Exploratory research on modulators of adipocyte differentiation

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First of all, four germacranolides, named caleanolactone A (compound 1), 2,3-epoxycalealactone A (compound 2), caleanolactone B (compound 3), caleanolactone C (compound 4) and three known germacranolide (compounds 5-7) were isolated by combination of chromatography on silica gel and preparative HPLC of acetone extracts from the leaves of Calea urticifolia (Compositae). Their structures were established on the basis of spectroscopic analysis including 2D NMR experiments. Compounds 5, 6 and 7 were identified as calein D, juanislamin and 2,3-epoxyjuanislamin, respectively. These germacranolides inhibited adipogenesis at the concentrations of 1.25 to 5μM. The cytotoxicity of eight germacranolides including parthenolide was investigated on 3T3-L1 cells by MTT method. Among those compounds, compound 3 showed the lowest cytotoxicity against 3T3-L1 cells, and did not affect cell proliferation at the dose of 5μM, a minimal inhibitory concentration against adipocyte differentiation. Parthenolide … More , a similar germacranolide having α-methylen-γ-lactone ring did not indicate inhibitory effect on 3T3-L1 cell differentiation, but suppressed cell proliferation at 5μM. Partenolide showed nonspecific binding to proteins and cystein whereas the effect of compound 3 was low. In addition, compound 3 did not affect the differentiation of other differentiation inducible cells, PC12, U937 and K562 cells. Secondly, we investigated the systematic synthesis of proanthocyanidins. Proanthocyanidins are oligomer of catechin or epicatechin which were suggested to be the inhibitors of 3T3-L1 cell differentiation. Although the isolation and semi-synthesis of procyanidin dimers and trimers have been reported, no efficient method for the stereoselective synthesis has not been reported. We succeeded to develop a simple and efficient method to synthesize pure proanthocyanidin oligomers. Using an intramolecular condensation method, 3,4-cis catechin dimmer was obtained. We could not find potent inhibitory activity of procyanidins tested against 3T3-L1 cell differentiation, epicatechin trimer showed powerful inhibitory activity against the Maillard reaction using our reported assay method. Third, mycophenolic acid (MPA) was isolated as an inhibitor of 3T3-L1 cell differentiation from a culture broth of a Penicillium sp. Although MPA was known as an inhibitor of inosine monophosphate dehydrogenase (IMPDH), it was concluded that IMPDH is not the real target of the inhibition of the differentiation of 3T3-L1 preadipocytes into mature adipocyetes. The real target of this case has not been known, however, MPA inhibited the master regulator of the adipocyte differentiation, PPARγ activated by pioglitazone under the reporter assay conditions. In addition, the direct interaction between PPARγ and MPA was detected on Biacore. We also succeeded to design and synthesis of the HDAC inhibitors derived from MPA as a lead compound. Less