La naissance avant terme constitue un important problème de santé périnatale partout dans le monde. Chaque année près de 15 millions de bébés naissent prématurément. Notre laboratoire s’intéresse depuis une décennie à la conception et le développement de nouvelles classes thérapeutiques (appelés agents tocolytiques) capables d’inhiber les contractions utérines et prolonger le temps de gestation. Due à son implication directe dans le déclanchement des contractions utérines, la prostaglandine F2α (FP) est devenue notre cible de choix. Le PDC 113.824 et aza-glycinyl-proline sont apparus comme des inhibiteurs allostériques puissants du récepteur de la prostaglandine F2α, capables de prolonger la durée de gestation chez les souris. Le travail réalisé au cours de ce mémoire a pour objectif d’étudier le tour β nécessaire pour la reconnaissance sur le récepteur de la prostaglandine F2α. Dans la conception de mime peptidique sélectif efficace et puissant, le repliement β est un élément structural essentiel pour le maintien ou l’amélioration de l’activité du mime peptidique. Les études conformationelles du PDC113.824 et l’aza-glycinyl-proline montrent que les squelettes centraux de ces mimes peptidiques pourraient adopter essentiellement un tour β de type I ou II`, ce qui suggère l’implication des deux types de tours dans l’activité biologique. La synthèse de mimes peptidiques adoptant le tour β de type I et II` est devenue une stratégie logique dans la recherche de nouveaux agents tocolytiques. Au cours de ces études quatre analogues du PDC113.824 ont été synthétisés, un azabicyclo[5.3.0]alkanone et un dipeptide macrocyclique (mimes du tours β de type I) pour étudier l’implication du tour β type I. Par la suite la synthèse de glycinyl-proline et le D-alaninyl-proline (des mimes du tour β du type II`) a été réalisée afin de valider l’implication du tour β type II` dans la reconnaissance sur le récepteur. Nous avons utilisé une stratégie de synthèse efficace pour obtenir les deux analogues (azabicyclo [5.3.0]alkanone et dipeptides macrocycles) à partir d’un intermédiaire commun, en procédant par une cyclisation transannulaire diastéréosélective du tétra-peptide. / Preterm birth is a major perinatal health problem. Worldwide each year, more than one baby on ten is born premature, accounting for approximately 15 million babies per year. Pursuing molecules that can delay labour, so called tocolytics, we have targeted the prostaglandin F2α receptor (FP), because of its intimate involvement with the stimulation of uterine contractions leading to birth and preterm birth. Our laboratory has previously demonstrated that PDC-113.824 and azapeptide can modulate FP function by an allosteric mechanism featuring biased signalling, and can delay labour in a mouse model. The objective of the present work is to study the active turn geometry responsible for the modulatory effects of our lead tocolytic agents on the prostaglandin F2α receptor. Crystal structure and computational analyses of indolizidin-2-one amino acid and aza-glycinyl-proline components have shown them to adopt geometry that mimic ideal type I and II’ β-turns suggesting the potential involvement of such turns in the biological activity. To study the relevance of a type I β-turn on activity, we have prepared analogs possessing macrocyclic and pyrroloazepinone dipeptide mimics, because of their reported propensities to mimic type I β-turns. The syntheses of analogs possessing glycinyl-proline and D-alaninyl-proline were conducted to validate the involvement of a type II` β-turn in receptor recognition. In addition, we reported an efficient strategy for preparing the macrocyclic and pyrroloazepinone dipeptide mimics from a common intermediate using a diastereoselective transannular cyclization on a tetrapeptide precursor.

Made available in DSpace on 2015-05-14T12:59:56Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2116236 bytes, checksum: d980c77ceebcbdbba85d747746d497be (MD5) Previous issue date: 2012-02-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The flavonoid 3,6-dimethyl ether galetin (FGAL) was isolated from aerial parts of Piptadenia stipulacea (Benth.) Ducke. In previous studies, Macêdo et al. (2011a) demonstrated that FGAL inhibited in a significant and concentration-dependent manner the phasic contractions induced by carbachol and oxytocin on rat uterus, in a higher potency to oxytocin. The aim of this study was to investigate the mechanism of tocolytic action of FGAL on rat uterus. Segments of rat uterus were suspended in organ bath and the isometric contractions were monitored. FGAL inhibited (pD 2 = 5.68 ± 0.06) oxytocin cumulative curves and these were shifted to the right, in a non parallel manner (slope = 3.59 ± 0.04), with Emax the reduction, suggesting a non-competitive antagonism. In relation to the tonic contractions, FGAL relaxed in a concentration-dependent and significant manner but not equipotent rat uterus pre-contracted both oxytocin (pD2 = 6.9 ± 0.1) and KCl (pD2 = 5.6 ± 0.06). How the opening channels for calcium-dependent voltage (CaV) is one of the common step to the signaling pathways of oxytocin and KCl to maintain the tonic phase of contraction, raised the hypothesis that FGAL could be acting by blocking the influx of Ca2+ through the CaV. Therefore, cumulative contractions were induced with CaCl2 in depolarizing medium nominally without Ca2+ in the presence and absence of several concentrations of FGAL. The flavonoid antagonized the contractions induced by CaCl2 evidenced by shift to the right of the control curve in a non parallel manner with reduction of Emax, suggesting that FGAL is blocking indirectly the CaV to promote tocolytic effect on rat uterus. The K+ channels play a key role in membrane potential regulation and CaV modulation. According to this premise, we decided to investigate the involvement of these channels in the tocolytic action of FGAL. The relaxing potency of FGAL (pD2 = 6.9 ± 0.1) were reduced approximately 20 folds in the presence of CsCl (pD2 = 5.6 ± 0.01), a non-selective K+ channels blocker, confirming K+ channels the participation to the relaxant effect of the flavonoid. To determinated which K+ channel(s) subtype(s) were involved, selective blockers of these channels were used. The relaxation curve induced by FGAL was shifted to right in the presence of 4-aminopyridine (pD2 = 5.4 ± 0.01), a selective blocker of voltage gated K+ channels (KV); of glibenclamide (pD2 = 5.3 ± 0.01), a selective blocker of ATP sensitive K+ channels (KATP); of apamin (6.3 ± 0.02), a selective blocker of small conductance calcium-activated K+ channels (SKCa) and tetraethylamonium (TEA+) 1 mM (pD2 = 5.0 ± 0.08), a big conductance calcium-activated K+ channels (BKCa), suggesting the involvement of these channels on tocolytic action mechanism of FGAL on rat uterus. The aminophylline, a non selective inhibitor of phosphodiesterases (PDE), did not potentialized (pD2 = 6.7 ± 0.03) the relaxation produced by FGAL on rat uterus, suggesting that the cyclic nucleotides pathway is not involved on relaxing effect induced by FGAL. Thus, we concluded that the relaxing action mechanism of FGAL in rat uterus involves positive regulation of K+ channels, that indirectly modulated the CaV, leading to a consequent reduction of [Ca2+]c and uterine smooth muscle relaxation. / O flavonoide 3,6-dimetil éter galetina (FGAL) foi isolado das partes aéreas de Piptadenia stipulacea (Benth.) Ducke. Em estudos anteriores, Macêdo et al. (2011a) demonstraram que FGAL inibiu de maneira dependente de concentração as contrações fásicas induzidas por carbacol e ocitocina em útero isolado de rata, sendo mais potente para ocitocina. Desta forma, o objetivo desse estudo foi investigar o mecanismo de ação tocolítica de FGAL em útero de rata. Para tanto segmentos de útero de rata foram suspensos em cuba para órgãos isolados e as contrações isométricas foram monitoradas. FGAL inibiu (pD´2 = 5, 68 ± 0,06) as curvas cumulativas à ocitocina e estas foram desviadas para direita, de forma não paralela ( slope = 3,59 ± 0,04), com redução do Emax, sugerindo um antagonismo não competitivo. Em relação às contrações tônicas, FGAL relaxou de maneira significante, dependente de concentração e não equipotente o útero pré-contraído tanto por ocitocina (pD2 = 6,9 ± 0,1) como por KCl (pD2 = 5,6 ± 0,06). Como a abertura dos canais para cálcio dependentes de voltagem (CaV) é um dos passos comum às vias de sinalização da ocitocina e do KCl para manutenção da fase tônica da contração, levantou-se a hipótese de que FGAL poderia estar agindo por bloqueio do influxo de Ca2+ através dos CaV. Assim, foram induzidas contrações cumulativas com CaCl2 em meio despolarizante e nominalmente sem Ca2+ na presença e na ausência de várias concentrações de FGAL. O flavonoide antagonizou as contrações induzidas pelo CaCl2 evidenciado pelo desvio da curva controle para a direita de maneira não paralela e com diminuição do efeito máximo. Sugerindo assim que FGAL bloqueia de maneira indireta os CaV para promover efeito tocolítico em útero de rata. Os canais para K+ desempenham um papel chave na regulação do potencial de membrana e modulação dos CaV. Diante dessa premissa, decidiu-se investigar a participação desses canais na ação tocolítica de FGAL. A potência relaxante de FGAL (pD2 = 6,9 ± 0,1) foi reduzida em aproximadamente 20 vezes na presença de CsCl (pD2 = 5,6 ± 0,01), bloqueador não seletivo dos canais para K+, confirmando a participação de canais para K+ no efeito relaxante do flavonoide. Para determinar qual(is) canal(is) para K+ estariam envolvidos usou-se bloqueadores seletivos desses canais. A curva de relaxamento induzida por FGAL foi desviada para direita na presença de 4 -aminopiridina (pD2 = 5,4 ± 0,01), um bloqueador seletivo dos canais para K+ ativados por voltagem (KV); de glibenclamida (pD2 = 5,3 ± 0,01), um bloqueador seletivo dos canais para K+ ativados por ATP (KATP); de apamina (6,3 ± 0,02), um bloqueador seletivo dos canais para K+ de pequena condutância ativados por cálcio (SKCa) e tetraetilamônio 1 mM (pD2 = 5,0 ± 0,08), que nesta concentração é um bloqueador seletivo dos canais para K+ de grande condutância ativados por cálcio (BKCa), sugerindo o envolvimento destes canais no mecanismo de ação tocolítica de FGAL em útero isolado de rata. A aminofilina um inibidor não seletivo de fosfodiesterases (PDE) não potencializou (pD2 = 6,7 ± 0,03) o relaxamento produzido por FGAL em útero de rata, sugerindo que não há participação da via dos nucleotídios cíclicos no efeito relaxante induzido por FGAL. Conclui-se que o mecanismo de ação tocolítica de FGAL em útero de rata envolve a modulação positiva de canais para K+, majoritariamente dos BKCa, que modulam indiretamente os CaV, levando ao relaxamento da musculatura lisa uterina.

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-09-11T13:17:28Z No. of bitstreams: 1 arquivototal.pdf: 2915806 bytes, checksum: 8e05efc416b7ae0bf16dc64a785206e4 (MD5) / Made available in DSpace on 2017-09-11T13:17:28Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2915806 bytes, checksum: 8e05efc416b7ae0bf16dc64a785206e4 (MD5) Previous issue date: 2014-12-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Rollinia leptopetala R. E. Fries species, popularly known as “pinha-brava”, “bananinha” and “pereiro”, and traditionally cited as digestive. From R. leptopetala leaves was extracted an essential oil (RL-OE) that showed tocolytic effect in carbachol- (CCh) and oxytocin-induced phasic contractions on rat uterus. Thus, we aimed characterize the mechanism of tocolytic action of RL-OE using by functional techniques. Since the mechanisms to induce the phasic contractions are distinct from the tonic ones, we decided to evaluate whether RL-OE would relax rat uterus pre-contracted with KCl or oxytocin. The essential oil relaxed pre-contracted with 60 mM KCl (EC50 = 22.4 ± 2.4 μg/mL) or 10-2 UI/mL oxytocin (EC50 = 4.1 ± 0.4 μg/mL). After, it was hypothesized that RL-OE would antagonize oxytocin receptors, and this hypothesis was confirmed since cumulative concentration-response curves to oxytocin were inhibited, shifted to the right, in a non-parallel manner and with Emax reduction, discarding a competitive antagonism. The participation of adrenergic receptors was also evaluated. For this, phentolamine (an antagonist of α-receptors) was used, but none change in RL-OE tocolytic potency was observed, being discarded the involvement of this receptor. Additionally, propranolol (an antagonist of β-receptors) was used and the relaxation curve induced by RL-OE, in the blocker presence, was shifted to the left, with potentiation of oil effect and rejecting the hypothesis of β-receptors activation. Other pathways that modulate the myometrium contractility are nitric oxide (NO) and Cyclooxygenase (COX) pathways. The involvement of them in tocolytic mechanism of RL-OE was investigated using L-NAME and indomethacin, inhibitors of NO and COX pathways, respectively. However, RL-OE effect was not altered in the presence of inhibitors, discarding their contribution. The participation of K+ channels was developed using non-selective and selective blockers of them. The tocolytic potency of RL-OE (EC50 = 4.1 ± 0.4 μg/mL) was reduced 2.2 fold in the presence of 5 mM CsCl (EC50 = 8.9 ± 1.1 μg/mL), a non-specific blocker K+ channels, indicating participation of these channels. To investigate which subtypes of K+ channels would be involved selective K+ channels blockers were used. In the presence of 1 mM TEA+, blocker of large conductance calcium-activated K+ channels (BKCa), apamin, blocker of small conductance calcium-activated K+ channels (SKCa) and glibenclamide, blocker of ATP-sensitive potassium channel (KATP) did not changed the tocolytic action of RL-OE, showing that BKCa, SKCa and KATP are not involved. Interestingly, the relaxation curve induced by RL-OE was shifted to the right in the presence of 4- aminopyridine, blocker of voltage-gated K+ channels (KV), with reduction of RL-OE potency (EC50 = 10 ± 0.6 μg/mL), indicating KV participation in the mechanism of tocolytic action of RL-OE on rat uterus. Therefore, the tocolytic mechanism of RL-OE involves the positive modulation the K+ channels, in special KV subtypes, that indirectly blockade of voltage-gated Ca2+ channels (CaV) leading to uterine smooth muscle relaxation. / A espécie Rollinia leptopetala R. E. Fries, conhecida popularmente como “pinha-brava”, “bananinha” e “pereiro”, é utilizada tradicionalmente como digestiva. Das folhas dessa espécie foi extraído o óleo essencial (RL-OE), que, em estudos anteriores, apresentou atividade tocolítica frente às contrações fásicas induzidas por carbacol (CCh) ou ocitocina em útero isolado de rata. Assim, o objetivo desse trabalho foi caracterizar o mecanismo de ação tocolítica do RL-OE, por meio de metodologias funcionais. Como os mecanismos para indução da contração fásica são diferentes dos que mantém a tônica, decidiu-se verificar se o RL-OE relaxaria o útero pré-contraído com KCl ou ocitocina. O óleo relaxou o útero pré-contraído com 60 mM de KCl (CE50 = 22,4 ± 2,4 μg/mL) ou com 10-2 UI/mL de ocitocina (CE50 = 4,1 ± 0,4 μg/mL), sendo mais potente para ocitocina. Em seguida, hipotetizou-se que o RL-OE estaria antagonizando os receptores de ocitocina, sendo essa hipótese confirmada, uma vez que, observou-se a inibição das curvas concentrações-resposta cumulativas à ocitocina, desviando-as para a direita, de maneira não paralela e com redução do seu Emax, descartando-se um antagonismo do tipo competitivo. A participação dos receptores adrenérgicos foi avaliada. Para isso, utilizou-se a fentolamina, um antagonista dos receptores α, entretanto não houve alteração da potência tocolítica do RL-OE, sendo descartado o antagonismo desse receptor. Também utilizou-se o propranolol, um antagonista dos receptores adrenérgicos β, constatando-se que a curva de relaxamento, na presença do bloqueador, foi desviada para a esquerda, com potencialização do RL-OE, descartando a ativação desses receptores. Outras vias que modulam a função contrátil do miométrio uterino são as vias do óxido nítrico (NO) e da ciclo-oxigenase (COX). Investigou-se a participação dessas vias no mecanismo de ação do óleo essencial utilizando L-NAME e indometacina, inibidores das vias do NO e da COX, respectivamente. Entretanto, o efeito do RL-OE não foi alterado na presença dos inibidores, descartando a participação dessas vias no seu mecanismo de ação tocolítica. Outra via investigada foi o modulação dos canais de K+. Para isso, utilizou-se bloqueadores não-seletivo e seletivos desses canais. A potência tocolítica do RL-OE (CE50 = 4,1 ± 0,4 μg/mL) foi reduzida 2,2 vezes na presença de 5 mM de CsCl (CE50 = 8,9 ± 1,1 μg/mL), bloqueador não seletivo desses canais, confirmando a participação dos canais de K+ no efeito tocolítico do RL-OE. Para verificar qual(is) canal(is) de K+ estariam envolvidos usou-se bloqueadores seletivos desses canais. O fato do TEA+ 1 mM, bloqueador dos canais de K+ de grande condutância ativados por cálcio (BKCa); da apamina, um bloqueador dos canais de K+ ativados por Ca2+ de pequena condutância (SKCa) e da glibenclamida, bloqueador dos canais de K+ sensíveis ao ATP (KATP) não alterarem o efeito tocolítico do RL-OE indica que os BKCa, SKCa e os KATP não estariam envolvidos em seu mecanismo de ação. Entretanto, a curva concentrações-resposta de relaxamento induzida pelo RL-OE foi desviada para direita na presença de 4-aminopiridina, bloqueador dos canais de K+ dependentes de voltagem (Kv), com redução da potência tocolítica do RL-OE (CE50 = 10 ± 0,6 μg/mL), sugerindo o envolvimento dos Kv no mecanismo de ação tocolítica do RL-OE em útero de rata. Conclui-se que o RL-OE exerce efeito tocolítico modulando positivamente os canais de K+, especificamente os Kv, que bloqueariam de modo indireto os canais de cálcio dependentes de voltagem (CaV), resultando no relaxamento da musculatura lisa uterina

Made available in DSpace on 2015-05-14T13:00:13Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1650279 bytes, checksum: 848f51aee13c80e0d670d1542b962211 (MD5) Previous issue date: 2010-03-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / 8(17),12E,14-labdatrien-18 oic acid (labdane-302), is a diterpene isolated from the stem bark of Xylopia langsdorfiana A. St.-Hil. & Tul. In a preliminary study, Ribeiro (2003) demonstrated that labdane-302 inhibited in an equipotent manner the phasic contractions induced by carbachol or oxytocin in rat uterus. The aim of the present study was to investigate the spasmolytic action mechanism of labdane-302 in that organ. Isometric and isotonic contractions were monitored and the parameters of relative potency and efficacy were determined from cumulative concentration-response curves. Labdane-302 inhibited the cumulative concentration-response curves to carbachol (pD´2 = 3.4 ± 0.1; r2 = 0.9 ± 0.05) and oxytocin (pD´2 = 3.8 ± 0.2; r2 = 0.9 ± 0.04) these were shifted to the right, in a non-parallel manner (Schild plot slope = 0.15 ± 0.04 and 1.13 ± 0.1 respectively), with reduction of the maximal effect (Emax), suggesting a noncompetitive antagonism. Labdane-302 was not effective in relaxing the uterus pre-contracted by 60 mM KCl (Emax = 9.75 ± 0.07%),on the other hand, relaxed in a significant and concentration dependent manner the rat uterus pre-contracted by oxytocin (pD2 = 4,3 ± 0,06), suggesting a possible involvement of the K+ channels in the spasmolytic effect caused by labdane-302. Because K+ channels play a major role in the regulation of membrane potential and modulation of CaV, we decided to investigate the participation of K+ channels in the spasmolytic action of labdane-302. The relaxant potency of labdane-302 (pD2 = 4.3 ± 0.06) was decreased about 16 times in the presence of CsCl (pD2 = 3.1 ± 0.06), a non-selective K+channels blocker, suggesting a possible involvement of the K+ channels in the tocolytic effect of the labdane-302. In order to verify which subtypes of K+ channels could be involved we used selectives blockers of these channels. The observation that 4-aminopyridine, a selective blocker of voltage-gated K+ channels (Kv), and that glibenclamide, a selective blocker of the ATP-sensitive K+ channels (KATP) did not change the relaxant effect of labdane-302 suggests that KV and KATP are not involved in its action mechanism. However, the log concentration-response curve induced by labdane-302 was shifted to the right in the presence of apamine (pD2 = 3.8 ± 0.03), a selective blocker of the small-conductance calcium-activated K+ channels (SKCa) and TEA+ 1 mM (pD2 = 3.6 ± 0.04), a selective blocker of the large conductance clacium-activated K+ channels (BKca). The involvement of BKCa was confirmed using a specific blocker of that channels iberiotoxin (IbTx) (pD2 = 3.8 ± 0.06) , suggesting the involvement of SKCa and BKCa in the tocolytic action mechanism induced by labdane-302 on uterus rat. The aminophylline a nonselective inhibitor of phosphodiesterases (PDE) potentiated (pD2 = 7.8 ± 0.1) in about 320 times the relaxation produced by labdane-302 in rat uterus. The results suggest that effect of labdane-302 on uterus rat, involves the activation of the SKCa e BKCa, which modulate indirectly the CaV, leading to a decrease the [Ca2+]c , and cyclic nucleotides like cAMP and cGMP may be involved in this tocolytic action / O ácido 8(17),12E,14-labdatrieno-18-óico (labdano-302) é um diterpeno isolado das cascas do caule de Xylopia langsdorfiana A. St.-Hil. & Tul. Em estudos anteriores Ribeiro (2003) demonstrou que o labdano-302 inibiu de maneira eqüipotente as contrações fásicas induzidas por carbacol e ocitocina em útero de rata. Assim, o objetivo desse estudo foi investigar o mecanismo de ação tocolítico do labdano-302. As contrações isométricas e isotônicas foram monitoradas e os parâmetros de potência e eficácia relativas foram determinados a partir de curvas de concentrações-resposta cumulativas. O labdano-302 inibiu as curvas cumulativas ao carbacol (pD´2 = 3,4 ± 0,1; r2 = 0,9 ± 0,05) e ocitocina (pD´2 = 3,8 ± 0,2; r2 = 0,9 ± 0,04) e estas foram desviadas para direita, de forma não paralela ( slope de Schild = 0,15 ± 0,04 e 1,13 ± 0,1 respectivamente), com redução do Emax, sugerindo um antagonismo não competitivo. O labdano-302 não foi eficaz em antagonizar as contrações induzidas por 60 mM de KCl apresentando um Emax = 9,75 ± 0,07%, por outro lado, labdano-302 relaxou de maneira significante e dependente de concentração quando o útero era pré-contraído por ocitocina (pD2 = 4,3 ± 0,06), sugerindo que este diterpeno deve estar agindo por uma modulação positiva de canais para potássio. Os canais para K+ desempenham um papel chave na regulação do potencial de membrana e modulação dos CaV, então decidiu-se investigar a participação desses canais na ação tocolítica do labdano-302. A potência relaxante de labdano-302 (pD2 = 4,3 ± 0,06) foi reduzida em aproximadamente 16 vezes na presença de CsCl (pD2 = 3,1 ± 0,06), bloqueador não seletivo dos canais para K+, confirmando a participação de canais para K+ no efeito relaxante do labdano-302. Para verificar qual(is) canal(is) para K+ estariam envolvidos usou-se bloqueadores seletivos desses canais. O fato da 4-aminopiridina, bloqueador seletivo dos canais para K+ abertos por voltagem (Kv), e da glibenclamida, bloqueador seletivo dos canais para K+ sensíveis ao ATP (KATP) não alterar o efeito relaxante do labdano-302 indica que os KV e os KATP não estariam envolvidos em seu mecanismo de ação tocolítico. Entretanto, a curva concentração-resposta de relaxamento induzida pelo labdano-302 foi desviada para direita na presença de apamina (pD2 = 3,8 ± 0,03), um bloqueador seletivo dos canais para K+ ativados por Ca2+ de pequena condutância (SKCa), ou de tetraetilamônio 1 mM (pD2 = 3,6 ± 0,04), que nesta concentração é um bloqueador seletivo dos canais para K+ de grande condutância ativados por cálcio (BKCa). A participação dos BKCa foi confirmada utilizando um bloqueador específico para esses canais a iberiotoxina (IbTx) (pD2 = 3,8 ± 0,06), sugerindo o envolvimento dos SKCa e dos BKCa no mecanismo de ação tocolítico do labdano-302 em útero isolado de rata. A aminofilina um inibidor não seletivo de fosfodiesterases (PDE) potencializou (pD2 = 7,8 ± 0,1) em cerca de 320 vezes o relaxamento produzido por labdano-302 em útero de rata. Conclui-se que o mecanismo de ação relaxante do labdano-302 em útero isolado de rata envolve a modulação positiva de canais para K+, mais especificamente os SKCa e BKCa, que modulam indiretamente os CaV, levando a uma conseqüente redução da [Ca2+]c , e que nucleotídios cíclicos como o AMPc e GMPc podem estar envolvidos nesta ação.

Les accouchements prématurés constituent un problème médical majeur en constante augmentation et ce, malgré tous les efforts mis en œuvre afin de contrer le déclenchement des contractions avant terme. Cette thèse relate du ''design'' rationnel d'un nouvel agent thérapeutique (i.e., tocolytique) qui serait capable de 1) arrêter les contractions, et 2) prolonger la gestation. Pour ce faire, une nouvelle cible, la prostaglandine F2α et son récepteur ont été sélectionnés et le peptidomimétisme a été choisi afin de résoudre cette problématique. L'introduction contient un historique rapide de la conception à la synthèse (''drug design'') du peptide parent, le PDC113, premier peptide a avoir démontré des aptitudes tocolytiques suffisantes pour faire du peptidomimétisme. La deuxième partie de l'introduction présente les concepts du peptidomimétisme appliqués au PDC113 qui ont permis d'accéder au PDC113.824, inhibiteur allostérique du récepteur de la prostaglandine F2α, et explique comment ce mime nous a permis d'élucider les mécanismes de signalisation intracellulaire impliqués dans la contraction musculaire lisse. Cette thèse présente la conception, la synthèse et l'étude structure-activité de mimes de repliement de tour β au sein du mime peptidique original (PDC113.824) dans lequel nous avons remplacé l'azabicycloalkane central (l'indolizidin-2-one) par une série d'autres azabicycloalcanes connus et des acides aza-aminés dont nous avons élaboré la synthèse. Dans un premier temps, une nouvelle stratégie de synthèse en solution de l'aza-glycyl-proline à partir de la diphényle hydrazone et du chloroformate de p-nitrophényle a été réalisée. Cette stratégie a permis d'éliminer les réactions secondaires de cyclisation intramoléculaires communément obtenues lors de l'introduction d'acides aza-aminés avec les protections traditionnelles de type carbamate en présence de phosgène, mais aussi de faciliter l'accès en une étape à des dérivés peptidiques du type aza-glycyle. L'élongation de l'aza-glycyl-proline en solution nous a permis d'accéder à un nouveau mime tetrapeptidique du Smac, un activateur potentiel de l'apoptose au sein de cellules cancéreuses. Par la suite, nous avons développé une stratégie de diversification sélective de l'azote α du résidu azaglycine en utilisant différents types d'halogénures d'alkyle en présence de tert-butoxyde de potassium. Afin de valider le protocole d'alkylation de l'aza-dipeptide, différents halogénures d'alkyle ont été testés. Nous avons également démontré l'utilité des aza-dipeptides résultants en tant que ''building block'' afin d'accéder à une variété d'azapeptides. En effet, l'aza-dipeptide a été déprotégée sélectivement soit en N-terminal soit en C-terminal, respectivement. D'autre part, la libération de l'amine de l'ester méthylique de l'aza-alkylglycyl-proline a conduit à une catégorie de composés à potentiel thérapeutique, les azadicétopipérazines (aza-DKP) par cyclisation intramoléculaire. Enfin, notre intérêt quant au développement d'un nouvel agent tocolytique nous a amené à développer une nouvelle voie de synthèse en solution du PDC113.824 permettant ainsi d'élucider les voies de signalisation intracellulaires du récepteur de la prostaglandine F2α. Afin de valider l'importance de la stéréochimie et d'étudier la relation structure/ activité du mime, nous avons remplacé l'indolizidin-2-one (I2aa) centrale du PDC113.824 par une série d'autres azabicycloalcanes et azadipeptides. Les azabicycloalcanes D-I2aa, quinolizidinone, et indolizidin-9-one ont été synthétisés et incorporés au sein du dit peptide ne donnant aucune activité ni in vitro ni ex vivo, validant ainsi l'importance du tour β de type II' pour le maintien de l'activité biologique du PDC113.824. Finalement, l'insertion d'une série de dérivés aza(alkyl)glycyl-prolyles a mené à de nouveaux inhibiteurs allostériques du récepteur de la PGF2α, l'un contenant l'azaglycine et l'autre, l'azaphénylalanine. Cette thèse a ainsi contribué, grâce à la conception et l'application de nouvelles méthodes de synthèse d'aza-peptides, au développement de nouveaux composés à potentiel thérapeutique afin d'inhiber le travail prématuré. / Premature birth is a steadily increasing major medical problem, in spite of efforts made to counter the onset of preterm contractions. This thesis describes the rational design of a new therapeutic agent (i.e., tocolytic) capable of 1) stopping uterine contractions, and 2) prolonging gestation. The prostaglandin F2α receptor was explored for tocolytic development and a peptidomimetic approach was developed to produce modulators of this novel target. A brief discussion introduces the impact of preterm birth and history on the design and synthesis of a peptide lead, PDC113, to address this unmet medical need. Subsequently, the peptidomimetic approach is described for converting PDC113 to the small molecule PDC113.824, which was shown to be an allosteric modulator of prostaglandin F2α receptor, which mediates intracellular signalling pathways involved in uterine contraction. This thesis presents the design, synthesis and structure-activity relationship study of the peptide mimic PDC113.824, in which we have replaced the central β-turn mimic moiety, the indolizidin-2-one amino acid, by a series of other previously reported azabicycloalcane turn mimics and novel aza-amino acids. To accomplish the latter, new solution-phase methods for synthesizing aza-glycyl-proline analogs were developed starting from diphenyl hydrazone and p-nitrophenyl chloroformate. This strategy has surmounted side reactions such as the intramolecular cyclization commonly obtained with traditional coupling reactions employing alkyl-carbazates and phosgene, facilitating access to aza-glycyl-proline derivatives by a one step reaction. The modification of the aza-glycyl-proline using conventional strategies led to a new Smac mimic (a proapoptotic molecule), with potential as a tetrapeptide activator of apoptosis in cancer cells. Subsequently, we focused on the diversification of the aza-glycine residue by selective alkylation using different alkyl halides in the presence of potassium tert-butoxide. To validate the alkylation protocol, various alkyl groups were employed, and the usefulness of the resulting aza-dipeptides as ''building blocks'' was examined. Conditions were developed for selectively unmasking the protecting groups at the N- and C-terminal of the aza-dipeptide. In addition, removal of the amine protection of aza-alkylglycyl-proline methyl ester gave access to a class of compounds with therapeutic potential, the aza-diketopiperazines (aza-DKP), by intramolecular cyclization. Finally, our interest in developing a new tocolytic agent led us to develop a new solution-phase synthetic route to PDC113.824 for elucidating the intracellular signalling pathways of prostaglandin F2α receptor. To explore the importance of stereochemistry and to study the relationship between structure and activity, we replaced the central indolizidin-2-one (I2aa) of PDC113.824 by a series of others azabicycloalcanes and aza-dipeptides. The D-I2aa, quinazolidinone and indolizidin-9-one analogs were synthesized and incorporated into the mimic; however, none exhibited activity neither in vitro nor ex vivo, thus indicating the importance of a type II' β-turn for maintaining biological activity of PDC113.824. Finally, the synthesis of a series of aza(alkyl)glycyl-prolyl analogs led to new allosteric modulators of the PGF2α receptor, one containing aza-glycine and another aza-phenylalanine. This results reported in this thesis have contributed to the development of novel agents with promise for inhibiting preterm labor by the conception and application of new methods for making aza-peptides.

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Les accouchements prématurés constituent un problème médical majeur en constante augmentation et ce, malgré tous les efforts mis en œuvre afin de contrer le déclenchement des contractions avant terme. Cette thèse relate du ''design'' rationnel d'un nouvel agent thérapeutique (i.e., tocolytique) qui serait capable de 1) arrêter les contractions, et 2) prolonger la gestation. Pour ce faire, une nouvelle cible, la prostaglandine F2α et son récepteur ont été sélectionnés et le peptidomimétisme a été choisi afin de résoudre cette problématique. L'introduction contient un historique rapide de la conception à la synthèse (''drug design'') du peptide parent, le PDC113, premier peptide a avoir démontré des aptitudes tocolytiques suffisantes pour faire du peptidomimétisme. La deuxième partie de l'introduction présente les concepts du peptidomimétisme appliqués au PDC113 qui ont permis d'accéder au PDC113.824, inhibiteur allostérique du récepteur de la prostaglandine F2α, et explique comment ce mime nous a permis d'élucider les mécanismes de signalisation intracellulaire impliqués dans la contraction musculaire lisse. Cette thèse présente la conception, la synthèse et l'étude structure-activité de mimes de repliement de tour β au sein du mime peptidique original (PDC113.824) dans lequel nous avons remplacé l'azabicycloalkane central (l'indolizidin-2-one) par une série d'autres azabicycloalcanes connus et des acides aza-aminés dont nous avons élaboré la synthèse. Dans un premier temps, une nouvelle stratégie de synthèse en solution de l'aza-glycyl-proline à partir de la diphényle hydrazone et du chloroformate de p-nitrophényle a été réalisée. Cette stratégie a permis d'éliminer les réactions secondaires de cyclisation intramoléculaires communément obtenues lors de l'introduction d'acides aza-aminés avec les protections traditionnelles de type carbamate en présence de phosgène, mais aussi de faciliter l'accès en une étape à des dérivés peptidiques du type aza-glycyle. L'élongation de l'aza-glycyl-proline en solution nous a permis d'accéder à un nouveau mime tetrapeptidique du Smac, un activateur potentiel de l'apoptose au sein de cellules cancéreuses. Par la suite, nous avons développé une stratégie de diversification sélective de l'azote α du résidu azaglycine en utilisant différents types d'halogénures d'alkyle en présence de tert-butoxyde de potassium. Afin de valider le protocole d'alkylation de l'aza-dipeptide, différents halogénures d'alkyle ont été testés. Nous avons également démontré l'utilité des aza-dipeptides résultants en tant que ''building block'' afin d'accéder à une variété d'azapeptides. En effet, l'aza-dipeptide a été déprotégée sélectivement soit en N-terminal soit en C-terminal, respectivement. D'autre part, la libération de l'amine de l'ester méthylique de l'aza-alkylglycyl-proline a conduit à une catégorie de composés à potentiel thérapeutique, les azadicétopipérazines (aza-DKP) par cyclisation intramoléculaire. Enfin, notre intérêt quant au développement d'un nouvel agent tocolytique nous a amené à développer une nouvelle voie de synthèse en solution du PDC113.824 permettant ainsi d'élucider les voies de signalisation intracellulaires du récepteur de la prostaglandine F2α. Afin de valider l'importance de la stéréochimie et d'étudier la relation structure/ activité du mime, nous avons remplacé l'indolizidin-2-one (I2aa) centrale du PDC113.824 par une série d'autres azabicycloalcanes et azadipeptides. Les azabicycloalcanes D-I2aa, quinolizidinone, et indolizidin-9-one ont été synthétisés et incorporés au sein du dit peptide ne donnant aucune activité ni in vitro ni ex vivo, validant ainsi l'importance du tour β de type II' pour le maintien de l'activité biologique du PDC113.824. Finalement, l'insertion d'une série de dérivés aza(alkyl)glycyl-prolyles a mené à de nouveaux inhibiteurs allostériques du récepteur de la PGF2α, l'un contenant l'azaglycine et l'autre, l'azaphénylalanine. Cette thèse a ainsi contribué, grâce à la conception et l'application de nouvelles méthodes de synthèse d'aza-peptides, au développement de nouveaux composés à potentiel thérapeutique afin d'inhiber le travail prématuré. / Premature birth is a steadily increasing major medical problem, in spite of efforts made to counter the onset of preterm contractions. This thesis describes the rational design of a new therapeutic agent (i.e., tocolytic) capable of 1) stopping uterine contractions, and 2) prolonging gestation. The prostaglandin F2α receptor was explored for tocolytic development and a peptidomimetic approach was developed to produce modulators of this novel target. A brief discussion introduces the impact of preterm birth and history on the design and synthesis of a peptide lead, PDC113, to address this unmet medical need. Subsequently, the peptidomimetic approach is described for converting PDC113 to the small molecule PDC113.824, which was shown to be an allosteric modulator of prostaglandin F2α receptor, which mediates intracellular signalling pathways involved in uterine contraction. This thesis presents the design, synthesis and structure-activity relationship study of the peptide mimic PDC113.824, in which we have replaced the central β-turn mimic moiety, the indolizidin-2-one amino acid, by a series of other previously reported azabicycloalcane turn mimics and novel aza-amino acids. To accomplish the latter, new solution-phase methods for synthesizing aza-glycyl-proline analogs were developed starting from diphenyl hydrazone and p-nitrophenyl chloroformate. This strategy has surmounted side reactions such as the intramolecular cyclization commonly obtained with traditional coupling reactions employing alkyl-carbazates and phosgene, facilitating access to aza-glycyl-proline derivatives by a one step reaction. The modification of the aza-glycyl-proline using conventional strategies led to a new Smac mimic (a proapoptotic molecule), with potential as a tetrapeptide activator of apoptosis in cancer cells. Subsequently, we focused on the diversification of the aza-glycine residue by selective alkylation using different alkyl halides in the presence of potassium tert-butoxide. To validate the alkylation protocol, various alkyl groups were employed, and the usefulness of the resulting aza-dipeptides as ''building blocks'' was examined. Conditions were developed for selectively unmasking the protecting groups at the N- and C-terminal of the aza-dipeptide. In addition, removal of the amine protection of aza-alkylglycyl-proline methyl ester gave access to a class of compounds with therapeutic potential, the aza-diketopiperazines (aza-DKP), by intramolecular cyclization. Finally, our interest in developing a new tocolytic agent led us to develop a new solution-phase synthetic route to PDC113.824 for elucidating the intracellular signalling pathways of prostaglandin F2α receptor. To explore the importance of stereochemistry and to study the relationship between structure and activity, we replaced the central indolizidin-2-one (I2aa) of PDC113.824 by a series of others azabicycloalcanes and aza-dipeptides. The D-I2aa, quinazolidinone and indolizidin-9-one analogs were synthesized and incorporated into the mimic; however, none exhibited activity neither in vitro nor ex vivo, thus indicating the importance of a type II' β-turn for maintaining biological activity of PDC113.824. Finally, the synthesis of a series of aza(alkyl)glycyl-prolyl analogs led to new allosteric modulators of the PGF2α receptor, one containing aza-glycine and another aza-phenylalanine. This results reported in this thesis have contributed to the development of novel agents with promise for inhibiting preterm labor by the conception and application of new methods for making aza-peptides.

Les accouchements prématurés restent un défi pour la médecine moderne. Malgré les efforts de préventions déployés, les taux de naissances prématurés sont en constante augmentation dans les pays industrialisés. Le récepteur interleukine-1 (IL-1R) a été étudié dans le but de développer des agents thérapeutiques pouvant prolonger la gestation et mener à des prognostiques néonataux. Dans cette optique, il y a été démontré que le peptide 101.10 possède a démontré une capacité à moduler le récepteur IL-1R, retarder les accouchements prématurés et réduire l’incidence de la rétinopathie prématurée par un mécanisme allostérique impliquant une signalisation biaisée. Dans le but d’étudier la conformation active du peptide 101.10 et d’en améliorer l’activité, nous avons développé une méthode pour introduire des α-amino γ-lactames β-substituées dans des séquences peptidiques. Appliquer cette stratégie au peptide 101.10 a permis d’améliorer la compréhension de la relation structure-activité pour la modulation allostérique du récepteur IL-1R. Les composés peptidomimétiques ont le potentiel de mimer la conformation et l’activité des peptides bioactifs. Ils offrent le potentiel d’améliorer la reconnaissance moléculaire, d’optimiser le transport à travers des différentes membranes biologiques et d’augmenter la résistance métabolique. Parmi les différentes classes de composés peptidomimétiques, les α-amino γ-lactames (Agl) permet de rigidifier par des liens covalents la chaine peptidique principale favorisant les structures secondaires de type tour β. Les analogues Agl β-substituées mimer et rigidifier la chaine latérale des acides aminés. Cette thèse introduit des méthodes efficaces pour la synthèse stéréocontrolée des résidus α-amino γ-lactames β-substituées possédant diverses fonctionnalités. L’introduction de ces résidus dans différents peptides bioactifs a été effectuée pour étudier leur relation structure-activité. En utilisant le peptide modulateur du récepteur IL-1R 101.10 comme peptide représentatif, la présente recherche a permis d’identifier de nouveaux agents tocolytiques qui peuvent prolonger la gestation et améliorer le pronostique néonatal. Dans le chapitre 2, des stéréo-isomères de (Agl) et β-hydroxy-α-amino-γ-lactam (Hgl) ont été utilisés pour étudier l’influence de la configuration du groupement hydroxyle sur la conformation et l’activité du peptide 101.10. L’orientation de ce groupement dans les peptides Agl et Hgl s’est avéré avoir une influence conformationnelle et sur l’activité. La spectroscopie par dichroïsme circulaire (CD) a illustré la propension de certains analogues, comme le [(3R,4S)-Hgl3]-101.10, à adopter des tours β. Les analogues Agl et Hgl ont été examinés dans une série d’essais in vitro et in vivo modélisant les accouchements prématurés. Dépendant de leur structure et configuration, les analogues lactames ont démontré une sélectivité fonctionnelle différente dans diverse processus biologiques, démontrant les particularités de divers phénotypes. Par exemple, l’inhibition des JNK et ROCK kinases s’est avérée importante respectivement dans leurs effets tocolytiques et dans la diminution de la vaso-oblitération. Notamment, parmi les douze analogues testés, [(3R,4S)-Hgl3]-101.10 s’est avéré démontrer la même activité in vitro et in vitro que le peptide parent. Dans le chapitre 3, des méthodes de déplacement du groupement hydroxyle des résidus Hgl ont permis l’introduction stéréosélective de substituent en position β des résidus Agl. Une combinaison de réaction de Mitsunobu sur les résidus trans Hgl et une ouverture nucléophile les sulfamidates cycliques dérivés des lactames cis, ont mené à l’obtention de mime rigidifiés de résidus Ser, Thr, Cys, Dap, Dab, His et Met. Dans le chapitre 4, différentes lactames β-substitués ont été introduits dans la séquence du peptide 101.10 par une combinaison de chimie en solution et sur support solide pour étudier d’avantage les éléments structurels nécessaire pour réguler l’activité et la signalisation de cette cytokine clef dans la médiation de l’inflammation. Considérant l’activité de l’analogue [(3R,4S)-Hgl3]-101.10, plusieurs analogues β-substitués possédant une orientation similaire pour la chaine principale et latérale ont été synthétisés. Certains analogues ont démontré une activité biologique prometteuse dans des modèles de rétinopathie et seront étudiés dans le futur. En conclusion, des méthodes de synthèse d’α-amino-γ-lactames et de leur contrepartie β-substitués et leur introduction dans des peptides d’intérêt pour étudier leur relation structure-activité ont été développés. En utilisant ces méthodologies sur le modulateur allostérique du récepteur IL-1R 101.10, le conformère actif in vivo responsable de l’activité tocolytique et protectrice contre la rétinopathie associée aux accouchements prématures ont été identifiés. Considérant l’utilité de la synthèse de lactames pour le développement d’agents susceptibles de prolonger la gestation et d’améliorer le prognostique associé aux accouchements prématurés, cette thèse a permis la conception de prototypes de médicaments pour traiter les accouchements prématurés ainsi que l’évaluation des contraintes structurelles pertinentes pour la biologie des peptides. / Preterm birth (PTB) is an unmet biomedical need. Despite efforts to counter the onset of preterm labor, the rate of premature birth has increased steadily in developed countries. The interleukin-1 receptor (IL-1R) has been pursued as a target for designing agents which can prolong labor and improve neonatal outcomes. Towards these goals, a lead peptide 101.10 had been shown to modulate the IL-1R, to delay PTB and to mitigate associated retinopathy of prematurity (ROP) by an allosteric mechanism featuring biased signaling. With the goals of understanding the active conformers and improving the activity of 101.10, methods were conceived for the synthesis and introduction of β-substituted α-amino γ-lactams into peptides. Applying such methods on 101.10 has provided insight into the structure-activity relationships required for allosteric modulation of the IL-1R. Peptidomimetics are promising structures that replicate peptide function and conformation. They offer the potential to improve molecular-recognition, to enhance transport across biological membranes, and to resist metabolism. Among peptidomimetic classes, α-amino γ-lactam (Agl) residues introduce covalent constraint to rigidify the peptide backbone and have been employed to favor turn secondary structures. β-Substituted Agl analogs offer additional potential to mimic and restrict peptide side-chain geometry. This thesis introduces effective methods for the stereo-controlled synthesis of β-substituted α-amino γ-lactams residues having various side chain functionality. Introduction of the parent Agl residue and β-substituted counterparts into biologically active peptides has been explored to study structure-activity relationships. Employing the IL-1R modulator 101.10 as a representative peptide, the described research has furnished novel labor delaying agents that can improve neonatal outcomes. In chapter 2, α-amino-γ-lactam (Agl) and β-hydroxy-α-amino-γ-lactam (Hgl) stereoisomers were employed to study the influence of configuration and hydroxyl group side chain on conformation and activity of the interleukin-1 receptor modulator peptide 101.10. The configuration and hydroxyl group side chain influenced the conformation and biological activity of Agl and Hgl-101.10 analogs. Circular dichroism (CD) spectroscopy illustrated β-turn conformers for specific analogs, such as [(3R,4S)-Hgl3]-101.10. The Agl and Hgl analogs were examined in a series of in vitro assays and in vivo models of PTB. Contingent on their structure vi and configuration, the lactam analogs exhibited different functional selectivity in the various biological pathways, and indicated the requirement for specific phenotypes. For example, inhibition of the JNK and ROCK kinase pathways were respectively shown to be important for delaying labor and diminishing vaso-obliteration in the PTB and ROP models. Notably, among the twelve analogs, [(3R,4S)-Hgl3]-101.10 was found to exhibit identical in vitro and in vivo activity as the parent peptide. In chapter 3, methods were developed for displacement of the β-hydroxy-α-amino-γ-lactam (Hgl) residue alcohol to introduce stereo-selectively different β-substituents on Agl residues. A combination of Mitsunobu chemistry on the trans Hgl residue, and nucleophilic ring opening of the cyclic sulfamidate derived from the cis lactam counterpart provided constrained mimics of Ser, Thr, Cys, Dap, Dab, His and Met residues. In chapter 4, various β-substituted lactams were introduced into the sequence of 101.10 by combination of solution and solid phases chemistry to further study the structural requirements for regulating the activity and signaling of this key cytokine mediator of inflammasome activation. Considering the activity of [(3R,4S)-Hgl3]-101.10, the β-substituted Agl analogs were synthesized possessing similar backbone and side chain configurations. Certain analogs exhibited promising biological activity in the ROP model meriting further study. In sum, methods were conceived for the synthesis and application of α-amino-γ-lactams and their β-substituted analogs to study peptide structure-activity relationships. Employing this chemistry on the IL-1R allosteric modulator 101.10 has identified the active conformer and in vitro activity responsible for ability to delay labor and mitigate retinopathy of prematurity. Considering the utility of the lactam synthesis methods for the development of improved agents for delaying labor and improving neonatal outcomes, this thesis has conceived useful prototypes for drugs to treat PTB, as well as useful methods for dissecting the structural requirements for peptide chemical biology.