As part of an International Cooperative Biodiversity Group (ICBG) program and a continuing search for antiproliferative natural products from the Madagascar rainforest, and a collaborative research project established between Virginia Tech and the Institute for Hepatitis and Virus Research (IHVR) focusing on searching for bioactive natural products from tropical forests in South Africa, 20 extracts were selected for investigation based on their antiproliferative activities against A2780 human ovarian cancer cell line or antimalarial activities against the Dd2 strain of Plasmodium falciparum. Bioassay-guided fractionation of seven of the extracts yielded twenty new compounds and twenty-four known compounds, and their structures were elucidated by using a combination of 1D (1H and 13C) and 2D NMR spectroscopy including COSY, HASQC, HMQC, HMBC, and NOESY sequences, mass spectrometry, UV, IR, ECD, optical rotation, and chemical conversions. In addition, ten known compounds were isolated from another five of the extracts, while studies on the remaining extracts were suspended due to loss of activity, unworkable small amounts of material, or low structural interest. The plants and their metabolites are discussed in the following order: five new antimalarial 5,6-dihydro-𝛼-pyrones and six bicyclic tetrahydro-𝛼-pyrone derivatives from Cryptocarya rigidifolia (Lauraceae); two new and five known antiproliferative lignans from Cleistanthus boivinianus (Phyllanthaceae); two new and two known antiproliferative sesquiterpenes lactones from Piptocoma antillana (Asteraceae); one new antiproliferative 1,4-naphthoquinone, one known antiproliferative isoflovonoid, and five known antiproliferative stilbenoids from Stuhlmannia moavi (Leguminosae); four known antiproliferative bisbenzylisoquinoline alkaloids from Anisocycla grandidieri (Menispermaceae); one new and two known antiproliferative butanolides, and two new antiproliferative secobutanolides from Ocotea macrocarpa (Lauraceae); one new antiproliferative and five known antiproliferative diterpenoids from Malleastrum rakotozafyi (Meliceae); and 10 known compounds from Monoporus sp. (Myrsinaceae), Premna corymbosa (Verbenaceae), Premna perplexanes (Verbenaceae), Epallage longipes (Asteraceae), and Cinnamosma fragrans (Canellaceae). / Ph. D.

La désymétrisation d’arylcyclohexa-2,5-diènes est une méthode très efficace pour obtenir en une seule étape des squelettes complexes à partir de synthons simples. Lors de cette thèse, une nouvelle approche à la synthèse d’arylcyclohexadiènes porteur d’un centre quaternaire a été développée. L’une des structures synthétisées par cette voie a ensuite été désymétrisée par une réaction d’hydroamination diastéréosélective, nous permettant de réaliser la première synthèse énantiosélective de l’épi-Elwesine. Notre démarche s’est ensuite orientée vers la synthèse et la désymétrisation de nouveaux types de diènes spirocycliques de type oxindoles. La présence dans ces diènes de deux faces très différenciées nous a permis de réaliser des processus complètement diastéréosélectifs. De plus une nouvelle séquence réarrangement-alkylation a été mise au point, nous permettant d’accéder efficacement à des squelettes de type phénanthridinones de façon régio- et diastéréosélective. / The desymmetrization of arylcyclohexa-2,5-dienes is a powerful method to synthesize complex structures from simple synthons in a single step. We first developed a new protocol to obtain arylcyclohexa-2,5-dienes bearing a quaternary center. One of these structures was desymmetrized via a diastereoselective hydroamination and further elaborated into epi-Elwesine, an Amaryllidaceae alkaloid. We next turned our attention towards the synthesis and desymmetrization of spirocyclic cyclohexadienes. Diastereoselective processes were carried out due to the presence of two well- differentiated faces. A new rearrangement-alkylation process was developed and provides efficient access to phenanthridinones regio- and diastereoselectively.

Our project was aimed at the development of novel CB1 cannabinoid receptor antagonists that may have clinical applications for the treatment of cannabinoid and psychostimulant addiction. In this study, we designed, synthesized, and established the CB1 affinity for the 1,5-diaryl-1,2,3- triazole esters, a series of 4,5-diaryl-1-substituted-1,2,3-triazole analogues and a series of 4,5- diaryl-2-substituted-1,2,3-triazoles. Our research group has been interested in the synthesis of amphibian alkaloids due to their interesting biological activities. We have recently developed a general synthetic strategy which can rapidly prepare a few amphibian alkaloids simply from the abundant natural product (-)- cocaine This strategy was first successfully applied to the synthesis of (-)-monomorine. More recently, this strategy has also been utilized in the syntheses of both of the enantiomers of cispyrrolidine 225H and (+)-gephyrotoxin 287C.

Solanum lycocarpum A. St.-Hil. (Solanaceae Solanum), popular lobeira, é espécie arbustiva nativa e característica do Cerrado brasileiro. Seus frutos são empregados na medicina caseira como diurética, calmante, anti-espasmódica, antiofídica e antiepilética. As espécies do gênero Solanum são produtoras de heterosídeos alcaloídicos, os quais possuem atividade antitumoral, incluindo-se anticâncer de pele. O câncer de pele tem preocupado as autoridades no mundo com os crescentes índices atuais e, por esse motivo, a busca por novos ativos anticâncer é primordial. Dos frutos da lobeira, pode-se produzir extrato alcaloídico, o qual se constitui de uma mistura de glicoalcalóides composta majoritariamente de solasonina e solamargina. Tais compostos têm sido estudados quanto ao potencial anticancerígeno, são relativamente seletivos para células tumorais e, em consequência, apresentam baixa toxicidade às células sadias. Por estes motivos, o potencial antitumoral desta espécie deve ser investigado. Para isso, os frutos foram coletados, secos e triturados. A droga vegetal resultante foi submetida à extração tipo ácido-base para obtenção do extrato alcaloídico. O método analítico empregado para quantificação dos heterosídeos alcaloídicos foi desenvolvida e validada em CLAE-UV. A partir do extrato alcaloídico padronizado, foram desenvolvidas formulações para uso tópico. Estas formulações foram avaliadas por meio de ensaios de permeação e retenção dérmica in vitro, cujo método analítico para quantificação de solasonina e solamargina foi validado. Para escolha da formulação mais promissora para ensaio anticâncer in vivo, as formulações de melhor desempenho in vitro foram avaliadas quanto à retenção dérmica in vivo em camundongos hairless. A formulação B, contendo 1% de extrato alcaloídico, 5% de monoleína (monoleato de glicerol), 5% de propilenoglicol em base de gel de Natrosol (pH 6,5), apresentou retenção dérmica total adequada para futuros ensaios anticâncer de pele in vivo. / Solanum lycocarpum A. St.-Hil. (Solanaceae - Solanum), popular wolf-fruit is a native shrub species and characteristic of the Brazilian Cerrado vegetation. Its fruits are used in folk medicine as diuretic, sedative, anti-spasmodic, antiepileptic and antiophidic. This species belongs to the genus Solanum, known to produce alkaloid heterosides, which possess antitumor activity, including skin anticancer. Skin cancer has concerned the authorities in the world because of its growing rates. Therefore, the search for new active anticancer is paramount. The wolf-fruit furnish an alkaloidic extract, which is rich in a mixture of heteroside alkaloids and composed mostly of solasonine and solamargine. Such compounds have been studied as potential anticancer, because they are relatively selective for tumor cells and, consequently, have low toxicity to health cell. For these reasons, the antitumor potential of this species should be investigated. For that, the fruits were collected, dried and crushed. The dried plant biomass was submitted to acid-base extraction to obtain the alkaloidic extract. The analytical method for the quantitation of the alkaloids in both crude alkaloid extract and plant biomass was developed and validated by HPLC-UV. From the alkaloidic extract, formulations were developed for topical use. These formulations were evaluated by in vitro tests of skin permeation and retention in Franz diffusion cell, for which an analytical method for quantifying solasonine and solamargine was developed, as well. The selection of the most promising formulation for anticancer assay in vivo was based in both, the permeation performance of the formulation in vitro and its skin retention in vivo in hairless health mice. The formulation B, containing crude alkaloid extract 1%, Monoolein 5%, propyleneglycol 5% in Natrosol gel (pH 6.5), showed total dermal retention, which is suitable for future trials of skin anticancer in animal models.

Les bactéries qui sont des sources prolifiques d'antibiotiques et des fournisseurs importants d’agents pharmaceutiques peuvent produire une grande variété de métabolites. Ainsi, la découverte de métabolites issus bactéries est un nouveau challenge pour les chimistes. Parmi ces sources, les lichens sont admis comme niches intéressantes de nouvelles bactéries et de nouveaux composés bactériens. Par conséquent, les communautés de micro-organismes associées aux lichens sont devenues des sources prometteuses pour la production de composés naturels actifs. Dans cette thèse, nous avons concentré notre travail sur l'isolement des bactéries de Rhizocarpon geogaphicum, l'un des lichens crustacés les plus populaires vivant sur la roche. Parmi les souches isolées, Paenibacillus odorifer a été sélectionnée pour poursuivre les travaux visant à produire des composés actifs. Après des étapes d’optimisation de culture, l’étude des extraits issus des cultures de P. odorifer soit par le bioréacteur soit en Erlenmeyer a permis l’isolement des métabolites : un polysaccharide antioxydant, deux dérivés tert-butylphénoliques cytotoxiques issus de la bioaccumulation et de la biotransformation de précurseurs, d'un nouvel alcaloïde cytotoxique, de deux diols, de deux dérivés de type furfural et quelques autres composés connus. Des hypothèses de biosynthèse ont pu être proposés pour certains composés. La diversité des métabolites isolés de P. odorifer indique que cette espèce possède un grand potentiel de production des composés actifs et est une bactérie utilisatrice de substrats tert-butyl phénoliques. / Bacteria which are prolific sources of antibiotics and important suppliers to the pharmaceutical agents can produce a wide variety of metabolites. Thus finding metabolites from the bacterial lineages represented new interests for chemists. Among that, lichens are admitted as a rich source of new bacterial lineages and novel bacterial compounds. Therefore, microorganism communities associated with lichens became significant subjects as great potential for the production of active natural compounds. In this thesis, we focus our work on the isolation of bacterial lineages from the lichen Rhizocarpon geographicum, one of the most popular crustose lichens dwelling on the rock. Among the strains isolated, Paenibacillus odorifer was selected for further work to produce active compounds. After the culture optimization steps, the study of extracts from the P. odorifer cultures either in the bioreactor or in Erlenmeyer flask led to the production of metabolites: an antioxidant polysaccharide, two cytotoxic tert-butylphenol derivatives which came from the bioaccumulation and biotransformation of precursors, a novel and cytotoxic alkaloid compound, two diol compounds, two furfural derivatives and some other known compounds. Putative biosynthetic pathways have been proposed for some compounds. The diversity of metabolites isolated from P. odorifer highlighted that this species possessed a great potential of the production active compounds and were a new case of tert-butyl phenol utilizing bacterium.

Base teórica: A artrite reumatoide (AR) é uma doença autoimune sistêmica onde a inflamação crônica da sinóvia articular e a subsequente erosão óssea e da cartilagem resultam em destruição articular, dor e incapacidade funcional. Apesar dos recentes progressos no tratamento da AR, estes ainda apresentam limitações e significativos efeitos adversos, salientando a necessidade de novas estratégias terapêuticas. Plantas da família das Amarilidáceas apresentam em seus bulbos um conjunto de alcaloides muito característicos e exclusivos com atividades farmacológicas, tais como atividade antiviral, anti-inflamatória e atividade anticolinérgica. A montanina é um alcaloide isolado da Rhodophiala bífida, uma planta da família das amarilidáceas utilizada na medicina popular, mas nunca antes testada como terapia para doenças inflamatórias. Objetivo: Avaliar o efeito da montanina como uma terapia anti-inflamatória in vivo em dois modelos de artrite e in vitro sobre a proliferação de linfócitos e sobre a invasão de fibroblastos sinoviais (FLS). Métodos: Artrite induzida por antígeno (AIA) foi realizada em camundongos Balb/C com albumina bovina sérica metilada e a nocicepção e a migração de leucócitos para a articulação do joelho foram os parâmetros avaliados. Artrite induzida por colágeno (CIA) foi realizada em camundongos DBA/1J e o desenvolvimento e severidade da artrite foi avaliado através de escore clínico, nocicepção articular e escore histológico. Montanina foi administrada via intraperitoneal, duas vezes ao dia. A proliferação de linfócitos estimulados por concanavalina A (conA) foi realizada pelo método de MTT e invasão de FLS em 24 horas foi avaliada em um sistema de insertos de Matrigel. Resutados: A administração de montanina diminuiu a migração articular de leucócitos (p0,001) e a nocicepção (p0,01) em camundongos com AIA. Em camundongos com CIA, o tratamento com montanina reduziu a severidade da artrite e o dano articular avaliado pelos escores clínico (p0,01) e histológico (p0,05) e melhorou a nocicepção articular (p0,05), sem causar nenhum dano hepático. Além disso, montanina inibiu in vitro a proliferação de linfócitos estimulados com conA (p0,01) e diminuiu a invasão de FLS (p0,05) em 54%, com uma ação independente de citotoxicidade. Conclusão: Esses resultados indicam que a montanina pode ser explorada para se tornar um possível fármaco para o tratamento de doenças inflamatórias e autoimunes, como a AR. / Background: Rheumatoid arthritis (RA) is an autoimmune and systemic disease where the chronic inflammation of articular synovia and the subsequent bone and cartilage erosion results in joint destruction, pain and functional disability. Despite recent progress in RA treatments, its still have limitations and significant side effects, emphasizing the need of new therapeutic strategies. Amaryllidaceae plants presenting at its bulbs a set of very characteristics and exclusives alkaloids with pharmacological activities such as antiviral, anti-inflammatory and anticholinergic activity. Montanine is an alkaloid isolated from the Rhodophiala bifida, an Amaryllidaceae plant used in alternative medicine but never before tested as a therapy for inflammatory diseases. Objective: To evaluate the effect of montanine as an in vivo anti-inflammatory therapy in two arthritis models and in vitro on lymphocytes proliferation and fibroblast-like synoviocytes (FLS) invasion. Methods: Antigen-induced arthritis (AIA) was performed in Balb/C mice with methylated bovine serum albumin and nociception and leukocytes migration into the knee joint were evaluated. Collagen-induced arthritis (CIA) was performed in DBA/1J mice and arthritis development and severity were assessed by clinical scoring, articular nociception and histological scoring. Montanine was administered intraperitoneally twice a day. Lymphocyte proliferation stimulated by concanavalin A in 48 hours was performed with MTT assay and FLS invasion in 24 hours was assayed in a Matrigel-coated transwell system. Results: Administration of montanine decreased leukocyte articular migration (p0.001) and nociception (p0.01) in mice with AIA. In mice with CIA, treatment with montanine reduced severity of arthritis and joint damage assessed by clinical (p0.01) and histological score (p0.05) and ameliorates articular nociception (p0.05), without causing any hepatic damage. Moreover, montanine inhibited in vitro lymphocyte proliferation stimulated with ConA (p0.01) and decreased FLS invasion by 54% (p0.05), with an action independent of cytotoxicity. Conclusion: These findings suggest that montanine can be explored to become a possible medicament to treat inflammatory and autoimmune diseases such as arthritis.

O estresse de calor prejudica o crescimento e reprodução dos organismos vegetais, ao alterar a permeabilidade de membranas biológicas e desnaturar proteínas, limitando o metabolismo primário. Dentre as respostas ao estresse abiótico, está a síntese de metabólitos secundários. Braquicerina é um alcaloide monoterpeno indólico com ação antioxidante, protetora contra UV e antimutagênica sintetizado por partes aéreas de Psychotria brachyceras. O objetivo deste trabalho é investigar o metabolismo de braquicerina sob estresse de calor. Assim, espera-se contribuir para o conhecimento acerca do metabolismo secundário nas respostas ao estresse de calor, descrever a função in planta do composto, e fornecer ferramentas para obtenção do alcaloide para fins farmacêuticos. O acúmulo de braquicerina foi induzido em discos foliares mantidos a 40ºC por três dias, tanto em regime de elevação abrupta como gradual da temperatura. Baixa temperatura (10ºC) não afetou o acúmulo do alcaloide. Discos foliares de Psychotria carthagenensis, uma espécie que não sintetiza alcaloides monoterpeno indólicos, foram também desafiados por estresse de calor. Clorofila total, teor de peróxido de hidrogênio e peroxidação lipídica foram quantificados em ambas as espécies. P. carthagenensis foi relativamente tolerante ao calor, o que pode estar relacionado à sua elevada concentração de antocianinas, fortemente induzidas por choque térmico de 50ºC por 6h. Peroxidação lipídica foi reduzida nas amostras de P. brachyceras sob estresse de calor agudo ou gradual em comparação à condição controle, sendo este parâmetro inalterado nas duas condições em P. carthagenensis. O teor de peróxido de hidrogênio foi menor em P. brachyceras submetida a choque de térmico em relação ao controle, enquanto o mesmo parâmetro não foi alterado em P. carthagenensis. Discos foliares das espécies sensíveis ao calor Brugmansia suaveolens e Brassica oleracea, pré-tratadas com braquicerina em concentrações similares às encontradas em P. brachyceras, adquiriram fenótipo tolerante ao choque térmico. A expressão do gene que codifica a enzima triptofano descarboxilase (TDC), envolvida na biossíntese de braquicerina em P. brachyceras, foi fortemente inibida em discos foliares submetidos à 40ºC por 6h, 12h e 24h, sugerindo que o efeito da temperatura na estimulação de acúmulo de alcaloide ocorra em nível pós-transcricional. Em conjunto, os dados indicam que a exposição ao calor é um meio efetivo de aumentar o rendimento de braquicerina, cuja acumulação contribui para proteção contra os danos oxidativos associados. / Heat stress impairs plant growth and reproduction by altering membrane permeability and promoting protein denaturation, which limits primary metabolism. Secondary metabolites often take part in protection against abiotic stress responses. Brachycerine is a monoterpene indole alkaloid with antioxidant, UV protectant, and antimutagenic activity synthesized by Psychotria brachyceras shoots. Brachycerine metabolism was analyzed under heat stress, in order to shed light on brachycerine‘s in planta function and to provide potential tools to improve alkaloid yields for pharmaceutical analysis. Accumulation was induced in leaf disks kept at 40ºC for three days, both by abrupt and stepwise temperature increase. Brachycerine concentration was not affected by low temperature (10ºC) exposure. Leaf disks of Psychotria carthagenensis, a species devoided of alkaloids, were also challenged by heat. Total chlorophyll, hydrogen peroxide and lipid peroxidation concentrations were determined in both species. P. carthagenensis was relatively tolerant to heat treatments which may be explained by its high anthocyanin concentration, strongly induced by heat shock of 50ºC for 6h. Brugmansia suaveolens and Brassica oleracea, pre-treated with brachycerine in concentrations equivalent to those found in P. brachyceras, had a heat shock tolerant phenotype, based on chlorophyll content. Expression of the TRYPTOPHAN DECARBOXYLASE gene, which encodes for an enzyme involved in alkaloid biosynthesis (TDC) was strongly repressed in leaf disks exposed to 40ºC for 6h, 12h e 24h, suggesting that temperature effect may occur at post-transcriptional level. Taken together, data indicate that heat exposure is an effective means to increase yields of brachycerine, whose accumulation contributes to protect against associated oxidative damage.

My objectives were to determine if nutritional benefits occur when animals are offered foods with compounds -- alkaloids, saponins, and tannins - that are potentially complementary. I hypothesized that food intake and digestibility increase when lambs consume plants such as alfalfa ALF that contain saponins or birdsfoot trefoil (BFT) that contain tannins when the basal diet is endophyte-infected tall fescue (TF) or reed canarygrass (RCG) both of which contain alkaloids. I predicted that the nutritional status of lambs would be enhanced if basal diets of alkaloid-containing grasses were supplemented with ALF or BFT. Lambs fed a basal diet of either endophyte-infected TF or RCG ate more food and consequently digested more dry matter, energy and nitrogen when supplemented with ALF or BFT. Lambs ingested more dry matter and digested more nutrients when fed a basal diet of RCG than one of TF, and supplementing with ALF and BFT was more beneficial for lambs fed TF than for lambs fed RCG. Increased intake of digestible nutrients was due to greater intake when lambs were offered more than one food, not due to an increase in digestibility. In pen trials meant to complement the field trials, lambs were offered an alkaloid-containing (either gramine or 5-methoxy-N,N-dimethyltryptamine) total mixed ration and supplemented with a food that contained saponins or tannins. All rations were isocaloric (3.3Mcal/kg) and isonitrogenous (14% CP). Lambs fed a ration with either alkaloid and offered a food containing saponin digested approximately the same amount of dry matter, energy, nitrogen, and NDF as lambs not offered saponin. When lambs were fed a ration with either alkaloid and supplemented with food that contained tannins, tannin consumption adversely affected dry matter, energy, and NDF digestibility, but lambs offered food with tannins increased dry matter intake, and as a result, they digested the same amount of dry matter, energy, and NDF as lambs not offered the food with tannins. Lambs offered tannin digested and retained more nitrogen than lambs not offered tannin. These findings indicate a nutritional advantage for sheep eating mixtures as opposed to monocultures of foods with different profiles of secondary compounds and nutrients.

Clavicipitaceous fungal endophytes of the genera Epichloë and Neotyphodium form symbioses with grasses of the family Pooideae in which they can synthesise an array of bioprotective alkaloids. Some strains produce the ergot alkaloid ergovaline, which is implicated in livestock toxicoses caused by ingestion of endophyteinfected grasses. Cloning and analysis of a plant-induced non-ribosomal peptide synthetase (NRPS) gene from Neotyphodium lolii and analysis of the E. festucae E2368 genome sequence revealed a complex gene cluster for ergot alkaloid biosynthesis. The EAS cluster contained a single-module NRPS gene, lpsB, and other genes orthologous to genes in the ergopeptine gene cluster of Claviceps purpurea and the clavine cluster of Aspergillus fumigatus. Functional analysis of lpsB confirmed its role in ergovaline synthesis and bioassays with the lpsB mutant unexpectedly suggested that ergovaline was not required for black beetle (Heteronychus arator) feeding deterrence from epichloë-infected grasses. Southern analysis showed the cluster was linked with previously identified ergot alkaloid biosynthetic genes, dmaW and lpsA, at a subtelomeric location. The ergovaline genes are closely associated with transposon relics, including retrotransposons, autonomous DNA transposons and miniature inverted-repeat transposable elements (MITEs), which are very rare in other fungi. All genes in the cluster were highly expressed in planta but expression was very low or undetectable in mycelia from axenic culture, including under nitrogen-, carbonor phosphate-limited conditions. Comparative analysis of the EAS gene cluster in four different epichloë strains showed marked differences in gene expression and ergot alkaloid synthesis. Gene order is conserved in each strain although evidence for recombination between two MITEs and expansion or reduction of a simple sequence repeat (SSR) at a single intergenic region was observed. Heterologous expression of a candidate regulatory gene, laeA, from Aspergillus nidulans, which is a global regulator of secondary metabolism in aspergilli, did not affect eas gene expression. This, along with phylogeny and microsynteny analysis, suggests there is not an orthologue of this gene in epichloë. This work provides a genetic foundation for elucidating biochemical steps in the ergovaline pathway, the ecological role of individual ergot alkaloid compounds, and the regulation of their synthesis in planta.

In this study, anaerobic treatability of opium alkaloid wastewater and the effect of radiation pretreatment (gamma-rays) on anaerobic treatability were investigated. Biochemical Methane Potential (BMP) assay was performed with alkaloid wastewater having initial COD values of 2400, 6000 and 9600 mg/L with and without basal medium (BM). The highest anaerobic treatment efficiency of 77% was obtained in the BMP reactor containing alkaloid wastewater with initial COD of 9600 mg/L and BM. Co-substrate use was investigated by using BMP assay. Alkaloid wastewater having initial COD concentrations of 9000, 13000 and 18000 mg/L were used with glucose, acetate and glucose-acetate as co-substrates. Results revealed that co-substrate use did not improve alkaloid removal efficiency significantly but it abrogated the acclimation period of anaerobic bacteria to alkaloid wastewater. Continuous reactor experiments were carried out in Upflow Anaerobic Sludge Blanket (UASB) reactors. Highest overall efficiencies (above 80%) were obtained in the reactor fed with co-substrate (R2) for all initial COD concentrations. Up to 78% removal efficiency was obtained in R1 (fed with alkaloid wastewater only) at initial COD concentration of 19 g/L. Effect of radiation was sought by using BMP assay with two initial COD concentrations of 14 and 25 g/L, and two radiation doses 40 and 140 kGy. At 14 g/L COD, there was no effect of radiation on gas production for both doses. However at initial COD of 25 g/L, reactors containing wastewater dosed with 140 and 40 kGy produced gas with higher rates above certain point with respect to raw wastewater.