Η Ωκυτοκίνη (ΟΤ) είναι ένα κυκλικό εννιαπεπτίδιο του υποθαλάμου, η οποία απελευθερώνεται στη γενική κυκλοφορία από τον οπίσθιο λοβό της υπόφυσης. Οι κύριες φυσιολογικές δράσης της είναι η επαγωγή συσπάσεων του μυομητρίου και η έκθλιψη του γάλακτος. Εν τούτοις, η μεγάλη διασπορά των υποδοχέων της ΟΤ στον εγκέφαλο αποδίδουν στην ορμόνη το ρόλο του νευροδιαβιβαστή, ο οποίο ρυθμίζει τις αναπαραγωγικές και κοινωνικές συμπεριφορές. Ο ρόλος της ΟΤ στην πρόκληση του πρόωρου τοκετού οδήγησε την έρευνα για το σχεδιασμό και τη σύνθεση πεπτιδικών ανταγωνιστών της ορμόνης ως δραστικούς τοκολυτικούς παράγοντες. Πλήθος τέτοιων αναλόγων συντέθηκε και εξετάστηκε, αλλά μόλις για ένα, το Atosiban, και μόνο στην Ευρώπη, επετράπη η κυκλοφορία για την πρόληψη του πρόωρου τοκετού με την εμπορική ονομασία Tractocile™. Ο σχεδιασμός νέων αναλόγων της ΟΤ βασίζεται στα συμπεράσματα δομής-δραστικότητας. Η ανταγωνιστική δράση εξαρτάται από τη διαμόρφωση και την υδροφοβικότητα του αμινοξέος στη θέση 2. Επιπρόσθετα, η απαλοιφή της τελικής αμινομάδας προσδίδει στο πεπτίδιο παράταση της δράσης. Ιδιαίτερη σημασία στην εκλεκτική πρόσδεση της ΟΤ στον υποδοχέα της έχει η Ιle στη θέση 3, μιας και είναι το μόνο αμινοξύ στο οποίο διαφέρει στον εικοσαμελή δακτύλιο η ΟΤ από το συγγενές μόριο της, τη Βασοπρεσίνη (VP). Βασιζόμενοι στα ανωτέρω συμπεράσματα και για διερεύνηση του ρόλου της θέσης 3 της ορμόνης, συνθέσαμε δεκατέσσερα νέα ανάλογα της ΟΤ. Όλα τα ανάλογα περιέχουν β-μερκαπτοπροπιονικό οξύ (Mpa) στη θέση 1 και D-O-αιθυλ-τυροσίνη [D-Tyr(Et)] ή D-1-ναφθυλ-αλανίνη [D-Nal(1)] στη θέση 2. Η ισολευκίνη (Ile) στη θέση 3 έχει υποκατασταθεί με γ-αμινοϊσοβουτυρικό οξύ (Aib), L- ή D-α-τερτβουτυλ-γλυκίνη [L-/D-Gly(But)], L- ή D-3-πυριδυλ-αλανίνη [L-/D-Pal(3)] και L- ή D-β-(2-θεϊενυλ)-αλανίνη (L-/D-Thi). Τα φάσματα μάζας των αναλόγων συμφωνούν με τα αναμενόμενα αποτελέσματα. Τα δεκατέσσερα ανάλογα εξετάστηκαν ως προς την ωκυτόκιο δράση σε απομονωμένο ιστό μήτρας αρουραίου, ως προς τη δράση επί της πίεσης και ως προς τη συγγένεια με τον ανθρώπινο ωκυτόκιο υποδοχέα. Επιπλέον, τα ανάλογα [Mpa1, D-Tyr(Et)2, Thi3]OT, [Mpa1, D-Tyr(Et)2, D- Thi3]OT], [Mpa1, D-Nal(1)2, Thi3]OT και [Mpa1, D-Nal(1)2, D- Thi3]OT] εξετάστηκαν και ως προς τη δράση τους επί του πολλαπλασιασμού των καρκινικών κυτταρικών σειρών MDA-MB-468 και MCF-7. Όλα τα ανάλογα δρουν ως ανταγωνιστές της φυσικής ορμόνης. Συγκεκριμένα, τα ανάλογα [Mpa1, D-Nal(1)2, Gly(But)3]OT και [Mpa1, D-Nal(1)2, Thi3]OT έχουν ισχυρή αντι-ωκυτόκιο δράση (pA2=8.34±0.30 και 8.50±0.24, αντίστοιχα). Όσο αφορά τα αποτελέσματα των βιολογικών δοκιμών επί του πολλαπλασιασμού των καρκινικών κυττάρων,αυτά δεν οδηγούν σε ασφαλή συμπεράσματα για τη δράση των αναλόγων. / Oxytocin (OT) is a cyclic nonapeptide hormone of hypothalamus that is released into the general circulation from the posterior lobe of the pituitary gland. Its major physiological roles are: a) the ability to induce uterine constructions and b) milk injection. However, widespread distribution of OT receptors in the brain and specific behavioral effects of centrally applied OT, have firmly established a role of OT as a neurotransmitter modulating reproductive and social behaviors. The role of OT in preterm labor led to the search for and design of synthetic peptide antagonists as potential tocolytic agents. A number of those OT analogues synthesized and studied. However, only Atosiban was approved, in Europe only, under the trade name Tractocile™ for the treatment of preterm labor. The design of new OT analogues is based on consequences from structure-activity studies. Antagonistic activity depends on the configuration and the hydrophobicity of the amino acid in position 2. Additionally, the deficiency of the amino-group in position 1 leads to prolongation of the activity. Furthermore, Ile3 is important for the selective binding of OT to its receptor. Based on these findings and for the investigation of the role of position 3 on biological activities, we synthesized by the Fmoc/But solid phase methodology fourteen new analogues of OT. All the analogues contain β-mercaptopropionic acid (Mpa) position 1 and D-O-ethyl-tyrosine [D-Tyr(Et)] or D-1-naphthyl-alanine [D-Nal(1)] in position 2. Isoleucine (Ile) in position 3 has substituted by γ-aminoisobutyric acid (Aib), L- or D-α-tertbutyl-glycine [L-/D-Gly(But)], L- or D-3-pyridyl-alanine [L-/D-Pal(3)] and L- or D-β-(2-thienyl)-alanine (L-/D-Thi). Electro-spray MS was in agreement with the expected results. The analogues were tested for uterotonic activity in the rat uterus in vitro test, for pressor activity in the rat pressor assay and for the affinity to human OT receptor using [3H]OT. Also, the analogues [Mpa1, D-Tyr(Et)2, Thi3]OT, [Mpa1, D-Tyr(Et)2, D- Thi3]OT], [Mpa1, D-Nal(1)2, Thi3]OT and [Mpa1, D-Nal(1)2, D- Thi3]OT] were tested for the activity on the proliferation of MDA-MB-468 and MCF-7 cells. All the new analogues are antagonists of the hormone. In particular, the analogues [Mpa1, D-Nal(1)2, Gly(But)3]OT and [Mpa1, D-Nal(1)2, Thi3]OT have potent anti-uterotonic activity (pA2=8.34±0.30 and 8.50±0.24, respectively). As regards the tests of proliferation, we are unable to come to a sfe conclusion as far as the activity of the analogues is concerned.

Anteriormente, foi identificada a atividade esquistossomicida da piplartina, um alcalóide-amida isolado em nossos estudos de bioprospecção em plantas da família Piperaceae. Neste trabalho, foi analisada a estrutura e as propriedades de uma série de análogos da piplartina para avaliar a relação estrutura-atividade. A atividade de análogos sintéticos da piplartina foi avaliada in vitro em vermes adultos de S. mansoni. Primeiro, os análogos foram testados a 50 e 100 μg/mL. Os compostos ativos foram selecionados para a determinação dos valores de IC50. Ao todo, 36 análogos com modificações em três regiões da piplartina foram avaliados. Todas as modificações tiveram uma influência negativa na atividade biológica; no entanto, uma seletividade específica entre os gêneros foi observada. Seis análogos tiveram os valores de IC50 determinados, variando entre 72,33 a 216,86 μM. As diferenças na atividade esquistossomicida entre a piplartina e seus análogos podem ser atribuídas a alterações na forma, equilíbrio hidrofílico-lipofílico e distribuição de carga eletrostática. / Previously, the schistosomicidal activity of piplartine, an alkaloid-amide isolated in our bioprospection studies with plants from Piperaceae species, was identified. In this work, we analyzed the structure and properties of a series of piplartine derivatives to propose a structure-activity relationship model. In vitro activity was evaluated in S. mansoni adult worms exposed in vitro to piplartine synthetic analogs. First, analogs were screened at 50 and 100 μg/mL. Active compounds were selected to the determination of IC50 values. A total of 36 synthetic piplartine analogs with modifications in three regions of the piplartine molecule were evaluated. All the modifications had a negative influence in the biological activity; nevertheless, a gender specific selectivity was observed. Of all the analogs, 6 had the IC50 values determined, ranging from 72.33 a 216.86 μM. Differences in the schistosomicidal activity between piplartine and its analogs could be attributed to changes in shape, hydrophilic-lipophilic equilibrium and electrostatic charge distribution.

Anteriormente, foi identificada a atividade esquistossomicida da piplartina, um alcalóide-amida isolado em nossos estudos de bioprospecção em plantas da família Piperaceae. Neste trabalho, foi analisada a estrutura e as propriedades de uma série de análogos da piplartina para avaliar a relação estrutura-atividade. A atividade de análogos sintéticos da piplartina foi avaliada in vitro em vermes adultos de S. mansoni. Primeiro, os análogos foram testados a 50 e 100 μg/mL. Os compostos ativos foram selecionados para a determinação dos valores de IC50. Ao todo, 36 análogos com modificações em três regiões da piplartina foram avaliados. Todas as modificações tiveram uma influência negativa na atividade biológica; no entanto, uma seletividade específica entre os gêneros foi observada. Seis análogos tiveram os valores de IC50 determinados, variando entre 72,33 a 216,86 μM. As diferenças na atividade esquistossomicida entre a piplartina e seus análogos podem ser atribuídas a alterações na forma, equilíbrio hidrofílico-lipofílico e distribuição de carga eletrostática. / Previously, the schistosomicidal activity of piplartine, an alkaloid-amide isolated in our bioprospection studies with plants from Piperaceae species, was identified. In this work, we analyzed the structure and properties of a series of piplartine derivatives to propose a structure-activity relationship model. In vitro activity was evaluated in S. mansoni adult worms exposed in vitro to piplartine synthetic analogs. First, analogs were screened at 50 and 100 μg/mL. Active compounds were selected to the determination of IC50 values. A total of 36 synthetic piplartine analogs with modifications in three regions of the piplartine molecule were evaluated. All the modifications had a negative influence in the biological activity; nevertheless, a gender specific selectivity was observed. Of all the analogs, 6 had the IC50 values determined, ranging from 72.33 a 216.86 μM. Differences in the schistosomicidal activity between piplartine and its analogs could be attributed to changes in shape, hydrophilic-lipophilic equilibrium and electrostatic charge distribution.

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No. of bitstreams: 1 daniellecristinalimmermannfranco.pdf: 2469355 bytes, checksum: e65f0823e3d0c81a2ef45352354878ca (MD5) Previous issue date: 2017-05-26 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / O resveratrol é um polifenol da classe dos estilbenos produzido por inúmeras plantas em resposta ao estresse do ambiente sendo reconhecido por apresentar inúmeras atividades biológicas, tais como antioxidante, anti-inflamatória, analgésica, antiviral, cardioprotetora, antitumoral, dentre muitas outras. Apesar disso, a reduzida biodisponibilidade do resveratrol tem sido fator limitante para a sua ampla utilização, fomentando a pesquisa por análogos quimicamente modificados que tenham suas atividades potencializadas e biodisponibilidade melhorada. Nesse contexto, o objetivo do presente estudo foi avaliar os potenciais anti-inflamatório e antitumoral de compostos análogos do resveratrol. Foram avaliados quinze análogos quanto às suas propriedades físico-químicas e interferência na viabilidade de células RAW 264.7 e de células tumorais da linhagem 4T1 (método de MTT). Os oito análogos com melhor resposta aos ensaios anteriores foram selecionados e testados sobre outra linhagem celular (J774.A1) e cultura primária de macrófagos peritoneais, confirmando sua inocuidade às mesmas. A LC50 sobre células RAW 264.7 também foi determinada. Em seguida, os análogos foram testados quanto ao potencial antioxidante (ensaio de captura do íon DPPH); influência na produção de óxido nítrico (NO) (reação de Griess), de citocinas (IL-1, IL-6, IL-10, IL-12, IFN- e TNF-α) e da quimiocina CCL-2 (ambos por Elisa). A atividade imunomoduladora foi determinada por meio da análise da expressão de MHCII e de moléculas costimuladoras CD80, CD86 e CTLA-4 (citometria de fluxo), além da influência na proliferação de linfócitos T e B (método de MTT). A partir destes dados, quatro análogos foram selecionados e avaliados in vivo pelo modelo de edema de orelha murino, revelando resultados satisfatórios para todos os análogos. No entanto, dois deles se sobressaíram e foram empregados em um modelo de artrite induzida por zymosan, confirmando que os mesmos são capazes de fornecer resultados comparáveis à dexametasona. Quanto ao potencial antitumoral, foi determinado a LC50 e índice de seletividade (IS) dos análogos frente a linhagens celulares de câncer de mama humano (MCF-7) e murino (4T1) e melanoma murino (B16). Os resultados obtidos revelaram que pelo menos cinco análogos podem ser explorados neste sentido, apresentando IS comparávelou superior ao de fármacos classicamente utilizados na terapia antineoplásica. Mais estudos devem ser conduzidos no intuito de determinar os mecanismos de ação envolvidos na atividade anti-inflamatória, a influência da imunomodulação na atividade antitumoral e a biodisponibilidade destes análogos. / Resveratrol is a polyphenolic phytoalexin stilbene produced by many plants in response to hostile environmental conditions, recognized to present innumerous biological activities, such as antioxidant, anti-inflammatory, analgesic, antiviral, cardioprotective, antitumor and others. However, the reduced bioavailability of resveratrol has been the limiting factor for its widespread use. Therefore, chemically modified resveratrol analogs that have its activities potentiated, eliminating the problem of bioavailability, are the subject of investigations. Thus, the aim of the present study was to evaluate the anti-inflammatory and antitumor potential of resveratrol analogues. Fifteen analogues were evaluated for physicochemical properties and influence on the viability of RAW 264.7 cells and 4T1 strain tumor cells (MTT method), determining the analogs with highest potential to remain in the study. Thus, the eight selected analogs were tested on another cell line (J774.A1) and a primary culture (peritoneal macrophages), confirming their innocuity to it. These analogs also had LC50 determined on RAW 264.7 cells. Next, it was tested for antioxidant potential (DPPH ion capture assay); evaluated on the influence on the production of nitric oxide (NO) (Griess reaction for nitrite dosing); cytokines (IL-1, IL-6, IL-10, IL-12, IFN- and TNF-α) and chemokine (MCP1) (both by Elisa); Expression of MHCII and CD80, CD86 and CTLA-4 (flow cytometry) and proliferation of T and B lymphocytes (MTT method). From these data, four analogs used topically in a model of acute inflammation of ear edema were found, revealing satisfactory results for all analogues. However, two of them excelled and were employed in a Zymosan-induced arthritis model, confirming that they are able to provide results comparable to dexamethasone. As for the direct antitumor potential of synthetic molecules, the LC50 and the selectivity index (SI) against human breast cancer cells (MCF-7) and murine (4T1) and murine melanoma (B16) were determined. The obtained results revealed that at least five analogs can be exploited in this way, presenting comparable or higher SI than drugs classically used in antineoplastic therapy. Further studies shall be conduct in order to determine the mechanisms of action involved in anti-inflammatory activity, the influence of immunomodulation on antitumor activity and the bioavailability of these analogs.

Em função da potente atividade antifúngica de piperolídeos (1 e 2) isolados do extrato das folhas de Piper malacophyllum foram sintetizados diversos análogos visando estabelecer indicativos sobre relações estrutura-atividade frente à Cladosporium cladosporioides e C. sphaerospermum e frente à forma epimastigota de Trypanosoma cruzi. Outros produtos naturais foram investigados nos extratos e foi constatada a predominância dos butenolídeos 1 e 2 no extrato metanólico de folhas. Foram isolados e caracterizados ainda dois sesquiterpenos [acetato de shizuka-acoradienolila (4) (inédito) e shizuka-acoradienol (5)] e dois compostos alquenilfenílicos [5-[(3E)-oct-3-en-1-il]-1,3- benzodioxol (3) (inédito) e gibbilimbol B (6)]. Esses dois ultimos foram observados como compostos predominantes nos extratos das raízes, caules e frutos. O alquenilbenzeno 3 foi também o produto majoritário no óleo essencial das folhas. Foi desenvolvido e validado um método em cromatografia eletrocinética micelar (MEKC) para a análise dos butenolídeos em extratos de folhas de P. malcophyllum. Os compostos sintéticos foram preparados partir da condensação da 4-metoxifuran-2(5H)-ona (24) com diversos benzaldeídos que resultaram na obtenção de butenolídeos 7-20, 22 e 23. Os piperolídeos naturais e sintéticos mostraram-se como classes promissoras como modelos para antifúngicos em ensaios contra C. sphaerospermum e C. cladosporioides, mas não foram promissores como tripanocidas. / Based on the powerful antifungal activity of piperolides (1 e 2) isolated from Piper malacophyllum leaves extracts, several analogues were synthesized aiming at establishing preliminary structure-activity relationship against Cladosporium cladosporioides, C. sphaerospermum and against epimastigote form of Trypanosoma cruzi. Phytochemical studies carried out on methanolic extract from leaves indicated butenolides 1 e 2 as major compounds in addition to two sesquiterpenes [shizukaacoradienol acetate (4) (new compound) and shizuka-acoradienol (5)] and two alkenylphenyl compounds [5-[(3E)-oct-3-en-1-yl]-1,3-benzodioxole (3) (new compound) and gibbilimbol B (6)]. The last two were detected as major compounds in the root, stem and fruit extracts. The alkenylbenzene 3 was also found as the major product in the essential oil from leaves. One validated method based on micellar electrokinetic chromatography (MEKC) was developed to analyse and validated the amount of butenolides the in P. malcophyllum leaves extracts. The butenolides 7-20, 22 e 23 were prepared by condensation of 4-methoxyfuran-2(5H)-one (24) with several benzaldehydes. The natural and synthetic piperolides were found as a promissing antifungal class against C. sphaerospermum and C. cladosporioides, although no trypanocidal activity was observed.

Em função da potente atividade antifúngica de piperolídeos (1 e 2) isolados do extrato das folhas de Piper malacophyllum foram sintetizados diversos análogos visando estabelecer indicativos sobre relações estrutura-atividade frente à Cladosporium cladosporioides e C. sphaerospermum e frente à forma epimastigota de Trypanosoma cruzi. Outros produtos naturais foram investigados nos extratos e foi constatada a predominância dos butenolídeos 1 e 2 no extrato metanólico de folhas. Foram isolados e caracterizados ainda dois sesquiterpenos [acetato de shizuka-acoradienolila (4) (inédito) e shizuka-acoradienol (5)] e dois compostos alquenilfenílicos [5-[(3E)-oct-3-en-1-il]-1,3- benzodioxol (3) (inédito) e gibbilimbol B (6)]. Esses dois ultimos foram observados como compostos predominantes nos extratos das raízes, caules e frutos. O alquenilbenzeno 3 foi também o produto majoritário no óleo essencial das folhas. Foi desenvolvido e validado um método em cromatografia eletrocinética micelar (MEKC) para a análise dos butenolídeos em extratos de folhas de P. malcophyllum. Os compostos sintéticos foram preparados partir da condensação da 4-metoxifuran-2(5H)-ona (24) com diversos benzaldeídos que resultaram na obtenção de butenolídeos 7-20, 22 e 23. Os piperolídeos naturais e sintéticos mostraram-se como classes promissoras como modelos para antifúngicos em ensaios contra C. sphaerospermum e C. cladosporioides, mas não foram promissores como tripanocidas. / Based on the powerful antifungal activity of piperolides (1 e 2) isolated from Piper malacophyllum leaves extracts, several analogues were synthesized aiming at establishing preliminary structure-activity relationship against Cladosporium cladosporioides, C. sphaerospermum and against epimastigote form of Trypanosoma cruzi. Phytochemical studies carried out on methanolic extract from leaves indicated butenolides 1 e 2 as major compounds in addition to two sesquiterpenes [shizukaacoradienol acetate (4) (new compound) and shizuka-acoradienol (5)] and two alkenylphenyl compounds [5-[(3E)-oct-3-en-1-yl]-1,3-benzodioxole (3) (new compound) and gibbilimbol B (6)]. The last two were detected as major compounds in the root, stem and fruit extracts. The alkenylbenzene 3 was also found as the major product in the essential oil from leaves. One validated method based on micellar electrokinetic chromatography (MEKC) was developed to analyse and validated the amount of butenolides the in P. malcophyllum leaves extracts. The butenolides 7-20, 22 e 23 were prepared by condensation of 4-methoxyfuran-2(5H)-one (24) with several benzaldehydes. The natural and synthetic piperolides were found as a promissing antifungal class against C. sphaerospermum and C. cladosporioides, although no trypanocidal activity was observed.