Synthesis And Activity Of Small Molecular Compounds Of Benzene Sulfonamides

Cardiovascular disease（CVD）is the leading cause of death in the world,and the incidence is still increasing steadily.Atherosclerosis（AS）is a chronic local inflammatory disease of arterial wall that is the main pathological basis of CVD.A large number of studies have shown that Low density lipoprotein cholesterol（LDL-C）in plasma is closely associated with AS cardiovascular disease.Lowering the level of LDL-C in the blood can effectively reduce the occurrence of cardiovascular disease and thus prevent cardiovascular events.Proprotein convertase subtilisin kexin type 9（PCSK9）is a protease involved in regulating cholesterol level in vivo,and its activity is closely related to LDL-C level.LDL-C in plasma is captured by the Low density lipoprotein receptor（LDLR）located in the liver cell membrane when it flows through the liver and enters into the cell for metabolism.LDLR then returns to the cell membrane for reuse.However,PCSK9 can bind to LDLR to form a compact complex and enter the lysosome for degradation,thus reducing the level of LDLR on the surface of liver cells,reducing the liver’s clearance of LDL-C,resulting in the increase of plasma LDL-C and dyslipidemia.Therefore,blocking PCSK9 can effectively reduce plasma LDL-C level,and PCSK9 inhibitors have become a new strategy for the treatment of hypercholesterolemia.In the previous study on the lipid-lowering activity of a series of benzothiazolylbenzoylsulfonamide compounds,our research group found that benzothiazole ring and benzoylsulfonamide had obvious inhibitory activity of PCSK9 when the connection part between benzothiazole ring and benzoylsulfonamide was 4-methoxy-1-methylpiperidine compound with ethane（1:1）.Therefore,in this study,4-methoxy-1-methylpiperidine compound with ethane（1:1）,a key pharmacodynamic group,was retained and substitutions on benzothiazole ring and benzene ring were investigated.The benzothiazole rings were replaced by electron-absorbing groups,electron-contributing groups,large volume groups and polar hydrophilic groups,and their effects on the activity,water solubility and safety of the compounds were investigated.33 small molecule target compounds of benzosulfonamides in two categories were designed and synthesized.In the first type of compound,the ester group on the sixth position of benzothiazole ring was changed into small substituting group for investigation.Benzo[d]thiazol-2-amine ring was used as raw material to generate2-chlorobenzo[d]thiazole intermediate through Sandmeyer reaction,and then reacted with three active 1-（phenylsulfonyl）piperidin-4-ol selected by previous work to obtain 15compounds in the first type.During the experiment,it was found that the water solubility of the first type of compound was poor,so the second type of compound was designed and synthesized by introducing polar hydrophilic groups into the sixth position of benzothiazole ring.Using ethyl 2-aminobenzo[d]thiazole-6-carboxylate as raw material,ethyl2-chlorobenzo[d]thiazole-6-carboxylate intermediate 3 was prepared by Sandmeyer reaction.Then the alkylation reaction with three 4-hydroxybenzoylsulfonyl piperidine was carried out to form ethyl 2-（（1-（phenylsulfonyl）piperidin-4-yl）oxy）benzo[d]thiazole-6-carboxylate intermediate 4a-c,after ester hydrolysis and condensation with different amines to obtain the second class of target compounds 18.In this paper,a total of 33 target compounds which have not been reported before were synthesized.The structures of the new compounds were confirmed by MS,¹H NMR and ¹³C NMR,respectively,and the key synthesis steps in the experiment were optimized.Using PCSK9-Luc HepG2 cell model based on luciferin reporter enzyme,33 target compounds were tested for their transcriptional inhibitory activities at 10μg/m L.The inhibition rates of L₁,L₂,L₃,L₄,L₅,L₆,L₇,L₈,L₉,L₁₀,L₁₁,L₁₂,L₁₃,L₁₄,L₁₅,L₃₁,L₃₂and L₃₃were 91%,91%,96%,87%,96%,95%,90%,56%,91%,52%,94%,93%,94%,70%,90%,99%,86%and 98%respectively,showed good transcriptional inhibitory activity against PCSK9.Cytotoxicity of 33 target compounds was tested,and the compounds with low toxicity and high inhibition rate were evaluated concentration-dependent.It was found that they could inhibit PCSK9 transcription concentration-dependent,with IC₅₀values between5.3 and 9.9μM.Western blotting method was used to evaluate the effects of five compounds with good activity on the levels of PCSK9 and LDLR proteins.It was found that L₄,L₆,L₁₁and L₁₂could significantly reduce the level of PCSK9 protein in HepG2 cells and increase the level of LDLR protein.L₁₃can only significantly increase LDLR protein level,but has no obvious effect on PCSK9.The above work identified 5 small molecule PCSK9 transcriptional inhibitors with further development value,which laid the foundation for the continuous research of PCSK9 transcriptional inhibitors.