The need for new antibiotics has been highlighted recently with the increasing pace of emergence of drug resistant pathogens (MRSA, XDR-TB, etc.). Modification of existing antibiotics with the additions of side chains or other chemical groups and genomics based drug targeting have been the preferred method of drug development at the corporate level in recent years. These approaches have yielded few viable antibiotics and natural products are once again becoming an area of interest for drug discovery. We examined the antimicrobial "Red Soils" of the Hashemite Kingdom of Jordan that have historically been used to prevent infection and cure rashes by the native peoples. Antimicrobial producing bacteria were present in these soils and found to be the reason for their antibiotic activity. After isolation, these bacteria were found to excrete their antimicrobials into the liquid culture media which we could then attempt to isolate for further study. Adsorbent resins were employed to capture the antimicrobial compounds and then elute them in a more concentrated solution. As part of a drug discovery program, we sought a way to quickly characterize other soils for potential antibiotic producing bacteria. The community level physiologic profile was examined to determine if this approach would allow for a rapid categorizing of soils based on their probability of containing antimicrobial producing microorganisms. This method proved to have a high level of variability that could not be overcome even after mixing using a commercial blender. The role of these antimicrobial producing bacteria within their natural microbial community has largely been confined to microbe-plant interactions. The role of antimicrobial-producing microorganisms in driving the diversity of their community has not been a focus of considerable study. The potential of an antimicrobial-producing bacterium to act as a driver of diversity was examined using an artificial microbial community based in a sand microcosm. The changes in the microbial assemblage indicate that antimicrobial-producing bacteria may act in an allelopathic manner rather than in a predatory role. / Ph. D.

As part of an International Cooperative Biodiversity Group (ICBG) program and a collaborative research project with the Natural Products Discovery Institute, four plant extracts were investigated for their antiproliferative and antiplasmodial activities. With the guidance of bioassay guided fractionation, two known antiproliferative terpenoids (2.1 and 2.2) were isolated from Hypoestes sp. (Acanthaceae), four known antiplasmodial liminoids (3.1-3.4) were isolated from Carapa guianensis (Meliaceae), one inactive terpenoid (4.1) was isolated from Erica maesta (Ericaceae), and four cerebrosides (4.2-4.5) were obtained from Hohenbergia antillana (Bromeliaceae). The structures of these compounds were elucidated by using 1D (1H and 13C), 2D (HMBC, HSQC, COSY, NOESY) NMR spectroscopy and mass spectrometry. The structures of the compounds were also confirmed by comparing them with reported values from the literature. Compounds 2.1 and 2.2 showed moderate antiproliferative activity against the A2780 human ovarian cancer cell line with IC50 values of 6.9 uM and 3.4 uM, respectively. They also exhibited moderate antiplasmodial activity against chloroquine-resistant Plasmodium falciparum strain Dd2 with IC50 values of 9.9 ± 1.4 uM and 2.8 ± 0.7 uM, respectively. Compounds 3.1 to 3.4 had moderate antiplasmodial activity against Plasmodium falciparum Dd2 strain with IC50 values of 2.0 ± 0.3 uM, 2.1 ± 0.1 uM, 2.1 ± 0.2 uM and 2.8 ± 0.2 uM, respectively. Compounds 4.1 and 4.2 showed very weak antiplasmodial activity against Plasmodium falciparum Dd2 strain, with IC50 values between 5 and 10 ug/mL. / Master of Science

Bioassay-guided fractionation of the ethyl acetate extract of Virola sp. (Myristicaceae) using the Sc7 yeast strain resulted in the isolation of the weakly cytotoxic biochanin A (an isoflavone compound). The bioassay of three mutant yeast strains (1138, 1104, and 1353) directed the isolation of DNA-damaging alkaloids from Solanum hostmannii. These alkaloids were identified as verazine and (20R) epimer verazine. In addition, three oxoaporphine alkaloids were isolated from the bark of Papualthia sp. (Annonaceae). Oxocrebanine and atherospermidine showed DNA-damaging activity whereas liriodenine had cytotoxic activity. / Master of Science

A piplartina (PPT) ou piperlongumine é um produto natural da classe dos alcaloides encontrada em espécies da família Pipereaceae. Devido a sua alta potência e seletividade na inibição de diversas linhagens de células tumorais, a PPT têm sido investigada como um potencial candidato à fármaco. Neste contexto, estudos relacionados à sua toxicidade e segurança devem ser realizados, incluindo a determinação do papel das enzimas do Citocromo P450 (CYP450) sobre o metabolismo da PPT. Esta família de enzimas é responsável pela biotransformação de cerca de 75% dos fármacos presentes no mercado. Os estudos pré-clínicos que visam avaliar o metabolismo podem ser realizados empregando modelos in vitro como uma ferramenta para predição de características farmacocinéticas in vivo. Assim, o presente estudo teve como objetivo a avaliação do perfil metabólico da PPT frente as enzimas do CYP450 empregando-se estudos in vitro com microssomas hepáticos humanos (HLM) e a posterior predição de parâmetros farmacocinéticos. Estes estudos incluíram a determinação de parâmetros enzimáticos, estudos de inibição da PPT sobre as principais isoformas do CYP450, elucidação estrutural de metabólitos gerados com a reação de metabolismo e, finalmente, estudos de fenotipagem enzimática. A metodologia geral de estudo de metabolismo in vitro envolveu o uso de técnicas cromatográficas acopladas a diversos detectores/analisadores, tais como arranjo de diodos, espectrometria de massa e ressonância magnética nuclear. O metabolismo foi avaliado pela medida da taxa de desaparecimento da PPT do meio microssomal. Após validação da metodologia para quantificação da PPT e determinação das condições de velocidade inicial de reação, um perfil sigmoidal foi obtido, indicando o metabolismo da PPT por enzimas contendo múltiplos sítios ativos e/ou catálise por diversas enzimas concomitantemente. Os parâmetros cinéticos calculados foram Vmax = 5,5 ± 0,5 nmol mg proteína-1 min-1, S50 = 127,70 ?mol L-1 e Coeficiente de Hill (h) = 3. O clearance intrínseco obtido foi de 22,68 ?L min -1 mg -1. A fração não ligada às proteínas plasmáticas e microssomais foi de 0,07 e 0,76, respectivamente. O clearance in vivo predito foi de 19,79 mL min -1 kg -1, o clearance hepático de 1,89 mL min -1 kg -1 e extração hepática de 0,09. Dentre quatro isoformas avaliadas, CYP3A, CYP2C9, CYP2D6 e CYP1A2, a PPT demonstrou um potencial em causar interação produto natural-medicamento apenas sobre a CYP1A2. A PPT é um inibidor competitivo dose-dependente da CYP1A2, apresentando um valor de Ki de 1,5 ?mol L-1. A razão [I]/Ki obtida de 9,1 prediz uma interação relevante in vivo. Além disso, a PPT apresentou uma inibição tempo-dependente sobre a CYP1A2 com valores de KI de 8 ?mol L-1 e kinact de 0,014 min-1. A inibição dose-, ii NADPH- e tempo-dependente confirmam uma inibição baseada no mecanismo em que o modo pelo qual a PPT liga-se à enzima é irreversível. Baseado nos dados obtidos pelas análises por espectrometria de massa e ressonância magnética nuclear, quatro metabólitos gerados após metabolismo da PPT com HLM tiveram suas estruturas propostas. Assim, foram caracterizados os metabólitos M1 (produto de uma desmetilação na posição meta do anel 3,4,5-trimetoxicinâmico), M2 (produzido por uma epoxidação entre o C3 e C4 do anel lactâmico), M3 (gerado através de uma oxidação no C5 do anel lactâmico) e, finalmente, M4 (produto de uma reação transdiidrodiol entre C3 e C4). O metabólito M4 é formado tardiamente (após 40 min de reação) e provavelmente é um metabólito secundário produzido a partir de M2 através de uma reação trans-diidrodiol. O estudo de fenotipagem demonstrou que as principais isoformas que contribuem para o metabolismo da PPT são a CYP1A2 (formação de M1) e a CYP3A4 (formação de M2 e M3). O emprego das isoformas recombinantes demonstrou a formação de M4 a partir da catálise por diversas isoformas, CYP2C19, CYP2C8, CYP2D6, CYP2B6 e CYP2E1. Portanto, o perfil metabólico do candidato a agente antitumoral PPT frente às enzimas do CYP450 foi demonstrado neste trabalho, proporcionando aspectos relacionados à segurança e eficácia desta substância. Os dados apresentados certamente servirão como guia em estudos clínicos futuros / Piplartine (PPT) or Piperlongumine is a naturally occurring alkaloid found in species of Pipereaceae family. Due its high potency and selectivity of inhibition of several cancer cell lines, PPT has been investigated as a potential drug candidate. In this context, studies related with toxicity and safety should be performed, including the role of the Cytochrome P450 (CYP450) enzymes in PPT metabolism. This family of enzymes is responsible for the biotransformation of 75% of the drugs in the market. The preclinical studies that aim to evaluate the drug metabolism can be performed by employing in vitro models as a tool for prediction of in vivo pharmacokinetic characteristics. Therefore, the aim of this work was to evaluate the metabolic profile of PPT after metabolism by CYP450 enzymes employing in vitro studies with human liver microsomes (HLM) and the ensuing prediction of pharmacokinetic parameters. These studies embraced the kinetic parameters determination, inhibition ability of PPT over the most important CYP450 isoforms, structural elucidation of the produced metabolites after metabolism reaction and, finally, the enzymatic phenotyping study. The general procedure for in vitro metabolism studies consisted of the use of chromatographic techniques coupled to different detectors/analyzers, such as diode array, mass spectrometry and nuclear magnetic resonance. The metabolism was evaluated measuring the rate of disappearance of the PPT from de microsomal medium. After method validation for PPT quantification and determination of initial velocity conditions, the enzymatic kinetics with a sigmoidal profile indicating a metabolism of PPT by enzymes with multiple active sites and/or metabolism by multiple CYP450 enzymes was observed. The following parameters were calculated: Vmax = 5.5 ± 0.5 nmol/mg protein/min, S50 = 127.7 ?mol/L, and Hill coefficient of 3.0. The intrinsic clearance was 22.68 ?L min -1 mg -1. The unbound fraction of PPT on plasmatic and microsomal proteins was 0.07 and 0.76, respectively. The predicted in vivo clearance was 19.79 mL min -1 kg -1, the hepatic clearance was 1.89 mL min -1 kg -1 and the hepatic extraction was 0.09. Among 4 isoforms evaluated, CYP3A, CYP2C9, CYP2D6 and CYP1A2, a potential natural product-drug interaction for only CYP1A2 isoenzyme by PPT was observed. PPT showed to be a competitive and dosedependent inhibitor of CYP1A2, showing a Ki value of 1.5 ?mol L-1. The ratio [I]/Ki of 9.1 predicts an important in vivo interaction. Furthermore, a time-dependent inhibition of CYP1A2 with a KI of 8 ?mol L-1 and a kinact of 0.014 min-1 by PPT was demonstrated. The dose-, time- and NADPH-dependent inhibition confirms an inhibition based on mechanism through an irreversible bond. Based on results obtained from the mass spectrometry analysis and from the nuclear magnetic resonance analysis, four metabolites were identified and characterized. The metabolites characterized were: M1 (product of a demethylation in the 3,4,5-trimethoxyphenyl portion, M2 (derived from an epoxidation between C3 and C4 on the lactone ring), M3 (product of a simple oxidation on C5 of lactone ring), and finally M4 (derived from a dihydrodiol reaction between C3 and C4). The metabolite M4 is produced later (after 40 min of reaction) and probably is a secondary metabolite produced from M2 through a dihydrodiol reaction. The phenotyping study demonstrated that the main isoforms involved in PPT metabolism are CYP1A2 (production of M1) and CYP3A4 (production of M2 and M3). The recombinant isoforms study demonstrated that several isoforms (CYP2C19, CYP2C8, CYP2D6, CYP2B6 and CYP2E1) catalyze the production of M4. In summary, a wide view about the metabolism of the promising drug candidate PPT by CYP450 enzymes was accomplished. These results, certainly, will be a useful guide for further clinical studies of PPT

Muamvatin (30) is a polypropionate natural product isolated from Siphonaria normalis by Ireland et al. in 1986. Muamvatin (30) is made from eight propionate units and contains an extraordinary trioxaadamantane ring system. This ring system exists in only one other naturally occurring polypropionate known as caloundrin B. Regarding the rare muamvatin trioxaadamantane ring system, it was hypothesized this ring system may not be formed via an enzymatic process and the actual natural product could be the linear precursor ent-71 which cyclizes to muamvatin (30) during isolation. The first total synthesis of muamvatin (30) by Paterson et al. confirmed its absolute and relative configuration, but the ambiguity regarding the origin of the trioxaadamantane ring system in this molecule remains unresolved. This work describes two approaches to make the linear precursor ent-71 from triol ketone 153. The carbon skeleton of muamvatin was synthesized through two iterative diastereoselective aldol reactions. In the first approach, “the thiopyran route”, the diene moiety of aldehyde 73 required protection to avoid reduction during desulfurization. Although use of the tircarbonyliron complex was successful, the trihydroxy ketone revealed upon desulfurization was unstable and spontaneously cyclized to bicyclic acetal 156. Molecular mechanics revealed that the relative configurations embedded in C3, C7, and C8 dramatically effected the stability of the corresponding bicyclic acetal. With that lesson learned, the fully assembled linear precursor 197 was made in our second approach “the acyclic route”. The oxidation state of the backbone oxygens were manipulated via an unusual chemoselective double Swern oxidation. Finally, revealing the sensitive 5-hydroxy-3,7,9-trione functionality formed the precursor 202. Efficient cyclization of precursor 202 and removal of the protecting group at C11-OH produced the desired natural product 30. The cyclization conditions tested on the linear precursor 202, suggested that although the cyclization to the trioxaadamantane is strongly favored thermodynamically, the process is very slow and unlikely to occur during the isolation process. Thus, formation of the trioxaadamantane ring system could be an enzyme-mediated process as was concluded for caloundrin B.

The work presented in this thesis mainly describes the discovery and development of methodology for the synthesis of anthecularin and anthecotulides, a family of unusual sesquiterpene lactones. Firstly, two 1,3-dipolar cycloaddition approaches toward anthecularin have been evaluated, using either oxidopyrylium ylide chemistry (Path A) or carbonyl ylides, generated by rhodium-catalysed decomposition of diazo ketones (Path B). Synthesis of the key precursor for the diazo strategy was achieved but unfortunately no desired cycloadduct was isolated. Secondly, an experimentally straightforward method to stereoselectively synthesise β-hydroxymethyl-α-methylene-γ-butyrolactones was developed using chromium or zinc. The synthetic utility of this methodology was demonstrated in syntheses of (±)-methylenolactocin, (±)-hydroxymatairesinol and, ultimately, (±)-hydroxyanthecotulide using a gold-catalysed Meyer-Schuster rearrangement. Finally, the first asymmetric synthesis of (+)-anthecotulide has been achieved, in 6 steps from commercially available materials. During this synthesis the absolute configuration was established. Furthermore, a novel rhodium-catalysed enantioselective ene-yne cycloisomerisation was used to form the α-methylene-γ-butyrolactone core.

<strong>Introduction</strong> The introduction describes the importance of arylamine compounds to society and provides a brief overview of the methods available for their synthesis. The application of metathesis catalysis to the de novo synthesis of heteroaromatic compounds is also described. <strong>Results and discussion</strong> The first section describes efforts towards the de novo synthesis of arylamines using a cross metathesis/oxidation protocol to form a 1,5-unsaturated dicarbonyl followed by an amine mediated cyclisation. The scope with respect to the 1,5-unsaturated dicarbonyl and amine is covered as well as the utility of some of the products. The section concludes with a modification of the Bohlmann Rahtz pyridine synthesis to furnish arylamines. The next section describes the applications of our methodology to the synthesis of naphthylamines, specifically using the palladium catalysed &alpha;-arylation reaction. A discussion of the α-arylation reaction is included as well as our efforts to explore the scope of the reaction. The third section follows our efforts to apply this methodologyy to the synthesis of five benzo[c]phenanthridine alkaloids including the first reported synthesis of maclekarpine B and C. The final section concludes with a discussion of our efforts towards the de novo synthesis of furans bearing a benzylic stereocentre.

This thesis describes the studies towards the synthesis of β-amino acids found in the microsclerodermins, a family of complex macrocyclic hexapeptides. These β-amino acids have four or five contiguous stereocentres and an aliphatic side-chain. The synthetic route utilised an aminohydroxylation reaction to install the most challenging moiety in the structure - the 1,2- amino alcohol. The work aimed to construct the core structure (five contiguous stereocentres) of the β-amino acids and install the side-chain later to enable the synthesis of multiple members (A, B, F, G, H and I) of the microsclerodermin family. The synthesis started with Roche ester, which contained the first methyl stereocentre. It was converted to the diene precursor in four high yielding steps. The next two stereocentres were installed via a Sharpless asymmetric dihydroxylation. With the appropriate protecting groups in place, the remaining two stereocentres were to be installed via a Sharpless asymmetric aminohydroxylation. Various reported reagents and conditions were used to effect the transformation, but the attempts were unsuccessful. This forced us to alter our synthetic plans, and the revised synthetic route involved the use of the tethered aminohydroxylation (TA) reaction developed by the Donohoe group. After the development of an efficient protocol to prepare the TA-precursor, the alkene, with three stereocentres already in place, was successfully converted to the desired stereopentad via the TA-reaction (10 steps, 11% overall yield). With the stereopentad in hand, installation of the side-chain of the β-amino acids through an alkenyl or alkyl linkage was investigated. For alkenyl-linked side-chains, the appropriate aldehyde was synthesised, but attempts to effect the transformation via a Horner-Wadsworth- Emmons reaction or a Witting reaction failed. With lessons learnt from those, we then focused our efforts on installing an alkyl-linked side-chain. In this case, we were able to install a side- chain via a copper-mediated displacement reaction, which gave us a protected form of the precursor of the β-amino acid of microsclerodermin B. Finally, various efforts to study the reactivity of the stereopentad and the investigations into finding the most effective set of protecting groups have been used to propose a synthetic route for the incorporation of the β- amino acid into the macrocycle.

In 2006, the secondary metabolite leiodolide A was isolated from a newly discovered deep-sea sponge of the genus Leiodermatium. The 19-membered macrolide represented a new class of mixed polyketide, nonribosomal, peptide synthetase natural products. A total synthesis of leiodolide A is yet to be achieved and is of specific interest, not only for its complex structure and undefined stereochemistry, but also the potent cytotoxic properties it possesses, particularly towards leukaemia, non-small cell lung and ovarian cancers. A synthetic strategy for leiodolide A must be flexible to overcome the currently unresolved stereochemistry and a convergent route towards the synthesis of the molecule required three subunits. Following the earlier synthesis of the C21-C25 vinyl stannane fragment, this work describes the synthesis of the C1-C10 subunit in the both possible diastereomeric forms. The synthesis of the two required C13 epimers of the C11-C20 subunit is also detailed accompanied by an investigation into potential fragment coupling, in preparation for total synthesis.

Várias espécies do gênero Fícus são muito utilizadas na medicina popular como agente anti-hemíntico, anti-reumático, antifúngico, antibacteriano, etc. Recentemente, um novo produto natural denominado Aripuanin foi isolado das folhas da espécie Fícus Aripuanensis. Neste trabalho, foi realizada a primeira síntese total do Aripuanin. / Some species of the Fícus are used in folk medicine for their anthelmintic, antiheumatic, antifungal, antimicrobial. Recently, a new natural product denominated Aripuanin, was isolated from the leaves of Fícus Aripuanensis. In this work, was performed the first total synthesis of Aripuanin.