Exploratory research on modulators of adipocyte differentiation

首先，从菊科产荨麻叶的丙酮提取物中分离得到4个大根香叶内酯，分别命名为caleanolactone A（化合物1）、2，3-epoxycalealactone A（化合物2）、caleanolactone B（化合物3）、caleanolactone C（化合物4）和3个已知的大根香叶内酯（化合物5-7）。它们的结构是建立在光谱分析的基础上，包括2D NMR实验。化合物5、6和7分别鉴定为calein D、juanislamin和2，3-epoxyjuanislamin。这些大根霉内酯在1.25至5μM浓度下抑制脂肪生成。采用MTT法研究了包括小白菊在内的8种吉大根内酯类化合物对3 T3-L1细胞的细胞毒性。在这些化合物中，化合物3显示出对3 T3-L1细胞的最低细胞毒性，并且在5μM的剂量（对脂肪细胞分化的最小抑制浓度）下不影响细胞增殖。Parthenolide ...更多信息 结果表明，含α-亚甲基-γ-内酯环的吉兰黄酮对3 T3-L1细胞的分化无抑制作用，但在5μM浓度下可抑制3 T3-L1细胞的增殖。Partenidine显示与蛋白质和半胱氨酸的非特异性结合，而化合物3的作用较低。此外，化合物3不影响其它分化诱导细胞，PC 12、U937和K562细胞的分化。其次，我们研究了原花青素的系统合成。原花青素是儿茶素或表儿茶素的低聚物，被认为是3 T3-L1细胞分化的抑制剂。虽然原花青素二聚体和三聚体的分离和半合成已被报道，但没有有效的立体选择性合成方法尚未报道。我们成功地开发了一种简单有效的方法来合成纯的原花青素低聚物。用分子内缩合法合成了3，4-顺式儿茶素二聚体。我们没有发现原花青素对3 T3-L1细胞分化的有效抑制活性，表儿茶素三聚体对美拉德反应显示出强大的抑制活性。第三，霉酚酸（MPA）作为3 T3-L1细胞分化的抑制剂从青霉属（Penicillium sp.）的培养液中分离。虽然已知MPA是肌苷一磷酸脱氢酶（IMPDH）的抑制剂，但可以得出结论，IMPDH不是抑制3 T3-L1前脂肪细胞分化为成熟脂肪细胞的真实的靶。该病例的真实的靶点尚不清楚，然而，MPA抑制脂肪细胞分化的主要调节因子，在报告基因测定条件下吡格列酮激活的PPARγ。此外，在Biacore上检测到了PPARγ与MPA之间的直接相互作用。以MPA为先导化合物，成功地设计合成了HDAC抑制剂。少

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