Synthesis Of Pentacyclic Triterpenoid Derivatives As Novel Pancreatic Lipase Inhibitors

Obesity quickly became popular globally and attracted concern,and easily lead to many metabolic diseases,seriously endangering the level of public health of human beings.In recent decades,researchers have undertaken extensive research on the mechanisms and treatment of obesity,The results indicate inhibition of pancreatic lipase activity is one of the most widely used methods of treating obesity.Currently,the only behalf of PL inhibitor drugs is orlistat,Which has some side effects,particularly,causing liver damage.Therefore,the safe,effective,non-toxic side effects and long-term use of inhibitors of PL has become a hot topic in the field of obesity research.Despite researchers has found a large number of PL inhibitors from abundant natural product resources currently,such as terpenes,saponins,polyphenols,flavonoids,etc.,but most of the inhibitors have no high activity and low bioavailability,Therefore obtaining a compound of high efficiency,low toxicity and targeted inhibitory activity by finding or structural modifications of existed PL inhibitors become the primary task of research and development of new drugs of PL inhibitors.Our group has established the method for determinate PL activity and further verified in previous work.The screening assay for some traditional Chinese herbs has tested,but due to the activity of inhibitor screened is low,or more chemical groups can be modified,lead to it is difficult to as lead compounds for structural modification of the inhibitor.In this paper,on the basis of our preliminary experiment through lots of literature research,we filtered Ursolic acid?Ursolic acid,UA?,Oleanolic acid,?Oleanolic acid,OA?and Maslinic acid?Maslinic acid,MA?from triterpene compounds as the research object which with high activity of PL inhibition,extensive sources,and easy to structural modifications,and carries on the confirmation of PL inhibition,C-28 determined as an object by molecular simulation for structural modification.Then,combining with the characteristics of Orlistat and antibiotic action,the C-28 of three kinds of compounds linked with?-lactone or ?,?-unsaturated ester structure,letting the electrophilic carbon atoms with hydroxyl oxygen atoms combine covalently on the active center of serine for PL,expectations by reversible inhibition into irreversible inhibition and improve the activity of PL inhibition significantly.As a result,although the activity is greatly improved,Covalent bond is not formed.Finally,the derivatives with high activity of PL inhibition was chosen for the further structural modification in order to form a covalent bond.The main research contents and results are as follows:On the basis of preliminary experiments and extensive literature Investigation,Screened UA,OA and MA,withing a good inhibitory activity,conducted verification experiments.First,doing enzyme assay experiments in vitro,IC₅₀ values of UA,OA,MA were 32 ?g/mL,44?g/mL and 54?g/mL respectively;Second,The type of inhibition of three kinds of compounds were found to be competitive inhibition,mixed inhibition,mixed inhibition respectively;third,selecting carboxyl group of C-28 as an object of transformation by molecule docking.Synthesis of three ?,?-unsaturated ester compounds and study of inhibitory activity.Firstly,UA,OA,MA as raw materials and brominated?,?-unsaturated ester,obtaining three kinds of triterpenoid ?,?-unsaturated ester derivatives,which were identified as new compounds by 'H NMR,13C NMR and MS.Secondly,IC₅₀ values of three compounds were 9.1 ?g/mL,10.8 ?g/mL and 25.0 ?g/mL respectively by doing enzyme activity assay in vitro,its inhibitory activity than the original compounds were increased by 4.4,5.1,2.7 times respectively,three compounds were reversible inhibition,not forming a covalent binding with PL,the inhibition type were competitive inhibition,Mixed inhibition and anti-competitive inhibition respectively.Synthesis of three,?-lactone derivatives and study of inhibitory activity.First,vinyl acetate as raw material and synthesized y-bromo-?-lactone by reaction of brominated lactone with bromine,desired intermediate?-bromo-?-lactone compound,then it reacted with UA,OA,MA synthesized three kinds of triterpenoid ?-lactone derivatives,which were identified as new compounds by 1H NMR,13C NMR and MS.Secondly,the IC₅₀ values of three compounds were 7.2 ?g/mL,31.2 ?g/mL and 70.0 ?g/mL respectively by doing enzyme activity assay in vitro,inhibitory activity of derivatives of UA,OA than the original compounds were increased by 5.4,1.7 times respectively,inhibitory activity of derivatives of MA reduced,three compounds were reversible inhibition,not forming a covalent binding with PL,the inhibition type were Mixed inhibition,competitive inhibition,and Mixed inhibition respectively.Combining the test results of the second and third part,Six kinds of derivatives is not formed covalent binding to PL.this section choose UA?-lactone derivatives with the highest inhibitory activity to further structural modification in order to achieve the purpose of covalently bound with PL.First,through molecular docking,UA-?-lactone derivatives with the highest inhibitory activity far away from active center group of PL.Second,UA as raw material,carboxy of UA was extended to transformation,and reacted with intermediate y-Bromo-?-lactone compound,Finally five kinds of triterpenoid derivatives were synthesized,and confirmed by the 'H NMR,and the final product was identified as a new compound.Third,,IC₅₀ values of five compounds were 36 ?g/mL,35.2 ?g/mL,21.3 ?g/mL,26.4 ?g/mL,11.2 ?g/mL respectively by doing enzyme activity assay in vitro,inhibitory activity of this five compounds than UA were increased by 0.1,1.0,2.1,1.5,4.1 times respectively,five compounds were reversible inhibition,not forming a covalent binding with PL,the inhibition type of five compounds were competitive inhibition,mixed inhibition,mixed inhibition mixed inhibition and competitive inhibition respectively.the study did not achieve the desired objectives,The study did not achieve the desired objectives,the follow-up work required further structural modification in order to achieve the ultimate goal of forming covalently bound with PL.