RESUMO
Scospirosins A (1) and B (2), two unprecedented spiro ent-clerodane dimers with 6/6/10/6 and 6/6/6/6/6 ring systems, respectively, were isolated from Isodon scoparius. Their structures were unambiguously established by spectroscopic, X-ray crystallographic, and chemical approaches. A bioinspired protecting-group-free strategy for their synthesis was achieved on a gram scale and featured the application of green methods, including neat reaction, sensitized photooxygenation, and electrochemical oxidation. 2 exhibited selective immunosuppressive activity against the proliferation of T lymphocytes (IC50 = 1.42 µM).
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Diterpenos Clerodânicos/síntese química , Diterpenos Clerodânicos/farmacologia , Imunossupressores/farmacologia , Isodon/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Biomimética , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Diterpenos Clerodânicos/análise , Imunossupressores/química , Imunossupressores/isolamento & purificação , Concentração Inibidora 50 , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Linfócitos T/efeitos dos fármacosRESUMO
The discovery of potent cytotoxic isolates from botanicals provides an opportunity to explore this viable tool for cancer chemoprevention. The antileukemic potential of clerodane diterpene from Polyalthia longifolia leaves has already been established. However, in this present study, utilizing chromatographic techniques we report for the first time, the isolation of a rare tetranorditerpene (compound 1) from P. longifolia. The structure of compound 1 was elucidated and confirmed by spectrophotometric data. UPLC-MS analysis was conducted on the methanolic extract, ethyl acetate fraction, and isolated tetranorditerpene showed that the tetranorditerpene is one of the major constituents of the plant with a retention time of 30.78 min. In addition, a methyl ester derivative (compound 2) of the isolated tetranorditerpene was synthesized. Using the CCK-8 assay, we compared the cytotoxic potential of isolated tetranorditerpene (1) and methyl ester derivative (2) with the previously isolated clerodane diterpenes. Our results showed that the methyl ester derivative (2) displayed the highest inhibitory activity against human leukemia HL-60 cells. The isolated tetranorditerpene (1) did not exhibit significant inhibitory effect against HL-60 cells. Morphological examination indicated chromatin condensation and nuclear fragmentation suggesting induction of apoptosis in compound 2 treated HL-60 cells. The methyl esterification of the isolated tetranorditerpene (1) conferred on it a significant level of antileukemic activity suggesting the possibility of a synergistic relationship between pure compound isolation and synthetic reaction in the discovery of new chemopreventive agents.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Diterpenos Clerodânicos/isolamento & purificação , Diterpenos Clerodânicos/farmacologia , Leucemia/patologia , Polyalthia/química , Apoptose , Diterpenos Clerodânicos/síntese química , Diterpenos Clerodânicos/química , Esterificação , Ésteres/síntese química , Ésteres/farmacologia , Células HL-60 , Humanos , Leucemia/prevenção & controle , Folhas de Planta/químicaRESUMO
The kappa opioid receptor (KOPR) has been identified as a potential drug target to prevent or alter the course of mood, anxiety and addictive disorders or reduce response to stress. In a search for highly potent and selective KOPR partial agonists as pharmacological tools, we have modified 12-epi-salvinorin A, a compound which we have previously observed to be a KOPR partial agonist. Five analogues of 12-epi-salvinorin A were synthesized and their effects on G protein activation as well as ß-arrestin2 recruitment were evaluated. Only 12-epi-salvinorin A (1) partially activated signaling through G proteins, yet acted as a full agonist in the ß-arrestin 2 DiscoveRx assay. Other salvinorin analogues tested in these functional assays were full agonists in both assays of KOPR activation. By comparison, the non-selective opioid ligand nalbuphine, known to be a partial agonist for G-protein activation, was also a partial agonist for the ß-arrestin mediated signaling pathway activated through KOPR.
Assuntos
Diterpenos Clerodânicos/farmacologia , Receptores Opioides kappa/agonistas , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Diterpenos Clerodânicos/síntese química , Diterpenos Clerodânicos/química , Relação Dose-Resposta a Droga , Humanos , Conformação Molecular , Receptores Opioides kappa/metabolismo , Relação Estrutura-AtividadeRESUMO
Salvinorin A, the most potent naturally occurring hallucinogen, has attracted an increasing amount of attention since the kappa-opioid receptor (KOR) was identified as its principal molecular target by us [Roth, B. L., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 11934-11939]. Here we report the design, synthesis, and biochemical characterization of novel, irreversible, salvinorin A-derived ligands suitable as active state probes of the KOR. On the basis of prior substituted cysteine accessibility and molecular modeling studies, C315(7.38) was chosen as a potential anchoring point for covalent labeling of salvinorin A-derived ligands. Automated docking of a series of potential covalently bound ligands suggested that either a haloacetate moiety or other similar electrophilic groups could irreversibly bind with C315(7.38). 22-Thiocyanatosalvinorin A (RB-64) and 22-chlorosalvinorin A (RB-48) were both found to be extraordinarily potent and selective KOR agonists in vitro and in vivo. As predicted on the basis of molecular modeling studies, RB-64 induced wash-resistant inhibition of binding with a strict requirement for a free cysteine in or near the binding pocket. Mass spectrometry (MS) studies utilizing synthetic KOR peptides and RB-64 supported the hypothesis that the anchoring residue was C315(7.38) and suggested one biochemical mechanism for covalent binding. These studies provide direct evidence of the presence of a free cysteine in the agonist-bound state of the KOR and provide novel insights into the mechanism by which salvinorin A binds to and activates the KOR.
Assuntos
Diterpenos Clerodânicos/química , Receptores Opioides kappa/química , Linhagem Celular , Diterpenos Clerodânicos/síntese química , Diterpenos Clerodânicos/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Sondas Moleculares , Estrutura Molecular , Mutagênese , Receptores Opioides kappa/efeitos dos fármacos , Receptores Opioides kappa/genéticaRESUMO
Tumoral cells are known to have a higher ascorbic acid uptake than normal cells. Therefore, the aim of this study was to obtain polymeric nanoparticles containing the antitumoral compound trans-dehydrocrotonin (DHC) functionalized with L-ascorbic acid 6-stearate (AAS) to specifically target this system tumoral cells. Nanoparticle suspensions (NP-AAS-DHC) were prepared by the nanoprecipitation method. The systems were characterized for AAS presence by thin-layer chromatography and for drug loading (81-88%) by UV-Vis spectroscopy. To further characterize these systems, in vitro release kinetics, size distribution (100-140 nm) and Zeta potential by photon-correlation spectroscopic method were used. In vitro toxicity against HL60 cells was evaluated by tetrazolium reduction and Trypan blue exclusion assays. Cell death by apoptosis was quantified and characterized by flow cytometry and caspase activity. Zeta potential analyses showed that the system has a negatively charged outer surface and also indicate that AAS is incorporated on the external surface of the nanoparticles. In vitro release kinetics assay showed that DHC loaded in nanoparticles had sustained release behavior. In vitro toxicity assays showed that NP-AAS-DHC suspension was more effective as an antitumoral than free DHC or NP-DHC and increased apoptosis induction by receptor-mediated pathway.