Design,Synthesis And Biological Evaluation Of 3,4-Dihydropyrimidine-2(1H)-ones

3,4-dihydropyrimidine-2?1H?-ones?DHPMs?had a wide range of biological effects,such as antiviral,anti-tumor,anti-inflammatory,anti-bacterial,calcium channel antagonist,antihypertensive agents and free radical scavenging.In recent years,DHPMs attracted more and more attention,and it was particularly important to obtain high yield and high stereoselective DHPMs by simple operations.Biginelli reaction was one of the most common multi-component reactions.In 1893,Pietro Biginelli firstly reported the synthesis of DHPMs using easily-accessible starting materials,benzaldehyde,ethyl acetoacetate and?thio?urea cyclization condensation reaction.DHPMs could be easily obtained through a simple"one pot"method.It was reported that most of the DHPMs are heat shock protein 70?Hsp70?inhibitors,but Christopher reported the SW02,which had no inhibitory effect on Hsp70,but has moderate intensity agonism.On the basis,the structure optimization and structure-activity relationship of compound SW02 were studied in this paper.Firstly,a series of dihydropyrimidine rings were obtained by using Biginelli reaction,and then N₁-alkylation substitution reaction was carried out.An orthogonal test table L₈?2⁷?was selected to analyze factors about the selectivity and yield of N₁-alkylation.25 dihydropyrimidine compounds were designed and synthesized;and the physical and chemical properties and activity evaluation were completed.In the anti-tumor activity,C57 mice were used to establish the xenograft tumor model to observe the inhibitory of the DHPMs on the tumor.SH-SY5Y human neuroblastoma cells line was induced into mature neuronal cells,and okadaic acid was used to simulate the damage model of AD neurons.MTT method,direct microscope observation and changes in synaptic length were used for the target.The expression of the protein was analyzed by Western blot,and the effect of the target compound on the reactive oxygen species of the cells was analyzed by flow cytometry.According to the results of animal experiments in vivo,thirteen substituted DHPMs with good anticancer activities were selected to generate pharmacophores and guide the design of novel DHPMs derivatives.These data indicated that in xenograft tumor model,compound 303 or 305 were able to significantly inhibit tumor growth,with inhibition ratios?IR?of 54.9%and 34.3%in100 mg/kg respectively and these compounds may be the inhibitor of Hsp70.This study had shown that the aryl chain in the R³,and 4-biphenyl low electron-donating group in the R¹ had a moderate growth inhibitory effected on xenograft tumor model.Compounds 303and 305 were available as further optimized lead compounds.15 models were generated in pharmacophores.The best pharmacophore model 1 was chosen with low energy and high value of steric and hydrogen bonding.Eight pharmacophoric features,namely three acceptor atoms,two donor atoms and three hydrophobic centers were identified.Two acceptor atoms were at the R² position and carbonyl position.Hydrophobic center was the DHPMs parent ring,R¹ position and R³ position.In anti-AD activity in vitro,the compounds 102?201?301?401?501?601?801 with oxyalkyl chain at R³ position had no obvious toxicity,on the contrary it had some effects that promote synaptic growth in the model of AD neuron damage,so these compounds may become the agonist of Hsp70,which provides an idea for the subsequent experiments on the binding ability of compounds to Hsp70 ATPase.Seven DHPMs compounds were selected that can maintain cell morphology.Western blot results showed that DHPMs compounds 102,601 can significantly improve Tau protein hyperphosphorylation,and reduce the expression of Hsp70 protein.The results of ROS in the cells by using DCFH-DA fluorescent probe detection showed that the degree of damage to cells was related to OA.Adding DHPMs at the same time can reduce the level of intracellular reactive oxygen species,of which 102was the most obvious improvement in ROS.The protective effect of DHPMs on nerve cells may be to reduce oxidative stress by inhibiting ROS levels in cells,but further research of deep mechanism was still needed.From the results of anti-tumor and anti-AD activity evaluation,some of the compounds may be the inhibitor of Hsp70,such as compounds 303 and 305.These compounds were characterized in that R¹ was biphenyl group and R³ was p-bromobenzyl group or long alkyl chain.Some of the compounds were agonist of Hsp70,such as compounds 102 and 601.These compounds were characterized in that R¹ was p-bromophenyl or p-pyridylphenyl and R³ was oxygenated alkyl chain.According to the above experimental results,binding experiments and structural optimization of Hsp70ATPase could be continued to obtain better activity results.