Structure-Activity Relationship (SAR) Studies to Maximize the Activity of Compounds Isolated from Octocorals

This chapter presents some significant study cases on octocoral organisms (Eunicea succinea, Eunicea mammosa, Eunicea knighti, Pseudoplexaura flagellosa, Eunicea laciniata, Antillogorgia elisabethae, Muricea austera, Paragorgia sp., Lobophyton sp., Sarcophyton glaucum and Sinularia lochmodes) that have been identified as a source of promising bioactive compounds and whose results have further been used for studies on structure-activity relationship (SAR) as a strategy to increase the value of the activity initially detected. The scientific literature data discussed here were obtained with the SciFinder tool during the period 2000–2016 and from the additional results here presented for the biofilm inhibition activity of compounds and synthetic analogs for the cases related with Eunicea knighti and Pseudoplexaura flagellosa (until now unpublished data of the authors of this chapter)

Compuestos tipo cembrano naturales y algunos análogos sintéticos como potenciales agentes antifouling

Los estudios de bioprospección en los octocorales del Caribe colombiano Pseudoplexaura flagellosa y Eunicea knighti permitieron aislar e identificar dieciséis compuestos tipo cembrano, encontrando que los ocho compuestos aislados de la fracción orgánica de P. flagellosa son epímeros en C-8 (1-8). Los compuestos 2, 4 y 6 son reportados por primera vez y aunque los compuestos 1, 3, 5, 7 y 8 han sido reportados previamente, sus estructuras y asignaciones de RMN fueron revisadas, completadas o corregidas en estos cembranos según cada caso. Las estructuras de estos compuestos fueron establecidas con base en los análisis detallados de sus datos espectroscópicos. Adicionalmente, la configuración relativa de los compuestos 1-3 y 7 fue confirmada mediante el análisis de difracción de Rayos-X de los cristales y la configuración absoluta de los compuestos 1-8 fue determinada usando una combinación del método modificado de Mosher e interconversiones químicas inequívocas. De la fracción orgánica del octocoral E. knighti fueron aislados como constituyentes menores tres nuevos diterpenos 9-11, junto con los cembranos conocidos 12-16. Las estereoestructuras de los compuestos nuevos fueron determinadas mediante un análisis detallado de los datos espectroscópicos, y una combinación de transformaciones químicas y el método modificado de Mosher. Todos los cembranos aislados (1-16) fueron evaluados para establecer sus propiedades antifouling usando los bioensayos de inhibición de quorum sensing (QS) e inhibición del biofilm bacteriano, estableciendo que los compuestos 3, 6, 7, 10, 11 y 14 tienen excelente actividad inhibitoria de QS contra la cepa biosensora Chromobacterium violaceum, la cual fue establecida mediante inhibición de la producción del pigmento violeta sin interferir en su crecimiento. Adicionalmente, los compuestos 3, 5, 6, 8, 9, 11 y 16 exhibieron inhibición de la maduración del biofilm bacteriano en las cepas Pseudomonas aeruginosa, Vibrio harveyi y Staphylococcus aureus. Por lo anterior se puede concluir que 3, 6, 7, 10, 11 y 14 fueron los compuestos más activos en los dos bioensayos, lo que los hace excelentes agentes antifouling y a su vez pueden ser considerados como potenciales aditivos ambientalmente amigables en las pinturas comerciales antifouling. Algunos compuestos tipo cembrano reportados en este trabajo presentan mejores valores de inhibición de QS y de biofilm bacteriano que los reportados para la mayoría de los compuestos aislados previamente de organismos marinos. También es importante mencionar que en esta investigación se hizo el primer reporte de compuestos tipo cembrano como inhibidores del biofilm bacteriano y como inhibidores de QS en C. violaceum. Sobre la base de lo anterior se pudo establecer que los cembranos naturales presentan propiedades antifouling, pero la interacción estructura-función no ha sido esclarecida, por lo cual treinta y cuatro análogos de cembranos fueron sintetizados a partir de seis cembranos naturales (1, 3, 4, 13, 15 y 16) los cuales fueron usados como cabezas de serie. Los análogos sintéticos (17-50) obtenidos a través de transformaciones químicas selectas fueron evaluados in vitro contra los sistemas de QS y como inhibidores de la formación y maduración del biofilm bacteriano en las cepas anteriormente mencionadas, encontrando que la mitad de los análogos ensayados presentaron una superior actividad inhibitoria de QS y del biofilm bacteriano que los compuestos precursores; principalmente los cembranos 17, 18, 20, 21, 23, 27, 28, 35-37 presentaron los mejores valores de inhibición y algunos compuestos (18, 23 y 28) mostraron una alta efectividad, hasta tres veces superior que los precursores naturales. Este selecto grupo de cembranos estructuralmente relacionados pueden ser usados como inhibidores de QS y del biofilm bacteriano, lo que los hace excelentes candidatos para ser usados como agentes activos en pinturas antifouling comerciales, ya que los compuestos inhibidores del biofilm e inhibidores de QS son a menudo considerados como potentes agentes antifouling. Teniendo en cuenta lo anterior se planeó un ensayo de campo diseñado por nosotros, el cual consistió en preparar diferentes formulaciones de pinturas antifouling utilizando el compuesto que presentó la mejor actividad inhibitoria de los sistemas de QS y de la formación del biofilm bacteriano en los ensayos in vitro, y que a su vez pudiera ser obtenido en cantidades del orden de gramos. De este modo se escogió el cembrano 28 para hacer una mejor aproximación a la realidad de sus propiedades antifouling al ser usado como aditivo en un recubrimiento industrial en condiciones naturales y de mayor interacción con diversos organismos del fouling en el océano. Los resultados permitieron establecer que los sustratos tratados con el compuesto 28 previnieron completamente el asentamiento del macrofouling durante el periodo de prueba y es importante notar que algunos cirripedios y bivalvos fueron observados en el lugar donde se realizó el experimento, pero no en los sustratos utilizados. La formulación con el compuesto 28 previno el asentamiento de microorganismos en un 50% durante los seis meses que duró la prueba, mientras que los controles fueron completamente cubiertos por organismos del fouling como algas filamentosas, esponjas, tunicados, gusanos, poliquetos, tubícolas y cianobacterias. Se estableció una aproximación sintética para la obtención del cembrano 19 el cual presentó la mejor actividad antifouling en los ensayos in vitro y en campo, a partir de la macrociclización del C9-alcohol propargílico 16 y el cloruro C10-alílico 6. Se logró desarrollar una ruta sintética que permitió obtener los precursores 15 y 6 con altos rendimientos y en cantidades del orden de gramos a partir de materiales de partida comercialmente disponibles, económicos y sencillos. Sin embargo, el paso final de macrociclización no fue desarrollado debido al bajo rendimiento en la obtención del compuesto 16, aunque es de resaltar que se desarrolló una ruta sintética exitosa en la obtención de los compuestos 15 y 6. Nuevos intentos para lograr la conversión de 15 en 16 con mejores rendimientos están siendo realizados en este momento en nuestro grupo de investigación, para lograr concluir la síntesis total de este compuesto, ya que la estrategia sintética ha sido desarrollada en más de un 90%. Finalmente, el trabajo desarrollado durante esta tesis tuvo unos alcances excelentes y sorprendentes que permitieron ir mas allá de los objetivos planteados inicialmente, logrando hacer un aporte significativo a la comunidad científica, principalmente en las áreas de productos naturales, síntesis, RMN y estereoquímica.Abstract: Bioprospecting studies in the Colombian Caribbean octocorals Pseudoplexaura flagellosa and Eunicea knighti, allowed to isolate and identify sixteen cembranoid compounds. Eight compounds isolated from the P. flagellosa organic fraction are epimers in C-8 (1-8). Compounds 2, 4 and 6 are reported for the first time. Although compounds 1, 3, 5, 7 and 8 have been reported previously, the cembranoid structures and NMR assignments were revised, completed or corrected. The structures of these compounds were established on the basis of detailed analysis of their spectroscopic data. Furthermore, the relative configurations of compounds 1-3 and 7 were confirmed by single-crystal X-ray diffraction. The absolute configurations of compounds 1-8 were determined using a combination of the modified Mosher method and unambiguous chemical interconversions. From the organic fraction of the octocoral E. knighti were isolated three new diterpenes 9-11 as minor constituents, along with the known cembranoids 12-16. The stereostructures of the new compounds were determined by detailed spectroscopic analysis and a combination of chemical transformations and modified Mosher´s methods. An evaluation of the antifouling properties for all the cembranoids isolated was made, using quorum sensing inhibition (QSI) assay and biofilm inhibition test. Compounds 3, 6, 7, 10, 11 and 14 showed excellent QSI activity against the biosensor Chromobacterium violaceum, as measured by inhibition of the production of violacein pigment, without interfering with its growth. Additionally, compounds 3, 5, 6, 8, 9, 11 and 16 exhibited inhibition of biofilm maturation without interfering in the growth of Pseudomonas aeruginosa, Vibrio harveyi, and Staphylococcus aureus. Therefore, the most active compounds were 3, 6, 7, 10, 11 and 14 for both assays, so, they are excellent antifouling agents that can be considered potential additives environmentally friendly to be used in commercial antifouling paints. Some compounds reported in this work showed higher values of antifouling activity stablished as inhibitors of QS and bacterial biofilm, in comparison with those reported for most of the compounds isolated previously from marine organisms. It is important to mention, that in this research was made the first report of cembranoid as inhibitors of bacterial biofilm and as compounds that interfere with QS in C. violaceum. . In this way, was established that the natural cembranoids show antifouling properties, but their structurefunction interactions are not well understood. For that reason, thirty four cembranoid analogues were synthesized using six natural cembranoids (1, 3, 4, 13, 15 y 16) as lead compounds. The analogues (17-50) obtained through the selected chemical transformations were tested in vitro against the QS systems and as inhibitors of the biofilm formation and maturation in the bacteria mentioned above. Half of the cembranoid analogues assayed showed superior QSI activity to the lead compounds, principally the cembranoids 17, 18, 20, 21, 23, 27, 28, 35-37 presented the highest inhibition values, and some compounds (18, 23 y 28) displayed remarkable potency up to three times higher than the natural compounds. Thereby, we have synthesized a pool of cembranoid with QSI activity and also as biofilm inhibitors that can be used in commercial antifouling paints due to their potential as antifouling agents. Taking into account the above, a field assay was designed in order to evaluate different formulations of antifouling paints, using the compound with the highest activity value against QS and the bacterial biofilm, and that could be obtained in a gram scale. Thereby, the cembranoid 28 was chosen and evaluated in more real conditions using it as an additive in an industrial coating in natural conditions and high interaction with the different fouling organisms in the sea. The results obtained showed that the substrates painted with the compound 28 avoid completely the macrofouling settlement during the six months of test. It is important to mention, that some barnacles were observed where the experiment took place, however, those organisms were not found in the substrates used. The formulation with the compound 28 avoid the microorganisms settlement in a 50% during the six months of the experiment, whereas the controls were completely covered by different fouling organisms as algae, sponges, tunicates, polychaetes, worms and cyanobacteria. A synthetic approximation was made in order to obtain the cembranoid 19, which showed the highest antifouling activity in vitro and in field assays, from the macrocyclization of C9-propargyl alcohol 16 with the C10-allylic chloride 6. A synthetic route was developed to obtain the precursors 15 and 6 in a high yield from commercial, cheap and simple. Although the final macrocyclization step could not be achieved due to the poor yield of the compound 16, a successful synthetic route was made to obtain the compounds 15 and 6. Different strategies are being undertaken in our research group to obtain the total synthesis of this compound, taking into account that at least 90% of this synthesis has been made. Finally, the research done during this thesis showed excellent achievements and surprising results, allowing the research to go beyond the objectives initially proposed. In this way, a significant contribution was made for the scientific community, principally in natural products, synthesis, NMR and stereochemistry fields.Doctorad

Corals in a Changing World

Corals comprise a wide variety of colonial marine invertebrates belonging to the Phylum Cnidaria. Their polyps form the most colorful, complete, and diverse communities on the Earth resembling underwater cities, commonly called coral reefs, which host a wide variety of invertebrates and fish species. They are highly productive ecosystems, contribute to the health of the biosphere, and offer a good number of economic and ecological services to coastal populations and to many people around the world. However, due to a diverse number of natural and anthropogenic stressors, corals have shown a severe decline over the past few decades. Being aware of the importance and relevance of the facts described, the book ""Corals in a Changing World"" offers new scientific information regarding the actual status and, in some cases, the resilience state of coral reef systems. Timely information is critical for managers and decision makers to implement sustainable management measures according to the ecological condition of coral reefs. In addition, the book also discusses the use of well-maintained coral microcosms to provide a good basis for performing experiments with natural fluctuations and to present studies dedicated to the coral diversity characterization and to their importance as a source of important biological compounds, which could be converted into industrial products

New Diterpenes Isolated from the Colombian Caribbean Soft Coral Pseudoplexaura flagellosa and Their Cytotoxic Properties

11 páginasStudies about secondary metabolites isolated from soft corals around the world have proven the potential of these organism as producers of compounds with a potent cytotoxic activity. In this work, we obtained the extract of Pseudoplexaura flagellosa collected in Santa Marta, Colombia, the cytotoxic activity of this extract, fractions and compounds was established against SiHa (ATCC® HTB-35™), MDA-MB-231 (ATCC® HTB26™), A549 (ATCC® CRM-CCL-185™), PC3 (ATCC® CRL1435), and L929 (ATCC® CCL1™) cell lines. The results showed that the extract of soft coral P. flagellosa has cytotoxic activity with an IC50 of 35.4, 72.3, 49.8 and 40.5 μg/mL against SiHa, A549, PC3 and MDA-MB-231 cell lines, respectively. Thus, this extract was repeatedly subjected to different chromatographic columns and final purification of these fractions afforded pure compounds 1 – 4, which were elucidated by 1D and 2D NMR experiments including 1H, 13C, COSY, HSQC, HMBC and NOESY, and HRESIMS. In addition, Mosher method was used to establish the stereochemistry of compound 2 and chemical interconversion allow establishing the stereochemistry of compound 1. These results let to conclude that compound 2 is a new stereoisomers of acetylated asperdiol previously reported on literature. Additionally, three analogues 5, 6, and 7 were synthesized from compound 1 and the cytotoxic activity of all compounds was evaluated using doxorubicin as positive control. The results showed that 6 (IC50 of 19.3, 23.7, 13.4 and 18.7 μg/mL against SiHa, A549, PC3 and MDA-MB-231, respectively) was the most active compound against all the cancer cell lines

Mechanisms of Rebaudioside A Degradation and Ingredient-Sweetener Interactions in Beverages during Storage

The instability of rebaudioside A (Reb A) in food product applications during storage challenges their utilization. The pathways of Reb A degradation in aged acidic beverages were investigated. Three Reb A degradation compounds of known sensory importance were monitored, consisting of (1) a rearrangement, (2) a hydration, and (3) an epoxidation/rearrangement product. Using deuterium-labeled water (D2O) experiments, compounds 1–2 were reported to be generated by acid-catalyzed mechanisms involving the formation of a carbocation on carbon position 16, followed by either deprotonation via E1 elimination on C15 to form the more thermodynamically stable trisubstituted alkene (compound 1), or by the Markovnikov addition of water via SN1 substitution to form a tertiary alcohol (compound 2). Compound 3 was generated by epoxidation of the exomethylene at the C16–17 positions, followed by the opening and rearrangement of the ring to form a new alkene bond between C15–C16 and a primary alcohol on C17. Further analysis of the effect of beverage ingredients indicated the addition of caramel color significantly increased (p < 0.0001) the concentrations of compounds 1–2 compared to the aged control by 89 and 83%, respectively, whereas a specific coffee flavor and caramel color were reported to significantly reduce (p < 0.0001) the formation of compound 3 compared to the aged control during storage by 90 and 79%, respectively