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Malaria affects hundreds of millions of people worldwide and equally claims hundreds of thousands of lives each year. With the current spread of drug resistance to standard antimalarial drugs like chloroquine and the emergence of artemisinin-resistant parasites, new antimalarial drugs and formulations are urgently needed. An ethnobotanical survey was carried out in this study in search of novel compounds with promising antiplasmodial activity. Using the ethnobotanical approach, a total of 61 plant species from 59 genera distributed in 34 plant families were found to be used traditionally for the treatment of malaria in Nigeria. Biological evaluation of the plant¿s methanolic extracts was assessed using the parasite lactate dehydrogenase (pLDH) assay against the chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of Plasmodium falciparum. A total of five (5) plant species showed more potent antiplasmodial activities against the malaria parasites. These are Acanthospermum hispidum, Cassia occidentalis, Kaempferia aethiopica Prosopis africana and Physalis angulata with MIC values ranging between 7.815µg/ml to 31.25µg/ml (3D7 strain) and 15.63µg/ml to 62.50µg/ml (K1 strain) against the malaria parasites, respectively. Two plants, Prosopis africana (Leguminosae-mimosoideae) and Physalis angulata (Solanaceae) were selected for further study. The phytochemical investigation of the active chloroform extracts of P. africana and P. angulata yielded several compounds with three known alkaloids, namely, prosopinine (I), prosopine (II) and acetamide (III). Their structures were confirmed by MS, 1D and 2D NMR spectroscopy. Compounds I, II and III have moderate in vitro antiplasmodial activity against the malaria parasites. Both chloroquine and artemether were used as standard control. / Association of Commonwealth Universities and the Commonwealth Scholarship Commission in the UK (Commonwealth Scholarship Reference Number: NGCS-2005-259).

No / A series of analogues of cryptolepine (1) have been synthesized and evaluated for their in vitro antiplasmodial and cytotoxic properties. The IC50 values of several compounds (11a, 11k¿m, 11o, 13) against Plasmodium falciparum (strain K1) were <0.1 ¿M, 5¿10-fold lower than that of 1 but their cytotoxicities were only 2¿4 times greater than that of 1. Compounds with a halogen in the quinoline ring and a halogen or a nitro group in the indole ring have enhanced antiplasmodial activity. In mice infected with P. berghei, the 7-bromo-2-chloro (11k) and 2-bromo-7-nitro (13) derivatives of 1 suppressed parasitemia by >90% at doses of 25 mg kg-1 day-1 with no apparent toxicity to the mice. 2,7-Dibromocryptolepine (15) was evaluated at several dose levels, and a dose-dependent suppression of parasitemia was seen (ED90 = 21.6 mg kg-1 day-1). The antimalarial mode of action of 1 appears to be similar to that of chloroquine and involves the inhibition of hemozoin formation. A number of analogues were assessed for their effects on the inhibition of ß-hematin (hemozoin) formation, and the results were compared with their antiplasmodial activities having taken account of their predicted accumulation into the acidic parasite food vacuole. No correlation was seen (r2 = 0.0781) suggesting that the potent antimalarial activity of compounds such as 15 involves other mechanisms in addition to the inhibition of hemozoin formation.

Made available in DSpace on 2014-12-17T14:10:25Z (GMT). No. of bitstreams: 1 GracielleRD_DISSERT.pdf: 641978 bytes, checksum: 570ea10863ca885dc9cff38e172d53f2 (MD5) Previous issue date: 2012-03-07 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Malaria is a major parasitic disease worldwide, accounting for about 500 million cases and causing 2 million to 3 million deaths annually. Four species are responsible for transmitting this disease to humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. The parasite resistance to antimalarial drugs and the usual limitations of the vector control implications are contributing to the spread of the disease. The most of significant advances in the search for new antimalarial drugs is based on natural components, the main ones being currently used antimalarial drugs derived from plants. Research on natural products of marine origin (particularly algae) show that some species possess antiplasmodial activity. Knowing that the coast of Rio Grande do Norte is home to several species of algae, the present study was to evaluate, for the first time, the antimalarial activity of ethanolic extracts of seaweed Spatoglossum schroederi, Gracilaria birdiae and Udotea flabellum against Plasmodium falciparum 3D7 strain tests and in vitro using the murine model (Plasmodium berghei) for evaluation in vivo. These species were ground, macerated with ethanol for 24 hours and the extracts concentrated in rotaevaporador (45 ? C ? 5 ? C). For in vitro tests, the extracts were diluted and tested at concentrations between 100 and 1.56 &#956;g/ml (seven concentrations in triplicate), in order to obtain IC50 of each extract. The cytotoxicity tests with macrophages and BGM were performed using the MTT colorimetric assay. BGM macrophages and cells were distributed in 96 wells per plate (1x 105 to macrophages and 1x104 cells per well for BGM) and incubated for 24h at 37 ? C. The ethanol extracts were diluted and tested at concentrations of 100 to 1,56 &#956;g/ml (seven concentrations in triplicate). After periods of 24 hours of incubation with the extracts, 100 &#956;g of MTT was added to each well, and 3 hours elapsed, the supernatant was removed and added 200 &#956;l of DMSO in each well. The absorbance of each well was obtained by reading on a spectrophotometer at 570 nm filter. To evaluate the acute toxicity in vivo, Swiss mice received a single dose (oral) 2000 mg/kg/animal of each extract tested. The parameters of acute toxicity were observed for 8 days. For in vivo tests, Swiss mice were inoculated with 1x105 erythrocytes infected with P. berghei. The treatment was given first to fourth day after infection with 0.2 ml of the extracts in doses of 1000 and 500 mg//g animal. The negative control group received 0.2 ml of 2% Tween-20, whereas the positive control group received sub-dose of chloroquine (5 mg/kg/animal). The assessment of antimalarial activity was done by suppressing suppressing the parasitemia at 5 and 7 days after infection. The growth inhibition of parasites was determined relative to negative control (% inhibition = parasitaemia in control - parasitemia in sample / parasitemia control x 100), the mortality of animals was monitored daily for 30 days The results showed that algae Spatoglossum schroederi and Udotea flabellum showed antimalarial activity in vitro, with reduced parasitemia of 70.54% and 54, respectively. The extracts of the three algae tested showed moderate to high cytotoxicity. Algae S. schroederi and U. flabellum were active against P. berghei only at doses of 500 mg / kg with reduction ranging from 54.58 to 52.65% for the fifth day and from 32.24 to 47.34% for the seventh day, respectively. No toxicity was observed in vivo at the dose tested, over the 8 days of observation. Although preliminary data, the bioactive components in those possible seaweed may be promising for the development of new anti-malarial drugs / A mal?ria ? a maior doen?a paras?tica mundial, respons?vel por cerca de 500 milh?es de casos e causando 2 a 3 milh?es de mortes anualmente. Quatro esp?cies s?o respons?veis pela transmiss?o dessa doen?a ao homem: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae e Plasmodium ovale. A resist?ncia do parasito aos antimal?ricos usuais e as limita??es existentes no combate ao vetor s?o implica??es que contribuem para a expans?o dessa parasitose. Os avan?os mais significativos na busca de novos medicamentos contra a mal?ria baseiam-se em componentes naturais, sendo os principais antimal?ricos atualmente utilizados derivados de plantas. Pesquisas com produtos naturais de origem marinha (particularmente as algas) mostram que algumas esp?cies possuem atividade antiplasm?dica. Sabendo que o litoral do Rio Grande do Norte abriga v?rias esp?cies de algas, o presente estudo consistiu em avaliar, pela primeira vez, a atividade antimal?rica dos extratos etan?licos das algas Spatoglossum schroederi, Gracilaria birdiae e Udotea flabellum contra a cepa 3D7 Plasmodium falciparum em testes in vitro e utilizando o modelo murino (P. berghei) para avalia??o in vivo. As algas foram trituradas, maceradas com etanol por 24 horas e os extratos concentrados em rotaevaporador (45? C ? 5?C). Para os testes in vitro, os extratos foram dilu?dos e testados nas concentra??es entre 100 e 1,56 &#956;g/ml (sete concentra??es em triplicata), com a finalidade de obten??o da CI50 de cada extrato. Os testes de citotoxicidade com macr?fagos e c?lulas BGM foram realizados usando o ensaio colorim?trico MTT. Macr?fagos e c?lulas BGM foram distribu?das em 96 po?os por placa (1x 105 para macr?fagos e 1x104 c?lulas por po?o para BGM), sendo incubadas por 24h a 37?C. Os extratos etan?licos foram dilu?dos e testados nas concentra??es de 100 at? 1,56 &#956;g/ml (sete concentra??es em triplicata). Ap?s per?odos de 24h de incuba??o com os extratos, 100 &#956;l de MTT foi adicionado a cada po?o, e decorridas 3h, o sobrenadante foi removido e adicionou-se 200 &#956;l DMSO em cada po?o. A absorb?ncia de cada po?o foi obtida atrav?s de leitura em espectrofot?metro com filtro de 570 nm. Para avaliar a toxicidade aguda in vivo, camundongos Swiss receberam dose ?nica (oral) de 2000 mg/kg/animal dos extratos testados. Os par?metros de toxicidade aguda foram observados durante 8 dias. Para os testes in vivo, camundongos Swiss foram inoculados com 1x105 hem?cias infectadas com Plasmodium berghei. O tratamento deu-se do primeiro ao quarto dia ap?s a infec??o, com 0,2 ml dos extratos em doses de 1000 e 500 mg/kg/animal. O grupo controle negativo recebeu 0,2 ml de Tween-20 2%, enquanto que o grupo controle positivo recebeu sub-dose de cloroquina (5 mg/kg/animal). A avalia??o da atividade antimal?rica foi feita atrav?s da supress?o da parasitemia no 5? e 7? dias ap?s infec??o. A inibi??o do crescimento dos parasitos foi determinada em rela??o ao grupo controle negativo (% inibi??o = parasitemia do controle parasitemia com amostra/ parasitemia do controle x 100); a mortalidade dos animais foi acompanhada diariamente por 30 dias. Os resultados mostraram que as algas Spatoglossum schroederi e Udotea flabellum apresentaram atividade antimal?rica in vitro, com redu??o da parasitemia de 70,54 e 54%, respectivamente. Os extratos das tr?s algas testadas mostraram citotoxicidade moderada a elevada. As algas S. schroederi e U. flabellum foram ativas contra o P. berghei apenas nas doses de 500 mg/kg com redu??o variando de 54,58 a 52,65% para o quinto dia e 32,24 a 47,34% para o s?timo dia, respectivamente. N?o foi observada toxicidade in vivo para a dose testada, durante os 8 dias de observa??o. Embora sejam dados preliminares, os poss?veis componentes bioativos presentes nessas algas marinhas podem ser promissores para o desenvolvimento de novas drogas antimal?ricas

Despite decades of intense research, malaria remains a deadly worldwide disease. Resistance of Plasmodium falciparum to chemical treatment still remains important. Efforts are now being directed towards the discovery and development of new chemically diverse anti-malarial agents. In the course of the search for new antimalarial compounds, a study of plants traditionally used against malaria by the people inhabiting the forests located near Bhubaneswar, Orissa, India was made, which permitted the identification of 34 plants currently used. Among these, 27 plants were selected for testing for antiplasmodial activity aimed at identifying the most effective plants for further research. Also, their activities were compared with 27 randomly collected plant species in order to asess the value of an ethno-medical approach.

Ce doctorat porte sur l’étude de l’activité antipaludique de la cépharanthine. Deux méthodes écologiques d’extraction, utilisant des micro-ondes et ultrasons, ont été proposées. L'activité antiplasmodiale a été évaluée par le calcul de la concentration qui inhibe 50 % de la croissance parasitaire. Les cibles potentielles ont été évaluées par l’étude de la variation d’expression des gènes. Au niveau microscopique, la cépharanthine a semblé inhiber le développement parasitaire et bloquer les parasites au stade anneau. Au niveau transcriptomique, la cépharanthine semble avoir un impact sur le transport de protéines plasmodiales à la surface du globule rouge ; sur des organelles nécessaires à la survie du parasite, et sur les interactions entre le globule rouge parasité et l’endothélium ou les globules rouges sains. Des études de combinaisons thérapeutiques ont montré que la cépharanthine semble potentialiser certains antipaludiques. Une analyse quantitative de la cépharathine plasmatique a permis d’effectuer une étude pharmacocinétique. La cépharanthine pourrait être un chef de file intéressant pour le développement de nouveaux antipaludiques. / This PhD focuses on the study of the antimalarial activity of cepharanthine. Two green extraction methods, using microwave and ultrasound, have been proposed. The antiplasmodial activity was evaluated by calculating the concentration inhibiting 50 % of parasite growth. Potential targets were evaluated by studying the variation of gene expression. At the microscopic level, cepharanthine seemed to inhibit the parasite growth and to block parasite at the ring stages. The transcriptomic assay showed that cepharanthine seems to have an impact on the transport of Plasmodium proteins to the red blood cell surface, on organellar functions necessary for the survival of the parasite, and on the interactions between infected red blood cells and the endothelium or healthy red blood cells. The study of combination therapies showed that cepharanthine appears to potentiate some antimalarial compounds. A quantitative analysis of cepharathine in mouse plasma allowed performing a pharmacokinetic study. Cepharanthine could be an interesting lead to the development of new antimalarial drugs.

Association of Commonwealth Universities. Commonwealth Scholarship Commission. United Kingdom.

L'objectif de ce travail consiste en la synthèse et l'évaluation antiparasitaire in vitro de nouveaux dérivés du thiazole et apparentés. Plusieurs stratégies de synthèse visant à une pharmacomodulation en séries mono- et polycycliques ont été étudiées : en série 2-méthyl-5-nitrothiazole, la pharmacomodulation anti-Trichomonas de la position 4 par stratégie SRN1 n'a pas permis d'améliorer l'activité déjà démontrée en série 2-méthyl-5-nitroimidazole, mais a conduit à des dérivés à activité antiproliférative in vitro, spécifique de la lignée HepG2. En série 4-arylsulfonylméthyl-2-méthylthiazole, la pharmacomodulation de la position 5, par couplage de Suzuki-Miyaura d'une part, et par arylation directe et réaction de Knoevenagel intramoléculaire d'autre part, a conduit à des dérivés mono- et polycycliques dont certains ont démontré une activité antiplasmodiale in vitro encourageante. En série 5H-thiazolo[3,2-a]pyrimidin-5-one, la réaction de double couplage de Suzuki-Miyaura a révélé l'importance du groupement phényle en position 6 pour l'activité antiplasmodiale de ces dérivés. Enfin, l'évaluation biologique in vitro de thiéno[3,2-d]pyrimidin-4(3H)-ones a permis de caractériser le pharmacophore responsable de l'activité antiplasmodiale significative de cette série. Les résultats préliminaires encourageants d'une étude mécanistique antiplasmodiale présentent l'inhibition spécifique des kinases plasmodiales comme un mécanisme d'action potentiel de ces composés. / The objective of this work consists of the synthesis and the antiparasitic in vitro evaluation of new thiazole derivatives and related structures. Several synthetic strategies aiming at the pharmacomodulation on mono- and polycyclic series have been studied: in 2-methyl-5-nitrothiazole series, the anti-Trichomonas pharmacomodulation on position 4 by SRN1 strategy did not improve the activity previously demonstrated in 2-methyl-5-nitroimidazole series, but led to derivatives displaying a selective in vitro antiproliferative activity toward the HepG2 cell line. In 4-arylsulfonylmethyl-2-methylthiazole series, the pharmacomodulation on position 5, by Suzuki-Miyaura cross-coupling reaction on the one hand, and by direct arylation and intramolecular Knoevenagel reaction on the other hand, led to mono- and polycyclic derivatives among which some displayed an encouraging in vitro antiplasmodial activity. In 5H-thiazolo[3,2-a]pyrimidin-5-one series, a double Suzuki-Miyaura cross-coupling reaction revealed that the phenyl group on position 6 contributes to the antiplasmodial effect of this series. Finally, the in vitro biological evaluation of the thieno[3,2-d]pyrimidin-4(3H)-one scaffold let to characterize the pharmacophore responsible for the significant antiplasmodial activity. Some preliminary encouraging results regarding a mechanistic antiplasmodial study show the specific inhibition of plasmodial kinases, as a potential mechanism of action of some of these compounds.

Le principal objectif de cette thèse, au caractère interdisciplinaire, concernait l'étude de l'éponge Biemna laboutei de Madagascar, connue pour sa toxicité (causant notamment des dermatites). Les travaux entrepris comprenaient en premier lieu, l'étude chimique de cette éponge incluant l'extraction, l'isolement et l'identification des métabolites secondaires par différentes techniques chromatographiques (CLMP, CLHP…) et spectroscopiques (UV-visible, HRMS, RMN 1D et 2D…). Dix-huit alcaloïdes appartenant à la classe rare des hétérocycles tricycliques (5,6,8b)-triazaperhydroacénaphtylène (i.e. ptilocaulines, mirabilines, nétamines) ont été isolés et identifiés. Douze de ces alcaloïdes tricycliques guanidiniques, de structures nouvelles, ont été nommées nétamines H-S. Ces composés ont été classés en fonction des insaturations de leur noyau tricyclique : type pyrimidine, insaturés en Δ8,8a et insaturés en Δ8a,8b. Pour deux des composés isolés, les nétamines I (58) et J (59), une étude configurationnelle reposant sur la comparaison de spectres UV et DCE, expérimentaux et théoriques (théorie de la fonctionnelle et de la densité), a permis la détermination de leurs configurations absolues : 5aS, 7R, 8R pour la nétamine I et 5aS, 7R, 8S pour la nétamine J. La valorisation des molécules isolées a ensuite été envisagée via d'une part la réalisation d'une étude chimiotaxonomique et d'autre part, l'évaluation de leurs activités biologiques (cytotoxicité, activité antipaludique, …). La nétamine M (62) a présenté une activité cytotoxique sur les cellules cancéreuses KB (CI50 = 1,0 μg/mL) et les nétamines O (64), Q (66) et K (60) se sont montrées actives contre le parasite Plasmodium falciparum, responsable du paludisme avec respectivement une CI50 de 4,66 ; 2,53 et 0,62 μg/mL. Enfin, au cours de ces travaux de thèse, ont été explorées trois nouvelles stratégies de synthèse conduisant au squelette des alcaloïdes guanidiniques tricycliques, suivant une approche biomimétique. / The main purpose of this interdisciplinary thesis was to study Biemna laboutei, a sponge from Madagascar, known to have toxic properties (dermatitis-producing). The chemical investigation of this sponge including extraction, isolation and identification of secondary metabolites was first undertaken using several chromatographic (HPLC, MPLC…) and spectroscopic (UV-visible, HRMS, NMR 1D et 2D…) techniques. Eighteen alkaloids belonging to the rare class of tricyclic (5,6,8b)-triazaperhydroacenaphtylene heterocycles (i.e. ptilocaulins, mirabilins, netamines) were isolated and identified. Twelve new alkaloids from this group of guanidine derivatives were named netamine H-S. These compounds were grouped on the basis of unsaturation and double bond regiochemistry, with pyrimidine, Δ8,8a and Δ8a,8b heterocycles. For two compounds, netamine I (58) and J (59), a joint theoretical (Density functional theory) and experimental study of UV and ECD spectra allowed the determination of their absolute configuration: 5aS, 7R, 8R for netamine I and 5aS, 7R, 8S for netamine J. The chemotaxonomic meaning of these alkaloids was discussed. Their biological activities were also evaluated. Netamine M (62) exhibited a cytotoxic activity towards KB cells (IC50 = 1,0 μg/mL) while netamine O (64), Q (66) and K (60) were active against the malaria parasite Plasmodium falciparum with IC50 value of 4,66; 2,53 and 0,62 μg/mL respectively. Finally, this work was also dedicated to the biomimetic synthesis of the tricyclic guanidine skeleton. Three new synthesis routes were explored.