Design And Synthesis Of4,4-dimethyl-3β,7β-dihydroxylithocholic Acid Derivatives And Studies On Their Ubiquitination And Degradation Of HMG-CoA Reductase

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is the key enzyme of cholesterol biosynthesis in vivo, the ubiquitination and degradation of HMGCR is a new way to reduce cholesterol levels. On the basis of the research group’s preliminary work, this dissertation was focused on finding novel compounds with high activity and specificity which can promote the ubiquitination and degradation of HMGCR, the research work was carried out as follows:Part I. Design and synthesis of4,4-dimethyl-3β,7β-dihydroxylithocholic acid derivatives and studies on their ubiquitination and degradation of HMGCR1. With lithocholic acid as the starting material and then forming a4,4-dimethyl-3β,7β-dihydroxy substituted unit, four compound libraries A, B, C, D, including totally49new compounds were designed and synthesized by modifying the D-ring of the side chain. All compounds’structures were characterized by spectroscopic methods.2.The results of promoting the ubiquitination and degradation of HMGCR showed that when the chain length has5,6,7carbon atoms and the side-chain functional group is hydrophilic, such as primary, secondary, tertiary hydroxyl group or carboxyl group, all the compounds have a better activity in promoting the ubiquitination and degradation of HMGCR. The D-ring side chain with five carbon atoms is relatively optimum length and the tertiary hydroxyl group is maybe the best functional group. Further tested showed that the activity of compound2-121is close to25-HC while compound2-74is stronger than25-HC.3. Incubation compounds2-74and2-121with CHO-7cells, it was preliminary confirmed that this type of target molecules can effectivity inhibit the endogenous synthesis of cholesterol in the cell by using cell growth experiments method.4. The preliminary mechanism of compounds2-74and2-121on ubiquitination and degradation of HMGCR was also studied. It was shown that compounds2-74and2-121almost do not activate LXR signaling pathway by using the Real-time PCR test to assay target genes ABCG5, SREBP-1, FAS, which indicated that4,4-dimethyl-3β,7β-dihydroxylithocholic acid derivatives do not activate LXR signaling pathway without25-HC. Compounds2-74and2-121may be potential lead compounds by promoting the ubiquitination and degradation of HMGCR to reduce cholesterol levels in vivo.Part Ⅱ. Design and Synthesis of small molecular probe based on compound2-691. In order to explore the potential targets of lithocholic acid derivatives on promoting the ubiquitination and degradation of HMGCR. The probe PALCAD was designed on the basis of SAR of lithocholic acid derivatives and with compound2-69as the active group, trifluoromethylphenyl diazirine as the photoaffinity labeling group, poly ethylene glycol chain as the connecting chain, and biotin as the reporting group.2. Several synthetic methods were tried and carried out, NMR and mass spectrometry of the crude product showed the characteristic signals of PALCAD although we failed in obtaining the high purity probe PALCAD.The synthesis and purification of the small molecular probe PALCAD is still ongoing. The expansion of functional group (NH2, NHR, NHCO, etc.) for D-ring side chain and the study of effectiveness of the hydrogen bond donating (accepting) will be continued on the basis of the previous SAR study.