Design, Synthesis And Activity Study Of 3-Phenylpropane-1,2-Diamine Derivates As Aminopeptidase N Inhibitors

ObjectiveAminopeptidase N(APN),as known as CD13,is a member of zinc dependent metalloproteinase.It is widely expressed on the surface of renal and intestinal brush border cells,synaptic membranes in central systems and APN is over-expressed on the surface of tumor cells.APN is demonstrated to play a key role in the process of tumor growth,invasion and metastasis.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.Additionally,World Health Organization(WHO) had reported that 70%terminal cancer patients usually suffer the pain.Synthetic analogues of morphine are applied as analgesics but they have undesirable side effects (e.g.addiction).Numerous researches have shown that one of the most promising approaches appears to potentiate the antinociceptive effected induced by the endogenous opioid substance enkephalin.However,enkephalin is limited in the clinical therapy for easily degradation by aminopeptidase N. More and more researches focus on the field of anti-aminopeptidase N drug as that enzyme has the close relationship with the cancer.The research mainly contained APN mono-antibody,APN ligand binding drug and aminopeptidase N inhibitor.APN mono-antibody is only for research at this time and some reports have showed that APN ligand NGR(asparagine-glycine-arginine) as the receptor of APN linked with anticancer drug can get well therapeutic effect.Most of the research on the anti-aminopeptidase N drug is to find aminopeptidase N inhibitor possessed inhibitory activity.Bestatin,the marketed drug as aminopeptidase N inhibitor,now is clinically used to prolong the survival of patients with acute adult nonlymphcytic leukemia.In recent years,many natural aminopeptidase N inhibitors have been reported,such as Probestatin,Amastatin and Curcumin.Moreover,numerous synthetic APN inhibitors have also been reported,such asα-aminophosphonates APN inhibitors,β-amino-thiols APN inhibitors.In our group,we also had reported numerous synthetic small molecular compounds targeted with aminopeptidase N in the past ten years.Based on the three dimensional crystal structure of E.Coli APN and enzyme-bestatin complex,this study used computer-aided drug design software to design and synthesize a series of compounds.Then we hope to find lead compounds which possess potential aminopeptidase N inhibitory activity.MethodsSo far the three dimensional structure of human APN has not been found and the crystal structure of E.Coli aminopeptidase N was reported in 2006.As the structure of lead compound bestatin,we build a virtual molecule library with the advantages of computer-aided drug design software.The following design strategy is screening the virtual molecule library to obtain the pharmacophore 3-phenylpropane-1,2-diamine, which could satisfy the requirement of the active site of E.Coli APN.We compared the mode of the pharmacophore and AHPA with the active site of APN by the Sybyl 7.0.The result showed that the electrostatic potential surface of 3-phenylpropane-1, 2-diamine and AHPA is very similar.Based on the pharmacophore 3-phenylpropane-1, 2-diamine,various fragments are introduced with the pharmacophore by the FlexX docking aided and virtual screened.As a result,the target compounds are design rationally and synthesized easily.In this research,the D-phenylalanine was used as the starting material.The phenylalanine was suffered esterification,sulfonylation,nitridation and reduction to get the key intermediate(2R)-2-[(tert-butoxycarbonyl)amino]-3-phenyl-propylamine. Then various fragments were coupled with the intermediate to generate a series of dipeptidemimatics.The compounds of seriesâ…¡are designed based on the optimization of the seriesâ… .The intermediate was coupled with different prepared dipeptide to generate various analogues of tripeptide.In addition,enzyme assay and HL-60 cell proliferation assay were processed in this research.ResultsIn this research,we obtained 79 target compounds and all of them are novel without any report by now with the structures identified by IR,¹H-NMR and ESI-MS.In series I,we designed and synthesized 38 analogous of dipeptide with the scaffold 3-phenylpropane-1,2-diamine.The enzyme assay showed that most compounds exhibited APN inhibitory activities and the most potential compounds are compound 11p and 21 and compound 11j,11k also exhibited well activity against APN.From the structure of compound 11j,11k and 11p,we know the polar group(e.g.-OH,-SH, -NH₂) of the compound can increase the activity of the compound.In addition, compound 21 is an analogue of tripeptide and shows the best APN inhibitory activity, maybe owing to the structure difference of the compound 21.Base on the results of the seriesâ… ,we optimized the structure of compounds with the compound 11p and 21 as lead compound.In this series,we obtained 41 target compounds and preliminary result showed that most of target compounds displayed better APN inhibitory activities than the seriesâ… .The compounds in seriesâ…¡structurally matched the active of the enzyme are better than these of compound in seriesâ… .When the compounds in seriesâ…¡were matched with the enzyme,the phenyl group of the pharmacophore could inserted to the S₁ pocket,and two side chains R,R₁ inserted to the S₁' and S₂', respectively.The IC₅₀ of compound 32d,44a,44d,44h are less than 10μM,which are similar to that of bestatin.The results of in vitro growth inhibition against HL-60 cells indicated that most potent APN inhibitor displayed good inhibitory effect against the growth of HL-60 cells.Compound 11p,21,32d,44a,44d and 44h,which showed well enzyme inhibitory activities against APN,also exhibited good potency against the proliferation of HL-60 cell.Moreover,compound 32d,44h exhibited the inhibitory effect of cell proliferation with IC₅₀ values of 0.29±0.06 mM and 0.27±0.08 mM,respectively, which showed better potency than that of bestatin.Additionally,based on the key role of APN in the process of degradation of enkephalin,we established two useful systems for the in vitro evaluation of the novel APN inhibitor.Potentiating effect of APN inhibitors on the twitch inhibition of enkephalin in guinea pig ileum experiment and effects of APN inhibitors on the hydrolysis of Tyr-Gly during the incubation with the striatal membrane of guinea pig experiment,both of them could be used to evaluate the activity of novel APN inhibitor.Finally,the structure activity relationship(SAR) was showed based on the structure and the activity data of the target compounds with the scaffold 3-phenylpropane-1,2-diamine.In order to investigate the interaction of our target compounds with APN, flexible docking of the compound 44h was built and docked into the active site of APN(PDB code:2DQM) using Sybyl7.0.The binding studies showed the phenyl group inserted to S₁ pocket,the thiol group orientated to S₁' pocket and the lysine side chain plunged into pocket S₂' Comparative Molecular Field Analysis(CoFAR) was utilized to establish the quantitative structure activity relationship(QSAR) of the target compounds.The steric contour map and the electrostatic contour map of the CoMFA model showed the model had good cross-validated coefficient q² and predictive potency.ConclusionsIn conclusion,based on the crystal structure of eAPN and the mode of bestatin with the enzyme,we utilize the computer-aided drug design software to design and synthesize the target compounds with the scaffold 3-phenylpropane-1,2-diamine.We reported a convenient and economical method of the synthesis of APN inhibitors. Preliminary activity assays showed most compounds possess APN inhibitory activity and the IC₅₀ of compound 32d,44a,44d,44h is less than 10μM which can be used in the further research.We also established a QASR model of target compounds which is beneficial for the design APN inhibitor in the future.