A Novel Class Of Low-Density-Lipoprotein Receptor Up-regulators: Synthesis, Structure-Activity Relationships And Cholesterol-lowering Effect Of Isoquinoline Analogues

By screening the pool of traditional Chinese herbs,we found in our previous studies that berberine(BBR) was a novel LDLR-up-regulator and cholesterol-lowering agent with a mechanism distinct from statins.BBR has definite chemical structure with a molecular weight of 371.5 and has a number of pharmacological activities.In the present study,we take BBR as a leading compound for the structure-activity relationship(SAR) analysis.By replacing the side chain of D ring with various substituents,alterrating the double bond of ring C and attaching different substitents at the 13-positon,45 new analogues were designed and synthesized.The LDLR- up-regulating activities of these compounds were determined and SAR of this group of chemical entities was elucidated accordingly.(1) By substituting 9-OCH₃ and/or 10-OCH₃ on D ring with OC₂H₅,OC₃H₇,the analogues with increasing volume of the side chain at the 9,10-positions had an activity lower than that of BBR.Removing 9-OCH₃,the analogue with 10-OH remained the activity,but substituting 10-OH with OCH₃,OC₂H₅ or OC₃H₇ made the acitity decreased partially.Removing 9-OCH₃ and 10-OCH₃ from the D ring made the activity decreased consumedly.(2) The analogue with a dimethoxyl group at the 10,11-positions of D ring had the best activity of 1.6-fold of that of BBR.Substituting 10-OCH₃ and/or 11-OCH₃ with OH,CH₃, OC₂H₅ or OC₃H₇ made the acitity decreased consumedly or even deminished.This result indicated that a dimethoxyl structure in an ortho distribution afforded the optimal activity.(3) Though an ortho dimethoxyl structure is essential for the activity,but increasing the number of the methoxyls at ring D did not enhance the activity,in fact,the analogues with 3 methoxyls at ring D had a regulating effect 60%of that in BBR.(4) The aromatic system of ring C was essential;reducing one or two of the double bonds in the ring C made a complete loss of the activity.(5) The hydrogen at 13-position was also essential;replacing the hydrogen with different alkoxyl or substituted benzyl group made the activity disappeared completely. In vitro,LDLR mRNA expression was up-regulated by 8.5-fold with 53,which was the best analogue among the compounds.Besides,53 also increased LDLR protein expression on cell surface dramatically.So,we evaluated the cholesterol-lowering effect of 53 in vivo.First,hypercholesterolemic Balb/c mice were used.Our results showed that 100 mg/kg of BBR or 53 orally administrated twice a day for 3 weeks effectively lowered serum CHO levels by 12.1%and 32.9%,LDL by 25%and 50%,respectively. Then the cholesterol-lowering effect of 53 was evaluated in hypercholesterolemic SD rats.100 mg/kg of BBR or 53 orally administrated twice a day for 30 days lowered serum CHO by 27.4%and 38.5%,LDL by 28.9%and 43.5%,respectively.The CHO-and LDL-lowering activities of 53 were significantly higher than that of BBR in animals (P＜0.01).In the animal experiments,53 increased liver LDLR mRNA expression level to 1.5-fold of that of BBR,consistent with the results in vitro.In the two animal models, liver and kidney functions were not affected by 53.These results indicated that 53 has better cholesterol-lowering activity than BBR and does not have liver or kidney toxicities.The acute toxicity experiment showed that the LD₅₀ of 53 was far more than 5000 mg/kg in mice.The pathological examination of cerebrum,heart,liver,kidney,lung and intestine of the mice indicated that 53 does not have toxicity on these organs.There could be at least three explaination for why 53 has better activity than BBR: Firstly,the results of Computer-assistant Drug Design showed that there were four active groups in these analogues:hydrogen-bonding acceptor,hydrophobic core,positive ion core and aromatic hydrophobic core.The molecule of BBR only mapped onto 3 active group sites of the modle,while 53 mapped well with all 4 active group sites.Secondly, 53 had an electropositivity in the 7-nitrogen ion more than that of BBR;the 7-N ion plays an important role in the interaction between the compound and the speculated target molecule.Thirdly,53 had a better water-solubility than BBR,which might increase its absorption in vivo.In this study,45 analogues were designed and synthesized.The physical and chemical information of these compounds have been confirmed by MS,¹H NMR and IR.All of these results indicated that 53 had a good cholestorel-lowering activity,unique mechanism of action different from that of statins,good safety and improved water-solubility.In addition,compound 53 could be synthesized successfully with a high yield via five steps.We consider the compound 53 a promising candidate for further preclinical investigation.