Development Of Novel Anti-cancer Agents Based On Aminopeptidase N/CD13

?.Research BackgroundAminopeptidase N(APN,CD13,EC 3.4.11.2),a Zn2+ dependent type II membrane-bound protein,is widely expressed on diverse cell surfaces.As an eco-peptidase,CD 13 could hydrolyze diverse peptides or oligopeptides.CD 13 plays a crucial role in cancer invasion,metastasis,angiogenesis,analgesia,immunoregulation and so on.Recenty,it has been confirmed that CD 13 is a biomarkers of semi-dormant liver cancer stem cells(CSCs)which is closely related to relapse,metastasis and drug resistance.Therefore,CD13 inhibitors deserve to be developed for the treatment of cancer.Ubenimex(Bestatin,Ube),the only marketed inhibitor of CD13,is widely used as immune-enhancement for the treatment of leukaemia and also used for complementary treatment of solid cancer after surgery,radiation and chemotherapy.It can significantly improve the survival rates of malignant tumor patients after surgery,radiation and chemotherapy.Many research showed that combination of CD 13 inhibitor Ubenimex and the traditional cytotoxic antitumor agents(such as fluorouracil(5-FU),cyclophosphamide,doxorubicin)could not only improve antitumor efficiency,but also reversed the drug resistance of tumor cells.Based on these research results and the concept of mutual prodrug,novel series of mutual prodrugs were designed and synthesized by integrating ubenimex and 5-FU or gemcitabine into one molecule.Further structural modification and optimization of these compounds led to one promising candidate compound 18a with excellent in vitro and in vivo anti-cancer activity.?.Design,synthesis and anti-cancer activity evaluation of novel mutual prodrugs of Ubenimex and FluorouracilCombination of Ubenimex and 5-FU exhibited significant synergistic efficiency.Therefore,we connected 5-FU and Ubenimex into one molecule to obtain a mutual prodrug by the chemical method.Firstly,we designed and synthesized a novel mutual prodrug BC-01(9)of Ubenimex and 5-FU by coupling 5-FU moiety to the carboxyl group of Ubenimex through ester.The CD13 inhibitory activity of BC-01 was comparable with that of Ubenimex.Meanwhile,the antiproliferative activity of BC-01 was better than the positive control Ubenimex and 5-FU.The in vivo antitumor results confirmed that BC-01 remarkably reduced tumor volume,and exhibited both orally and intravenously antitumor activity.However,the extremely poor stability of BC-01 in human plasma hindered its further development as an acceptable prodrug,so the structure of BC-01 was optimized.We designed and synthesized six BC-01 derivatives 18a-18f with different kinds of amino acids as linker between Ubenimex and 5-FU.Biological evaluation showed that all of these compounds expressed surprisingly more potent CD 13 inhibitory activity than BC-01 and Ubenimex,especially,the IC50 values of representative compound 18f was 0.096 ?M.The preliminary stability test of compound 18a and 18f revealed that 18a and 18f were more stable than BC-01 in human plasma.Pharmacokinetic study showed that compound 18a could release Ubenimex and 5-FU in two steps after the intravenous administration.Moreover,the pharmacodynamics study of 18a in vitro showed that 18a decreased the number of PLC/PRF/5 cells expressing CD13,which is a kind of cells related to tumor relapse and drug resistance.The anti-angiogenesis results suggested that compound 18a could inhibit HUVECs tube formation and microvessels sprouting.The anti-invasion assay results showed that 18a could suppress the invasion of cells.The pharmacodynamics study of 18a in vivo showed that compound 18a exhibited both orally and intravenously antitumor activity.The antitumor activity of 18a was better than that of BC-01 and the positive control Capecitabine.Meanwhile,18a was effective in 5-FU-resistant H22 tumor transplant model.The anti-metastasis study of compound 18a showed that the number of metastatic pulmonary nodes in mice treated with 18a was much fewer.Compared with the control,18a exhibited superior lifespan extension effect.Some mechanism researches suggested that 18a exhibited immunostimulating potency via improving the macrophage phagocytic activity.Meanwhile,a new synthesis route of 18a was designed and developed.The new synthesis process not only improved the yield,reduced the steps of the synthesis,simplified the post-processing,but also improved the product purity to 99.4%.?.Design,synthesis and anti-cancer activity evaluation of novel mutual prodrugs of Ubenimex and GemcitabineIn view of the superior activity of mutual prodrugs of 5-FU and Ubenimex,Ubenimex and gemcitabine were integrated into one molecule via ester bond and amide bond to get a series of mutual prodrugs.This series of compounds displayed good CD13 enzyme inhibitory activities,among which,the IC50 values of compound 38b was 0.26 ?M,which was better than that of Ubenimex(3.70 ?M).On the other hand,the antiproliferative results indicated that the attachment of 4-(N)-and 5'-sites of gemcitabine to the carboxyl group of Ubenimex maintained the antiproliferative activity of gemcitabine.The preliminary stability test of representative compound 32 revealed that it could release gemcitabine completely within 120 min in vitro.The pharmacodynamics study of 32 in vitro showed that compound 32 was an orally active prodrug of gemcitabine.?.Design,synthesis and anti-cancer activity evaluation of Novel dual-target Anticancer Agents based on HDACWe designed and synthesized a novel dual-target antitumor agent 46 by replacing the pyridine cap group of MS-275 with its bioisostere,the cytotoxic agent 5-FU.Compound 46 showed similar HDAC inhibitory activity with MS-275.The antiproliferative results indicated that 46 expressed the antiproliferative activity toward the examined cell lines,but the inhibitory activity was less potent than positive control MS-275.Meanwhile,the preliminary stability test of compound 46 revealed that it was very stable in artificial gastric juice,artificial intestinal juice,and human plasma.On the basis of these premises,we guess that the good stability of compound 46 affect its synergistic effect.?.Conclusion and perspectiveIn conclusion,based on the liver cancer stem cells biomark CD 13,and in the guidance of computational chemistry,medicinal chemistry,biochemistry and pharmacokinetic,two series of total 19 novel mutual prodrugs were designed and synthesized by combining the CD13 inhibitor Ubenimex and other marketed cytotoxic drugs(5-FU and gemcitabine)into one molecular structure through an ester bond or an amido bond using mutual prodrugs method.All of these compounds exhibited effective CD13 enzyme inhibitory activities.Then we conducted CD13 inhibitory assay(including CD 13 on the surface of cancer cell lines),in vitro and in vivo anti-cancer assays,pharmacokinetic assay and synthesis route optimization of 18a.Our comprehensive in vitro and in vivo antitumor activity evaluation supported further research and development of 18a as a promising mutual prodrug of Ubenimex and 5-FU.Meanwhile,the proof of concept described in this research is valuable toward the further development of traditional cytotoxic chemotherapy drugs.