctor of Philosophy / The most severe form of malaria disease is caused by the parasite, Plasmodium falciparum, which gives rise to over 200 million infections and more than 400 thousand deaths every year, the majority of which affect young children. In recent years, the effectiveness of clinically used antimalarial medicines decreased due to an increase in drug-resistant strains of P. falciparum. Therefore, there is an urgent need for new antimalarial agents that could bypass the emerging resistance. A promising candidate for a new antimalarial drug is a molecule named MMV008138. This molecule exists in four distinct forms called stereoisomers. Stereoisomers are molecules with the same chemical formula, but the atoms in each molecule are positioned differently. Only one of MMV008138's four stereoisomers (1a) was effective in killing the P. falciparum. The second chapter of this work discusses a new method for identifying stereoisomers in molecules like MMV008138. We demonstrate that the new method is both reliable and simpler than the previously used procedures. The third chapter of this dissertation discusses the preparation of two new compounds based on the structure of 1a that contain modifications promising improved biological activity. Unfortunately, neither of these two molecules was able to kill the P. falciparum efficiently.

Ces travaux ont consisté à optimiser une voie de synthèse des spirotétrahydro-β-carbolines. Une stratégie en trois étapes à partir du 3-(2-nitrovinyl)indole a été élaborée avec la séquence linéaire suivante : addition de Michael, réduction de la fonction nitro en amine et cyclisation de Pictet-Spengler. L’addition de Michael a été développée sans protection préalable de l’indole sous activation ultrasons. La réduction de la fonction nitro en amine a été menée en présence d’hypophosphites. Au cours de cette étude, la désoxygénation des cétones aromatiques a été identifiée comme réaction secondaire de la réduction et a fait l’objet d’un développement approfondi. Une nouvelle voie d’accès à des isatines fonctionnalisées en position C-5 par substitution des sels de diazonium a été mise au point notamment par la réaction de Heck-Matsuda qui a fait l’objet d’une étude plus complète. La réaction finale de Pictet-Spengler entre tryptamines et isatines aboutit aux spiroindolones. Finalement, l’activité biologique des dérivés spirotétrahydro-β-carbolines synthétisés a été évaluée et l’un d’entre eux a montré une très bonne activité envers le Plasmodium falciparum / The aim of this work was to optimize a new way of synthesis of spirotetrahydro-β-carbolines. A three-steps strategy from the 3-(2-nitrovinyl)indole was elaborated with the following linear sequence : Michael addition, reduction of the nitro function into amine and Pictet-Spengler cyclisation. Michael addition was developed without protection of the indole under sonication activation. Reduction of the nitro function into amine was carried out with hypophosphites. During this study, desoxygenation of aromatic ketones has been identified as a side reaction of the reduction and has been the subject of specific development. A new synthetic pathway to functionalized isatins on C-5 position via substitution of diazonium salts was developed, and Heck-Matsuda reaction has been more particularly studied. The final Pictet-Spengler reaction between tryptamines and isatins led to tetrahydro-β-carbolines. Finally, biological activity of spirotetrahydro-β-carbolines was evaluated and one of these compounds showed good activity against Plasmodium falciparum

Resumé<p><p>Le cancer reste une maladie grave car il représente une des causes principales de décès dans les pays développés. Plus d'un tiers de cancers solides réagi très faiblement à la chimiothérapie conventionnelle et/ou développe rapidement une résistance au traitement. Des thérapies ciblées, utilisées en association avec les traitements conventionnels, pourraient augmenter la survie des patients. C’est dans le cadre des thérapies ciblées que ce travail de thèse s’inscrit.<p><p>Nous nous sommes intéressés à synthétiser de nouvelles molécules qui pourraient être efficaces contre les cancers résistants à l'apoptose et donc aux traitements conventionnels. La principale cible de notre projet était la kinase DYRK1A, qui a été décrite comme étant impliquée dans la prolifération cellulaire et la résistance à l'apoptose. Dans ce but, une série de nouvelles molécules, principalement des dérivés de la tétrahydro-β-carboline, a été synthétisée et leurs propriétés antitumorales ont été caractérisées in vitro. En effet, ces structures ressemblent à celle de l’harmicine, un alcaloïde apparenté à l’harmine, l’inhibiteur de DYRK1A le plus sélectif et le plus puissant connu à ce jour. <p><p> Une méthodologie "one-pot" très efficace, développée au Laboratoire de Chimie Organique (ULB), a été utilisée pour obtenir les squelettes de type tétrahydro-β-carboline. Le deuxième chapitre de cette thèse détaille cette méthodologie et décrit la librairie de 47 dérivés qui ont été synthétisés. <p><p>Un second objectif de ce travail était de développer une version énantiosélective de cette méthodologie afin de la rendre encore plus intéressante. Cette partie, décrite dans le troisième chapitre, a été réalisée avec succès en collaboration avec l’Unité de Recherche en Chimie Organique et Macromoléculaire de l'Université du Havre (Le Havre, France). Les expériences que nous avons réalisées ont permis, non seulement d'obtenir le composé le plus actif avec un bon excès énantiomérique, mais également de mieux comprendre les aspects mécanistiques qui constituent la base de l'énantiosélectivité. <p><p>L'évaluation des propriétés anticancereuses des composés synthétisés est ensuite détaillée dans le quatrième chapitre. Les analyses toxicologiques et pharmacologiques ont montré que trois molécules présentent une bonne activité antitumorale in vitro avec une sélectivité prometteuse entre les cellules cancéreuses et les cellules normales. D’une manière inattendue, les tests biologiques plus poussés, que nous avons réalisés, ont suggéré que ces molécules n'agissent pas comme des inhibiteurs de kinases. Elles interfèrent en fait sur la prolifération cellulaire, en ciblant des facteurs de transcription spécifiques, par des mécanismes qui doivent encore être élucidés. Ces expériences biologiques ont été réalisées en collaboration avec le Laboratoire de Toxicologie et Cancérologie Expérimentale (ULB).<p><p>/<p><p>Summary<p><p>Cancer is a devastating disease which remains one of the major causes of death in developed countries. More than one third of adult solid cancers respond very poorly to chemotherapy and/or rapidly develop resistance to treatment. Targeted therapies, used in combination with conventional treatments could be used to increase the survival of cancer patients.<p><p>In this work we were interested in developing new molecules related to the targeted therapy concept that could be effective against cancer types that are resistant to apoptosis and thereby to conventional treatments. The leading target of our project was the DYRK1A kinase, which was described as being involved in cell proliferation and resistance to apoptosis. For this purpose, a series of new molecules, mainly tetrahydro β carboline derivatives, has been synthesized and their antitumoral properties were characterized in vitro. Indeed these structures resemble harmicine, an alkaloid similar to harmine, the most selective and potent DYRK1A inhibitor known to date.<p><p> An efficient “one-pot” methodology, developed in the Laboratoire de Chimie Organique (ULB) was used to obtain the tetrahydro β carboline scaffolds. Chapter II of this work describes the use of this methodology for the synthesis of a library of 47 derivatives.<p><p>A second goal of this work was to further improve this methodology by developing an enantioselective version. This part, described in chapter III, was carried out successfully in collaboration with the Research Unit in Macromolecular and Organic Chemistry of Université du Havre (Le Havre, France). The experiments we have performed enabled us not only to obtain the most active compound with a good enantiomeric excess, but also to gain insight of the mechanism responsible for the enantioselectivity.<p><p>The fourth chapter details the evaluation of the anti cancer properties of the synthesised compounds. The pharmacological and toxicological analyses showed that 3 molecules display actual anti-tumor activity in vitro with a promising selectivity between cancerous and normal cells. Surprisingly, further biological assays we have performed suggested that these molecules do not act as kinase inhibitors but influence cell proliferation through the targeting of specific transcription factors by mechanisms that remain to be deciphered. The biological experiments were performed in collaboration with the Cancerology and Experimental Toxicology Laboratory (ULB).<p><p><p><p><p><p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished