Design, Synthesis And Anticancer Activity Study Of Cyclic-imide Peptidomimetics As Metalloproteinase Inhibitors

Partâ… .Research backgroundMetalloproteinase is a big family of metal-ion containing hydrolases.It is discovered that two zinc-dependent metalloproteinase(Aminopeptidas N,APN/CD13 and Matrix Metalloproteinase,MMP) are involved in the invasion and metastasis of malignant tumors.Among which APN also plays critical roles in tumorgenesis,regulation of immunological function and virus infection as well.a.APN is over-expressed on the surface of tumor cells and the possible function in the process of tumor growth is:1) Degrading extracellular matrix,promoting the release of most growth factor and so speeding up the proliferation of tumor cells;2) Promoting the invasion and metastasis of tumor cells;3) Stimulating the release of cellular factors associated with the neovascularization by vascular endotheilal cells and promoting the angiogenesis of tumor;4) Enhancing the adhesiveness of tumor cells,and in turn the tumor cells with high adhesiveness will stimulate the release of metalloproteinase and enhancing its ability of ECM degradation;b,APN is also over-expressed on the surface of granulocyte and lymphocyte and participate the T-lymphocyte dependent inflammatory reaction as CD13 antigen.APN also has been implicated in the processing and trimming of antigenic peptides that protrude out of major histocompatibility complex(MHC) classâ…¡molecules on the cell surface.APN is also involved in the down-regulation of signal peptides,such as enkephaline in the brain and can cleave bioactive proteins,including several cytokines and antigen delivering peptides,degrading lots of immunoactive substances,impairing the immunological functions,depressing the recognization of macrophage and NK cells to surface antigen on tumor cells and the ability to kill these cells directly;c.APN was shown to be the major receptor for the transmissible gastroenteritis virus(TGEV),which causes a severe gastroenteritis in newborn pigs,and for the human coronavirus 229E (HCV229E) which causes upper respiratory infections;d.APN also play a role in the entry of HIV-1 into cells by degrading fMLP,a chemokine associated with the desensitization of CCR5,the co-receptor for HIV-1 entry and up-regulating the expression of CCR5;e.Furthermore,APN is also involved in the down-regulation of signal peptides,such as enkephaline in the brain and will be a promising target for analgesia;f.In recent years,APN has been elucidated to participate in the enzymatic cascade of rennin-angiotensin system in the brain and periphery by cleaving angiotensinâ…¢to antiotensinâ…£and is associated with the regulation of blood pressure.Therefore,APN will be a good target for the design of novel therapeutic agents for the treatment of diseases,such as cancer,leukemia,rheumatoid arthritis, diabetic nephropathy and central nervous system diseases,such as Alzheimer's disease.The anticancer drug research targeting to APN is closely watched in recent years because of it role in the tumor growth.The research fields include three aspects:APN mono-antibody and APN ligand binding drugs,both of which are in the initial stage; Small molecular APN inhibitors,either natural or synthetic.Bestatin,firstly isolated from a culture filtrate of Streptomyces olivoreticuli,has been used in clinics as an immunoenhancer in Japan in 1987.In recent years,many bestatin analogues were discovered,among which Actinonin is a more active inhibitors.Partâ…¡.The Virtual Screen and Rational Design of Target Compounds Although there are lots of small molecular inhibitors reported in recent years,however, there were no any reports of APN crystal structure until 2006.Most researches are focused on natural products and bestatin analogues.And the process of synthetic APN inhibitors is really slow,resulting in few report associated with target structure-based drug design toward APN.The characteristics of this study are the utilization of mechanism-based and structure-based drug design comprehensively.That is,based on the inference that the major components of ECM are Pro-Gly,we build a virtual molecule library with heterocycle as scaffold according to the conformational constraint strategy.And with advantages of computer-aided drug design software,we also build a four-emelment pharmacophore model according to the information of inhibitors with known activity and structure.The cyclic-imide scaffold was obtained by virtual screening of the heterocycle library with this pharmacophore.The following design strategy is structure-based drug design.That is,to design the side chain of cyclic-imide according to the information of the requirement of the active site of E.Coli APN and enzyme-inhibitor complex.Flexsible docking was used for modeling the interaction between target compounds and enzyme.The side chain of cyclic-imide can be designed rationally and synthesized easily.The compounds in three series are a result of optimization step by step.It is discovered that there is some relevance between part of the quantitative parameters (such as docking score) and the in vitro or in vivo activity of compounds.Furthermore, based on the analysis of the difference between the active site of target enzymes,we found the change of the selectivity from MMP to APN,indicating that the design strategy of this study is rational and is significant for the design of novel target compound.In addition,the experimental results can be well predicted by all kinds of screen model constructed.Additionally,the matching of the structure of compounds with the active site of enzyme is not the only consideration of this study.The fragments used in the side chain of the target compounds also apply the fragments with anticancer activity and natural amino acid side chain,which can be compatible with tissues. In addition,the ADME and pharmacokinetics properties of the target compounds have also been considered in advance.The compounds exist in the form of hydrochloride salt with better water solubility and bioavailability and so increasing the hit rate.Partâ…¢.The Synthesis of Newly Designed Target CompoundsOn the basis of virtual screening and target structure-based drug design.We obtained 94 structural confirmed compounds with varieties in the side chain of cyclic-imide. The polypeptide synthesis advantages are thoroughly reflected in the synthesis scheme,most of which are novel and feasible.Two strategy are applied for the synthesis of target compounds:Firstly,the 3-trimethoxybenzoic-2,6-piperidinedione-N-acetic acid derivatives are synthesized with L-Glu as starting material and the important intermediate 6 was obtained after acylation,cyclization by dehydration and aminolysis by Gly.The target compounds are obtained with EDCI as condensing agent for polypeptide synthesis from 6 with different amino acid derivatives.The methyl group can also be deprotected by Lewis acid BBr₃.Secondly,the intermediates that can be used for the variety of structure were synthesized in advance.12 and 27 can be obtained by the protection of amino group of L-Gln or L-Asn by Boc and then cyclization of amide withα-carboxyl group. The NH of cyclic-imide can be alkalization by halogenated hydrocarbon in the existence of phase transfer catalyst TBAI to form N-substituted intermediates of 13, 18 and 28.The Boc group of these three compounds can be deprotected by TFA and then the free amino group was acylated by different acyl chloride or mixture anhydride.The cyclic-imide-N-acetic acid derivatives can be obtained from its benzyl ester derivatives by catalytic transfer hydrogenation to deprotect the benzyl group. And the hydroxamate acids derivatives can be obtained according to the methods of mixture anhydride from its acid with isobutyl chloroformate.All the targeted compounds are novel without any report by now with the structures identified by IR, ¹H-NMR and ESI-MS.The purity of part compounds are analyzed by HPLC,with the area%of more than 96%. Partâ…£.The Biological Evaluation and SAR & QSAR StudyThe in vitro and in vivo preliminary activity evaluations were performed as for the newly synthesized 94 target compounds.The in vitro enzyme inhibitory activity were determined against APN and MMP,both of which are zinc-dependent metalloproteinase and closely associated with the invasion and metastasis of tumor cells,The cells assay is performed toward leukemia cells(HL-60 and K562) which express APN and SKOV3(which can express MMP-2 and MMP-9).The in vitro enzyme assay displayed that the compounds in Seiresâ… are all selective to MMP(the IC₅₀ ratio of 7j is 150).However,the inhibitory activity of 71,7o,9a,9b against APN is pretty high,and the IC₅₀ value of most active compound 9a(3.0μM) is equal to that of bestatin(IC₅₀=2.4μM),indicating that the introduction of conformational constraint can indeed improved the selectivity toward target enzyme. The free group in Seiresâ…¡and Seriesâ…¢is a decisive factor for the inhibition of APN, as the activity increased by an order of magnitude.Among which 17j,22a,22g in Seriesâ…¡and 32a,32c,32f,32i in Seriesâ…¢and all hydroxamate acid compounds 24/34 in both series displayed potent activity and high selectivity toward APN with the IC₅₀ ratio about 20.The IC₅₀ of 34d was 2.9μM.24f and 34f is more potent that bestatin against APN with IC₅₀ value of 1.8μM and 1.0μM,respectively. The SAR and QSAR analysis displayed that the substituents in P1' site in Seriesâ… is important for the selectivity of MMP and the Try residue is more favorable.The introduction of aliphatic residue can improve the activity against APN with a loss of selectivity.As for the compounds in Seriesâ…¡and Seriesâ…¢,free amino group is a necessary condition for the inhibitory activity of APN and the activity tendency of target compounds with different substituents in P1 is CONHOH＞COOH＞COOEt,and aromatic ring without a hydroxyl group is more favorable.The results of in vitro growth inhibition against leukemia cells indicated that most potent APN inhibitor displayed good inhibitory effect against the growth of HL-60 cells, among which 7g,9a,24f,24i,34f is more potent than bestatin.The possible mechanism may be that the downstream factor is influenced when cyclic-imide derivatives interact with APN,and then the DNA synthesis is inhibited followed by the promotion of apoptosis of tumor cells.The cells assay was also performed to leukemia cell K562.The inhibitory rate is still lower than 25%when the concentration of compounds is nearly saturated.The inhibitory effect can not be discovered even in the higher concentration and there was no phenomenon of abnormality about the shape of cells,indicating that the compounds are not cytotoxtic.The sensitivity of K562 to the inhibitors is less than that of HL-60 because of its lower expression of APN,in some degree suggesting that the newly synthesized compounds can target APN well.The cells assay against the growth of SKOV3 display that the compounds(7i,7r) with moderate activity against MMP can strongly inhibit the expression of MMP-2, MMP-9,and this may be the major reason for their anticancer effect. We used the anti-metastasis model of mice bearing H22 tumor cell to evaluate the inhibiting activities of cyclic-imide derivatives in vivo.All tested compounds were orally administered(p.o.) at a dose of 50 mg/kg,6 days/week for two weeks.The test results demonstrated that most of potent APN inhibitors(9a,24f,24i,34f,34i)showed significant anticancer activities(inhibitory rate＞50%) and were devoid of toxicological effects,among which the best compounds were 24f and 9a with the inhibitory rate as high as 72.91%and 81.56%,respectively,showing that they might be a promising lead compound and is worth of further study.The possible mechanism of anti-metastasis effect may be:1) to inhibit the activity of APN directly,and then inhibit the degradation of ECM;2) Some cellular factor that can regulate the transcription of gene was activated or inhibited following the inhibition of APN,and the expression of MMP or other molecules was influenced and in turn inhibiting the ECM degradation and the location of tumor cells in lung tissues. The five compounds used in in vivo experiment assay possess potent inhibitory effect in in vitro enzyme and cells assay and both of the experimental results can be fully consistent.All of these discoveries prove the following points:1) The screening ability of the pharmacophore constructed in advance is strong enough to screen out the potent cyclic-imide scaffold from the virtual library;2) The methods used for in vitro and in vivo activity assay are stable and can be reproduced with high degree of confidence;3) The design strategies applied in this study is identical to the novel ideas in modem medicinal chemistry fields,and the pre-consideration of ADME properties result in successful achievements.Partâ…¤.ConclusionThe cyclic-imide peptidomimetics derivatives designed in this study displayed potent metalloproteinase inhibitory activity.And some potent compounds can be screened in the in vitro and in vivo experiments and is promising and valuable of further study and used in pre-clinical study.The results of activity assay experiments can manifest quantitatively the tendency of the relationship between the structure and activity and meanwhile build the SAR and QSAR model.All of these discoveries provide a beneficial theoretical basis for the optimization of lead structure and lay a solid foundation for the discovery of novel more potent anti-metastasis agents in future.