Design, Synthesis And Activity Study Of L-Arginine Derivatives As Aminopeptidase N Inhibitors

Aminopeptidases N, a membrane-bound zinc-dependent exopeptidase, is expressed in many tissues including intestine, kidney, liver as well as central nervous system. APN participates in the final hydrolysis of nutrients and the degradation of bioactive molecules such as enkaphalin and endorphin. Furthermore, APN serves as a receptor for corona viruses and it is also involved in the process of antigen presentation. More importantly, Saiko and coworkers have found that APN is highly expressed in tumor cells and it is regarded as tumor marker or fos-related antigen CD13. APN plays an important role in tumor cell growth, invasion, metastasis and neovascularity. All these findings make this enzyme an interesting target for anti-tumor drugs research.Now the three dimensional structure of human APN has not been found, but the crystal structures of E. Coli aminopeptidase N and enzyme-Bestatin complex were reported in 2006. The crystal structure of eAPN showed that the catalytic region of APN consisted of one negative ion binding site and three hydrophobic pockets. The three hydrophobic pockets are S₁ pocket, which lies in the left of the zinc ion, and S₁' and S₂', which lie in the right of the zinc ion. This supplied basement for our rational drug design.Our study targeted on APN for many years. Based on the binding mode of Bestatin with eAPN, the pharmacophore of APN inhibitors was bulit. And then combined with the literature retrieval and our lab's achievements, we chose L-arginine as our scaffold. According to the requirements of the 3D-structure of eAPN, various fragments are introduced in the scaffold. Before synthesizing, we docked our compound with eAPN model via FlexX of sybyl 7.0. The result showed that almost all of the compounds had similar or higher score than Bestatin. This, to some extent, ensured the rationality of our design strategy and supplied basement for our study.In the synthesis, L-arginine was used as the starting material. The guanidinium group of L-arginine was protected by nitro group and the carboxy group was esterified to get the key intermediate. Then the intermediates were condensated with aromatic acyl chloride, aromatic sulfonyl chloride, aromatic carboxylic acid, Boc protected amino acid or Boc protected di-peptide analogues. The methyl ester groups of the former compounds were converted to hydroxymate group. The compounds contained Boc groups were treated with HCl/EtOAC to get the target compounds.In this study, 107 target compounds were obtained and all of them were identified by IR, ¹H-NMR, ESI-MS and HRMS. Literature retrieval proved that all the compounds were new and not reported. Additionally, in vitro anti-APN assay, in vitro anti-proliferation assays of HL-60, K562, ES-2, A549, PLC and H7402 cells were processed in this research. Finally, in vivo anti-metastasis assay in rat was conducted for some selected high activity compounds.The result of enzyme activity assay in vitro showed that B series of compounds had better activities than A series of compounds. And both of the two series showed better activities towards APN than towards MMP-2. A18 and A21 of A series exhibited better activities than others, which IC₅₀ came to 5.3 and 5.1μM (Bestatin, IC₅₀=3.1μM). B47, B50 and B52 of B series had potential activities, which IC₅₀ was 64.8, 53.4 and 59.0μM respectively, near to the positive control Bestatin (IC₅₀=51.8μM).11 compounds were selected and assayed for their anti-proliferation activity in vitro towards tumor cells which highly express APN/CD13, for example HL-60, ES-2, A549 and PLC cells, and tumor cells which lowly express APN, for example K562 and H7402 cells. MTT method was employed. The result showed that the designed compounds did have anti-proliferation activity towards tumor cells which highly express APN/CD13. But to tumor cells which lowly express APN, our compounds showed low activities to H7402 but high activities to K562. The reason why they showed good activities toward K562 remains to be discussed. Compound A22 showed excellent activities against all the 6 kinds of tumor cells, it should be studied more deeply.A22, B46, B50 and B52 were selected to conduct anti-metastasis activity assay against H₂₂ tumor cells in vivo. The result showed that A22 and BSO can decrease the number of nodes on the lung surface of mice bearing H₂₂ tumor cell, that is to say which can inhibit the metastasis of tumor cells. Their inhibition rates are higher than Bestatin (61.50%, 71.39% and 58.04%, respectively).Finally, according to the structure and activity of the compounds, we conducted QSAR research via computer assisted software. The QSAR model we built may direct the structure optimization in the future.The creativity points of our research are as follows:â‘ Based on the crystal structure of eAPN and the binding mode of Bestatin with the enzyme, utilizing the computer-assisted drug design software, we designed and synthesized the target compounds with the scaffold L-arginine. The scheme was feasible and the materials were economical.â‘¡Preliminary enzyme activity assay and anti-proliferation assays of HL-60, K562, ES-2, A549, PLC and H7402 cells showed most compounds possessed potential APN inhibitory activities. Compound A22 and B50 also showed better anti-metastasis activities against H₂₂ tumor cell in vivo than Bestatin.â‘¢We also bulit a QASR model of the target compounds, which can be used in the future for APN inhibitor design.