Design,Synthesis And Anti-tumor Activity Study Of Novel Aminopeptidase N(APN/CD13)Inhibitors

?.BackgroundAminopeptidase N(APN)is a type II transmembrane zinc dependent metalloproteinase,also called "moonlighting enzyme".APN can cleave the N-terminal amino acides of the various biologically active peptides.Dysregulated expression of APN has been observed in various tumors.In fact,APN has been reported to be associated with cancer invasion,metastasis and angiogenesis.Recently,APN has been considered as a cell-surface marker for the hepatoma cancer stem cells.Therefore,APN has been considered as a promising drug target for the cancer therapy.APN inhibitors(APNIs)included natural products and synthetic compounds.Most of them are the peptidomimetics,while some are the non-peptidomimetics.Bestatin,a dipeptide,was isolated from the culture of Streptococcus by Japanese scientists.After that,many natural products have also been reported,such as Probestin,Phebestin,Amastatin,Lapstatin,Leuhistin and AHPA-VaL,et al.However,the sources of natural products are limited,and most of them have multiple chiral centers.So the total synthesis of natural APNIs is difficult.Therefore,many researchers devoted the development of synthetic APNIs.Many synthetic APNIs have been reported,such as aminophosphonic acids derivatives,amino mercaptans derivatives,succinic acids derivatives and ureido peptidomimetics derivatives.Based on the three-dimensional crystal structure of the complex of biomacromolecules and small molecular inhibitors,we can conduct the rational drug design.In 2006,the three-dimensional crystal structure of the complex of E.coli APN and Bestatin was resolved.Bestatin can interact with Si and S1' pockets of APN by the terminal phenyl group and leucine side chain,respectively.The hydroxyl group and adjacent carbonyl group can chelate the zinc ion.The promising APNIs at least include two parts:a zinc binding group and a hydrophobic group,which can at least bind one of three hydrophobic pockets.II.Design,Synthesis and Anti-tumor Activity Study of Ureido peptidomimetics with 1,2,3-Triazole Moiety as APNIsThe most of APNIs are peptidomimetics,which can mimic the transition state of the hydrolysis process.They all have a hydrophobic side chain and a zinc binding group.Based on the structure-activity relationship of the ureido-based APNIs,we designed a novel ureido peptidomimetics with 1,2,3-triazole moiety as APNIs.The 1,2,3-triazole moiety can be formed via click chemistry.The click chemistry can rapidly construct a library with various chemical entities.In this paper,we designed and synthesized 80 new ureido peptidomimetics,including A-series compounds and B-series compounds.1H-NMR,13C-NMR and HRMS were used to identify the target compounds.According to the literature,all the compounds were not reported before.Subsequently,we screened the activities of the target compounds at the molecular level,cellular level and animal level.We firstly evaluated the inhibitory potencies of the target compounds against APN.Then we evaluated the inhibitory potencies of the selected compounds against the proliferation of the tumor cells using MTT method.After those,we evaluated the anti-angiogenic activities of the selected compounds using the HUVECs tubular structure formation model and the rat aortic ring micro-vessel growth model.The in vivo anti-metastatic activities of the selected compounds were assayed using the mice hepatoma H22 lung metastasis model and the in vivo anti-tumor activities of the selected compounds were evaluated using the mice hepatoma H22 xenograft model.The inhibitory activities of the most of the target compounds against APN were better than the positive control Bestatin,which rationalize our design strategy.B-series compounds containing a conformational restriction phenyl displayed better inhibitory activities against APN than A-series compounds.The compounds with an ortho-substituent presented better inhibition against APN than the counterparts with a meta-substituent or a para-substituent.Moreover,compound 44v containing a benzyloxyl group in the ortho position of the terminal phenyl group displayed improved APN inhibitory activities,possibly resulting from the additional interaction of the benzyloxyl group with the S2' pocket.Satisfactorily,the best compound 44v among A-series compounds and the best compound 65f among B-series compounds with IC50 values of 0.089 ± 0.007 ?M and 0.032 ± 0.003 ?M,respectively,were 104-fold and 292-fold more potent than the positive control Bestatin(APN ICso= 9.4± 0.5 ?M),respectively.It was reported before that the anti-proliferative activities of the most of APNIs were weak.However,the anti-proliferative activities of the compound 65f against PLC/PRF/5 cells and H7402 cells were better than that of 5-Fu,respectively.Then,the combination of 65f plus 5-Fu presented synergistic anti-proliferative activities agansist all the tested tumor cells.The in vivo anti-tumor acitivity of 65f plus 5-Fu was better than that of 5-Fu alone.The anti-tumor effect of 65f plus 5-Fu was similar with that of Bestatin plus 5-Fu.In the 65f plus 5-Fu treated group,no significant body loss was seen.suggesting little systematic toxicity.The combination of 65f plus 5-Fu may be used for the treatment of cancer.Moreover,we also evaluated the anti-angiogenic and anti-metastatic activities of compounds 44v and 65f.It was found that compounds 44v and 65f inhibited the micro-vessel growth in a dose-dependent manner,respectively.Interestingly,they could not inhibit the proliferation of HUVECs.Insteadly,they inhibited the tubular structure formations of HUVECs in a dose-dependent manner.Noticeably,in the mice hepatoma H22 lung metastasis assay,the number of nodes in mice treated by 44v(60 mg/kg,i.p.)and 65f(60 mg/kg,i.p.)were lower than that of mice treated by Bestatin(60 mg/kg,i.p.),respectively.So 44v and 65f may be further developed as anti-metastatic and anti-angiogenic candidate drugs.III.Design,Synthesis and Anti-tumor Activity Study of Pyrazoline Derivatives as APNIsThere were little reports about non-peptidomimetics as APNIs.This may be due to the lack of effective pharmacophore model.In this paper,according to the literature,we replaced the aromatic scaffolds of the lead compounds by a larger diphenylpyrazoline moiety.The zinc binding group and the linker moiety were retained.35 novel pyrazoline derivatives were designed and synthesized as APNIs,which can be divided into A-series compounds and B-series compounds.Moreover,compounds containing the pyrazoline moiety showed various activities,such as anti-tumor,anti-bacteria and anti-inflammation.1H-NMR,13C-NMR and HRMS were used to identify the target compounds.According to the literature,all the compounds were not reported before.Subsequently,we evaluated the inhibitory activities of the target compounds against APN and the inhibitory activities of the selected compounds against the proliferation of tumor cells.Moreover,we also evaluated the anti-metastatic activity of the selected compound using the mice hepatoma H22 lung metastasis model.The inhibitory activities of B-series compounds against APN were better than those of A-series compounds.Among A-sereis compounds and B-series compounds,the compounds with the hydroxamic acid side chain in the ortho position showed better inhibitory activities against APN than the counterparts with the side chain in the meta or para position.Moreover,the NH2CO group was the optimal substituent on the pyrazoline nucleus.This may be due to that the NH2CO group can form additional hydrogen bonds with the active center of APN.Among B-series compounds,the compounds with ortho-substituents presented better APN inhibitory activities than the counterparts with the meta-substituents or papa-substituents.Moreover,the compounds with the ortho-disubstituents presented better APN inhibitory activities than the counterparts with the ortho-monosubstituent.Among them,compound 76t(APN IC50 = 0.064 ± 0.01 ?M)displayed the best APN inhibitory activity,which was 140-fold more potent than the positive control Bestatin.The IC50 values of the selected target compounds 76b,76e and 76t against the proliferation of various tumor cell lines were lower than 200 ?M.Noticeably,the IC50 values of the three compounds against the K562 proliferation were 10-30?M.In the mice hepatoma H22 lung metastasis assay,the number of nodes in mice treated by 76t(60 mg/kg,i.p.)was lower than that of mice treated by Bestatin(60 mg/kg,i.p.).Encouraged by the pyrazoline-based compounds,compounds containing zinc binding group,a hydrophobically aromatic group and a suitable linker may be endowed with the activities against APN.To be true,we need more data to verify the rationality of the model.