Structure-Activity Relationship Of BBR Analogues In Upregulating Low Density Lipoprotein Receptor Expression And A New Generation Of BBR With Improved Bioavailability

Our previous study has shown that natural compound berberine (BBR) and its isomer pseudoberberine (Y53) were both novel cholesterol-lowering agents with a high safety and a new mechanism distinct from statins. Acting on the post-transcription level, the compounds significantly upregulate expression of the low-density lipoprotein receptor (LDLR) by stabilizing its mRNA and display significant cholesterol-lowering activity in vivo. The present investigation covers two parts.1. Structure-activity relationship (SAR) analysisIn an attempt to systematically elucidate SAR of berberine analogues in LDLR upregulation, 52 derivatives were designed and synthesized. The LDLR up-regulating activity of the compounds was examined and the SAR of this group of chemical entities is summed up.(1) Methylenedioxo cylic group (ring E) was essential for the activity. Enlarging, or opening the five-member ring, or substituting the newly generated hydroxyl with alkyloxy groups made the activity decreased or disappeared.(2) The hydrogen at 13-position was also important. Replacing the 13-hydrogen of BBR or Y53 with different alkyl or benzyl groups decreased the activity consumedly. Retaining 13-hexyl and focusing the structure modification on the skeleton, the resultant derivatives had no up-regulatory effect as well.(3) The planar-like steric configuration was necessary for the activity. Reduction of the double bonds in the ring C resulted in a flexation of the planar skeleton, and caused disappearance of the activity. Introducing substitutes to N7-position to recover its positive charge did not restore the activity.(4) The analogues with increased volume of the side chain at 9-position had lower activity. 9-hydroxyl derivative (M1, berberrubine), one of the main metabolites of BBR (9-methyloxy), owned an activity similar to that of BBR. Furthermore, M1 offers an opportunity of making prodrugs to improve the bioavailability of BBR.2. Improving bioavailability of BBR by making prodrugsIn order to overcome the poor bioavailability of BBR's (only 5% statistically), BBR's metabolite berberrubine (M1) was employed to make prodrugs. We introduced lipophilic groups to the naked hydroxyl group in M1 to prepare prodrug compounds, which would deliberate M1 in vivo through enzyme catalyzing. 17 prodrugs (including 8 ester, 3 ether and 6 dual-prodrugs) were designed and synthesized refering to lipid-water distribution coefficient (ClogP) prediction data. A novel compound with improved bioavailability and cholesterol-lowering activity—berberrubine palmitate (13) endured the in vitro hydrolyzation test and in vivo evaluation. 13 displayed a moderate hydrolyze rate in the hydrolyzation test, and its cholesterol-lowering effect was confirmed in three hypercholesterolemic animal models. In the SD rat model, our results showed that 100 mg/kg of BBR or 13 orally administrated twice a day for a month effectively lowered serum CHO levels by 27.4% and 33.1%, LDL-c by 28.9% and 43.0%, TG by 32.9% and 36.5%, respectively. The lipid-lowering activities of 13 were significantly higher than that of BBR in the animals (P＜0.01). Liver and kidney functions were not altered by 13 in the tests. These results indicated that 13 had a better cholesterol-lowering activity than did BBR and did not have toxicity on liver or kidney.69 compounds (including 52 BBR analogues and 17 prodrugs) were designed and synthesized, and their chemical structures were confirmed by MS and ~1H NMR. By systematically elucidating their SAR for LDLR upregulation, we basically fulfilled the chemical study of BBR analogues for their activity on LDLR, and found an active compound M1. Part of the work has been published on Journal of Medicinal Chemistry (2009).Prodrug 13 was composed of M1 and palmitate vector, which had been used in clinic and proved to be safe. 13 had been PCT patent protected (publication No.: WO/2006/029577). Compared with the known cholesterol-lowerering drug statins, 13 is a novel cholesterol-lowering candidate with new mechanism, good safety and high activity, thus promising for preclinical investigation.