Structure Design And Activity Of Scutellaria Flavone Cyclane-Aminol Mannich Base Derivatives

Activated lymphocytes abound in the body will result in the untermination of immune responses, causing inflammation, autoimmune diseases, and promoting the development of cancer and AIDS. Scutellaria flavones have immune-modulating effects, which can selectively induce apoptosis in activated lymphocytes, and are of low toxicity, therefore receiving more and more attention. However their clinical use is limited because of their poor solubility and low bioavailability.Baicalein, Wogonin, and Oroxylin were isolated from the total aglycones of Scutellaria Flavonoids prepared by the two-phase emzyme hydrolysis method. Norwogonin was prepared through demethylation, using WO as the raw material. Scutellarein was got by acid hydrolysis of scutellarin. And commercial Chrysin was purchased. All these Scutellaria Flavones used as lead compounds,18 Scutellaria Flavone Cyclane-Aminol Mannich base derivatives were semi-synthesized through Mannich condensation reaction with cyclane aminol and formaldehyde solution.10 of them,8-L-prolinol(+)-methyl-baicalein,8-L-prolinol (+)-methyl-wogonin,8-L-prolinol(+)-methyl-oroxylin,8-L-prolinol(+)- methyl-scutellarein, 8-((4-(2-hydroxyethyl)piperazin-1-y1)-methyl-scutellarein,8-L-prolinol (+)-norwogonin, 8-((4-(2-hydroxyethyl)piperazin-1-y1)-methyl-norwogonin,6,8-bis(hydroxypiperidine-methyl)-chrysin,6,8-bis(L-prolinol(+)-methyl)-chrysin,6,8-bis(((4-(2-hydroxyethy1) piperazin-1-y1)-methyl)-chrysin, were new structures. Spectroscopy was used to confirm and characterize the structures.Peripheral Blood Mononuclear Cells were isolated from rabbit peripheral blood, and activated by Phytohemagglutinin and Lipopolysaccharide, respectively, into activated T lymphocytes and activated B lymphocytes. Selective assay method of intracellular H2O2 levels was established using PF1, selective H2O2 fluorescent probe. The effect on H2O2 levels of these derivatives in activated lymphocytes was screened by PF1 method combined with fluorescence imaging. The results showed that Baicalein and Scutellarein were superior to the other lead compounds, and piperidinol was stronger than L-prolinol and N-hydroxyethyl piperazine. Among all the derivatives,8-hydroxypiperidine-methyl- baicalein (BA-j) was the most potent.12.5?M BA-j can induce 67.5?M H2O2, which is 2 times the amount BA induces.BA-j could selectively increase the intracellular H2O2 levels in activated lymphocytes without affecting PBMCs, and the increase was in a significant concentration-dependent manner. Further studies showed that, ROS regulation mechanism of BA-j was completely different from that of the cytotoxic drug 10-hydroxy campothecin (HCPT). BA-j can capture O2- and NaOC1, and release H2O2, thus can selectively induce apoptosis in cells, whereas HCPT increases the total amount of intracellular ROS, leading to cell inflammation and necrosis. BA-j can also inhibit the activation of PBMCs into actived lymphocytes by PHA and LPS.In summary, among the Scutellaria Flavone Cyclane-Aminol Mannich base derivatives, BA-j was the most effective in selectively increasing intracellular H2O2 level in activated lymphocytes, and is expected to be developed into new anti-abnormal immune response drug. This result provides new ideas for the treatment of cancer, inflammation, AIDS and other autoimmune diseases.