Semisynthesis and Evaluation of Anti-Inflammatory Activity of the Cassane-Type Diterpenoid Taepeenin F and of Some Synthetic Intermediates

A new strategy for the semisynthesis of the aromatic cassane-type diterpene taepeenin F (6) is reported. The introduction of the methyl group at C-14, characteristic of the target compound, was achieved via dienone 13, easily prepared from abietic acid (10), the major compound in renewable rosin. Biological assays of selected compounds are reported. The antiproliferative activity against HT29, B16-F10, and HepG2 tumor cell lines has been investigated. Salicylaldehyde 21 was the most active compound (IC50 = 7.72 μM). Products 16 and 21 displayed apoptotic effects in B16-F10 cells, with total apoptosis rates of 46 and 38.4%, respectively. This apoptotic process involves a significant arrest of the B16-F10 cell cycle, blocking the G0/G1 phase. Dienone 16 did not cause any loss of the mitochondrial membrane potential (MMP), while salicylaldehyde 21 caused a partial loss of the MMP. The anti-inflammatory activity of the selected compounds was investigated with the LPS-stimulated RAW 264.7 macrophages. All compounds showed potent NO inhibition, with percentages between 80 and 99% at subcytotoxic concentrations. Dienone 16 inhibited LPS-induced differentiation of RAW 264.7 cells, by increasing the proportion of cells in the S phase. In addition, salicylaldehyde 21 had effects on the cell cycle, recovering the cells from the G0/G1 full arrest produced in response to LPS action.Junta de Andalucia B-FQM-278-UGR20 B-FQM-650-UGR20 FQM-348 BIO-157Universidad de Granada/CBU

Synthesis of Tricyclic Pterolobirin H Analogue: Evaluation of Anticancer and Anti-Inflammatory Activities and Molecular Docking Investigations

This research was funded by grants from the Regional Government of Andalusia (Projects B-FQM-278-UGR20, B-FQM-650-UGR-20), and assistance was provided to the group FQM-348.Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules28176208/s1Pterolobirin H (3), a cassane diterpene isolated from the roots of Pterolobium macropterum, exhibits important anti-inflammatory and anticancer properties. However, its relatively complex tetracyclic structure makes it difficult to obtain by chemical synthesis, thus limiting the studies of its biological activities. Therefore, we present here a short route to obtain a rational simplification of pterolobirin H (3) and some intermediates. The anti-inflammatory activity of these compounds was assayed in LPS-stimulated RAW 264.7 macrophages. All compounds showed potent inhibition of NO production, with percentages between 54 to 100% at sub-cytotoxic concentrations. The highest anti-inflammatory effect was shown for compounds 15 and 16. The simplified analog 16 revealed potential NO inhibition properties, being 2.34 higher than that of natural cassane pterolobirin H (3). On the other hand, hydroxyphenol 15 was also demonstrated to be the strongest NO inhibitor in RAW 264.7 macrophages (IC50 NO = 0.62 ± 0.21 μg/mL), with an IC50NO value 28.3 times lower than that of pterolobirin H (3). Moreover, the anticancer potential of these compounds was evaluated in three cancer cell lines: HT29 colon cancer cells, Hep-G2 hepatoma cells, and B16-F10 murine melanoma cells. Intermediate 15 was the most active against all the selected tumor cell lines. Compound 15 revealed the highest cytotoxic effect with the lowest IC50 value (IC50 = 2.45 ± 0.29 μg/mL in HT29 cells) and displayed an important apoptotic effect through an extrinsic pathway, as evidenced in the flow cytometry analysis. Furthermore, the Hoechst staining assay showed that analog 15 triggered morphological changes, including nuclear fragmentation and chromatin condensation, in treated HT29 cells. Finally, the in silico studies demonstrated that cassane analogs exhibit promising binding affinities and docking performance with iNOS and caspase 8, which confirms the obtained experimental results.Regional Government of Andalusia B-FQM-278-UGR20, B-FQM-650-UGR-20, FQM-34

Synthesis and Biological Evaluation of Cassane Diterpene (5 alpha)-Vuacapane-8(14), 9(11)-Diene and of Some Related Compounds

A set of thirteen cassane-type diterpenes was synthesized and an expedient synthetic route was used to evaluate 14-desmethyl analogs of the most active tested cassane. The anti-inflammatory activities of these 13 compounds were evaluated on a lipopolysaccharide (LPS)-activated RAW 264.7 cell line by inhibition of nitric oxide (NO) production, some of them reaching 100% NO inhibition after 72 h of treatment. The greatest anti-inflammatory effect was observed for compounds 16 and 20 with an IC50 NO of 2.98 +/- 0.04 mu g/mL and 5.71 +/- 0.14 mu g/mL, respectively. Flow-cytometry analysis was used to determine the cell cycle distribution and showed that the inhibition in NO release was accompanied by a reversion of the differentiation processes. Moreover, the anti-cancer potential of these 13 compounds were evaluated in three tumor cell lines (B16-F10, HT29, and Hep G2). The strongest cytotoxic effect was achieved by salicylaldehyde 20, and pterolobirin G (6), with IC50 values around 3 mu g/mL in HT29 cells, with total apoptosis rates 80% at IC80 concentrations, producing a significant cell-cycle arrest in the G0/G1 phase, and a possible activation of the extrinsic apoptotic pathway. Additionally, initial SAR data analysis showed that the methyl group at the C-14 positions of cassane diterpenoids is not always important for their cytotoxic and anti-inflammatory activities.Junta de Andalucia BFQM-278-UGR20 B-FQM-650-UGR2

Evaluation of Anticancer and Anti-Inflammatory Activities of Some Synthetic Rearranged Abietanes

This research was funded by grants from the Regional Government of Andalusia (Projects B-FQM-278-UGR20, and B-FQM-650-UGR-20), and assistance was provided to the group FQM-348.Supplementary Materials: The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms241713583/s1Synthesis of the rearranged abietane diterpenes pygmaeocins C and D, viridoquinone, saprorthoquinone, and 1-deoxyviroxocine has been successfully achieved. The anticancer and anti-inflammatory activities of selected orthoquinonic compounds 5, 7, 13, and 19, as well as pygmaeocin C (17), were evaluated for the first time. The antitumor properties were assessed using three cancer cell lines: HT29 colon cancer cells, Hep G2 hepatocellular carcinoma cells, and B16-F10 murine melanoma cells. Compounds 5 and 13 showed the highest cytotoxicity in HT29 cells (IC50 = 6.69 ± 1.2 µg/mL and IC50 = 2.7 ± 0.8 µg/mL, respectively). Cytometric studies showed that this growth inhibition involved phase S cell cycle arrest and apoptosis induction, possibly through the activation of the intrinsic apoptotic pathway. Morphological apoptotic changes, including nuclear fragmentation and chromatin condensation, were also observed. Furthermore, the anti-inflammatory activity of these compounds was evaluated on the basis of their ability to inhibit nitric oxide production on the lipopolysaccharide activated RAW 264.7 macrophage cell line. Although all compounds showed high anti-inflammatory activity, with percentages between 40 and 100%, the highest anti-inflammatory potential was obtained by pygmaeocin B (5) (IC50NO = 33.0 ± 0.8 ng/mL). Our results suggest that due to their dual roles, this type of compound could represent a new strategy, contributing to the development of novel anticancer agents.Regional Government of Andalusia B-FQM-278-UGR20, B-FQM-650-UGR-20, FQM-34

Síntesis de terpenoides en búsqueda de nuevos productos antitumorales

Esta Tesis doctoral ha sido realizada gracias a un proyecto concedido por el ministerio de Economía y Competitividad (proyecto CTQ2014-56611-R/BQU), y a la financiación a cargo del grupo de investigación FQM-348 “Productos Naturales y Síntesis Orgánica Aplicada”El hombre, desde sus comienzos, ha utilizado las virtudes de la naturaleza para su beneficio. En ella existe una enorme diversidad de organismos vivos que biosintetizan todo tipo de sustancias químicas durante su metabolismo, siendo el reino vegetal una amplia fuente de nuevos agentes terapéuticos que han ayudado a mejorar la salud y el bienestar del ser humano. Estos compuestos pueden tener funciones esenciales para la vida, como es el caso de los hidratos de carbono, lípidos, proteínas y ácidos nucleicos, llamados metabolitos primarios. Otros, en cambio, no son esenciales para la vida, pero cumplen importantes funciones de supervivencia: son los denominados metabolitos secundarios. Estos participan en mecanismos adaptativos de defensa, comunicación y autoconservación, entre otros y normalmente se generan en pequeñas cantidades, pero sus funciones son vitales para la supervivencia de los seres vivos. Los metabolitos secundarios pueden ser clasificados según su origen biosíntetico, en alcaloides, policétidos, terpenoides…etc. Entre ellos, los terpenoides, pertenecen a la amplía familia de isoprenoides (se construyen a partir de unidades de cinco carbonos denominados isopreno). Normalmente procedentes del ácido mevalónico, destacan por su abundancia en la naturaleza y su nutrida variedad de estructuras aisladas tanto de especies vegetales (sintetizan más de 22000 derivados isoprénicos 3) como de especies animales, insectos o microorganismos. Los diterpenos, que constituyen un extenso grupo de terpenoides ampliamente distribuidos en el reino vegetal y en diversos microorganismos, forman parte de estos metabolitos bioactivos. Sus espectros de actividades biológicas, variados y potentes en muchos casos, suponen un estímulo tanto para la investigación académica como para sectores industriales como el farmacéutico, agroalimentario, cosmético o incluso el de perfumería. Sin embargo, y a pesar de su interés netamente demostrado, la difícil obtención de estos metabolitos y su escasa proporción en sus fuentes naturales, supone una limitación considerable, no sólo para llevar a cabo estudios detallados de sus propiedades biológicas, sino también para conseguir aplicaciones industriales capaces de rentabilizar el potencial de estos metabolitos. Ello, junto al continuo aislamiento de nuevas estructuras, supone un desafío para muchos investigadores del campo: el llevar a cabo procesos de síntesis orgánica o metodologías que permiten la obtención de cantidades suficientes de estos productos con el fin de profundizar en los estudios aplicados, y así, conseguir evaluar el potencial verdadero de estos metabolitos en sectores industriales como es el sector farmacéutico. Una de las líneas principales de investigación de nuestro grupo “Productos Naturales y Síntesis Orgánica Aplicada”, consiste en el desarrollo de nuevas rutas sintéticas y metodologías para la síntesis de terpenos y compuestos relacionados de alto valor añadido a partir de compuestos naturales mucho más asequibles y económicamente rentables, e incluso comerciales. En paralelo, nuestro grupo posee otra línea de investigación, basada en la preparación de derivados de los productos naturales con objeto de realizar un screening biológico en búsqueda de potenciales aplicaciones. Dentro de este contexto se enmarca la presente tesis doctoral, Investigación y desarrollo de nuevas rutas sintéticas hacia diterpenos naturales: Furanocassanos aromáticos, a partir de compuestos naturales más abundantes y comerciales más económicos como el ácido (-)-abiético (50) y (+)-esclareolida (99). Paralelamente, se describen las síntesis de análogos monoterpénicos relacionados estructuralmente con estos diterpenos de tipo cassano, a partir de β-ciclocitral (124), con el objeto de estudiar la relación estructura-actividad (SAR) de estas sustancias naturales. Finalmente se llevará a cabo la investigación del potencial biológico de los compuestos importantes obtenidos, mediante ensayos de actividad anticancerígena y antiinflamatoria.Tesis Univ. Granada.ministerio de Economía y Competitividad (proyecto CTQ2014-56611-R/BQU)FQM-348 “Productos Naturales y Síntesis Orgánica Aplicada

Unprecedented Elimination Reactions of Cyclic Aldols: A New Biosynthetic Pathway toward the Taiwaniaquinoid Skeleton

The acid treatment of 6,7-seco-abietane dialdehydes gives, in high yield, the corresponding derivatives with the 4a-methyltetrahydrofluorene skeleton of taiwaniaquinoids. A mechanism involving the elimination of formic acid from the cyclic aldol intermediate is proposed here. This process can be postulated as a new biogenetic pathway from abietane diterpenes to taiwaniaquinoids. Using this novel reaction, the first enantiospecific synthesis of bioactive natural cupresol and taxodal has been obtained

Unprecedented Elimination Reactions of Cyclic Aldols: A New Biosynthetic Pathway toward the Taiwaniaquinoid Skeleton

The acid treatment of 6,7-seco-abietane dialdehydes gives, in high yield, the corresponding derivatives with the 4a-methyltetrahydrofluorene skeleton of taiwaniaquinoids. A mechanism involving the elimination of formic acid from the cyclic aldol intermediate is proposed here. This process can be postulated as a new biogenetic pathway from abietane diterpenes to taiwaniaquinoids. Using this novel reaction, the first enantiospecific synthesis of bioactive natural cupresol and taxodal has been obtained

Synthesis and Biological Evaluation of Cassane Diterpene (5α)-Vuacapane-8(14), 9(11)-Diene and of Some Related Compounds

A set of thirteen cassane-type diterpenes was synthesized and an expedient synthetic route was used to evaluate 14-desmethyl analogs of the most active tested cassane. The anti-inflammatory activities of these 13 compounds were evaluated on a lipopolysaccharide (LPS)-activated RAW 264.7 cell line by inhibition of nitric oxide (NO) production, some of them reaching 100% NO inhibition after 72 h of treatment. The greatest anti-inflammatory effect was observed for compounds 16 and 20 with an IC50 NO of 2.98 ± 0.04 μg/mL and 5.71 ± 0.14 μg/mL, respectively. Flow-cytometry analysis was used to determine the cell cycle distribution and showed that the inhibition in NO release was accompanied by a reversion of the differentiation processes. Moreover, the anti-cancer potential of these 13 compounds were evaluated in three tumor cell lines (B16-F10, HT29, and Hep G2). The strongest cytotoxic effect was achieved by salicylaldehyde 20, and pterolobirin G (6), with IC50 values around 3 μg/mL in HT29 cells, with total apoptosis rates 80% at IC80 concentrations, producing a significant cell-cycle arrest in the G0/G1 phase, and a possible activation of the extrinsic apoptotic pathway. Additionally, initial SAR data analysis showed that the methyl group at the C-14 positions of cassane diterpenoids is not always important for their cytotoxic and anti-inflammatory activities