Synthesis And Preliminary Biological Activity Evaluation Of Enzyme Inhibitors Based On Dynamic Combinatorial Chemistry

Over the past two decades,dynamic combinatorial chemistry(DCC)has become a powerful tool for the discovery of biologically active lead compounds.In DCC,dynamic combinatorial library(DCL)is generated spontaneously through covalent or non-covalent reversible reactions between basic units with matching functional groups,which are governed by thermodynamic principles.Due to the adaptive nature of DCL,its original equilibrium can be changed by the addition of external stimuli,which in turn can disrupt the original equilibrium and further change the composition of the molecular library.Proteins have been widely used as external templates for screening bioactive components.Often driven by the stimulus of an external template,DCL achieves the amplification of components with higher binding affinity at the expense of depleting other less favorable combinations,thus enabling a thermodynamically controlled screening process at the molecular level.Fragment-based drug design(FBDD)has been widely used in the field of drug discovery in recent years,and a variety of clinical drugs have been developed by this strategy.FBDD starts with low-affinity binding fragments and eventually evolves into highly active compounds through fragment modification.Compared with conventional high-throughput screening(HTS),FBDD has the advantages of target specificity and cost-effectiveness.Nevertheless,it still involves tedious synthesis and biological validation as other conventional methods.The inherent disadvantage of FBDD can be effectively overcome by combining it with DCC,thus significantly improving the efficiency of drug development.This thesis explores the development of novel inhibitors of urease,α-amylase and α-glucosidase using the concepts of DCC and FBDD in three main areas.(1)An acylhydrazone compound 1 with moderate urease inhibitory activity was firstly screened by a fragment-based drug design strategy as an initial fragment,on which fragment growth was performed to optimize the enzyme activity.Starting from the initial fragment,three acylhydrazides and five aldehydes were devised and produced.A thermodynamically controlled DCL was constructed by the reversible acylhydrazone generation reaction of the acylhydrazide with the aldehyde group.Two acylhydrazone compounds in the DCL underwent significant amplification under the action of urease as the protein template,and further activity testing screened for an optimal ligand H1J2.Subsequently,based on the optimal compound screened in the first DCL,four additional aldehydes and two acylhydrazides were added to construct a second DCL to further optimize the ligand structure in order to obtain a more active urease inhibitor.The acylhydrazone compounds H1J2 and H1J6 were amplified in DCL-2.Commercial standards AHA and thiourea were used as controls to test the activity of the amplification fraction.The activity results showed that the optimal ligand H1J6 was a more powerful and less toxic urease inhibitor,and its activity was not only higher than that of the control but also more than 10 times higher than that of the initial fragment 1.And the potential interaction mechanism of the ligand and urease was further investigated by enzymatic reaction kinetic studies and molecular docking simulations;(2)By referring to the structure of a compound 2c5 c with moderate α-amylase inhibitory activity,five aldehyde derivatives with different substituents and four acylhydrazides were devised and produced on the basis of their structures,and DCL were constructed by reversible reactions.three acylhydrazone compounds were amplified in the molecular library in the presence of α-amylase.These three compounds were then synthesized individually and their inhibition of α-amylase was further explored.The results showed that two of the acylhydrazone fractions were significantly more active than the lead compound 2c5 c.Notably,the activity results correlated well with the amplification fold,with the compound with the best activity,2d5 d,exhibiting better α-amylase inhibition than the commercial control acarbose.Follow-up kinetic experiments as well as molecular docking simulations further investigated the possible mechanism of action between receptor and ligand;(3)Five starting fragments were screened by fragment-based drug design,and the fragments were further optimized for bioactivity by combining fragment growth with DCC.Aldehyde fractions of the five fragments were designed as five different aldehyde derivatives and mixed with three acylhydrazide derivatives containing different reactive groups to construct a DCL-1 containing 15 acylhydrazone components by reversible acylhydrazone reaction.α-Amylase and α-glucosidase were used as protein templates for the regulation of dynamic equilibrium,respectively.The liquid phase results showed that four amplification fractions were present under the action of α-glucosidase,while the distribution of α-amylase as a template fraction did not change significantly.Further activity tests led to the existence of a selective inhibition mechanism of the optimal amplification ligand C3E5,i.e.,selective inhibition of α-glucosidase but not α-amylase.Subsequently,a second DCL was constructed based on the structure of the optimal ligand to optimize the results,and the amplification ligand of DCL-2 exhibited the same selective inhibition mechanism as the optimal amplification ligand in DCL-1.Among them,the optimal ligand C4E8 in DCL-2 showed triple the α-glucosidase inhibitory activity over acarbose and no α-amylase inhibitory activity.Subsequent enzymatic reaction kinetic studies and molecular docking simulations further investigated the possible mechanism of action between the receptor and ligand.