Orientadores: João Ernesto de Carvalho, Ronaldo Aloise Pilli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-09-28T18:41:42Z (GMT). No. of bitstreams: 1 Costa_DeboraBarbosaVendramini_D.pdf: 5920460 bytes, checksum: d6638ad4d3d0a96e4bbcdb55823f3254 (MD5) Previous issue date: 2012 / Resumo: A carcinogênese é um processo longo, multi-etapas, onde células normais progressivamente adquirem um fenótipo neoplásico. Sua origem é favorecida por fatores genéticos, exposição à carcinógenos químicos, infecções crônicas e incorporação de mutações em genes envolvidos com a regulação da homeostase celular. O crescente entendimento da biologia tumoral tem fornecido alvos moleculares para a triagem orientada de quimioterápicos e de agentes quimiopreventivos, geralmente de origem natural ou sintetizados com base em produtos naturais. A evolução da química orgânica permitiu que compostos naturais fossem sintetizados de forma econômica e aperfeiçoados quanto às suas propriedades físico-químicas, favorecendo sua disponibilidade. Esse trabalho teve como foco a goniotalamina, uma estiril-lactona naturalmente encontrada na forma enantiomérica (R) em plantas do gênero Goniothalamus (Annonaceae). Foram sintetizadas as formas (R), (S) e racêmica, que em painel de nove linhagens tumorais humanas levaram à inibição de crescimento e morte celular com elevada potência. Apesar da potência sobre a MCF-7 (carcinoma mamário responsivo ao estrógeno), estudos com estimulação estrogênica revelaram que a atividade da goniotalamina é independente da via hormonal. De qualquer maneira, a goniotalamina racêmica inibiu em 65% a proliferação da MCF-7 implantada em fibras semipermeáveis (hollow fiber) em camundongos. A citometria de fluxo revelou que o racemato de goniotalamina induz apoptose na linhagem tumoral de cólon HT-29 em baixa concentração (10 ?g/mL), com participação das caspases 8 e 9, sugerindo ativação das vias extrínseca e intrínseca. Houve também indução de apoptose na linhagem de melanoma humano SK-MEL-2, mas esse efeito diminuiu quando essa linhagem foi transfectada com Bcl-XL, inibidor apoptótico ligado à mitocôndria, sugerindo ativação principalmente da via apoptótica extrínseca, com ativação da via intrínseca para amplificação do sinal. A goniotalamina em suas três formas comprovou in vivo a atividade apresentada in vitro, pela inibição do desenvolvimento tumoral em modelo de tumor sólido e ascítico de Ehrlich, sem sinais de toxicidade nas doses efetivas. Esses resultados, aliados aos bons rendimentos na rota sintética, favoreceram a continuidade dos estudos com a forma racêmica. Considerando que aproximadamente 25% dos tumores estão relacionados com inflamações crônicas, a goniotalamina racêmica foi avaliada em modelos de edema de pata induzido por carragenina e por diversos mediadores (fosfolipase A2, histamina/serotonina, prostaglandina E2 e bradicinina), apresentando atividade anti-edematogênica em todos os modelos. Corroborando com esses resultados, a goniotalamina apresentou efeito antinociceptivo em modelos de algesia induzidos por processos inflamatórios. A goniotalamina inibiu o desenvolvimento de colite induzida por TNBS e o tratamento oral (30 mg/kg, três vezes por semana/três meses) preveniu o desenvolvimento de inflamação e câncer em animais deficientes em interleucina 10, sem apresentar sinais de toxicidade. Tais dados foram revelados após análise de dano macroscópico e microscópico (histologia) do cólon, havendo diminuição da expressão relativa dos genes para TNF? e MMP9, importantes no processo inflamatório e de invasão e metástase. Além disso, a goniotalamina diminuiu a incidência de metástase pulmonar em modelo de melanoma metastático. Os resultados obtidos e o perfil multi-alvos apresentado pela goniotalamina sugerem sua avaliação em terapias combinatórias para tratamento de processos inflamatórios crônicos e de câncer / Abstract: Carcinogenesis is a long process involving several steps, in which normal cells progressively acquire a neoplastic phenotype. Its origin is favored by genetic factors, exposure to chemical carcinogens, chronic infections and incorporation of mutations into genes involved in regulation of cellular homeostasis. The increasing understanding of tumor biology has provided molecular targets for screening of targeted chemotherapeutic and chemopreventive agents, usually from natural sources or synthesized based on natural products. The improvement of organic chemistry allowed the synthesis of natural products in an economic and refined way, with improvement on their physicochemical properties and availability. This work focuses on goniothalamin, a styryl-lactone naturally found on its enantiomeric form (R), in plants of Goniothalamus (Annonaceae) genus. The (R), (S) and racemic forms were synthesized and led to growth inhibition and cell death in a panel of nine human tumor cell lines, with high potency. Despite the potency against MCF-7 (breast carcinoma responsive to estrogen), estrogen stimulation studies revealed that the goniothalamin activity is independent of the hormone pathway. Anyway, racemic goniothalamin inhibited on 65% the proliferation of MCF-7 implanted into semi permeable fibers (hollow fiber) in mice. Flow cytometry analysis showed that low concentration of racemic goniothalamin (10 ?g/mL) leads colon tumor cell line (HT-29) to apoptosis with involvement of caspase 8 and 9, suggesting the activation of the extrinsic and intrinsic pathways. Apoptosis was also induced in human melanoma cell line SK-MEL-2, but the activity was decreased in SK-MEL-2 transfected with Bcl-XL, a mitochondrial related apoptotic inhibitor, suggesting mainly activation of the extrinsic apoptotic pathway, with activation of the intrinsic pathway to amplify the signal. Goniothalamin on its three forms comproved in vivo the in vitro activity by inhibiting tumor development in models of Ehrlich solid and ascitic tumor in mice, without signals of toxicity at the effective doses. These results, combined with good yields in the synthetic rout favored the racemic form to continue the in vivo studies. Considering that about 25% of tumors are related to chronic inflammation, racemic goniothalamin was evaluated in models of paw edema induced by carrageenan and various mediators (phospholipase A2, histamine/serotonin, prostaglandin E2 and bradykinin) displaying anti-edematogenic activity in all models. Corroborating these results, goniothalamin showed antinociceptive effect in models of algesia induced by inflammatory processes. It also inhibited the development of TNBS-induced colitis and oral treatment (30mg/kg, three times/week, three months) prevented the development of inflammation and cancer in interleukin-10 deficient mice, without signals of toxicity. These data were revealed after macroscopic and microscopic (histology) colon damage analysis, with decreased expression of TNF? and MMP9, which are important in inflammation, invasion and metastasis process. Furthermore, goniothalamin reduced the incidence of lung metastasis in a metastatic melanoma model. These results and the multitarget profile presented by goniothalamin suggest its evaluation in combinatory therapies for treatment of chronic inflammation and cancer / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Orientador: Lucia Helena Brito Baptistella / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-08T22:53:36Z (GMT). No. of bitstreams: 1 Jorge_AndredeCarvalho_M.pdf: 8483799 bytes, checksum: cbe192b8fe9d903f29cd3c2171475ffc (MD5) Previous issue date: 2006 / Resumo: O ácido quínico 1 é um metabólico de planta de ampla ocorrência natural que teve seu uso limitado no passado como precursor quiral em síntese de múltiplas etapas. Essa aplicação mudou em anos mais recentes quando várias publicações demonstraram toda a potencialidade sintética do seu esqueleto quiral altamente funcionalizado. O objetivo deste trabalho é a utilização de 1 para a síntese de biciclos furanofurona saturados e/ou insaturados com substituintes na junção dos anéis. O interesse nesses esqueletos bicíclicos se deve ao enorme potencial de atividade biológica que eles possuem, já que são idênticos àqueles encontrados em alguns compostos naturais isolados de plantas da família Annonaceae. Esses compostos são pertencentes a um grupo de moléculas conhecido como estiril lactonas, cujos membros, em sua grande maioria, apresentam significantes atividades citotóxicas em relação a células tumorais humanas. A seqüência proposta pode abrir novas alternativas para a preparação de análogos inéditos na serie das estiril lactonas, explorando a possibilidade de um aumento de cadeia através de uma reação de Wittig em carbonila de lactona. Estes novos análogos devem também ser submetidos a teste antiproliferativos, visando a análise de suas possíveis atividades farmacológicas. Os estudos efetuados permitem afirmar que a transformação do sistema ácido quínico em furanofuronas saturadas é bastante viável. A partir de 1 não foi possível a preparação do biciclo furanofurona insaturado 2, mas a rota proposta para os derivados furanofurona saturados 3, 4 e 5 se mostrou bastante versátil, abrindo possibilidades para a preparação de outros tipos de derivados. / Abstract: Quinic acid 1 is a plant metabolite widespread in nature that had in the past a limited use as a chiral precursor on organic synthesis. This application changed in recent years, and now several publications has shown the enormous synthetic potential of its highly functionalized skeleton. The purpose of this work is the use of 1 for the synthesis of furanefurone saturated and/or unsaturated bicycles with substitutes in the ring junction. The interest in these bicyclic skeletons is due to their enormous potential for biological activity, since they are identical to those found in some isolated natural compounds found in Annonaceae family plants. These compounds belong to a group of molecules known as styryl lactones, whose members, in its great majority, present significant cytotoxicity against human tumor cells. The proposed sequence can open new alternatives for the preparation of unknown analogous of this series, always exploring the possibility of carbon chain extention through a Wittig reaction in the lactone carbonyl group. These new analogues must also be submitted to some antiproliferative essays. The present studies allow confirming that the transformation quinic acid to furanefurone saturated systems is feasible. From 1, it was not possible the preparation of the unsaturated furanefurone 2, but the proposed sequence for 3, 4 and 5 has shown new possibilities for the synthesis of other derivatives. / Mestrado / Quimica Organica / Mestre em Química

Orientador: Ronaldo Aloise Pilli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-04T13:47:35Z (GMT). No. of bitstreams: 1 Fatima_Angelode_D.pdf: 6353488 bytes, checksum: a8d593bc18e70b70bdac0ab5c7789f96 (MD5) Previous issue date: 2005 / Doutorado / Quimica Organica / Doutor em Quimica

<p style="text-align: justify;">Reverzno-faznom tečnom hromatografijom pod visokim pritiskom primenom dva sistema<br />rastvarača ispitano je pona&scaron;anje i hromatografska lipofilnost prirodnih stiril laktona 7-(+)-<br />goniofufurona, 7-epi-(+)-goniofufurona, krasalaktona B i C i dvadeset njihovih<br />novosintetizovanih derivata i analoga. U ranijim ispitivanjima pokazalo se da ova jedinjenja<br />imaju veliki biolo&scaron;ki potencijal jer pokazuju zapaženu citotoksičnost prema vi&scaron;e humanih<br />tumorskih ćelijskih linija. Hromatografsko pona&scaron;anje jedinjenja uglavnom je u skladu sa<br />njihovom strukturom. Ustanovljene su linearne veze između hromatografskih retencionih<br />konstanti i većine in silico parametara lipofilnosti. Primenom hemometrijske QSRR analize<br />utvrđeni su veoma dobri multi linearni regresioni prediktivni modeli kvantitativne zavisnosti<br />između eksperimentalno dobijene hromatografske retencione konstante, koja defini&scaron;e<br />retenciju jedinjenja u čistoj vodi i in silico molekulskih deskriptora odnosno strukture<br />jedinjenja. Lipofilnost jedinjenja ima najveći uticaj na njihove farmakokinetičke, tj. ADME<br />(apsorpcija, distribucija, metabolizam, eliminacija) osobine. Definisani su i statistički<br />potvrđeni najbolji multi linearni regresioni modeli zavisnosti farmakokinetičkih parametara<br />stiril laktona i od drugih molekulskih deskriptora. In vitro citotoksična aktivnost jedinjenja<br />evaluirana je prema četiri nove humane maligne ćelijske linije: kancer prostate (PC3), kancer debelog creva (HT-29), melanom (Hs294T), adenokancer pluća (A549). Najaktivnije<br />novosintetizovano jedinjenje je triciklični 4-fluorocinamatni analog, koji ispoljava<br />nanomolarnu aktivnost (IC₅₀ 2,1 nM) prema ćelijama melanoma i aktivniji je preko 2250 puta od komercijalnog antitumorskog agensa doksorubicina (DOX). SAR analizom utvrđena je zavisnost između strukture i biolo&scaron;ke aktivnosti jedinjenja. Molekulskim dokingom ispitana je veza stiril laktona i ciljanog proteina značajnog za kancer prostate. Jedinjenja sa visokom inhibitornom aktivno&scaron;ću prema ćelijama kancera prostate imaju visok doking skor i mogu graditi koordinativno-kovalentnu vezu sa Fe²+jonom prisutnim u aktivnom centru enzima. 3D-QSAR analizom, koja je izvedena metodama komparativnih polja CoMFA i CoMSIA, formiran je značajan prediktivni model između hemijske strukture i biolo&scaron;ke aktivnosti stiril laktona.</p> / <p>The behavior and the chromatographic lipophilicity natural styryl lactone 7-(+)-<br />goniofufurone, 7-epi-(+)-goniofufurone, crassalactones B and C and twenty of their newly<br />synthesized derivatives and analogs were examined using reverse-phase high performance liquid chromatography in the two solvent systems. In previous studies it has been shown that these compounds have great biological potential toward several human tumor cell lines. Chromatographic behavior of the compounds is generally in accordance with their structure. The relationships between the chromatographic retention constants and the majority of their in silico lipophilicity parameters are linear. The application of chemometric QSRR analysis determined very good multiple linear regression predictive models of quantitative correlation between experimentally obtained chromatographic retention constant, which determines the retention of the compound in pure water and in silico molecular descriptors, i.e. the structure of the compound. The lipophilicity of the compounds has a major influence on their pharmacokinetics, i.e. ADME (absorption, distribution, metabolism, elimination) properties. The best multi-linear regression models depending on the pharmacokinetic parameters of styryl lactone and other molecular descriptors have been defined and statistically validated. In vitro cytotoxic activity of the compounds was evaluated according to four novel human malignant cell lines: prostate cancer (PC3), colon cancer (HT-29), melanoma (Hs294T), lung adenocarcinom (A549). The most active compound was tricyclic 4-fluorocinnamic analog, which exhibits a nanomolar activity (IC50 2,1 nM) toward melanoma cells. This compound is over 2250 times more active than commercial antitumor agent doxorubicin (DOX). SAR analysis has revealed a correlation between the structure and the biological activity of the compounds. Using the molecular docking the relationship of the styryl lactone and the target protein important for prostate cancer was examined. The compounds with high inhibitory activity against prostate cancer cells have a high docking score and are capable to form a coordinative-covalent bond with a Fe2+ ion present in the active centre of the enzyme. 3DQSAR analysis, which was performed by methods of comparative CoMFA and CoMSIA fields, has formed a good predictive model between chemical structure and biological activity of the styryl lactone.</p>

<p>U&nbsp; radu&nbsp; su&nbsp; ostvarene&nbsp; vi&scaron;efazne&nbsp; sinteze&nbsp; većeg&nbsp; broja&nbsp; analoga&nbsp; prirodnih&nbsp; stiril-laktona&nbsp; (+)-goniofufurona&nbsp; i&nbsp; 7-epi-(+)-goniofufurona&nbsp; polazeći&nbsp; iz&nbsp; D-glukoze.<br />Ispitana&nbsp; je&nbsp; in&nbsp; vitro&nbsp; citotoksičnost&nbsp; sintetizovanih&nbsp; analoga&nbsp; prema&nbsp; devet<br />malignih&nbsp; i&nbsp; jednoj&nbsp; zdravoj&nbsp; ćelijskoj&nbsp; liniji.&nbsp; Uspostavljeni&nbsp; su&nbsp; korelacioni&nbsp; odnosi<br />izmedju&nbsp; strukture&nbsp; i&nbsp; antiproliferati vne&nbsp; aktivnosti&nbsp; sintetizovanih&nbsp; proizvoda, pored&nbsp; toga&nbsp; uradjeni&nbsp; su&nbsp; i&nbsp; dodatni&nbsp; biolo&scaron;ki&nbsp; testovi&nbsp; koji&nbsp; se&nbsp; odnose&nbsp; na dokazivanje&nbsp; mehanizma&nbsp; citotoksičnog&nbsp; dejstva&nbsp; pomenutih&nbsp; stiril-laktona&nbsp; i analoga.</p> / <p>Multistep&nbsp; synthesis&nbsp; of&nbsp;&nbsp; a&nbsp; number&nbsp; of&nbsp; natural&nbsp; styryl&nbsp; lactones&nbsp;goniofufurone&nbsp; and&nbsp; 7-epi-goniofufurone&nbsp; analogues&nbsp; was&nbsp; achieved&nbsp;starting&nbsp; f rom&nbsp; D-glucose.&nbsp; In&nbsp; vitro&nbsp; cytotoxicity&nbsp; of&nbsp; newly&nbsp; synthetized analogues&nbsp; against&nbsp; nine&nbsp; human&nbsp; tumour&nbsp; cell&nbsp; lines&nbsp; and&nbsp; against&nbsp; a single normal cell line was evaluated. Structure-activity relationships were&nbsp; established&nbsp; for&nbsp; both&nbsp; natural&nbsp; products&nbsp; and&nbsp; analogues.&nbsp; Some additional&nbsp; biological&nbsp; tests&nbsp; related&nbsp; to&nbsp; the&nbsp; cell mechanisms&nbsp; underlying the&nbsp; cytotoxicity&nbsp; of&nbsp;&nbsp; the&nbsp; mentioned&nbsp; styryl&nbsp; lactones&nbsp; and&nbsp; analogues, were also carried out.</p>

<p>U radu je ostvarena sinteza četiri prirodna proizvoda, goniobutenolida A i B,<br />krasalaktona D i 3-deoksi-kardiobutanolida i 32 derivata i analoga polazeći iz<br />D-glukoze. Sinteza prirodnog stiril-laktona, kardiobutanolida&nbsp; i njegova 4<br />derivata je izvedena polazeći iz&nbsp; D-ksiloze.&nbsp; Ispitan je uticaj sintetizovanih<br />jedinjenja&nbsp; na inhibiciju rasta 10 tumorskih i jedne zdrave ćelijske linije.<br />Uspostavljene su i&nbsp; korelacije izmedju strukture i antiproliferativne aktivnosti&nbsp;(SAR) i ispitan je mehanzam antitumorskog dejstva.</p> / <p>Synthesis of four natural products: goniobutenolide A and B,&nbsp;crassalactone D, 3-deoxy-cardiobutanolide and their 32 analogues&nbsp;and derivatives is accomplished starting from&nbsp; D-glucose. Synthesis of natural styryl lactone cardiobutanolide and its four derivatives is &nbsp;achieved&nbsp; starting&nbsp; from&nbsp; D-xylose.&nbsp; Synthesized natural lactones, their analogues and derivatives were evaluated for their antiproliferative activity against ten malignant cell lines&nbsp; as well as against a single normal cell line. Influence of functional groups on antitumour activity was established by correlating structures of synthesized compounds&nbsp; with&nbsp; their&nbsp; biological&nbsp; activity (SAR).&nbsp; Also, mechanism of antitumour action was investigated.</p>

<p>U radu je ostvarena enantiodivergentna totalna sinteza oba enantiomera goniofufurona, 7-epi-goniofufurona i krasalaktona C polazeći iz D-glukoze. Ključne faze u sintezi 7-epi-(+)-goniofufurona bile su stereoselektivna adicija fenilmagnezijum bromida na aldehidnu grupu pogodno za&scaron;tićene dialdoze, i stereospecifično formiranje furano-laktonskog prstena ciklokondenzacijom odabranog hemiacetalnog derivata sa Meldrum-ovom kiselinom. Sinteza (+)-goniofufurona i (+)-krasalaktona C zahtevala je inverziju konfiguracije na C-5<br />u zajedničkom intermedijeru, koja je efikasno ostvarena u uslovima Mitsunobu-ove reakcije, ili alternativno oksidacijom benzilne hidroksilne grupe u prohiralni keton, uz naknadnu stereoselektivnu redukcijom sa borohidridom. Sličan pristup je zatim primenjen za sintezu neprirodnih (&minus;)-enantiomera goniofufurona, 7-epi-goniofufurona i krasalaktona C, dva nova konformaciono ograničena analoga (+)- i (&minus;)-goniofufurona (oksetani 36 i ent-36), kao i odgovarajućih 7-deoksigenovanih derivata (31 i ent-31). Takodje je razvijena i prva totalna sinteza prirodnog (+)-krasalaktona B (3) i alternativna sinteza (+)-krasalaktona C (4) polazeći iz D-glukoze. Selektivni pristup molekulima 3, odnosno 4 omogućen je promenom uslova za TBDPS deprotekciju u finalnom intermedijeru 53. Osnovna karakteristika pomenutih pristupa je njihova generalnost i fleksibilnost. Na taj način je omogućena sinteza serije analoga i derivata (+)-goniofufurona, ili 7-epi-goniofufurona, uključujući i do sada nepoznate 7-epi-(+)-krasalaktone B (6) i C (7), 5,7-di-O-cinamoil derivate 8 i 9, 5,7-di-O-izopropilidenske derivate 5 i 10, kao i vi&scaron;e lipofilnih derivata (jedinjenja 26, 30, 33, 65, ent-30 i ent-33). Konačno, u drugom delu rada, ispitan je uticaj sintetizovanih stiril-laktona na rast odabranih tumorskih ćelijskih linija in vitro.</p> / <p> Enantiodivergent total syntheses of both (+)- and (&minus;)-enantiomers of goniofufurone, 7-epi-goniofufurone and crassalactone C have been accomplished starting from D-glucose. The key steps of the synthe-sis of 7-epi-(+)-goniofufurone were a stereo-selective addition of&nbsp;<br /> phenyl magnesium bromide to a protected dialdose, followed by a stereospecific furano-lactone ring formation by condensation of a partially protected lactole with Meldrum&rsquo;s acid. The synthesis of (+)-goniofufurone and (+)-crassalactone C required a configurational inversion at C-5 in the common intermediate that was efficiently achieved under the standard Mitsunobu conditions, or alternatively through a sequential oxidation of the benzylic hydroxyl group followed by a stereo-selective reduction with borohydride. A similar approach was applied to the synthesis of the unnatural enantiomers of goniofufurone, 7-epi-goniofufurone and crassalactone C, two novel, conformationally constrained analogues of both (+)- and (&minus;)-goniofufurone (oxetanes 34 and ent-34). as well as the corresponding 7-deoxygenated derivatives (31 and ent-31). We have also developed the first total synthesis of (+)-crassalactone B (2) and an alternative synthesis of (+)-crassalactone C (3) starting from D-glucose. Finally, the synthesized styryl-lactones were evaluated for their antiproliferative activity against a panel of human tumor cell lines.</p>