Milanova R, Moore M
Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada.
Arch Biochem Biophys. 1993 May 15;303(1):165-71. doi: 10.1006/abbi.1993.1268.
Triepoxide diterpenes isolated from the perennial herb, Tripterygium wilfordii, have shown promising antileukemic and anti-inflammatory activity in vitro and in vivo. However, the toxicity of these epoxides has limited their clinical usefulness and analogues are sought which possess the pharmacological actions but have a lower toxicity. In this study, we have generated oxidized analogues of these diterpenes from a synthetic lactone precursor (19-hydroxy-18 (4-3) abeo-abieta-3,8,11,13-tetraen-18-oic acid lactone) by incubating the lactone with the Zygomycete fungus, Syncephalastrum racemosum. Incubation of lactone with S. racemosum in liquid media yielded four distinct polar metabolites as determined by thin-layer chromatography of ethyl acetate extracts. Column purification followed by structural analysis by 1H NMR and mass spectroscopy revealed these compounds to be 7-beta-hydroxylactone, 7-alpha-hydroxylactone, 15-hydroxylactone, and 7-ketolactone. When lactone was added to 48-h-old cultures, 7-beta-hydroxylactone appeared after 30 min whereas there was a 4-h lag period before the 7-alpha-hydroxy and 15-hydroxy metabolites were detected suggesting that enzyme induction was necessary before metabolism occurred. The 7-ketolactone was not detected until 24 h of incubation and this may represent a nonenzymatic oxidation of 7-hydroxylactone. The addition of various cytochrome P450 inhibitors (ketoconazole, miconazole, alpha-naphthoflavone, aminobenzothiazole, and carbon monoxide) to liquid cultures prevented the 15-hydroxylation of lactone. Interestingly, in contrast to the other agents, ketoconazole and miconazole (20 microM) were not able to inhibit 7-hydroxylation even though 15-hydroxylation was inhibited by these agents. With the exception of carbon monoxide, cell growth was unaffected by these agents. These data suggest that the biotransformation of diterpenes by S. racemosum was mediated by an inducible hydroxylase system which is sensitive to cytochrome P450 inhibitors and that more than one hydroxylase enzyme may be responsible for the observed products.
从多年生草本植物雷公藤中分离出的三环氧化二萜类化合物，在体外和体内均显示出有前景的抗白血病和抗炎活性。然而，这些环氧化物的毒性限制了它们的临床应用，因此人们在寻找具有药理作用但毒性较低的类似物。在本研究中，我们通过将内酯与接合菌纲真菌总状共头霉一起孵育，从合成内酯前体（19-羟基-18(4-3)去甲枞酸-3,8,11,13-四烯-18-酸内酯）生成了这些二萜类化合物的氧化类似物。在内酯与总状共头霉在液体培养基中孵育后，通过乙酸乙酯提取物的薄层色谱分析确定产生了四种不同的极性代谢产物。柱纯化后通过1H NMR和质谱进行结构分析，揭示这些化合物为7-β-羟基内酯、7-α-羟基内酯、15-羟基内酯和7-酮内酯。当内酯添加到48小时龄的培养物中时，7-β-羟基内酯在30分钟后出现，而在检测到7-α-羟基和15-羟基代谢产物之前有4小时的延迟期，这表明在代谢发生之前需要酶诱导。7-酮内酯直到孵育24小时才被检测到，这可能代表7-羟基内酯的非酶促氧化。向液体培养物中添加各种细胞色素P450抑制剂（酮康唑、咪康唑、α-萘黄酮、氨基苯并噻唑和一氧化碳）可防止内酯的15-羟基化。有趣的是，与其他试剂相比，酮康唑和咪康唑（20μM）即使能抑制15-羟基化，也不能抑制7-羟基化。除一氧化碳外，这些试剂对细胞生长没有影响。这些数据表明，总状共头霉对二萜类化合物的生物转化是由一种可诱导的羟化酶系统介导的，该系统对细胞色素P450抑制剂敏感，并且可能有不止一种羟化酶负责观察到的产物。
