Submitted by Carlos Alberto Zanutto Bassetto Júnior null (cbjunior@fc.unesp.br) on 2016-06-30T16:14:01Z No. of bitstreams: 1 DocNãoPublicação_carlosbassettojr.pdf: 427722 bytes, checksum: fe38077e0c0376645f906842534a3f88 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-07-05T16:31:24Z (GMT) No. of bitstreams: 1 bassettojunior_caz_dr_bauru.pdf: 201721 bytes, checksum: afaa803c4d84d32fc5d6b4fc9fe23555 (MD5) / Made available in DSpace on 2016-07-05T16:31:25Z (GMT). No. of bitstreams: 1 bassettojunior_caz_dr_bauru.pdf: 201721 bytes, checksum: afaa803c4d84d32fc5d6b4fc9fe23555 (MD5) Previous issue date: 2016-06-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Esta tese teve como objetivo estudar as moléculas orgânicas (N-alquilbenzenossulfonamidas) como inibidoras de canais para potássio do tipo KV3.1, heterologamente expressos em células L-929. Com o presente estudo constatou-se que as moléculas, N-alquilbenzenossulfonamidas, produzem efeitos inibitórios sobre KV3.1. Através da técnica de whole cell patch clamp, observou-se que os valores de IC50 para as moléculas que bloquearam o canal foram 13,5 μM, 16,9 μM, 25,9 μM, 34,2 μM, 34,9 μM e 60 μM, respectivamente, para 4-cloro-3-nitro-N-butilbenzenossulfonamida (SMD2), 4-cloro-3-nitro-N-furfutilbenzenossulfonamida (SMD3), 4-[N-(3’aminopropil)-2-pirrolidona]-3-nitro-N-butilbenzenossulfonamida (SMD2_APP), 4-[N-(3’aminopropil)-2-pirrolidona]-3-nitro-N-furfurilbenzenossulfonamida (SMD3_APP), 4-cloro-N-butilbenzenossulfonamida (SMD2_SN) e 4-cloro-N-furfurilbenzenossulfonamida (SMD3_SN). O efeito de todas as moléculas mostrou-se reversível quanto à ligação com o canal e todas atuaram como bloqueadores de canal aberto. Em SMD2, molécula que mostrou o menor valor de IC50, observou-se um deslocamento de -8 mV em relação ao controle, nas curvas de condutância versus voltagem, nas cinéticas de ativação e na recuperação a partir da inativação em relação à voltagem. O SMD2 não alterou as constantes de tempo de desativação, embora tenha mudado as constantes de ativação e inativação, além de ter induzido o fenômeno de tail crossover. Observou-se que para potenciais mais despolarizados, ocorreu o alívio do bloqueio (Block Relief). Não foi observado o efeito da dependência do pH para o bloqueio e SMD2 não mudou a seletividade do canal. Constatou-se que pulsos despolarizantes de curta duração induzem efeitos menos intensos, ao passo que pulsos despolarizantes mais longos, produzem efeitos mais intensos de SMD2 sobre o canal. Além disso, foi observado que, quanto mais o canal é usado, ou seja, aberto, mais ele é bloqueado por SMD2. Todos esses dados sugerem que SMD2 não interage com o estado fechado e nem com o estado inativado do canal, mas sim com seu estado aberto, apresentando também um efeito dependente de uso. De um ponto de vista farmacológico, isso indica que SMD2 pode ser uma molécula importante na modulação da atividade dos canais KV3.1, presentes em células com altas frequências de disparos de potencial de ação, podendo constituir uma nova classe de moduladores farmacológicos desses canais. / This thesis had the aim of studying the organic molecules (N-alkylbenzenesulfonamides) that block KV3.1 potassium channel heterologously expressed in L-929 cells. It was found that N-alkylbenzenesulfonamides have restrained effects on KV3.1. Through the whole cell patch clamp technique, it was observed that the values of IC50, for molecules that block the channel, were 13,5 μM, 16,9 μM, 25,9 μM, 34,2 μM, 34,9 μM and 60 μM, respectively 4-Chloro-3-nitro-N-butylbenzenesulfonamide (SMD2), 4-Chloro-3-nitro-N-furfurylbenzene-sulfonamide (SMD3), 4-[N-(3′-Aminopropyl)-2-pyrrolidone]-3-nitro-N-butylbenzenesulfona-mide (SMD2_APP), 4-[N-(3′-Aminopropyl)-2-pyrrolidone]-3-nitro-N-furfurylbenzene-sulfonamide (SMD3_APP), 4-Chloro-N-butyllbenzenesulfonamide (SMD2_SN) e 4-Chloro-N-furfurylbenzenesulfonamide(SMD3_SN). The effect of all molecules was reversible regards to the linking with the channel and all act as open channel blocker. In SMD2, molecule which showed the smallest value of IC50, it was observed a displacement of -8 mV compared to control, for conductance curves versus voltage, for the kinetics of activation and for the recovery from inactivation in relation to voltage. SMD2 did not change the deactivation of time constants, although it changed the activation and inactivation constants, and more, SMD2 have induced tail crossover phenomenon. It was observed that, for more depolarized potentials, there was a block relief. It was not observed the effect of pH dependence for the block and SMD2 did not change the channel selectivity. It was observed that, short duration depolarizing pulses prompt less intense effects, whereas long duration depolarizing pulses prompt more intense effects of SMD2 on the channels. Furthermore, it was observed that the more the channel is used, in an open state, the more it is blocked by SMD2. All of these data suggest that SMD2 does not interact neither with the closed state nor the inactivated state of channel, but with its open state presenting an use-dependent manner, also showing a use-dependent effect. In a pharmacological point of view, this indicates that SMD2 may be an important molecule in the modulation of the activity in the KV3.1 channels, presents in cells with high frequency of firing of action potential and may constitute a new class of pharmacological modulators.

Stilbene phytoalexins such as resveratrol, 1, and the arachidins, including arachidin-1,2, are naturally synthesised by peanut (Arachis hypogaea) plants. The peanut phytoalexins are polyphenolic compounds consisting of a stilbene backbone, with a number of derivatives also possessing a prenyl moiety. These distinctive phytoalexins have gained attention, as they exhibit various biological activities, for instance arachidin-1, 2, has been reported to be more potent than resveratrol, 1, in the inhibition of lipopolysaccharide-induced expression of cyclooxygenase-2 (COX-2) and COX-2 mRNA, in vitro at doses that were low in cytotoxicity. Additionally the various arachidins have recently been shown to exhibit their anti-inflammatory properties, through the inhibition of a number of inflammatory mediator pathways. In this work, various routes into the synthesis of arachidin-1, 2, are described, via use of the Horner-Wadsworth-Emmons (HWE) reaction. Three different methodologies were explored, the first approach involving silyl ether (TIPS or TBDMS) protected benzaldehydes, proved unsuccessful due to cleavage of the silyl ether protecting groups, in basic and/or acidic conditions. This led to an alternative approach, whereby formation of the stilbene backbone proceeded via the regioselective demethylation of an acetal in the presence of sodium metal, subsequent electrophilic substitution using iodomethane and finally acetal hydrolysis of the acetal, gave the isolated aldehyde in moderate yield (52 %). Coupling of the aldehyde with the substituted benzylphosphonate, via the HWE reaction gave the desired trans-stilbene in good yield (86 %), however incorporation of the prenyl side chain proved to be challenging via the Wohl-Ziegler bromination. Further adaptation of the aforementioned route, whereby alkylation using diethyl iodomethylphosphonate, enabled the incorporation of the prenyl moiety and the subsequent construction of the trans-stilbene backbone, gave the 4-(3-methyl-but-1- enyl)-3,5,3',4'-tetramethoxystilbene, 3, albeit in poor yield (47 %). The final step involving demethylation using BBr3 gave arachidin-1, 2, also in poor yield (30 %), nevertheless this approach has been proved to be a successful route for the total synthesis of arachidin-1, 2, however optimised studies are required in order to obtain the desired compound in quantitative yields. Synthetic analogues of resveratrol, 1, are also known for their biological activities, including anti-inflammatory and chemopreventative properties. Recently, the anti-proliferative activity of a number of stilbenesulfonamides, against the National Cancer Institute's 60 (NCI-60) human tumour cell line has been reported. Furthermore, the anti-inflammatory effects of novel heterocyclic methylsulfone and sulfonamide analogues, via inhibition of the COX-2 protein have also been published, however both synthetic routes described require a total of six or seven steps, from the sulfanilamide and are limited to the synthesis of primary sulphonamides (SO2NH2). In this work, an efficient three step synthesis has been designed and successfully implemented, proceeding via chlorosulfonation of diethyl benzylphosphonate, to form the sulfonyl chloride intermediate. Aminolysis of the sulfonyl chloride intermediate was then performed, using a range of primary, secondary and cyclic alkyl amines, as well as aromatic amines; including ammonia, dimethylamine, morpholine and diphenylamine. Finally, formation of the stilbene backbone with various substituted aldehydes, via the HWE reaction offered a short, versatile and alternative route to the synthesis of novel primary, secondary and tertiary trans-stilbene benzenesulfonamides and heterocyclic analogues, in yields of 42 - 100 %. The activity of a selection of the synthesised stilbene benzenesulfonamides was evaluated against the human lung adenocarcinoma epithelial cell line (A549). Amongst the compounds tested, analysis of the data showed that the novel analogue, 4, was found to be the most potent compound, with a GI50 of 0.1 μM. Comparison with the previously published data found analogue, 4, to be approximately 500-fold more potent than the lead compound resveratrol, 1, (GI50 = 51.64 μM) and approximately twice as potent than 5-fluorouracil (GI50 = 0.189μM), a chemotherapy drug used to treat various forms of cancer 8. Overall, these results demonstrate that the total synthesis of trans-arachidin-1, 2, can be achieved via a five step methodology. A versatile route to the synthesis of novel stilbene benzenesulfonamides has also been successfully achieved, amongst the compounds synthesised one appears to show promising anticancer activity, and warrants further investigation (i.e. in vitro studies using other cancer cell lines, and the synthesis of additional compounds using analogue, 4, as a lead compound).