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Thesis (M.S.)--University of Hawaii at Manoa, 2006. / Includes bibliographical references (leaves 140-151). / xii, 151 leaves, bound ill., maps 29 cm

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 197-201). Also available on the World Wide Web.

Thesis (M.Sc. (Chemistry)) - Rhodes University, 2007.

This project forms part of a collaborative effort between the marine natural products chemists at Rhodes University and the medical biochemists at the University of Cape Town’s School of Medicine. Our UCT collaborators tested the cytotoxicity of a group of toluhydroquinones and toluquinones (9-15) against the oesophageal cancer cell line WHCO1 and revealed that the triprenylated toluhydroquinone 11 and it’s oxidised analogue 12 were the most active. This thesis presents an investigation into the role of the polyprenyl side-chain in the cytotoxicity of compound 11 and it’s oxidised analogue 12 by synthesizing and testing the cytotoxicity of simplified analogues of this compound. The synthesis of the two ortho-prenylated toluhydroquinone analogues 5-methyl-2-[(2'E,6'E)-3',7' -dimethyl-2',6'-octadienyl]-1,4-benzenediol (19) and 5-methyl-2-[(2'E,6'E)-3',7',11'-trimethyl-2',6',10'-dodecatrienyl]-1,4-benzenediol (21) and their two ortho-prenylated toluquinone analogues, 5-methyl-2-[(2'E,6'E)-3',7'-dimethyl-2',6'-octadienyl]-2,5-cyclohexadiene-1,4-dione (20) and 5-methyl-2-[(2'E,6'E)-3',7',11'-trimethyl-2',6',10'-dodecatrienyl]-2,5-cyclohexadiene-1,4-dione (22) is described. Our initial attempts to couple geranyl bromide, farnesyl bromide and farnesal to the aromatic precursors m-cresol and 1,4-dimethoxy-2-methylbenzene using directed ortho-prenylation and phenoxide carbon-alkylation were unsuccessful. The four target analogues were eventually synthesized via the initial metal halogen exchange reaction between 1-bromo-2,5-dimethoxy-4-methylbenzene and geranyl bromide/farnesyl bromide using n-BuLi and TMEDA in ditheyl ether at 0 °C to yield 92 and 104 respectively in moderate yield. The demethylation of both compounds preceded smoothly using AgO giving the target analogues 20 and 22 in good yield (approx. 90 %). The reduction of quinones 20 and 22 with sodium dithionite gave 19 and 21 in quantitative yield. The synthesis reported here is the first regioselective synthesis of these compounds. The anti-oesophageal cancer activity of 19-22 and two commercially available non-prenylated analogues 17 and 18 were tested against WHCO1. The conclusion drawn from the anti-oesophageal cancer study was that the polyprenyl side-chain plays a negligable role in the cytotoxicity of compounds such as 11 and 9 against the oesophageal cancer cell line WHCO1.

Recentemente foram isolados da esponja marinha Axinella cf. corrugata dois compostos derivados da esculetina: o éster metílico do ácido 4-esculetínico e o éster etílico do ácido 4-esculetínico. Este último apresentou importante atividade contra o vírus da SARS. Este projeto teve como meta isolar e cultivar as linhagens de fungos associadas à esponja Axinella cf. corrugata, bem como analisar seus extratos por HPLC-PDA-MS, objetivando a possível detecção desses compostos (ou derivados) nesses extratos. Avaliações preliminares levaram à obtenção de 11 amostras potencialmente relacionadas a esses compostos. Dentre elas, uma apresentou espectros no UV e de massas muito similares aos obtidos para o aduto de sódio do éster metílico do ácido 4-esculetínico. No entanto análises espectroscópicas mais detalhadas por RMN - 1H, RMN - 13C, HSQC, HMBC e COSY da amostra purificada permitiram identificar o composto isolado, a 1,3,6-trihidroxi-8-metil-9H-xanten-9-ona. Simultaneamente às análises químicas, os extratos obtidos a partir das linhagens fúngicas isoladas da esponja Axinella cf. corrugata tiveram suas atividades biológicas avaliadas frente a microrganismos e células tumorais humanas, resultando em mais de 20% dos extratos com alguma atividade biológica. / Recently two compounds derived from esculetin have been isolated from the marine sponge Axinella cf. corrugata: the methyl ester of esculetin-4-carboxylic acid and the ethyl ester of esculetin-4-carboxylic acid. The latter displayed antiviral activity against the SARS virus. This project aimed the isolation and the growth of fungal strains associated to the sponge Axinella cf. corrugata, and the subsequent analysis of the fungal extracts by HPLC-PDA-MS, aiming the possible detection of the esculetin compounds (or derivatives) in those extracts. Preliminary analysis yielded 11 samples potentially related to these compounds. Among these extracts, one presented UV and MS spectra very similar to the spectra obtained for the sodium adduct of the methyl ester of esculetin-4-carboxylic acid. However, a detailed spectroscopic analysis of a pure compound isolated by RMN - 1H, RMN - 13C, HSQC, HMBC e COSY allowed the identification of the compound, which is 1,3,6-trihydroxy-8-methyl-9H-xanthen-9-one. Simultaneously to the chemical analysis of the fungal crude extracts, the biological activities of the obtained extracts were evaluated against microrganisms and human tumoral cell lines. More than 20% of the extracts displayed some biological activity.

A ocorrência de fármacos no meio ambiente têm despertado o interesse de pesquisadores, uma vez que podem causar efeitos adversos à comunidade biótica. O norfloxacino (NOR) é um fármaco amplamente empregado no tratamento de infecções bacterianas tanto em humanos como em animais, e devido as suas propriedades físico-químicas, tem sido alvo de estudos envolvendo o seu monitoramento e efeitos toxicológicos em micro-organismos, principalmente em ambientes aquáticos. Neste contexto, o presente trabalho teve como objetivo a investigação da biotransformação/biodegradação do fármaco NOR por fungos derivados de ambiente marinho. Primeiramente, foi realizada uma triagem a partir de 7 cepas fúngicas, das quais 4 foram selecionadas - Penicillium raistrickii CBMAI 931, Cladosporium sp. CBMAI 1237, Aspergillus sydowii CBMAI 1241 e Penicillium raistrickii CBMAI 1235 - para avaliar a influência da adição do fármaco na inoculação dos mesmos. Os experimentos foram realizados na presença do fármaco (0,1 mg mL-1), e em caldo nutritivo (malte 2% em água do mar artificial) por 35 dias (32°C, 130 rpm). A adição do fármaco no dia, e após a inoculação, não influenciou no crescimento dos fungos. No entanto, a formação de produtos de biotransformação foi observada para o experimento com adição do NOR no dia da inoculação, os quais foram identificados por LC-QqTOF, baseando-se na similaridade entre as massas obtidas experimentalmente e teórica, assim como os produtos já reportados na literatura. A porcentagem de biodegradação do fármaco foi determinada para o experimento com adição do fármaco após a inoculação, para os fungos: P. raistrickii CBMAI 931 (34,07%) e A. sydowii CBMAI 1241 (58,91%). Para os experimentos realizados em meio mineral (59,35%) e na presença de um consórcio de fungos (57,05%), não foram observadas diferenças significativas. Não foi possível elucidar a estrutura do produto isolado na presença do fungo A. sydowii CBMAI 1241, devido a sua baixa concentração e a possibilidade de conjugação com substâncias endógenas. Um método analítico foi desenvolvido e validado para a determinação da porcentagem de biodegradação do fármaco nos experimentos com os fungos marinhos. / The occurrence of drugs on the environment has called attention to researchers, since they can cause adverse effects to the biotic community. The norfloxacin (NOR) is a compound widely used for treatment of serious bacterial infections in human and animals. Due to the physicochemical properties of this compound it has been focus of studies concerning about its monitoring and toxicological effects on microorganisms, mainly in aquatic environment. Thus, in the present study the biotransformation/biodegradation of NOR by marine-derived fungi was investigated. Firstly, it was performed a screening with 7 strain of marine fungi, in a which 4 were selected - Penicillium raistrickii CBMAI 931, Cladosporium sp. CBMAI 1237, Aspergillus sydowi CBMAI 1241 and Penicillium raistrickii CBMAI 1235 - to evaluate the influence of NOR addition in the inoculation. The experiments were carried out in the presence of NOR (0,1 mg mL-1) in nutritive broth (malt 2% in artificial sea water) for 35 days (32°C, 130 rpm). The NOR addition on the first day and after inoculation, did not affect the fungal growth. Nevertheless, the formation of biotransformation products was observed to the experiment with addition on the first day. These products were identified by LC-QqTOF, based on the similarity between experimental and theoretical mass, as the products already reported on the literature. The percentage of drug biodegradation was determined for the fungi P. raistrickii CBMAI 931 (34,07%) and A. sydowi CBMAI 1241 (58,91%) for the experiment carried out with NOR addition after inoculation. For the experiments performed in mineral medium (59,35%) and in the presence of fungal consortium (57,05%) no differences were observed for the biodegradation. It was not able to elucidate the structure of isolated product, in the presence of A. sydowii CBMAI 1241, due to its low concentration and probable conjugation with endogenous substances. The analytical method was developed and validated to determine the percentage of drug biodegradation in the experiments with marine fungi.

Este trabalho envolveu reações de biocatálise com objetivo de obter compostos enantiomericamente puros. Assim foram realizadas reações de redução de derivados de acetofenonas, resolução enzimática de alcoóis e azido-alcoóis e imobilização de células fúngicas em suportes sólidos para aplicação em biocatálise. Foi realizada a redução enantiosseletiva da 1-(4-metoxifenil)etanona (1) através da triagem com nove fungos marinhos (Aspergillus sydowii CBMAI 935, A. sydowii CBMAI 934, A. sclerotiorum CBMAI 849, Bionectria sp. CBMAI 936, Beauveria felina CBMAI 738, Cladosporium cladosporioides CBMAI 857, Mucor racemosus CBMAI 847, Penicillium citrinum CBMAI 1186, P. miczynskii CBMAI 930). Os fungos A. sydowii CBMAI 935 e Bionectria sp. CBMAI 936 catalisaram a biorredução estereosseletiva da 1-(4-metoxifenil)etanona (1) para o correspondente (R)-1-(4-metoxifenil)etanol (1a) com excelentes excessos enantioméricos (>99%). Os fungos B. felina CBMAI 738 e P. citrinum CBMAI 1186 catalisaram a biorredução estereosseletiva da cetona 1 para o correspondente S-álcool 1a com 69% de excesso enantiomérico. Os fungos marinhos (A. sclerotiorum CBMAI 849, A. sydowii CBMAI 934, B. felina CBMAI 738, M. racemosus CBMAI 847, P. citrinum CBMAI 1186, P. miczynskii CBMAI 931, P. miczynskii CBMAI 830, P. oxalicum CBMAI 1185, Trichoderma sp. CBMAI 932) foram utilizados na bioconversão assimétrica das iodoacetofenonas 2-4 para os correspondentes iodofeniletanois 2a-4a. Todos os fungos marinhos produziram exclusivamente (S)-o-iodofeniletanol (2a) e (S)-m-iodofeniletanol (3a) com diferentes valores de excessos enantioméricos (62-99%). Os fungos B. felina CBMAI 738, P. miczynskii CBMAI 830, P. oxalicum CBMAI 1185 e Trichoderma sp. CBMAI 932 produziram o correspondente (R)-p-iodofeniletanol (4a) com excessos enantioméricos de 32-99%. A bioconversão da p-iodoacetofenona (4) com células microbianas do P. oxalicum CBMAI 1185 mostrou uma competição entre a reação de redução e oxidação. Também foram realizadas as reduções das ceto-azidas 13-16 com fungos marinhos fornecendo bons resultados de seletividade (28-99% ee). As células microbianas dos fungos A. sclerotiorum CBMAI 849 e P. citrinum CBMAI 1186 foram imobilizadas em suportes de sílica gel, xerogel de sílica e quitosana. As células do P. citrinum CBMAI 1186 imobilizadas em quitosana catalisaram a redução da 1-(4-metoxifenil)-etanona (1) para o correspondente (S)-1-(4-metoxifenil)-etanol (1a) com excelente excesso enantiomérico (>99%). O fungo P. citrinum CBMAI 1186 imobilizado em quitosana também catalisou a biorredução de 2-cloro-1-feniletanona (7) para o 2-cloro-1-feniletanol (7a), mas neste caso, sem seletividade. Neste trabalho também foram realizadas as resoluções quimio-enzimáticas dos (±)-o-iodofeniletanol (2a), (±)-m-iodofeniletanol (3a), (±)-p-iodofeniletanol (4a), (±)-2-azido-1-feniletanol (13a), (±)-2-azido-1-(4-metoxifenil)etanol (14a), (±)-2-azido-1-(4-bromofenil)etanol (15a), (±)-2-azido-1-(4-nitrofenil)etanol (16a) e (±)-2-azido-1-(4-clorofenil)etanol (17a) com a lipase CALB. Os (S)-m-iodofeniletanol (3a) e (S)-p-iodofeniletanol (4a) foram obtidos com excelentes excessos enantioméricos (>99%) e posteriormente foram utilizados na síntese de compostos bifenílcos quirais por reação de acoplamento Suzuki fornecendo bons rendimentos (63-65%). A resolução quimio-enzimática dos azido-alcoóis 13a-17a foram realizadas com lipase Candida atarctica e os (R)-2-azido-1-feniletanol (13a), (R)-2-azido-1-(4-metoxifenil)etanol (14a), (R)-2-azido-1-(4-bromofenil)etanol (15a), (R)-2-azido-1-(4-nitrofenil)etanol (16a) obtidos foram utilizados na síntese dos triazóis quirais (R)-2-(1H-benzo[d][1,2,3]triazol-1-il)-1-feniletanol (13), (R)-2-(1H-benzo[d][1,2,3]triazol-1-il)-1-(4-metoxifenil)etanol (14), (R)-2-(1H-benzo[d][1,2,3]triazol-1-il)-1-(4-bromofenil)etanol (15) e (R)-2-(1H-benzo[d][1,2,3]triazol-1-il)-1-(4-nitrofenil)etanol (16) e (R)-2-(1H-benzo[d][1,2,3]triazol-1-il)-1-(4-clorofenil)etanol (17), obtidos com ótimos rendimentos (79-85%). / This work involved reactions of biocatalysis in order to obtain enantiomerically pure compounds. Thus reactions were performed reduction of acetophenones derivatives, enzymatic resolution of azido-alcohols, secondary alcohols and immobilization of fungal cells on solid supports for use in biocatalysis. We performed the enantioselective reduction of 1-(4-methoxyphenyl)ethanone (1) by screening with nine marine fungi (Aspergillus sydowii CBMAI 935, A. sydowii CBMAI 934, A. sclerotiorum CBMAI 849, Bionectria sp. CBMAI 936, Beauveria felina CBMAI 738, Cladosporium cladosporioides CBMAI 857, Mucor racemosus CBMAI 847, Penicillium citrinum CBMAI 1186, P. miczynskii CBMAI 930). The fungi A. sydowii CBMAI 935 and Bionectria sp. 936 CBMAI catalyzed stereoselective bioreduction of 1-(4-methoxyphenyl)ethanone (1) to the corresponding (R)-1-(4-methoxyphenyl)ethanol (1a) with excellent enantiomeric excess (>99%). Fungi B. felina CBMAI 738 and P. citrinum 1186 CBMAI catalyzed stereoselective bioreduction of ketone 1 to the corresponding S-alcohol 1a with 69% enantiomeric excess. The marine fungi (A. sclerotiorum CBMAI 849, A. sydowii CBMAI 934, B. felina CBMAI 738, M. racemosus CBMAI 847, P. citrinum CBMAI 1186, P. miczynskii CBMAI 931, P. miczynskii CBMAI 830, P. oxalicum CBMAI 1185, Trichoderma sp. CBMAI 932) were used in the bioconversion of asymmetric iodoacetophenones 2-4 to the corresponding iodophenylethanols 2a-4a. All marine fungi produced exclusively (S)-o-iodophenylethanol (2a) and (S)-m-iodophenyletanol (3a) with different values of enantiomeric excess (62-99%). Fungi B. felina CBMAI 738, P. miczynskii CBMAI 830, P. oxalicum CBMAI 1185 and Trichoderma sp. CBMAI 932 produced the corresponding (R)-p-iodophenylethanol (4a) with enantiomeric excess of 32-99%. The bioconversion of p-iodoacetophenone (4) with microbial cells of P. oxalicum CBMAI 1185 showed a competition between oxidation and reduction reaction. Were also performed reductions of azido-ketones 13-16 with marine fungi providing good results of selectivity (28-99% ee). Microbial cells of fungi A. sclerotiorum CBMAI 849 and P. citrinum CBMAI 1186 were immobilized on supports of silica gel, silica xerogel and chitosan. Whole cells of P. citrinum 1186 CBMAI immobilized on chitosan catalyzed the reduction of 1-(4-methoxyphenyl)ethanone (1) to the corresponding (S)-1-(4-methoxyphenyl)ethanol (1a) with excellent enantiomeric excess (>99%). The fungus P. citrinum 1186 CBMAI immobilized on chitosan also catalyzed the bioreduction of 2-chloro-1-phenylethanone (7) to 2-chloro-1-phenylethanol (7a), but in this case without selectivity. In this work were also performed chemo-enzymatic resolutions of (±)-o-iodophenylethanol (2a), (±)-m-iodophenylethanol (3a), (±)-p-iodophenylethanol (4a), (±)-2-azido-1-phenylethanol (13a), (±)-2-azido-1-(4-methoxyphenyl)ethanol (14a), (±)-2-azido-1-(4-bromophenyl)ethanol (15a), (±)-2-azido-1-(4-nitrophenyl)ethanol (16a) and (±)-2-azido-1-(4-chlorophenyl)ethanol (17a) with the lipase Candida atarctica. The (S)-m-iodophenylethanol (3a) and (S)-p-iodophenylethanol (4a) were obtained with excellent enantiomeric excess (>99%) and were subsequently used in the synthesis of chiral biphenyl compounds by the Suzuki reaction with good yields (63-65%). Chemoenzymatic resolution of azido-alcohols 13a-17a were carried out using lipase CALB and (R)-2-azido-1-phenylethanol (13a), (R)-2-azido-1-(4-methoxyphenyl)ethanol (14a), (R)-2-azido-1-(4-bromophenyl)ethanol (15a), (R)-2-azido-1-(4-nitrophenyl)ethanol (16a) obtained were used in the synthesis of chiral triazoles (R)-2-(1H-benzo[d][1,2,3]triazol-1-yl)-1-phenylethanol (13), (R)-2-(1H-benzo[d][1,2,3]triazol-1-yl)-1-(4-methoxyphenyl)ethanol (14) (R)-2-(1H-benzo [d][1,2,3]triazol-1-yl)-1-(4-bromophenyl)ethanol (15) and (R)-2-(1H-benzo[d][1,2,3]triazol-1-yl)-1-(4-nitrophenyl)ethanol (16) and (R)-2-(1H-benzo[d] [1,2,3]triazol-1-yl)-1-(4-chlorophenyl)ethanol (17) obtained in good yields (79-85%).

Neste trabalho descreve-se o estudo química dos extratos de quatro esponjas e dois fungos de origem marinha oriundos da costa do Brasil. Os extratos de três esponjas (Petromica ciocalyptoides, Topsentia ophiraphidites e Callyspongia sp.) apresentaram atividade inibitória à enzima adenosina fosforribosil transferase de Leishmania tarentolae. A partir desses extratos foram isolados 4 compostos. O trissulafato de halistanol, isolado das esponjas P. ciocalyptoides e T. ophiraphidites, e o ilhabelanol, ilhabreno e isoakaterpina, isolados da esponja Callyspongia sp. A partir do extrato bruto da esponja Axinella cf corrugata foram isolados dois derivados cumarínicos, provavelmente artefatos de isolamento do ácido 4-esculetínico, o qual é inédito como produto natural. O extrato bruto da esponja Axinella cf. corrugata apresentou atividade citotóxica, mas os compostos puros não apresentaram esta atividade. Os dois compostos puros foram testados ainda quanto sua atividade contra o vírus da SARS, na qual o éster etílico do ácido 4-esculetínico se apresentou ativo. A partir de dois extratos oriundos do fungo Beauveria felina, isolado da alga marinha Caulerpa sp, foram isoladas 17 frações puras que após diversas análises foram agrupadas em seis compostos conhecidos na literatura: a (Phe3, N-Val5) destruxina B, a cloroidrina da destruxina E, a roseotoxina B, a roseocardina, a isariina e a isariina B. Além disso, foram isolados dois compostos inéditos, a pseudodestruxina C e a cloloidrina Beta-Me-Pro da destruxina E. Os extratos brutos de Beauveria felina apresentaram atividades em bioensaios de atividade antituberculose e de citotoxicidade em linhagem de células de câncer. Os compostos puros avaliados no bioensaio antituberculose não foram ativos. Somente o composto roseotoxina B apresentou atividade citotóxica in vitro para quatro linhagens de células: mama, cólon, sistema nervoso e leucemia. / In this work we report the chemical investigation of bioactive crude extracts obtained from four sponges and two fungal strains of marine origin. The crude extracts of three sponges species (Petromica ciocalyptoides, Topsentia ophiraphidites and Callyspongia sp.) displayed inhibitory activity towards the enzyme adenine fosforribosyl transferase of Leishmania tarentolae (L-APRT). Four compounds have been isolated from these extracts: the known halistanol sulfate was isolated of sponges P. ciocalyptoides and T. ophiraphidites, while the novel ilhabelanol, ilhabrene and isoakaterpin have been isolated from the sponge Callyspongia sp. All compounds exhibited inhibition of L-APRT at micro M concentrations. Two coumarin derivatives have been isolated from the crude extract of the sponge Axinella cf. corrugata, probably as artifacts of isolation: esculetin-4-carboxylic acid methyl ester and esculetin-4-carboxylic acid ethyl ester. While the crude extract of the sponge Axinella cf. corrugata presented cytotoxic activity, the pure compounds were inactive in these assays. The esculetin-4-carboxylic acid ethyl ester was found to be an in vitro inhibitor of SARS virus. The crude extract obtained of a marine-derived Beauveria felina strain, isolated from the alga Caulerpa sp., displayed antituberculosis activity against Mycobacterium tuberculosis H37Rv and cytotoxic activity against MCF-7 (breast), HCT-8 (colon) and B16 (murine melanoma) cancer cell lines. Chemical fractionation of the crude extract led to the isolation of two new cyclodepsipeptides pseudodestruxin C and [Beta-Me-Pro] destruxin E chlorohydrin, and of the known destruxin E chlorohydrin, [Phe3, N-Me-Val5] destruxin B, roseotoxin B, roseocardin, isariin and isariin B. The depsipeptides [Phe3, NMe- Val5] destruxin B and rosetoxin B, have been tested against M. tuberculosis H37 Rv and in cytotoxicity bioassays against SF 295 (human CNS) MDA-MB435 (human breast) HCT8 (colon) and HL60 (leukemia) cancer cell lines. Only roseotoxin B displayed moderate cytotoxicity against the cancer cell lines.

Thesis (Ph. D.)--University of Canterbury, 2008. / Typescript (photocopy). "January 2008." Includes bibliographical references (leaves 201-215). Also available via the World Wide Web.