Design, Synthesis And Preliminary Activity Study Of Small Molecule Peptidomemitics As Aminopeptidase N (APN/CD13)Inhibitors

Objective:Aminopeptidases N (APN/CD13), a membrane bound zinc dependent type II exopeptidase, is widely distributed in many tissues of mammalian such as: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. What is more, APN is highly expressed in many tumor cells and was considered as the tumor marker which plays a crucial role in tumor cell growth, invasion, metastasis and angiogenesis. So APN/CD13is considered as a useful clinical marker for anti-tumor drugs research.To date, many natural or synthetic small molecule APN inhibitors have been reported. Natural APN inhibitors include Bestatin, Amastatin, Lapstatin, Probestin, Leuhistin, AHPA-Val, Psammaplin A, Betulinic acid, and so on. Synthetic APN inhibitors include β-amino-thiols, a-aminoaldehydes, α-aminophosphinic acids, AHPA derivatives, L-isoglutamines, a-aminoboronic acids, cyclic-imide derivatives, L-lysine derivatives, chloramphenicol amine derivatives, and so on. Howevere, Bestatin is the only one APN inhibitor that used in clinic for the treatment of adult acute nonlymphocytic leukemia. Bestatin, as a natural product, is limited in the source. Besides APN, Bestatin also strongly inhibits many other enzymes in vivo such as APB, LAP and so on, so it is necessary to find more selective and lower toxic APN inhibitors by using the methods of medicinal chemistry. Methods:Based on the previous work of our lab, by studying the active sites of eAPN and using the strategies of rational drug design and peptidomemitics, we designed and synthesized four series of94target compounds as APN inhibitors, by preliminary in vitro APN and MMP-2inhibition assays, in vitro anti-proliferation assays of HL-60, ES-2, MDA-MB-231and B16BL6cells, in vitro anti-angiogenesis assay and in vivo anti-metastasis assay in rat in order to find more active APN inhibitors.Results:In this study, four series of94target compounds were synthesized, and all the structures were identified by ESI-MS, HRMS and1H-NMR. All of the compounds were novel and have not been reported in the literature.The results of preliminary enzyme activity assay in vitro showed that:In A series, most compounds displayed good APN inhibitory activities, and compound7c showed to be the best one in this series, with the IC50value to5.00μM, which is comparable to the positive control Bestatin (IC50=4.18μM). In B series, we introduced the conformation constrained structure Tic, but the activities of these compounds in this series were not significantly improved comparing with the compounds in A series. Only compound23h with phenylglycine hydroxamic acid residue in the structure which may match better with the active sites of APN, had the best APN inhibitory activity in this series, with the IC50value to6.28μM, which is also comparable to the positive control Bestatin (IC50=5.55μM). Moreover, compounds in this series with phenylalanine, tyrosine and methionion hydroxamic acid residues such as compounds23i,23j and23k also had good activities. In C series, considering the structure similarity between3-phenylisoserine and AHPA, we introduced3-phenylisoserine scaffold in the design of APN inhibitors, but these compounds didn't show good enzyme inhibition activity except for compounds with hydroxamic acid group such as compounds30a,30b and30e. In this series, compound30e showed to be the best active APN inhibitor with the IC50value to1.26μM, which is better than that of Bestatin (IC50=2.55μM). Compounds with the best APN inhibitory activities in B and C series such as compounds30e and38d both have phenylglycine hydroxamic acid residue, we supposed that the phenylglycine hydroxamic acid residue may be more suitable for APN interaction. So we designed the D series compounds by maintaining the phenylglycine hydroxamic acid residue and coupling the amino group with different aromatic acids. Compared with compounds in previous work, compounds in this series showed improved APN inhibitory activities with the IC50values in the range of1.36-19.94μM. Compound38d with the IC50value to1.36μM, which is much better than that of Bestatin (IC50=6.40μM), showed to be the best one in this series.Further more, compounds with good APN inhibition activities were tested for the anti-proliferation activities towards tumor cells HL-60, ES-2, MDA-MB-231and B16BL6in vitro, the results showed that compounds with better APN inhibition activities in vitro also showed better anti-proliferation activities towards highly expressed APN tumor cells such as HL-60, ES-2and B16BL6. Compounds30e and38d showed better anti-proliferation activities than that of Bestatin towards ES-2and B16BL6cells, of which compound38d showed to be the best one.The results of in vitro HUVECs tubular structure formation assay and rat thoracic aorta ring assay demonstrated that both compounds30e and38d showed significantly anti-angiogenesis effect in vitro. Compound38d showed to be the best one in rat thoracic aorta ring assay; there was almost no microvascule formation around the rat thoracic aortic ring at the concertration of200μmol/L.In the anti-metastasis activity assay against B16BL6cells in C57BL/6, with the dose of100mg/kg/d, both compounds30e and38d could significantly inhibite the metastasis of B16BL6cells to lung, with the inhibition rates35.59%and46.89%respectively. And in the same dose, the inhibition rate of Bestatin is33.33%.Conclusions:Based on the crystal structure of E. coli APN, the interaction model of Bestatin with APN and the previous work of our lab, four series of94target small molecule peptidomimetics were designed and synthesized as APN inhibitors. By preliminary bioactivity assay in vitro and in vivo, we found several potencial compounds such as compounds30e and38d, which need to be further developed in the future. The QASR and binding models of these compounds were also studied, which would be beneficial for further design and development of novel small molecule peptidomemitics as APN inhibitors in the future.