Muitas naftoquinonas como o lapachol, podem ser encontradas em plantas da família Bignoniaceae e são conhecidas por desempenharem diversas atividades biológicas, acompanhadas, entretanto, por efeitos indesejáveis. A atividade citotóxica apresentada pelas naftoquinonas está relacionada ao aparecimento de espécies reativas de oxigênio in vivo que causam severo estresse oxidativo no interior das células. O isolapachol e a atovaquona são análogos estruturais do lapachol, sendo que a atovaquona é comercializada como fármaco para o tratamento de malária e certos tipos de pneumonia. Devido ao grande potencial biológico apresentado pelas naftoquinonas, várias tentativas no sentido de obtenção de derivados desprovidos de efeitos colaterais vêm sendo realizadas. Além disso, a determinação da segurança e eficácia dos fármacos está intimamente ligada ao estudo da formação de derivados in vivo por ocasião do metabolismo. A utilização de fungos filamentosos na predição do metabolismo que os fármacos sofreriam após administração oral, bem como de bactérias do trato gastrointestinal, pode contribuir substancialmente para a elucidação da rota metabólica de fármacos fornecendo informações sobre a geração de substâncias farmacologicamente ativas, inativas ou tóxicas e ainda sobre a produção de substâncias capazes de inibir a biotransformação de outros fármacos. Estudos de biotransformação também podem contribuir para a obtenção de novos esqueletos químicos. Dessa forma, o presente trabalho relata estudos do metabolismo microbiano do lapachol e do seu sal de potássio por bactérias do trato gastrointestinal e fungos filamentosos, além da correlação desses com as reações que ocorrem quando o isolapachol e a atovaquona são utilizados como substratos para os mesmos micro-organismos. Os experimentos de biotransformação utilizando lapachol e seu sal de potássio foram conduzidos por até dez dias, em diferentes meios de cultura, empregando-se quatro linhagens de bactérias presentes no trato gastrointestinal, além de 11 linhagens de fungos filamentosos. Foram obtidos sete metabólitos, sendo dois inéditos e dois anteriormente detectados em estudos sobre o metabolismo do lapachol em mamíferos. Durante a realização dos experimentos com o fungo filamentoso Aspergillus brasiliensis verificou-se a capacidade desse fungo em mimetizar uma reação muito importante em química orgânica, conhecida como oxidação de Hooker. As condições mais promissoras para a biotransformação do lapachol foram utilizadas nos estudos com a atovaquona e o isolapachol. A biotransformação da atovaquona possibilitou, pela primeira vez, a caracterização estrutural de um metabólito desse fármaco. Já os estudos realizados com o isolapachol permitiram inferências sobre a especificidade enzimática apresentada pelos micro-organismos avaliados. Todos os metabólitos obtidos foram submetidos aos ensaios de citotoxicidade frente a linhagens celulares normais e tumorais, o que possibilitou obter conclusões sobre a relação estrutura-atividade e sobre a citotoxicidade seletiva apresentada pelos metabólitos. Destaca-se o resultado obtido com um dos metabólitos do lapachol, ?-xiloidona, o qual se mostrou mais tóxico para a linhagem tumoral que o lapachol e não apresentou toxicidade frente à linhagem normal. O metabólito obtido a partir da biotransformação da atovaquona apresentou maior toxicidade não seletiva que a substância de partida. / Several naphthoquinones, as lapachol, can be found in the Bignoniaceae family and they present several biological activities with some unwanted effects. The cytotoxic activity displayed by naphthoquinones is correlated to the presence of reactive oxygen species, which are formed in vivo and cause severe oxidative stress within cells. Isolapachol and atovaquone are structural analogs of lapachol, and atovaquone is in the market as a drug for the treatment of malaria and some types of pneumonia. Because of the great biological potential presented by naphthoquinones, several studies have been carried out to obtain derivatives without side effects. Furthermore, the drug safety and efficacy are closely related to the study of the formation of in vivo derivatives during metabolism. The filamentous fungi and the bacteria from the gastrointestinal tract can be used in the prediction of drug metabolism after oral administration, which is an interesting tool to elucidation of the metabolic pathway of drugs, providing information on the generation of pharmacologically active, inactive or toxic substances and still on the production of compounds able to inhibit the biotransformation of other drugs. Biotransformation studies can also contribute to the obtention of new chemical skeletons (hits). Thus, the present work reports the study about the microbial metabolism of lapachol and its potassium salt by filamentous fungi and bacteria from the gastrointestinal tract, beyond the correlation of the reactions that occur when the isolapachol and atovaquone are used as substrates for the same microorganisms. The biotransformations of lapachol and its potassium salt were evaluated for up to ten days, in different culture media, catalyzed by four bacteria from the gastrointestinal tract and 11 filamentous fungi strains. Seven metabolites were obtained, from which two are new and two were previously detected in the mammals metabolism of lapachol. The filamentous fungus Aspergillus brasiliensis showed to be capable of mimicking the Hooker oxidation, an important organic chemistry reaction. The best conditions for the lapachol biotransformation have been used in the studies with isolapachol and atovaquone. The atovaquone biotransformation provided, for the first time, the structural characterization of a metabolite from this drug. The studies with isolapachol allowed inferences about the enzyme specificity shown by the evaluated microorganisms. All obtained metabolites were submitted to cytotoxicity assays against human cancer and tumoral cell lines. Several conclusions about the structure activity relationship and about the selective cytotoxicity showed by the metabolites were taken. It should be highlighted the obtained result with a lapachol metabolite, ?-xyloidone, which showed to be more toxic than lapachol against tumoral cell line and did not show cytotoxicity to normal cell line. The atovaquone metabolite displayed higher toxicity than pattern structure, and this activity was not selective.

Mesoporous aggregates of nanocrystalline zeolites with MFI and BEA frameworks have been synthesized using a one-pot and single structure directing agent method. The effect of different reaction conditions, such as temperature, time, pH and water content, on the particle size, surface area and mesopore volume has been studied. Nanocrystalline and mesoporous ZSM-5, β and Y zeolites were modified with different transition metals and the resulting single- and double metal containing catalyst materials were characterized. Nanocrystalline Silicalite-1 zeolite samples with varying particle size were functionalized with different organosilane groups and the cytotoxic activity of the zeolite nanocrystals was studied as a function of particle size, concentration, organic functional group type, as well as the type of cell line. Framework stability of nanocrystalline NaY zeolite was tested under different pH conditions. The synthesized zeolites used in this work were characterized using a variety of physicochemical methods, including powder X-ray diffraction, Solid State NMR, nitrogen sorption, electron microscopy, Inductively Coupled Plasma - Optical Emission Spectroscopy and X-ray Photoelectron Spectroscopy.

Ce travail de thèse s’intègre dans la stratégie développée et brevetée par l’UMR 990 INSERM/UdA, qui consiste à exploiter l’affinité d’un vecteur ammonium quaternaire chargé positivement pour les protéoglycanes, afin d’acheminer des agents de diagnostic et/ou de thérapie, pour la prise en charge du chondrosarcome. Du fait de son abondante matrice extracellulaire de nature chondrogénique, de sa faible vascularisation et de son environnement hypoxique, le chondrosarcome est une tumeur chimio- et radio-résistante. Le seul traitement efficace à ce jour reste la chirurgie qui possède un taux de survie à 10 ans de seulement 21% en cas de rechute et dans les formes les plus graves. Dans ce contexte, une prodrogue cytotoxique activable en hypoxie dérivée du cyclophosphamide, de type nitroimidazole (8-QA) et possédant un ammonium quaternaire, a été développée et évaluée in vitro et in vivo, comparativement à son équivalent non « quaternisé » (8) et à un équivalent « quaternisé » mais non activable en hypoxie (10-QA). In vitro, les études par résonance plasmonique de surface (SPR) ont montré que les deux prodrogues « quaternisées », 8-QA et 10-QA possédaient une affinité plus importante pour l’agrécane que la prodrogue 8 ne possédant pas le vecteur ammonium quaternaire. Par la suite, nous avons développé un modèle de culture en 3 dimensions (sphéroïdes) des cellules de chondrosarcome humain HEMC-SS qui reproduit le microenvironnement hypoxique et riche en protéoglycanes retrouvé dans les tumeurs du cartilage in vivo. Les molécules activables en hypoxie 8-QA et 8 ont une activité cytotoxique supérieure en situation d’hypoxie qu’en normoxie, avec une résistance aux traitements augmentée pour le modèle de sphéroïde comparativement aux cellules cultivées en 2-D. In vivo, sur un modèle de xénogreffe de cellules de chondrosarcome humain HEMC-SS implantées sur des souris SCID, modèle préalablement caractérisé en terme d’hypoxie (imagerie photoacoustique, échographie de contraste, Doppler) et de protéoglycanes, la molécule 8-QA entraine (i) une inhibition significative de la croissance tumorale de 62,1%, alors que la molécule 8 et la molécule 10-QA n’ont aucune activité in vivo ; (ii) une diminution des effets indésirables, améliorant ainsi l’index thérapeutique. La caractérisation de l’activité antitumorale sur les tumeurs prélevées a mis en évidence une diminution de l’activité mitotique, une augmentation de l’apoptose (p53S15) et une diminution de la prolifération (PCNA) pour les échantillons traités avec la molécule 8-QA. De plus les études d’immunohistochimie ont montré que cette molécule entraine une augmentation des dommages à l’ADN (marquage γH2Ax) retrouvés majoritairement dans les régions hypoxiques (marquage au pimonidazole). La dernière partie de ce travail de thèse a consisté à optimiser, chez des souris NUDE, de nouveaux modèles in vivo de chondrosarcome humain exprimant à la fois un environnement hypoxique et une matrice extracellulaire riche en protéoglycanes pour affiner la caractérisation de l’activité anticancéreuse de la molécule 8-QA. Pour cela nous avons développé un modèle de xénogreffe de sphéroïdes HEMC-SS implantés en situation sous cutanée et un modèle orthotopique de cellules de chondrosarcome humain HEMC-SS implantées en position paratibiale. Après caractérisation, les premiers résultats d’efficacité antitumorale sur ces deux modèles ont confirmé une activité prometteuse de la molécule 8-QA. En conclusion, ces travaux de thèse auront permis de démontrer que l’exploitation des caractéristiques phénotypiques du chondrosarcome par une approche bi-spécifique est une stratégie prometteuse pour la prise en charge thérapeutique de cette pathologie, certes rare mais redoutable. Il a également contribué à montrer l’importance de l’interaction des cellules tumorales avec leur environnement pour la réponse aux traitements de chimiothérapie. / This work takes place in the strategy developed and patented by our lab UMR 990 INSERM/UdA, that exploits the affinity of positively charged quaternary ammonium to proteoglycans, in order to address diagnostic and/or therapeutic agents for chondrosarcoma management. Due to its abundant chondrogenic extracellular matrix, its poor vascularization and its hypoxic microenvironment, chondrosarcoma is chemo- and radio-resistant. The only effective treatment remains surgical resection that could be at the origin of a 10-years survival rate of 21% in case of local relapse of high-grade tumors. In this context, a quaternary ammonium-conjugated hypoxia-activable prodrug, 8-QA, has been developed and studied in vitro and in vivo respectively to its non-quaternary ammonium conjugated derivative, 8, and to a quaternary ammonium-conjugated non-hypoxia activable 10-QA. In vitro, surface plasmon resonance (SPR) studies evidenced that the two quaternary ammonium-conjugated derivatives, 8-QA and 10-QA, highlighted a higher affinity for aggrecan than non-quaternary ammonium conjugated prodrug 8. We have then developed a 3-D culture model (spheroid) of human chondrosarcoma HEMC-SS cell line that was demonstrated to reproduce the hypoxia and proteoglycan-rich microenvironment similar to in vivo cartilaginous neoplasms. Hypoxia-activated prodrugs, 8 and 8-QA, exhibited a higher cytotoxic activity in hypoxia as compared to normoxia, associated to an increased resistance for 3-D culture respectively to 2-D cells. In vivo, on human HEMC-SS xenograft model implanted on SCID mice and previously characterized in term of hypoxia (photoacoustic and contrast echographic imaging, Doppler), 8-QA derivative induced (i) a significant inhibition of tumor growth of 62,1% while 8 and 10- QA derivatives did not exhibit any antitumor activity; (ii) a decrease of side effects, both improving the therapeutic index. Mechanistic analysis on tumors sampled evidenced a decrease of mitotic activity and proliferation (PCNA), an increase of apoptosis (p53S15) for 8-QA treated tissues. Interestingly, immunohistochemistry studies highlighted that 8-QA caused DNA damages (evidenced by γH2Ax marker) which are mainly localized in hypoxic areas (pimonidazole staining). Finally, we optimized, on NUDE mice, new in vivo human chondrosarcoma models highly expressing simultaneously a hypoxic and proteoglycan-rich microenvironment in order to refine the characterization of 8-QA antitumor activity. For this, we developed a xenograft model of HEMC-SS spheroid implanted subcutaneously and an orthotopic model of HEMC- SS cells inoculated in paratibial location. After characterization, first results of antitumor efficacy on these two models confirmed the promising activity of 8-QA derivative. To conclude, this work demonstrates the proof of concept that exploiting the phenotypic features of chondrosarcoma by a bi-selective approach, is a promising innovative strategy for the therapeutic management of this pathology, relatively rare but redoubtable. This work also demonstrates the role of tumor cell interactions with their environment for tumor growth and response to chemotherapy treatments.

Plant-derived natural products continue to be a valuable source of useful therapies for cancer as well as other diseases. As part of a continuing mission to obtain anticancer agents from natural sources, researchers at the National Cancer Institute (NCI) established the 60 human tumor cell line anticancer screen. The schweinfurthins are one family of unique natural products discovered through this screening process. Most of these natural compounds exhibit potent and differential activity in the 60-cell screen. Importantly, the pattern of activity displayed by the schweinfurthins shows no correlation to any clinically used anticancer drug, indicating that this family of natural products probably acts via a novel mechanism or at a novel target. Our group has conducted extensive structure-activity relationship studies in an effort to illuminate the mechanism of action of the schweinfurthins. In this thesis, the preparation and biological activity of a number of new schweinfurthin F analogues possessing variations in the D-ring alkyl chain and stilbene moiety will be discussed. These studies have clarified the importance of the D-ring to the schweinfurthins' pharmacophore. Based on the results obtained from the exploration of the structural requirements of these natural products, it was determi-ned that the right-half of the schweinfurthins would be an appropriate site for attachment of a molecular probe to be used in affinity experiments. The synthesis of these biotinylated probes will be examined in detail, and their use in pull-down assays will be summarized. The preparation of key schweinfurthin intermediates has involved the extensive use of Lewis acid-mediated cationic cascade cyclizations terminated by MOM-protected phenols. Those successes have inspired investigations of additional applications of these cyclizations. In particular, a variant of these cyclizations using "MOM-protected" enol ethers as reasonable substitutes for β-keto ester terminating moieties has been studied. These interrelated studies involving the synthesis of schweinfurthin analogues and the exploration of cascade cyclizations will be discussed in detail.

Amyloidoses comprise a group of diseases where normal or mutated protein precipitates into amyloid fibrils. The deposition of fibrils causes dysfunction of organs and toxicity to nervous tissue. Up to date, 24 different proteins and peptides are known to be able to form amyloid fibrils. The most well known are Amyloid beta peptide and Prione protein causing Alzheimer’s disease and Creutzfeld Jacob’s disease respectively. The aims of this thesis were to investigate the structural properties of cytotoxic amyloid and examine the mechanisms involved. The model protein mostly used in the studies was the plasma protein transthyretin (TTR). Familial Amyloidotic Polyneuropathy (FAP) is a hereditary, autosomal-dominant neurodegenerative disease caused by point mutations in the TTR gene. One of the most common variants of FAP is a mutation in position 30 where alanine is exchanged for methonine. This gives rise to “Skellefteåsjukan” in Sweden. TTR is secreted into the plasma as a tetramer. Point mutations destabilize the tetramer leading to disassembled monomers, which undergo partial denaturation as an initiation step to aggregation and amyloid fibril formation. In vivo amyloidogenesis takes a long time and does not occur until late in adult life. Most of the clinical TTR mutations do not form amyloid in vitro under physiological conditions. We have created amyloidogenic TTR mutants that are prone to aggregate and form fibrils under physiological conditions. This provides us with a model system on the cellular level for studies of the mechanisms of amyloid associated cytotoxicity as we can control the aggregation process and capture defined stages in the TTR amyloidogenic pathway. We used Atomic Force Microscopy (AFM) to follow the morphology of aggregates during fibril formation. Initially, amorphous aggregates were formed that subsequently matured into fibrillar structures, denoted protofilaments. This observation was interpreted as an optimisation of ß-strand registers. In addition we identified a correlation between the presence of early-formed aggregates of TTR and cytotoxicity. The toxic response was mediated via an apoptotic mechanism. We were not able to more carefully determine the structure and size of the toxic TTR species. To address this problem we turned to another amyloidogenic protein, equine lysozyme (EL). Intermediate samples corresponding to the aggregation and growth phase of amyloid fibrils of EL were collected. These samples were subjected to cytotoxicity assays as well as monomeric starting material and mature amyloid fibrillar species. The results clearly showed that the soluble oligomers were cytotoxic in contrast to the monomers and fibrils. Our data indicate that the toxic properties of the oligomers are size dependent. In this thesis we asked the question whether all mutated forms of TTR can be expressed and secreted or if there is a selection against the most aggressive mutations in vivo? We transfected hematopoetic K562 cells with wild type or mutant TTR, with or without the N-terminal signal peptide, responsible for secretion, to generate both extra- and intracellular TTR. We show that the post-translational quality control of the cells does not allow intracellular mutant TTR outside the secretory pathway, possibly due to the cytotoxic effects, while translocated to the secretory pathway made it escape the quality control permitting secretion and amyloid formation outside the cells. We have further analyzed the cytotoxic mechanisms induced by TTR oligomers with a focus on intracellular apoptotic signalling pathways. We show that TTR oligomers bind to the surface of the target cells but are not taken up, that is in contrast to mature fibrils that do not bind them at all. The apoptotic response occurred in a caspase-independent and a free radical dependent way.

The occurrence of mould in food and animal feed is a severe problem due to the secondary metabolites, called mycotoxins, which can possess toxic activity. Aspergillus fumigatus is a common fungus found in improperly stored animal feed and the abundance of spores of the fungus is frequently spread into the air. Gliotoxin has been identified as one of the most toxic second metabolites produced by A. fumigatus. Although A. fumigatus is known to produce mycotoxins that induce neurological syndromes, the neurotoxic properties of gliotoxin have not previously been studied. In this thesis a neurotoxic activity of gliotoxin was demonstrated by using differentiated human neuroblastoma SH-SY5Y cells as a surrogate for the nervous system. The major findings were as follows: i. Gliotoxin is highly toxic to SH-SY5Y cells and there is a correlation between the toxicity and the cellular redox status. ii. Gliotoxin reduces the number of neurites, but does not affect the cell bodies morphologically, at non-cytotoxic concentrations. This indicates that the toxin may induce peripheral axonopathy in vivo. iii. The intracellular free Ca2+ concentration is increased after exposure to gliotoxin, an effect that is the most ubiquitous feature of neuronal cell death. Simultaneously, calpains and caspases, proteases known to be involved in neuronal death and axonal degeneration, are activated. iv. The observed irreversible neurite degenerative effects of gliotoxin are mainly dependent on caspase activation, whereas calpains are involved in the gliotoxin-induced cytotoxicity. v. Gliotoxin induces a decreased rate of protein synthesis at non-cytotoxic concentration, which may contribute to the degeneration of neurites. vi. We did also succeed in developing an in vitro method for determination of toxic activity in animal feed. This study was done in collaboration with National Veterinary Institute (SVA) in Uppsala, and the method is today established and in use at Department of Animal Feed, SVA.

A number of diseases are linked to protein folding problems which lead to the deposition of insoluble protein plaques in the brain or other organs. These diseases include prion diseases such as Creutzfeld-Jakob disease, Alzheimer's disease, Parkinson's disease and type II (non-insulin dependent) diabetes. The protein plaques are found to consist of amyloid fibrils - cross-beta-sheet polymers with the beta-strands arranged perpendicular to the long axis of the fibre. Studies of ex vivo fibrils and fibrils produced in vitro showed that amyloid structures possess similar tinctorial and morphological properties. These suggest that the ability to form amyloid fibrils is an inherent property of polypeptide chains. The aims of this thesis were to investigate the structural properties of cytotoxic amyloid and examine the involved mechanisms. The model proteins used in the studies were the equine and hen lysozymes and de novo designed protein albebetin. Lysozymes are naturally ubiquitous proteins. Equine lysozyme belongs to an extended family of structurally related lysozymes and α-lactalbumins and can be considered as an evolutional bridge between them. Hen lysozyme is one of the most characterized protein and its amyloidogenic properties were described earlier. De novo protein albebetin and its constructs are designed to perform the function of grafted polypeptide sequence. Fibrils of equine lysozyme are formed at acidic pH and elevated temperatures where a partially folded molten globule state is populated. We have shown that lysozyme assembles into annular and linear protofilaments in a calcium-dependent manner. We showed that albebetin and its constructs are inherently highly amyloidogenic under physiological conditions. Fibrillation proceeds via multiple pathways and includes a hierarchy of amyloid structures ranging from oligomers to protofilaments and fibrils, among which two distinct types of oligomeric intermediates were characterized. Pivotal oligomers comprise of 10-12 monomers and on-pathway amyloid-prone oligomers constitute of 26-30 molecules. We suggest that transformation of the pivotal oligomers into the amyloid-prone ones is a limiting stage in albebetin fibrillation. Cytotoxic studies of albebetin amyloid species have revealed that initial, pivotal oligomers do not effect on cell viability while amyloid-prone ones induce cell death. We suggest that oligomeric size is important for the stabilizing cross-beta-sheet core which is crucial for cell toxicity. Cytotoxic studies of both oligomers and fibrils of hen lysozyme have revealed that both species induce cell death. The amyloid sample containing cross-β-sheet oligomers induces an apoptosis-like cell death. The oligomers without cross-β-sheet appeared to be non-toxic, indicating that the stabilization of this structural pattern is critical for the induced toxicity. In contrast, the fibrils induce more rapid, necrosis-like death. These studies gained insights into a structure–function relationship of different forms of amyloid and general pathways of cell death. This is an important step in understanding the mechanisms of amyloid-associated degeneration and defining specific therapeutic targets.

As fish cell cultures continue to be explored as alternatives to whole fish for evaluating the toxicity of environment chemicals, technical issues have emerged that influence results and thus need to be understood and standardized. These include carrier solvents, dosing protocols, exposure vessel, exposure media, viability endpoints, and cell lines. Some of these factors have been explored in this thesis for eight reactive contaminants exhibiting varied physicochemical properties using the rainbow trout cell lines RTgill-W1 and RTL-W1. Sodium dodecyl sulphate (SDS) was used as a reference (control) chemical. Cell viability was evaluated with alamar Blue, carboxyfluoroscein diacetate acetoxymethyl ester and neutral red as measures respectively of metabolic activity, plasma membrane integrity, and lysosomal function. Experimental in vitro EC50 values were compared to 1) pre-existing in vivo LC50s from the fathead minnow database and 2) pre-existing in vitro EC50s from the Halle database. Results point to good in vitro/in vivo correlations for menadione, dichlorophene, hexachlorophene, and acrolein. Poor correlations were observed for allyl alcohol, 4-fluoroaniline, acetaldehyde, and 2,3-dimethyl-1,3-butadiene due to a combination of solubility and volatility problems. Overall, the results suggest that the impact of different technical approaches on the evaluation of acute toxicity in vitro depends very much on the chemical class being investigated and less on the characteristics of the cell line. The in vitro cytotoxicity of reactive chemicals is challenging due to the nature of the chemicals’ physicochemical properties. Further improving the in vitro toxicity of reactive chemicals is a prerequisite for the ultimate goal of using fish cell cultures as acceptable, standard alternatives to the use of fish acute lethality assays.

As fish cell cultures continue to be explored as alternatives to whole fish for evaluating the toxicity of environment chemicals, technical issues have emerged that influence results and thus need to be understood and standardized. These include carrier solvents, dosing protocols, exposure vessel, exposure media, viability endpoints, and cell lines. Some of these factors have been explored in this thesis for eight reactive contaminants exhibiting varied physicochemical properties using the rainbow trout cell lines RTgill-W1 and RTL-W1. Sodium dodecyl sulphate (SDS) was used as a reference (control) chemical. Cell viability was evaluated with alamar Blue, carboxyfluoroscein diacetate acetoxymethyl ester and neutral red as measures respectively of metabolic activity, plasma membrane integrity, and lysosomal function. Experimental in vitro EC50 values were compared to 1) pre-existing in vivo LC50s from the fathead minnow database and 2) pre-existing in vitro EC50s from the Halle database. Results point to good in vitro/in vivo correlations for menadione, dichlorophene, hexachlorophene, and acrolein. Poor correlations were observed for allyl alcohol, 4-fluoroaniline, acetaldehyde, and 2,3-dimethyl-1,3-butadiene due to a combination of solubility and volatility problems. Overall, the results suggest that the impact of different technical approaches on the evaluation of acute toxicity in vitro depends very much on the chemical class being investigated and less on the characteristics of the cell line. The in vitro cytotoxicity of reactive chemicals is challenging due to the nature of the chemicals’ physicochemical properties. Further improving the in vitro toxicity of reactive chemicals is a prerequisite for the ultimate goal of using fish cell cultures as acceptable, standard alternatives to the use of fish acute lethality assays.

The soft corals of the species Paralemnalia thyrsoides was found to be a rich source of sesquiterpenoids, such as nor-nardosinane, nardosinane, neolemnane, and eremophilane, and other related skeletons. Continuing investigation on the chemical constituents of Paralemnalia thyrsoides has led to the isolation of nine new compounds, including one dinor-nardosinane 1, one neolemnane 2, five nardosinanes 4, 6¡V9, and two nor-nardosinanes 10 and 11, along with eleven known compounds, 2-deoxy-7-O-methyllemnacarnol (3), 2-deoxy-12£\-methoxy-7-O-methyllemnacarnol (5), paralemnolin Q (12), paralemnolin R (13), 4-acetoxy-2,8-neolemnadien-5-one (15), paralemnolin E (16), flavalins E (17), isoparalemnanone (18), paralemnolin K (19), and nor-nardosinane sesquiterpenoids (14 and 20). The structures of these compounds were determined on the basis of their spectroscopic analysis (1H, 13C NMR, 1H¡V1H COSY, HSQC, HMBC, IR and HRESIMS) and by comparison of the physical and spectral data with those of the related known compounds. The relative stereochemistry and assignments of 1H NMR chemical shifts were determined by NOESY and coupling constants. The absolute stereochemistry of dinor-nardosinane 1 was further determined by application of the Mosher¡¦s method. The cytotoxicity against of P-388 (murine lymphocytic leukemia), HT-29 (human colon adenocarcinoma), and A-549 (human lung epithelial carcinoma) cells as well as the anti-HCMV (human cytomegalovirus) activities of metabolites 1, 2, 4, 6¡V11 were evaluated. Metabolites 2, 3, 5, 6, 8 and 9 exhibited significant activity against P-388 cell in vitro ( ED50 ¡Ø 4 £gg/ mL) Keywords: Paralemnalia thyrsoides, sesquiterpenoids, cytotoxicity, anti-HCMV