Rozprawy doktorskie na temat „Synthesis Novel Bioactive Steroids”

Nowadays there’s a growing interest in biologically active peptides for the development of new therapeutics; however in some cases, they could not directly use as drugs, due to their inherent limitations, such as rapid metabolism and low oral activity. As a result, peptides are modified into peptidomimetics with specific characteristics, in a rational design. The present PhD project is focused on the synthesis of several peptides and peptidomimetics, structurally different and presenting individual features, properties, targets and pharmaceutical applications. In particular, two are the research studies we’ve developed during the three years, these are the design of novel Carnosine-like derivatives and of new Farnesyl Transferase Inhibitors (FTIs). Concerning the first topic, we investigated how Carnosine (β-alanyl-L-histidine) structural changes influence its role as scavenger of HNE (4-hydroxy-trans-2,3-nonenal) and other toxic aldehydes. For this reason we modified the carnosine structure firstly replacing the Hystidinil- portion with different aromatic system, secondly substituting the β-alanyl portion with ten different amino acids, chosen in order to cover exhaustively the available chemical space. Finally we rigidified the whole structure, inserting a 2-oxazolidinone; the entire compound underwent biological evaluation, testing their ability to quench HNE. As a result, some of the twenty dipeptides showed impressing scavenging activities and great selectivity towards toxic aldehydes, suggesting us that they can represent truly promising candidates for the design of improved carnosine derivatives. Regarding the second subject, we designed, synthesized and tested several peptidomimetics of the CAAX box, where CAAX is the sequence Cysteine-Valine-Isoleucine-Methionine, able to block the farnesylation of RAS proteins and therefore cell proliferation. The design started from a nanomolar range FTI, previously synthesized by our group, where the central dipeptide (AA) is replaced with a 4-amino-2-o-tolylbenzoyl spacer and the Cysteine (C) with the residue 2-amino-4-thiazolylacetyl. The synthesis of the novel FTIs followed two separate approaches; at first we kept the aromatic spacer and modified the N-terminal residue with other heterocycles; the unimproved antiproliferative activity suggested us to apply other kind of modification. Therefore we replaced the o-tolyl with six heteroaromatic residues, in addition the synthesized compounds presented, as N- terminal residue, the 2-amino-4-thiazolylacetyl itself or the 1,4-benzodioxan-2-ylmethyl or the 1,4-benzodioxan-2-ylformyl. In all the three series of compounds, the 2-thienyl, 1-naphtyl and the 3-furanyl derivatives showed the highest FTase inhibition, at low micromolar level. Taken together, our biological activities provide interesting results, confirming that peptides and peptidomimetics should be employed as therapeutics.

At the present there are 36 approved antiviral drugs in the UK of which half are nucleoside analogues. However, the emergence of drug resistance and of new virus strains necessitates new drugs. In particular in this thesis, different nucleoside analogues were studied as potential antivirals. One of the major issues related to nucleoside analogues is the emergence of resistance due to a lack of bioactivation to the monophosphate form. To overcome this issue, the phosphoramidate ProTide technology can be applied. This strategy allows the delivery of the monophosphate form directly inside the cell. Bicyclic nucleoside analogues are a new class of anti-varicella zoster agents of which Cfl743 is the most potent anti-varicella zoster compounds reported to date. Its 5'-valyl derivative, FV100, is currently in phase II clinical trials. A series of derivatives to increase the activity and to investigate the mechanism of action of this new class of compound are reported. Moreover, attempts to improve the scale up synthesis of FV100 are described. Ribavirin is a broad spectrum antiviral drug. The application of the ProTide approach to this compound was not successful. Enzymatic and molecular modelling studies have been performed in order to understand the lack of activity. Acyclovir and its esters are currently the treatment of choice for herpes simplex and varicella-zoster infections. The application of the ProTide technology gave surprising results. In fact, these compounds have been found to be active against HIV, whilst ACV itself did not show any activity. Moreover, these compounds retained activity versus thymidine kinase deficient strains against which acyclovir lost activity. These striking results prompted us to investigate other different nucleoside analogues, through a virtual screening using reverse transcriptase, guanylate or adenylate kinase and human polymerase y. The selected nucleoside analogues from this study include: ganciclovir, penciclovir and their derivatives. ProTides of these are thus pursued.

Throughout history natural products have served as a valuable resource for the treatment of various diseases. To this day, many of these ancient remedies and their derivatives are still in use. However, in recent years, antibiotic discovery has stagnated at a time where there has also been the alarming emergence of antibiotic resistance which threatens to undermine the effectiveness of existing therapies. As such, novel antimicrobial agents are urgently needed that possess unique mechanisms of action to those currently in use. A promising class of natural products that exhibit antimicrobial activity are the non-ribosomal peptides (NRPs). This thesis describes the synthesis and biological evaluation of a number of NRP-derived molecules with a view to discovering new antibiotic leads. Chapters 2 & 3 describe the design and synthesis of potent anti mycobacterial derivatives based on the sansanmycin natural product scaffold. This involved the development of a late stage diversification route, thereby providing a platform for expedient access to a diverse library of analogues. These analogues were subjected to biological evaluation, from which key structure activity relationships were elucidated for inhibition of the growth of Mycobacterium tuberculosis – the etiological agent of tuberculosis (TB). These studies ultimately led to the identification of a new lead compound which was further evaluated in two animal models of TB infection. Chapter 4 describes efforts towards the synthesis of the janthinocins, a class of NRPs with potent activity against Gram positive bacteria. The synthetic strategy relied on late stage, chemoselective transformations to install key functionalities namely, selective oxidation of tryptophan to afford β ketotryptophan and elimination of β methylcysteine to form dehydrobutyrine. A deoxy-variant of janthinocin B was successfully synthesised which was subjected to late stage oxidation to the parent natural product, albeit on a small scale.

Hepcidin is a 25 amino-acid peptide hormone with an essential role in the systemic regulation of iron. The dysregulation of hepcidin is linked to a number of iron loading disorders such as hereditary haemochromatosis and anaemia of inflammation. Procuring synthetic, recombinant or endogenous hepcidin is challenging and expensive; therefore strategies to improve the synthesis of hepcidin and add modifications to extend its short half-life in vivo are required for potential therapeutic applications. Industrial scale production of a peptide such as hepcidin for therapeutic use would likely be performed using a convergent approach, involving the ligation of peptide fragments. Native chemical ligation (NCL) is a powerful tool in peptide and protein synthesis, allowing peptide fragments to be joined together through a chemoselective ligation between a peptide-thioester and a cysteinyl peptide. Preparation of peptide¬thioesters is challenging, however thioester formation via 1 U 6 acyl transfer at cysteine residues allows generation of thioesters in both synthetic and recombinant peptides and proteins. Here, investigations into optimising the preparation of peptide-thioesters via 1 U 6 acyl transfer have been carried out to extend its application to the synthesis of complete hepcidi n, analogues and labelled variants. This led to the development of a novel, faster route to peptide thioesters via 1 U 6e acyl transfer, which was applied to the cyclisation of peptides via in situ NCL following thioester formation. Additionally a novel methodology via 1 U 6 acyl transfer has been established for the preparation of peptide hydrazides, which are stable alternatives to peptide-thioesters in NCL. These novel strategies have been applied to full-length hepcidin and fragments of hepcidin, which in conjunction with NCL, have been used to generate a variety of hepcidins. A measurable reduction in the iron efflux membrane protein, ferroportin, in cells treated with these analogues, confirmed their biological activity, indicating hepcidin-induced internalisation of ferroportin, including for hepcidin biotinylated using the hydrazide-NCL strategy.

A stereoselective synthesis of (25S)-Δ1-, (25S)-Δ1,4-, (25S)-Δ1,7-, (25S)-Δ8(14)-, (25S)-Δ4,6,8(14)-dafachronic acid, methyl (25S)-Δ1,4-dafachronate and (25S)-5α-hydroxy-3,6-dioxocholest-7-en-26-oic acid is described. (25S)-Δ1,4-Dafachronic acid and its methyl ester are natural products isolated from corals and have been obtained by synthesis for the first time. (25S)-5α-Hydroxy-3,6-dioxocholest-7-en-26-oic acid represents a promising synthetic precursor for cytotoxic marine steroids
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

A stereoselective synthesis of (25S)-Δ1-, (25S)-Δ1,4-, (25S)-Δ1,7-, (25S)-Δ8(14)-, (25S)-Δ4,6,8(14)-dafachronic acid, methyl (25S)-Δ1,4-dafachronate and (25S)-5α-hydroxy-3,6-dioxocholest-7-en-26-oic acid is described. (25S)-Δ1,4-Dafachronic acid and its methyl ester are natural products isolated from corals and have been obtained by synthesis for the first time. (25S)-5α-Hydroxy-3,6-dioxocholest-7-en-26-oic acid represents a promising synthetic precursor for cytotoxic marine steroids.
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

It is desirable for a dental restorative material to have bioactive and bonding properties. This study focuses on the synthesis of a covalently-linked polyurethane/nanohydroxyapatite (PU/nHA) composite and evaluates its chemical, physical, thermal and biochemical characteristics. nHA powder was produced from the sol-gel and novel composite material was chemically prepared by utilising solvent polymerisation. The resulting composites were analysed by chemical, thermal, and mechanical characterisations and electrospun to form fibre mats. The composites were hydrolytically degraded in deionised water and phosphate buffer solution (PBS) and were analysed. Bioactive behaviour was determined in modified-simulated body fluid. The bioadhesion with dentine was analysed in distilled water and artificial saliva. Cell growth and proliferation was measured and number of adhering bacteria was determined and serial dilution followed by plating for colony forming units per disc. Spectral analyses showed the grafted isocyanate and ether peaks on nHA indicating that urethane linkage was established. Covalent-linkage between nHA and PU were found in this novel composite with no silane agent. The physical and thermal properties were enhanced by nHA. These composites had high resistance toward hydrolysis and little degradation was observed. Bioadhesion and bioactivity analysis showed the composite adhered firmly on the tooth surface (dentine) and bond strength was similar to existing obturating material. Higher nHA content composite showed a thicker layer of adhesion. Cells were proliferated although at a lower rate of growth compared to PU, whereas, there was reduction in bacteria adhering to the grafted composite compared to PU. With its low bacterial adhesion and biocompatibility it may provide a promising solution to reduce infections. The electrospun nano-fibres were successfully developed and revealed no loose nHA particles. Hence, this novel composite has the potential to be used as a bioactive dental restorative material.

Améliorer la stabilité de l’interface dentine adhésif est primordial si on veut prolonger la durée de vie et la longévité des restaurations en composite. La reminéralisation utilisant des matériaux relarguant des ions semble etre une approche prometteuse pour protéger la couche hybride de la dégradation hydrolytique et enzymatique. Les verres bioactifs mésoporeux (MBG) présentent des caractéristiques de surface intéressantes qui permettent de les utiliser comme charges au sein de l’adhésif qui favorisent d’une part la reminéralisation par la libération d’ions, d’autre part la fonctionnalisation des pores par des médicaments antibactériens. Ces caractéristiques permettent de lutter contre la reprise de carie. Le présent travail porte sur la synthèse et l'évaluation de nouveaux MBG pouvant être utilisés comme charges dans les adhésifs dentaires. Les MBG ont été préparés dans un système sol-gel à base d'acétate avec des précurseurs industriellement sûrs et non toxiques. Les MBG préparés à grande échelle (50g) offrent des caractéristiques de surface améliorées par rapport aux MBG à petite échelle (10g). L'étude de l'influence des modificateurs de réseau (CaO: Na2O) sur les caractéristiques de surface des MBG a révélé que la porosité était influencée par la teneur en CaO dans la composition. Une surface spécifique très élevée (535 m2g-1) et un volume de pores (0,33 cm3) ont été atteints dans le MBG avec la plus forte teneur en CaO. Par la suite, l'ordre de l'effet d'addition des précurseurs sur les caractéristiques de surface des MBG a été étudié. En maintenant la composition fixée et en faisant varier l'ordre d'addition du précurseur pendant la synthèse sol-gel, on a obtenu une augmentation de 1,5 fois du volume des pores et une diminution de 1,2 fois de la taille moyenne des pores. La méthode démontrée est une voie simple et directe pour améliorer la porosité et l'homogénéité des MBG. De plus, la modulation de la taille moyenne des pores pour une composition fixe est également utile pour une éventuelle application d’administration de médicament. En ce qui concerne la bioactivité, les charges de MBG ayant la plus forte teneur en CaO ont augmenté le précipité de phosphate de calcium dans le SBF après 7 jours, par opposition au MBG avec une teneur élevée en Na2O. De plus, tous les échantillons testés étaient non cytotoxiques pour les fibroblastes gingivaux humains (HGF) in vitro. De manière positive, les MBG traités à basse température ont significativement augmenté l'activité métabolique des HGF. Le broyage à boulets a été utilisé pour réduire la taille des particules primaires de MBG à moins de 3 µm. Le fraisage a visiblement eu un effet néfaste sur la porosité des charges MBG. Néanmoins, une certaine porosité est restée. L'adhésif commercial a été mélangé avec 3, 10, 20 et 30% en poids de charge MBG. L'adhésif rempli de MBG jusqu'à 10% en poids de charge a une viscosité fluide compatible avec l’application d'un adhésif. Le MBG ainsi développé avec une haute porosité et un contenu en CaO représentent des charges prometteuses pour des futures applications en dentisterie restaurative et régénérative
Improving the stability of adhesive dentin interface is crucial to extend the longevity of composite restorations. Remineralization through use of ion releasing materials is a promising approach to protect the hybrid layer from hydrolytic and enzymatic degradation. Mesoporous bioactive glasses (MBGs) offer attractive surface features (enhanced surface area and porosity) to use them as fillers in dental adhesives to promote remineralization through ions release. Moreover, the functionalization of pores with antibacterial drugs is a good way to combat secondary caries. The present work focused on the synthesis and evaluation of novel MBGs suitable to be used as fillers in dental adhesives. The MBGs were prepared in an acetate based sol-gel system with industrially safe and non-toxic precursors. MBGs prepared in large scale (50g) offered enhanced surface characteristics in comparison to small scale (10g) MBGs. The investigation on the influence of network modifiers (CaO:Na2O) on the surface characteristics of MBGs revealed that the porosity was driven by CaO content in the composition. Notable, very high surface area (535m2g-1) and pore volume (0.33cm3g-1) was attained in the MBG with highest CaO content. Next, the order of precursor addition effect on the surface characteristics of MBGs has been studied. By Keeping the composition fixed and varying the order of precursor addition during sol-gel synthesis a doubling of surface area, 1.5 times increase in pore volume and 1.2 times decrease in mean pore size was obtained. The demonstrated method is a simple and straightforward route to improve the porosity and homogeneity of MBGs. Furthermore, modulation of mean pore size for a fixed composition is also useful to tailor the pores of the fillers for drug delivery application. With regards to bioactivity, the MBG fillers with highest CaO content had increased calcium phosphate precipitate in SBF after 7 days as opposed to MBG with high Na2O content. Furthermore, all tested samples were non-cytotoxic to Human Gingival Fibroblasts (HGFs) in vitro. Positively, MBGs treated at lower temperature significantly enhanced the metabolic activity of HGFs. Ball milling was employed to reduce the primary particle size of MBG to less than 3μm. Milling seemingly had an adverse effect on the porosity of the MBG filler. Nevertheless, some porosity remained. The commercial adhesive was mixed with 3, 10, 20 and 30 weight percentage of MBG filler. MBG filled adhesive up to10 weight percent filler content had flowable viscosity suitable for adhesive application. The developed MBG with high porosity and CaO content appears as a new step in the development of dental adhesives and also other bioactive dental materials

During my research career in Prof. V.J.Hruby's laboratory I worked on two different projects. The first project, which was initiated by the author, was planned to serve the need of our laboratory for a novel method of peptide cyclization. This method was planned to use recent advances in Pd0-catalyzed asymmetric synthesis combined with the structural richness offered by the Baylis-Hillman chemistry which could open new ways to diverse areas of drug design, molecular immunology and chemotherapy. This approach would provide cyclic peptides featuring N-alkylated amino acids that would confer high resistance to degradation by proteases. Because of numerous synthetic problems imposed, this strategy was not of considerable current use in peptide synthesis, especially on solid supports. However, despite a substantial amount of effort invested, this method faced serious drawbacks such as multistep synthesis and side reactions when applied to solid supports. Moreover, recent introduction of microwave technology which has helped to solve a great number of problems has led to a renaissance in the classical lactam and thioester bond cyclizations which overshadowed our quest for a novel methodology. The second project was focused on application of 4-anilidopiperidines for the synthesis of chimeric bioactive peptides. It was an effort towards the development of novel analgesics with reduced toxicity and enhanced potency. This project linked small molecule and multimeric ligand designs that were ongoing in our laboratory at the time. Major accomplishments in this project were made possible by successful resolution of several research challenges. I was able to find a straightforward, convenient and economical approach for the synthesis of novel analogues on a solid support. These developments led to novel compounds which showed substantial increases in their binding affinity relative to corresponding opioid analogues. To illustrate, compounds PET25, 26, 27, 29, 30, 31, and 32 showed high bioactivity and sub-nanomolar binding affinity to opioid receptors. Most of the peptides generated in the second project are still being investigated for their biological activities by our colleagues at the Department of Pharmacology, but the results to date indicate that some highly potent novel compounds have been made.

碩士
國立高雄海洋科技大學
水產食品科學研究所
97
Pregna-1,20-dien-3-one (Pregna), one of pregnane steroids, was obtained from column chromatography of chloroform extract of Umbellulifera striata on Silica gel. Our results showed the Pregna exhibited more growth inhibitory effect on HL-60 than other tested human leukemia cells, such as U937, THP-1, Jurkat. Pregna inhibited cell proliferation and induced apoptosis in a concentration- and time-dependent in HL-60 cells. Treatment of HL-60 cells with pregna, it resulted in dissipation of mitochondrial membrane potential, release of mitochondrial cytochrome C into cytosolic, activation of caspases 9 and -3, disruption of DNA integrity, and induction of apoptosis Moreover, treatment with Pregna caused a rapid loss of intracellular glutathione content and stimulation of reactive oxygen species (ROS) generation. N-acetylcysteine (NAC), an antioxidant, and the pan-caspase inhibitor, z-Val-Ala-Asp-fluoromethyl ketone (z-VAD-FMK), significantly inhibited Pregna-induced cell death. Taken together, it suggests that induction of apoptosis by Pregna may be modulated by generation of ROS and activation of caspase activity. According to our results, Pregna may be a promising anti-cancer drug and need to further study the molecular mechanism of anti-cancer effect.

Carbohydrates are natural aliphatic polyhydroxy aldehydes and ketones with an empirical formula of Cn(H2O)n that are distributed throughout our planet. This class of compounds have a central role in numerous physiological events, such as in cellular communication, and are part of the basic structure of some endogenous biomolecules. It also presents several chiral carbons that confers them stereogenic centres, which is an advantage for the selective interaction with the biological targets. In fact, carbohydrates are considered privileged scaffolds and can be linked to several pharmacophores, such as heterocycles, forming pseudo-C-nucleosides, which are composed by a heterocyclic ring attached to the carbon 4 of the furanose ring by a carbon-carbon bond. Therefore, they are largely used at the therapeutic level in the treatment of several illnesses. As an important example the association between carbohydrates and the treatment of bipolar disorder occurred through the discovery of an anticonvulsive compound, topiramate. Bipolar Disorder (BD), also known as manic-depressive illness, affects between 1 to 4% of the world's population and is characterised by recurrent mood changes, named manic or depressive episodes. In a manic episode, the patient is extremely euphoric, while in depressive episodes the patient feels depressed and may present suicidal tendencies. Currently, the most effective drug on the market for the treatment of this disorder is Lithium Carbonate. However, this inorganic compound has very significant adverse effects. Despite a large group of studies that have been developed in this context, it has not yet been possible to clearly identify the pathological basis of this disease as well as the mechanism of action of Lithium Carbonate. Thus, as presently there is a clear lack of available pharmacological alternatives for the treatment of this disease, the objective of this dissertation was to synthesise pseudo-C-nucleosides with potential interest for the treatment of BD and subsequent evaluation of cytotoxicity. For this purpose, three different techniques to synthesise the pseudo-C-nucleosides were performed, the Click Chemistry, the cyclization to form pyrazolidin-3-ones and the cyclization to form 5-hydroxypyrazoles. From these three strategies seven different compounds, six of which are pyrazolidin-3-one derivatives and one 5-hydroxypyrazole, have been obtained. Finally, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assays were performed to evaluate the cytotoxicity of the final compounds synthesised in two different cell lines, NHDF (Normal Human Dermal Fibroblasts) and N27 (Rat Mesencephalic Dopaminergic Neural Cells). In both cell lines, the compounds under study did not show relevant cytotoxicity. Thus, even if it was not possible to infer their potential for the treatment of BD, the results obtained for cytotoxicity are positive indicators to continue to develop tests to evaluate the activity of this compound for the treatment of this disorder.
Os açúcares são compostos naturais que se encontram disponíveis no nosso planeta. Estes compostos são constituídos por uma cadeia carbonada alifática com um grupo carbonilo, na forma de cetona ou de aldeído, e com um grupo hidroxilo ligado a cada um dos restantes carbonos. Acrescem ainda os compostos obtidos por reações de oxidação e de redução. São considerados derivados de glúcidos os compostos obtidos por substituição de um grupo hidroxilo por um halogéneo, um hidrogénio, uma cadeia carbonada, entre outras modificações. Esta classe de compostos começou a ser estudada no século XVIII, mas a maior parte dos avanços de elucidação estrutural e funcional só aconteceram no século XIX, com os estudos desenvolvidos por Emil Fischer. No entanto, só a partir do século XX, e com os avanços da ciência e da tecnologia é que se conseguiu estudar esta classe de compostos no seu expoente máximo. Consequentemente, foram descobertos inúmeros processos fisiológicos nos quais os açúcares ou os seus derivados participavam e, por isso, começou a tentar-se desenvolver compostos derivados de glúcidos com propriedades farmacológicas. Este interesse surgiu, naturalmente, não só pela função dos glúcidos no organismo mas também devido à sua estrutura, que evidencia um elevado número de carbonos quirais e que, consequentemente, tem um elevado número de centros estereogénicos que permitem a interação seletiva com alvos biológicos. Contudo, devido à quantidade de grupos hidroxilo livres, os açúcares, por si só, não são moléculas adequadas para serem utilizados como fármacos, por se revelarem demasiado polares, e, portanto, as suas propriedades farmacocinéticas não são as mais apropriadas, tendo, por isso, de ser objeto de modificações estruturais. Por todos estes motivos, os açúcares são considerados scaffolds privilegiados e podem ser ligados a diferentes farmacóforos, como por exemplo anéis heterocíclicos, formando nucleósidos e seus derivados. Dentro dos derivados dos nucleósidos, estão incluídos os pseudo-C-nucleósidos, que são constituídos por um anel heterocíclico que se encontra ligado ao carbono 4 do anel de furanose através de uma ligação carbono-carbono. Atualmente, já se encontram disponíveis vários fármacos com propriedades farmacológicas completamente distintas que têm na sua base estrutural um derivado de açúcar. Dentro das classes de fármacos representadas por, pelo menos, um composto glucídico estão os anticancerígenos, os antivirais, os agentes anti-Alzheimer e os anticonvulsivantes. Um composto importante que pertence à classe dos anticonvulsivantes é o topiramato. Este é constituído por um anel de piranose, com dois grupos isopropilideno, um ligado na posição 2 e 3 e outro na posição 4 e 5, e um grupo sulfamida ligado ao carbono 1. O topiramato bloqueia canais de sódio, potencia a ação do neurotransmissor inibitório GABA (ácido ?-aminobutírico) e é considerado um antagonista do neurotransmissor excitatório glutamato por atuação nos recetores AMPA (ácido a-amino-3-hidroxi-5-metil-4-isoxazolepropiónico) e cainato. Neste âmbito já existem estudos que confirmam a atividade deste composto no tratamento do transtorno bipolar (BD, do inglês, Bipolar Disorder), sendo este um ponto-chave do presente trabalho de investigação. O BD, também conhecido como transtorno maníaco-depressivo, afeta entre 1 a 4% da população mundial e carateriza-se por mudanças de humor recorrentes, denominadas como crises maníacas ou crises depressivas. As crises maníacas são, normalmente, fases em que a pessoa se apresenta extremamente eufórica em que o tratamento pode ser efetuado através de fármacos antipsicóticos, enquanto que as crises depressivas são fases em que a pessoa se sente deprimida, podendo, inclusivamente, apresentar tendências suicidas, e em que o tratamento pode ser efetuado através de fármacos antidepressivos. Atualmente, o fármaco mais eficaz que se encontra disponível no mercado para o tratamento deste transtorno é o Carbonato de Lítio. Este é um composto inorgânico e origina efeitos adversos muito significativos. Quando o tratamento em monoterapia não resulta, este fármaco pode ser combinado com o ácido valpróico ou com agentes antioxidantes. Contudo, este tipo de estratégia não é a mais adequada, uma vez que cerca de 37% dos pacientes medicados têm, pelo menos, uma crise de mania ou de depressão em menos de 1 ano, elevando-se para cerca de 60% em menos de 2 anos. Efetuando uma análise objetiva dos dados disponíveis, poder-se-á concluir que os fármacos que se encontram disponíveis no mercado são inefetivos contra este transtorno. Apesar dos estudos que se têm desenvolvido, ainda não foi possível identificar claramente as bases fisiopatológicas do BD. Contudo, sabe-se que existem alterações a nível de atividade de determinadas proteínas, sendo que a mais marcada é a hiperatividade na família das GSK-3 (Glicogénio Sintase Cinase 3), particularmente da GSK3B. Esta enzima é conhecida por estar envolvida no metabolismo energético celular e no desenvolvimento neuronal e, portanto, a sua hiperatividade desencadeia uma cascata de sinalização celular que pode originar danos neuronais irreparáveis e, inclusivamente, ser causa de morte neuronal. Apesar de ser ainda parcialmente desconhecido, tem-se assumido que o mecanismo de ação do Carbonato de Lítio se baseia no desencadeamento de múltiplos sinais e cascatas intracelulares que ocorrem através da inibição ou da ativação de recetores membranares. A principal via de atuação envolve a inibição dos canais de cálcio associados aos recetores NMDA (N-metil-D-aspartato), que são recetores do neurotransmissor excitatório glutamato. A inibição destes recetores, neste caso, permite proteger, a nível neuronal, o cérebro da excitotoxicidade1 provocada pelo excesso deste neurotransmissor. Atualmente, por falta de alternativas eficazes, a indústria farmacêutica tem vindo a utilizar técnicas de HTS (do inglês, High Throughput Screening) para avaliar fármacos comercializados para outras doenças do sistema nervoso central, tais como a esquizofrenia e a epilepsia, natentativa de encontrar compostos potencialmente bioativos destinados ao tratamento do BD. Os resultados têm sido promissores, particularmente no caso dos fármacos utilizados para o tratamento da epilepsia, que têm demonstrado maior eficácia, uma vez que a maior parte deles também atua em vias igualmente relevantes no BD. Como já referido, um dos exemplos mais relevantes neste contexto é o fármaco glucosídico topiramato. Assim sendo, existe clara necessidade de alargar as alternativas farmacológicas disponíveis para o tratamento desta doença, contribuindo para a melhoria da qualidade de vida daqueles que padecem desta doença. Além disso, estruturas glicosídicas com ação anticonvulsivante, podem ser um bom ponto de partida no desenvolvimento de compostos com esta atividade farmacológica. Desta forma, o objetivo desta dissertação foi sintetizar pseudo-C-nucleósidos, com potencial atividade para o tratamento do transtorno bipolar e posterior avaliação da citotoxicidade. Assim, tendo em conta a estrutura base do topiramato, foram realizadas alterações estruturais que incluíram a passagem do anel glucídico de piranose para furanose e a ligação de anéis heterocíclicos em vez de uma cadeia carbonada. Os triazóis, as pirazolidin-3-onas e os 5-hidroxipirazóis foram os heterociclos escolhidos para serem ligados aos açúcares. A pertinência da escolha destes substituintes é justificada pela presença de átomos de azoto na sua estrutura, que está descrita na literatura como vantajosa para a atividade biológica requerida, bem como na, já existente, atividade reportada que as mesmas estruturas apresentam a nível de doenças neuropsiquiátricas. Para a ligação do anel triazol ao açúcar, a técnica utilizada foi a Click Chemistry. No entanto, apesar de todas as tentativas realizadas não foi possível sintetizar nenhum composto através desta via. Foram utilizadas diversas estratégias, incluindo a proteção do grupo hidroxilo da posição 3 do anel de açúcar por três grupos protetores diferentes, benzilo, metilo e acetilo, bem como duas modificações estruturais que consistiram na substituição do grupo hidroxilo por iodo, num caso, e por hidrogénio no outro. Para a síntese do derivado pirazolidin-3-ona, foi efetuada uma ciclização de um éster a, ß-insaturado com a hidrazina monohidratada. Com este anel foram sintetizados dois conjuntos de três compostos, um par de isómeros, o S e o R, e um tautómero hidratado do heterociclo. [...]

DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL PSEUDOPEPTIDES AS INHIBITORS OF RHODESAIN OF T. brucei rhodesiense Human African Trypanosomiasis (HAT) is caused by a protozoan of Trypanosoma genus. There are two forms of HAT, the chronic form caused by Trypanosoma brucei gambiense and the acute form caused by Trypanosoma brucei rhodesiense. This infection is transmitted to humans by the bite of a fly belonging to Glossina genus (tsetse fly). There is an urgent need to find new targets for HAT treatment because current therapy shows many problems. In this research area, rhodesain, a cysteine protease that plays a key role in Trypanosoma brucei rhodesiense life cycle, can be considered a promising target for HAT treatment. Our research team has been involved in the last decade into the development of rhodesain inhibitors as novel agents for HAT treatment. In particular, I focused my research on the development of peptidomimetics or pseudopeptides as inhibitors of rhodesain. Starting from the structure of the reversible rhodesain inhibitors 1a-c, we designed a new series of peptidomimetics 2a-g maintaining the benzodiazepine scaffold as a β-turn mimetic. We introduced a characteristic peptide sequence for rhodesain inhibition, i.e. Phe-HomoPhe P2-P1, finally the 3-bromoisoxazoline warhead was replaced with a vinyl ester moiety, able to react as Michael acceptor, while at the P1’ site we selected a panel of aliphatic or aromatic nuclei to evaluate the size of the corresponding S1’ pocket. Moreover, starting from the potent irreversible rhodesain inhibitor 3, we designed a new series of dipeptide nitriles 4-5 a-f , showing a Phe or a Leu residue at the P2 site, strongly preferred by rhodesain. The amino group of the P2 substituent was protected with a series of variously decorated substituents, spanning into the P3 region, with the aim to optimize the interactions with the S3 pocket. While the hPhe residue at the P1 site was kept unchanged, due to the strong affinity for rhodesain S1 pocket; on the contrary, the vinyl ketone warhead was replaced with a nitrile group, to explore its reactivity towards the catalytic cysteine residue. All the new synthesized inhibitors were tested against rhodesain to evaluate their inhibitory properties. Their selectivity profile for the target protease was also evaluated by testing inhibitors against the cathepsin L, a human cysteine protease belonging to papain family, like rhodesain. The most active compounds were also tested against Trypanosoma brucei brucei to evaluate their antitrypanosomal activity. Docking studies on the most active inhibitors will allow us to clarify the binding mode of the new class of inhibitors. During my PhD work I considered the possibility to combine different inhibitors, e.g. a natural product and a synthetized inhibitor, to evaluate the combination effect on the rhodesain inhibition. The first study has been based on the combination of curcumin and RK-52, which showed an impressive potency (k2nd = 67 × 106 M−1 min−1) and a picomolar binding affinity of 38 pM against rhodesain. Curcumin is a natural product obtained from Curcuma longa L. endowed with many biological properties, more recently curcumin was shown to inhibit rhodesain of T. b. rhodesiense with an IC50 value of 7.75 µM. In the present work, we designed drug-combination studies at five selected combined doses by Chou and Talalay method for the evaluation of the potential synergistic or additive effects for the inhibition of rhodesain. Another combination study that has been carried out is based on the combination of quercetin and PS-1. The first one is a polyphenolic compound with a well-known antiparasitic activity. The second one is a rhodesain inhibitor with a Ki in a picomolar range. Also in this case, we evaluated by Chou and Talalay method if the combination PS-1 and quercetin could exert a synergistic or additive effects for the inhibition of the trypanosomal protease. In conclusion, the combination study let us to identify if the combination of two drugs induces an antagonist, additive or synergic effect. If an additive or synergic effect occurs, this could reduce some problems related to the toxicity because in this way the two inhibitors could be administer at a lower dose.
DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL NONPEPTIDE MOLECULES AS ANTILEUKEMIC AGENTS Diarylpentanoids are natural products extracted from several vegetables, fruits, teas and other plants. The chemical structure of these compounds is characterized by two aromatic rings linked by a five carbon (C5) bridge. These compounds could be considered as monocanrbonyl anlogues of curcumin (Figure 45), a natural product isolated from Curcuma Longa L., that has several pharmacological properties. These derivatives are characterized by the replacement of C7 bridge of curcumin with a C5 bridge, for this reason they are also called C5-curcuminoids. The diarylpentanoids are more stable either chemically or metabolically than curcumin, because of the replacement of the unstable β-diketone moiety. Among the various activities of the diarylpentanoids the most important is the antitumor activity. During my PhD work, I synthesized a new series of symmetrical diarylpentanoids 112a-p which were characterized by a dienone moiety and by a different pattern of substitution on the two phenyl rings. In this regard, we investigated the impact on cytotoxicity of electron-donating (methoxy) or electron-withdrawing (halogen) groups, which were introduced in ortho, meta or para position of both the aromatic rings. This choice was based on literature data, which attest that the antitumor activity is generally influenced by the number of substituents on the phenyl rings. In fact, the general cytotoxicity seems to enhance with an increasing number of substituents, while the cytotoxicity decreased, if the number of substituents reached eight. These compounds were tested against human acute lymphocytic CCRF-CEM leukemia cells and CEM/ADR5000 cells, a multidrug-resistant sub-line derived from drug-sensitive, parental CCRF-CEM cells developed in vitro. The results of this investigation will be reported and discussed in the next sections.

碩士
國立臺灣海洋大學
生物科技研究所
98
Biomaterial researches have been improved in past years. Mesoporous silica has stable structure, high surface area, size-controllable pores, functionalized surfaces, nontoxic and high biocompatibility. Mesoporous silicas also have some advantages in drug delivery system（DDS）,for example：the reappearance of the drug release curve, nontoxic after collapsed of the material, and not easily attacked by immune system. Mesoporous bioactive glass development has been rising in recent years, it combined mesoporous silica and bioactive glass together. The combined material maintained high surface area characteristics and with its highly bioactive property, it can form hydroxyapatite in bone tissue. By collocation with functionalized surface and osteogenesis components, MBGs open the door of the designing of novel mesoporous materials to the special medicinal applications. This project tries to modify the pore size by changing different synthesis temperature, and using 4-Acetamidophenol as a model drug to discuss the drug storage and release behavior of different pore size, and to study if they can be a proper carrier in DDS. This thesis utilizes in situ hydrothermal to synthesize new lanthanide crystal [Ln(μ1-tzbc)(μ2-tzbc)(OH)(H2O)4]2（tzbc = Tetrazole benzylcarboxylate）, containing carboxyl functional group. The crystal system in this series of lanthanide compounds are monoclinic and space group P-1. For Gadolinium: a(Å)=8.3531(3),b(Å)=10.4026(4)，c (Å)=13.4249(3), α(°)=109.913(3),β(°)=101.288(2),γ(°)=101.114(3).This crystal is constructed by two lanthanide atoms as a structure center which linked four carboxyl functional group, two μ1 mode and two μ2 mode. In physical test we did element analysis, FT-IR, thermal gravimetric analysis, luminescent tests, and in bio-test we discussed the interaction between compound and double helix.