Design, Synthesis, And Preliminary Biological Studies On The Novel N-Cinnamoyl-L-aspartic Acid Derivatives As Aminopeptidase N Inhibitors

Aminopeptidase N(APN/CD13),one of membrance-bound znic-dependent exopeptidase,is widely distributed in mammalian tissue cells including kidney, in(?)estine,liver as well as the central nervous system.APN can effect the immunologic function and the primary bioprocess,such as cell proliferation,secretion,invasion and Vasculogenesis.This enzyme is involved in the metabolism of angiotensinâ…¢in the brain and peripheral organs,in the degradation of nociceptin and in the inactivation of enkephalins.APN has also been proved to behave as a receptor for corona-viruses TEGV and 229E in humans.In addition,APN is demonstrated to play a key role in the process of tumorigenesis,regulation of immunologic function,and virus infection. The functional aspects are briefly summarized:(1) Degrading extracellular matrix, promoting the growth and invasion of tumor cells;(2) As a regulator of novel vessels, promoting the angiogenesis of tumor tissues;(3) Degrading immunoactive substance and bioactive peptides,such as interleukin,thymopentin,enkephalin and so on.Methods:Based on the three dimensional crystal structure of E.Coli APN and enzyme- bestatin complex,we used computer-aided drug design software to design and synthesize a series of compounds.We hope to find some antitumor lead compounds which possess potential aminopeptidase N inhibitory activity. In this reseach,the L- aspartic acid was used as the starting material.The aspartic acid was suffered esterification,condensation reaction,hydrolysis,ring-closure reaction to get the key intermediate cyclic acid anhydride.The target compounds are synthesized using the cyclic acid anhydride as material through a reaction sequence including ammonolysis reaction and condensation reaction.The chemical structure of target compounds are novel without any report by now with the structure identified by ESI-MS and ¹H-NMR.Results:In this reaearch,we designed and synthesized 88 target compounds with the scaffold N-cinnamoyl-L-aspartic acid.Preliminary bioactivity assays are carried out in vitro.The enzyme activity assay showed that most compounds exhibited APN inhibitory activities in vitro and the most potential compounds are compound B₂,B₄, B₁₁,B₁₅,B₁₆,B₁₇,B₁₈ and D₁.Structure-activity relationships of 88 tested compounds are elucidated and compounds containing aromatic heterocyclic substituted by halogen atom such as fluorine,chlorine,bromine and iodine had excellent inhibiting activity.Compounds containing carbonyl and hydroxamates show higher inhibiting activity than compounds containing carboxyl,may be the former matched the activity of the enzyme better and inserted to the hydrophobic pocket than the latter.The IC₅₀ of compounds B₂,B₁₅,D₁ is 5.9μM,11.1μM and 6.7μM respectively,which is similar to that of bestatin(IC₅₀:3.6±0.6μM).The results of in vitro growth inhibition against HL-60 cells indicated that most potent APN inhibitors displayed good inhibitory effect against the growth of HL-60 cells.Compounds B₂,B₁₁,B)(12) and D₁,which showed well enzyme inhibitory activities against APN,also exhibited good potency against the proliferation of HL-60 cell.Moreover,compounds B₂,B₁₁ and D₁ exhibited the inhibitory effect of cell proliferation with IC₅₀ values of 0.59±0.06 mM,0.47±0.04mM and 0.57±0.08mM, respectively,which showed better potency than that of Bestatin(IC₅₀=0.66±0.07 mM). Conclusion:In conclusion,with advantages of computer-aided drug design software,we builded a pharmacophore model according to the information of inhibitors with known activity and structure.The N-cinnamoyl-L-aspartic acid scaffold was obtained by virtual screening of the small molecular library with this pharmacophore.The following design strategy is structure-based drug design.That is, to design the side chain of N-cinnamoyl-L-aspartic acid according to the information of the requirement of the active site of E.Coli APN and enzyme-inhibitor complex. Flexsible docking was used for modeling the interaction between target compounds and the enzyme.The side chain of N-cinnamoyl-L-aspartic acid can be designed rationally and synthesized easily.We reported a convenient and economical method of the synthesis of APN inhibitors.And moreover,they have no evident toxicological effects in tested cell culture.Preliminary activity assays showed some compounds possess preferable APN inhibitory activity.We also established a QASR model of target compounds which can be used in the further research and be supposed to have potential anti-tumor activity in vivo and might be provided leading compounds or new scaffold.