TRPM3 (melastatin-related transient receptor potential 3) is a calcium-permeable nonselective cation channel that is expressed in various tissues, including insulin-secreting β-cells and a subset of sensory neurons from trigeminal and dorsal root ganglia (DRG). TRPM3 can be activated by the neurosteroid pregnenolone sulphate (PregS) or heat. TRPM3α2 mice display an impaired sensation of noxious heat and inflammatory thermal hyperalgesia. A calcium-based screening of a compound library identified four natural compounds as TRPM3 blockers. Three of the natural compounds belong to the citrus fruit flavanones (hesperetin, eriodictyol and naringenin), the forth compound is a deoxybenzoin that can be synthesized from an isoflavone of the root of Ononis spinosa (ononetin). The IC50 for the substances ranged from upper nanomolar to lower micromolar concentrations. Electrophysiological whole-cell measurements as well as calcium measurements confirmed the potency of the compounds to block TRPM3 in DRG neurones. To further improve the potency and the selectivity of TRPM3 block and to identify the pharmacophore within the flavanone structure, we conducted a hit optimisation procedure by re-screening a focussed library. The library composed of several flavanones with different substitutions on relevant chemical positions and of representatives from different flavonoid subgroups. Within this secondary screen, we identified isosakuranetin and liquiritigenin as active blockers of PregS-induced Ca2+ entry through TRPM3. Isosakuranetin, a flavanone that can be found in blood oranges and grapefruits, displayed an IC50 of 50 nM, and is the most potent inhibitor of TRPM3 identified so far. The novel compounds exhibited a marked specificity for TRPM3 compared with other thermosensitive TRP channels, and blocked PregS-induced [Ca2+]i signals and ionic currents in freshly isolated DRG neurones. Furthermore, isosakuranetin and hesperetin reduced the sensitivity of mice to noxious heat and PregS-induced chemical pain. Since the physiological functions of TRPM3 channels are still poorly defined, the development and validation of potent and selective blockers is expected to contribute to clarifying the role of TRPM3 in vivo. Considering the involvement of TRPM3 in nociception, TRPM3 blockers may represent a novel concept for analgesic treatment.

TRPM3 (melastatin-related transient receptor potential 3) is a calcium-permeable nonselective cation channel that is expressed in various tissues, including insulin-secreting β-cells and a subset of sensory neurons from trigeminal and dorsal root ganglia (DRG). TRPM3 can be activated by the neurosteroid pregnenolone sulphate (PregS) or heat. TRPM3α2 mice display an impaired sensation of noxious heat and inflammatory thermal hyperalgesia. A calcium-based screening of a compound library identified four natural compounds as TRPM3 blockers. Three of the natural compounds belong to the citrus fruit flavanones (hesperetin, eriodictyol and naringenin), the forth compound is a deoxybenzoin that can be synthesized from an isoflavone of the root of Ononis spinosa (ononetin). The IC50 for the substances ranged from upper nanomolar to lower micromolar concentrations. Electrophysiological whole-cell measurements as well as calcium measurements confirmed the potency of the compounds to block TRPM3 in DRG neurones. To further improve the potency and the selectivity of TRPM3 block and to identify the pharmacophore within the flavanone structure, we conducted a hit optimisation procedure by re-screening a focussed library. The library composed of several flavanones with different substitutions on relevant chemical positions and of representatives from different flavonoid subgroups. Within this secondary screen, we identified isosakuranetin and liquiritigenin as active blockers of PregS-induced Ca2+ entry through TRPM3. Isosakuranetin, a flavanone that can be found in blood oranges and grapefruits, displayed an IC50 of 50 nM, and is the most potent inhibitor of TRPM3 identified so far. The novel compounds exhibited a marked specificity for TRPM3 compared with other thermosensitive TRP channels, and blocked PregS-induced [Ca2+]i signals and ionic currents in freshly isolated DRG neurones. Furthermore, isosakuranetin and hesperetin reduced the sensitivity of mice to noxious heat and PregS-induced chemical pain. Since the physiological functions of TRPM3 channels are still poorly defined, the development and validation of potent and selective blockers is expected to contribute to clarifying the role of TRPM3 in vivo. Considering the involvement of TRPM3 in nociception, TRPM3 blockers may represent a novel concept for analgesic treatment.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Croton rhamnifolius (MÃll. Arg.) à uma espÃcie comum do sertÃo nordestino, principalmente nos estados de Pernambuco e CearÃ. O espÃcime foi coletado na cidade de Salgueiro â PE onde à popularmente conhecido por âquebra-facaâ, cujas as folhas sÃo empregadas para o tratamento de mal-estar gÃstrico, febre e Ãnsia de vÃmitos. Este trabalho apresenta uma abordagem fitoquÃmica, visando o isolamento e caracterizaÃÃo estrutural dos constituintes quÃmicos de C. rhamnifolius. A investigaÃÃo quÃmica foi realizada com os extratos hexÃnico e etanÃlico do c aule, e extrato etanÃlico da raiz. Os extratos foram submetidos a partiÃÃes lÃquido-lÃquido, cromatografias convencionais (adsorÃÃo-gel de sÃlica e exclusÃo - gel de dextrana) e atuais (HPLC - fase normal e reversa), resultando no isolamento e identificaÃÃo de dez terpenos, um alcaloide, um flavonoide e uma cumarina. Dentre as substÃncias isoladas, temos dois diterpenos de esqueleto clerodÃnico: Ãcido hardwickico e o Ãcido cleroda-3,13-dien-16,15-olido-18-Ãico; alÃm do diterpeno de esqueleto beierano: beiereno-3,12-diol; atiseno: 13 -hidroxi-atisen-3-ona; caurano: caur-16-eno; labdano rearranjado: rhamnifa, do sesquiterpeno: senecrassidiol; trÃs triterpenos pentacÃclicos: Ãcido acetil aleuritÃlico (AAA), lupeol e lupenona; o alcaloide de esqueleto quinolÃnico quinorhamina, um flavonoide 3', 4â, 5-trihidroxi-,7-dimetÃxiflavona; e uma cumarina escopoletina. O alcaloide indÃlico, rhamnifolina, obtido durante a dissertaÃÃo de mestrado e o diterpeno 13-hidroxi-atisen-3-ona foram avaliados quanto Ãs suas atividades citotÃxicas, tripanocida e leishmanicida, e os resultados obtidos evidenciaram uma excelente atividade do primeiro composto nos trÃs testes farmacolÃgicos. Para o segundo, foram obtidos resultados positivos contra Leishmania amazonensis. As substÃncias quÃmicas isoladas tiveram suas estruturas elucidadas por mÃtodos fÃsicos (ponto de fusÃo e rotaÃÃo Ãptica) e espectroscÃpicos (Espectroscopia na regiÃo do Inf ravermelho, Espectrometria de Massas e Espectroscopia de RessonÃncia MagnÃtica Nuclear de 1H e 13C, incluindo tÃcnicas uni e bidimensionais), alÃm de comparaÃÃo com dados da literatura. / Croton rhamnifolius (MÃll. Arg.) is a very common shrub of the interior northeastern region of Brazil, particularly in the states of Pernambuco and CearÃ. A specimen was harvested at Salgueiro County â Pernambuco where it is known as âquebra-facaâ (Pat. lit. = knife breaker) and is used to treat gastric problems. This work reports the phytochemical approach to isolate and characterize its chemical constituints. The chemical investigation was perfomed with hexane and ethanol extracts of the think, and the ethanol extract for the roots. Liquid-liquid partitioning, conventional (si-gel adsorption and dextran gel exclusion) and modern chromatography normal and revers phase (HPLC) lead to the isolation and characterization of ten terpenoids, an alkaloid, a flavonoid, and coumarin. Of the isolation compounds two were of the clerodane type hardwickic acid and cleroda-3,13-dien-16,15-olide-1 8-Ãico acid; a beyerene: beier-15-eno-3,12-diol; a atisane: 13-hydroxy-atisen-3-one; a kaurene: kaure-16-ene; a labdane: rhamnife; diterpene senecrassidiol; three were pentacyclic triterpenes: lupeol, lupenone and aleuritolic acid; an alkaloid quinoline: quinorhamine; a flavonoid 3', 4â, 5-trihydroxy-3,7-dimethoxy-flavone; and coumarin: scopoletin. The indole alkaloid rhamnifoline, previously isolated and the diterpene 13-hydroxy-atisen-3-one were evaluated concerning to their cytotoxic, tripanocidal and leishmanicidal activities. Just the alkaloid showed positive results for all assays. The diterpene showed activity just on the leishmania test. The structure of all isolated compounds were determined through physical methods (melting points and optical rotati on) and spectroscopic means (IR, MS and NMR) including two dimensional techniques and comparision to the literature.

Diese Dissertation beschreibt die Synthese der vier Flavanone Lonchocarpol A, 6-Dimethylallylnaringenin, Glabrol und Euchrenon A7. Des weiteren konnten drei dieser Naturstoffe enantiomerenrein mit Hilfe einer kinetischen Racematspaltung hergestellt werden. Dabei wurden die Carboxylkohlenstoffe selektiv mit einer asymmetrischen Transferhydrierung nach Noyori reduziert.

Diese Dissertation beschreibt die Synthese der vier Flavanone Lonchocarpol A, 6-Dimethylallylnaringenin, Glabrol und Euchrenon A7. Des weiteren konnten drei dieser Naturstoffe enantiomerenrein mit Hilfe einer kinetischen Racematspaltung hergestellt werden. Dabei wurden die Carboxylkohlenstoffe selektiv mit einer asymmetrischen Transferhydrierung nach Noyori reduziert.