br Results The newly synthesized leucine ureido

Results The newly synthesized leucine ureido derivatives were firstly evaluated for their enzymatic inhibition using porcine kidney APN (Microsomal, Biocol) with bestatin as the positive control (Table 1). One isoleucine ureido derivative 14 and phenylglycine ureido derivative 15 were also synthesized and evaluated, which exhibited much less potent APN inhibition than their corresponding leucine ureido derivatives 13a and 13d, respectively, suggesting that the leucine residue is more suitable for APN recognition. Introduction of various substituents on phenyl group of 13a led to mono-substituted analogs 13b–13w, 20a–20b and multi-substituted analogs 13aa–13ff. Comparing the mono-substituted analogs, it was found that for chlorine (13b–13d), bromine (13e–13g), methyl (13h–13j), methoxyl (13k–13m), ethyoxyl (13s–13u), benzyloxyl (13v–13w) and amino (20a–20b) substituted analogs, their APN inhibitory activity order is ortho-substituted > meta-substituted > para-substituted. However, the similar trend was not observed in nitro (13n–13p) or cyano (13q–13r) substituted analogs. Among multi-substituted analogs 13aa–13ff, compounds containing two ortho-substituents displayed more potent activity than their counterparts with other substitutions patterns (13aavs13dd, and 13ccvs13ee). Moreover, compounds containing two ortho-substituents are more potent than their counterparts containing one single ortho-substituent (13aavs13b, 13bbvs13h, and 13ccvs13k). Replacement of the benzyl group of 13a using other functional groups led to derivatives 25a–25h with comparable or better APN inhibitory activities to 13a, indicating that different substituents on the triazole were generally well tolerated. It was worth noting that all our newly synthesized leucine ureido derivatives exhibited better APN inhibitory potency than the positive control bestatin. The most potent cyp450 inhibitors 13v (IC50 = 0.089 ± 0.007 μM) was over 100-fold more potent than bestatin (IC50 = 9.4 ± 0.5 μM). Furthermore, some compounds with potent enzyme inhibition activity were selected to evaluate their inhibitory capacities against human APN stemming from ES-2 and PLC/PRF/5 cell lines. The results in Table 2 show that compound 13v was still the most potent compound with IC50 values over an order of magnitude lower than that of bestatin. MTT assay was performed to evaluate the in vitro cytotoxicity of compounds 13v and 13w. The results listed in Table 3 show that both 13v and 13w presented better proliferation inhibition than bestatin against all the cell lines tested. Compound 13v was further progressed to human umbilical vein endothelial cells (HUVECs) tubular structure formation assay to evaluate its anti-angiogenesis activity. The results in Fig. 2 show that, compound 13v could reduce the capillary tube formation in a dose-dependent manner. It was worth noting that 10 μM of 13v presented similar anti-angiogenesis activity with 100 μM of bestatin. It is considered that the rat aortic ring model could simulate the in vivo angiogenesis environment better than the HUVECs tube formation model. Our results in Fig. 3 show that compound 13v could potently reduce micro-vessel growth in a dose-dependent manner. Moreover, similar with the results in HUVECs tubular structure formation assay, 10 μM of compound 13v demonstrated comparable micro-vessel growth inhibition relative to 100 μM of bestatin. Based on the significant roles of APN in tumor metastasis5, 6, 7, we used the mouse H22 hepatoma cell pulmonary metastasis model to evaluate the in vivo anti-metastasis activity of 13v. The number of metastasis nodes on the surface of pulmonary lobes was counted to evaluate the anti-metastasis effect of tested compounds. As depicted in Fig. 4, the number of nodes in mice treated by 13v (60 mg/kg, ip) was lower than that of mice treated by bestatin (60 mg/kg, ip). The inhibitory rates of metastasis of 13v and bestatin were 71% and 64%, respectively. There were no apparent loss of body weight and no evident toxic signs in liver and spleen in the group treated by 13v.