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 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

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

Diclofenac N-Derivatives as Therapeutic Agents with Anti-Inflammatory and Anti-Cancer Effect

A series of diclofenac N-derivatives (2, 4, 6, 8c, 9c, 10a-c) were synthesized in order to test their anti-cancer and anti-inflammatory effects. The anticarcinogen activity has been assayed against three cancer cell lines: HT29, human colon cancer cells; Hep-G2, human hepatic cells; and B16-F10, murine melanoma cells. First, we determined the cytotoxicity of the different compounds, finding that the most effective compound was compound 8c against all cell lines and both compounds 4 and 6 in human Hep-G2 and HT29 cell lines. Compounds 4 and 8c were selected for the percentage of apoptosis determination, cell cycle distribution, and mitochondrial membrane potential measure because these products presented the lowest IC50 values in two of the three cancer cell lines assayed (B16-F10 and HepG2), and were two of the three products with lowest IC50 in HT29 cell line. Moreover, the percentages of apoptosis induction were determined for compounds 4 and 8c, showing that the highest values were between 30 to 60%. Next, the effects of these two compounds were observed on the cellular cycle, resulting in an increase in the cell population in G2/M cell cycle phase after treatment with product 8c, whereas compound 4 increased the cells in phase G0/G1, by possible differentiation process induction. Finally, to determine the possible apoptosis mechanism triggered by these compounds, mitochondrial potential was evaluated, indicating the possible activation of extrinsic apoptotic mechanism. On the other hand, we studied the anti-inflammatory effects of these diclofenac (DCF) derivatives on lipopolysaccharide (LPS) activated RAW 264.7 macrophagesmonocytes murine cells by inhibition of nitric oxide (NO) production. As a first step, we determined the cytotoxicity of the synthesized compounds, as well as DCF, against these cells. Then, sub-cytotoxic concentrations were used to determine NO release at different incubation times. The greatest antiinflammatory effect was observed for products 2, 4, 8c, 10a, 10b, and 9c at 20 µg·mL−1 concentration after 48 h of treatment, with inhibition of produced NO between 60 to 75%, and a concentration that reduces to the 50% the production of NO (IC50 NO) between 2.5 to 25 times lower than that of DCF. In this work, we synthesized and determined for the first time the anti-cancer and anti-inflammatory potential of eight diclofenac N-derivatives. In agreement with the recent evidences suggesting that inflammation may contribute to all states of tumorigenesis, the development of these new derivatives capable of inducing apoptosis and anti-inflammatory effects at very low concentrations represent new effective therapeutic strategies against these diseases.MINISTERIO DE ECONOMÍA Y COMPETITIVIDAD, PID2019-106222RB-C32/SRA (State Research Agency, 10.13039/501100011033)“Consejería de Economía, Conocimiento, Empresas y Universidad. Junta de Andalucía”, grant number B1-BIO-281-UGR1

Efficient In Vitro and In Vivo Anti‐Inflammatory Activity of a Diamine‐PEGylated Oleanolic Acid Derivative

Recent evidence has shown that inflammation can contribute to all tumorigenic states. We have investigated the anti‐inflammatory effects of a diamine‐PEGylated derivative of oleanolic acid (OADP), in vitro and in vivo with inflammation models. In addition, we have determined the sub‐cytotoxic concentrations for anti‐inflammatory assays of OADP in RAW 264.7 cells. The in‐ flammatory process began with incubation with lipopolysaccharide (LPS). Nitric oxide production levels were also determined, exceeding 75% inhibition of NO for a concentration of 1 μg/mL of OADP. Cell‐cycle analysis showed a reversal of the arrest in the G0/G1 phase in LPS‐stimulated RAW 264.7 cells. Furthermore, through Western blot analysis, we have determined the probable molecular mechanism activated by OADP; the inhibition of the expression of cytokines such as TNF‐α, IL‐1β, iNOS, and COX‐2; and the blocking of p‐IκBα production in LPS‐stimulated RAW 264.7 cells. Finally, we have analyzed the anti‐inflammatory action of OADP in a mouse acute ear edema, in male BL/6J mice treated with OADP and tetradecanoyl phorbol acetate (TPA). Treatment with OADP induced greater suppression of edema and decreased the ear thickness 14% more than diclofenac. The development of new derivatives such as OADP with powerful anti‐inflammatory effects could represent an effective therapeutic strategy against inflammation and tumorigenic processes

Caracterización de la capacidad anticancerígena y antiinflamatoria del OADP, derivado aminopegilado semisintetico del ácido oleanólico y aproximación a estas actividades en n-derivados del diclofenaco

En los últimos años, ha existido un gran interés científico en el estudio de las propiedades terapéuticas de compuestos orgánicos naturales destacando actividades biológicas tan importantes como sus efectos antiinflamatorio y anticancerígeno. Dentro de este abanico de compuestos orgánicos encontramos los compuestos de origen natural como son los terpenos, metabolitos segundarios que se encuentran presentes en el reino vegetal. Dentro de los terpenos destacamos a los triterpenos que se caracterizan por presentar 30 átomos de carbono constituyendo uno de los grupos con mayor cantidad de compuestos bioactivos. Por otro lado, encontramos otro tipo de compuestos orgánicos como son los antiinflamatorios no esteroideos destacando el diclofenaco que se encuadra dentro de los compuestos fenólicos (derivado del ácido fenilacético) con propiedades antiinflamatorias. En base a esto, esta tesis doctoral se divide en dos partes, la primera corresponde a la extracción, purificación del ácido oleanólico (OA), síntesis del derivado aminoPEGilado de ácido oleanólico (OADP) y la caracterización de las actividades anticancerígenas y antinflamatorias de este derivado, determinando sus mecanismos moleculares de acción. La segunda parte, corresponde a un estudio preliminar de las actividades anticancerígenas y antinflamatorias de diferentes amino-derivados del diclofenaco.Tesis Univ. Granada.Proyectos concedidos por la Consejería de Economía, Conocimiento, Empresas y Universidad, Junta de Andalucía: B1-BIO- 281-UGR18 y B1-FQM-217-UGR18Proyectos de desarrollo tecnológico (prototipos y pruebas de concepto), Universidad de Granada, P30-

A Diamine-PEGylated Oleanolic Acid Derivative Induced Efficient Apoptosis through a Death Receptor and Mitochondrial Apoptotic Pathway in HepG2 Human Hepatoma Cells

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Our recent studies have shown that the diamine-(PEG)ylated oleanolic acid (OADP) has strong anti-tumor effects in HCCs. In this study, we evaluated the anti-tumor mechanisms of OADP in the HepG2 HCC cell line. The cytotoxicity results showed that HepG2 cell viability was markedly reduced, with a very low 50% of cell growth inhibitory concentration (IC50, 0.14 µg/mL). We then investigated the anti-tumor mechanisms of OADP in HepG2 cells. The flow-cytometry analysis was used to evaluate cell apoptosis, indicating that 74–95% of cells were apoptotic. OADP caused cell cycle arrest in the G0/G1 phase and the loss of the mitochondrial membrane potential (MMP). Western blot analysis was performed to assess the expression levels of key proteins associated with the underlying molecular mechanism. The results showed the clear upregulation of caspase-8, caspase-9, caspase-3, Bak, p21, and p53, accompanied by the downregulation of Bcl-2. Similar results were obtained by the cotreatment with OADP and the c-Jun N-terminal kinase (JNK) inhibitor SP600125. Agents such as OADP, which are capable of activating extrinsic and intrinsic apoptotic pathways, may represent potential HCC cancer therapies.Junta de Andalucia B1-BIO-281-UGR18 B1-FQM-217-UGR1

Anti-cancer and anti-inflammatory activities of a new family of coordination compounds based on divalent transition metal ions and indazole-3-carboxylic acid

A new family of mononuclear coordination compounds has been synthetized and characterized: [M(3-ind)(HO)] (M = Co (1), Ni (2), Zn (3), Fe (4), Mn (5); 3-ind = indazole-3-carboxylate). These materials are mononuclear coordination compounds that possess strong hydrogen bond interactions. The anti-inflammatory effects of these compounds were assayed in lipopolysaccharide activated RAW 264.7 macrophages by inhibition of NO production. Moreover, the cytotoxicity of the complexes and the ligand in RAW 264.7 cells were determined for the first time. The most significant results were obtained for the compounds 4 and 5 reaching values of NO inhibition close to 80% at 48 h, and above to 90% at 72 h of treatment. The highest inhibitory effects on NO production were showed at the range 7–23 μg/mL for compounds 4 and 5. As a consequence, compounds 4 and 5 could be potential drugs due to the interesting anti-inflammatory properties showed. The anti-cancer potential of these compounds has been also tested against different tumor cell lines. The cytotoxicity of the ligand and of compounds 2 and 3 were assayed in three cell lines: HT29, colon cancer cells, Hep-G2, hepatoma cells and B16-F10 melanoma cells. The best results have been achieved with compound 2 in HepG2 and B16-F10 cell lines, being between 1.5 and 2 times more effective that the ligand in HepG2 cells, and B16-F10 cells. All in all, indazole-3-carboxylic acid is a promising ligand for the formation of coordination compounds with biochemical properties

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

5-Aminopyridine-2-carboxylic acid as appropriate ligand for constructing coordination polymers with luminescence, slow magnetic relaxation and anti-cancer properties

Five new coordination polymers (CPs) constructed of aminopyridine-2-carboxylate (ampy) ligand have been synthesized and fully characterized. Three of them correspond to metal-organic chains built from the coordination of ampy to sodium and lanthanides with formulae [MNa(ampy)4]n (M = terbium (2), erbium (1) and ytterbium (3)) resembling a previously reported dysprosium material which shows anticancer activity. On another level, the reaction of Hampy with cobalt and copper ions ({[CoK(ampy)3(H2O)3](H2O)3}n (4) and [Cu(ampy)2]n (5)) lead to CPs with variable dimensionalities, which gives the opportunity of analyzing the structural properties of this new family. Lanthanide materials display solid state intense photoluminescent emissions in both the visible and near-infrared region and exhibit slow relaxation of magnetization with frequency dependence of the out-of-phase susceptibility. More interestingly, in our search for multifunctional materials, we have carried out antitumor measurements of these compounds. These multidisciplinary studies of metal complexes open up the possibility for further exploring the applications in the fields of metal-based drugs.This work has been funded by Red Guipuzcoana de Ciencia, Tecnología e Innovación (OF188/2017), University of the Basque Country (GIU 17/13), Gobierno Vasco/Eusko Jaurlaritza (IT1005-16), Junta de Andalucía (FQM-1484 and FQM-394) and the Spanish Ministry of Economy and Competitiveness (MCIU/AEI/FEDER, UE) (PGC2018-102052-A-C22, PGC2018-102052-B-C21). The authors thank for technical and human support provided by SGIker of UPV/EHU and European funding (ERDF and ESF).Peer reviewe