Synthesis,Antitumor Activity And Structure-activity Relationship Of Cyclopentapeptide Galaxamide Analogues

Galaxamide is a cyclic pentapeptide from Galaxaura filamentosa,preliminary studies indicated the effective inhibition effect of Galaxamide on several types of tumor cels like Hep G2,MCF-7 and MDA-MB-231.In this thesis,we synthesized series of Galaxamide analogs,revealed the structure-reactivity relationship and systematically investigated the anticancer activity of analogs.Basically,we designed three types of Galaxamide analogs which were numbered as Analog-1?Analog-36.The Galaxamide analogs were synthesized through methylation of?-amino acid,introduction of non-natural d-amino acid or change of amino acid residue position on parent molecular structure of Galaxamide,based on the considerations of spatial configuration,steric hindrance and molecular conformation.Based on our previous work,here we optimized the synthetic strategy for preparation of Galaxamide analogs.Firstly,liquid phase synthetic method together with a classic“3+2”strategy was used to synthesize 6 types of Galaxamide cyclic pentapeptides Analog-1?Analog-6.In the synthesis,using amino acid and amino acid monomer with protected terminal carboxyl groups as the raw materials and DEPBT as the condensation reagent,linear pentapeptides Analog-1?Analog-6 was first synthesized in mixed solutions of THF and DIEA.Then cyclic pentapeptides Analog-1?Analog-6 were prepared in mixture of THF/CH₂Cl₂/CH₃CN,where HATU/DEPBT/TBTU were used as the mixed condensation reagents.On the contrary,cyclic pentapeptides Analog-7?Analog-36 were synthesized using a solid phase synthetic route?the so-called Fmoc method?.In this synthesis,linear pentapeptides Analog-7?Analog-36 were first synthesized using 2-chlorotrityl chloride resin as the solid phase,where DEPBT-DIEA as the condensation reagent,DMF as the reacting solvent,20%piperidine/DMF as the deprotective reagent.Then,cyclic pentapeptides Analog-7?Analog-36 were synthesized in highly diluted solution with Py BOP as condensation reagent,DCM as reacting solvent,DIEA as reagent for adjusting p H?p H was adjusted to 8?.Finally,structure and components of all the resultant products were carefully characterized by ESI-HRMS,¹H NMR and ¹³C NMR.Results indicated the successful synthesis of Galaxamide derivates as designed.Next,we evaluated the tumor inhibitory effects of all the obtained Galaxamide analogues on several types of cancer cels like Hep G2,Hela?MCF-7,MDA-MB-231,A549,A549-DPP,SW1990,Panc1,SMMC-7721,QGY-7701.Results indicated a structure and cancer cell type dependent anticancer activity of those Galaxamide analogues.In particular,IC₅₀of Analog-6against Hep G2,Hela,MCF-7 and MDA-MB-435 was 6.53±0.32?M,5.45±0.40?M,12.99±0.29?M and 10.77±0.35?M,respectively.Both Analog-8 and Analog-10 displayed better inhibition effects on SMMC-7721 and QGY-7701 cancer cells than that of parent Galaxamide and DPP.Specifically,IC₅₀of Analog-8 against SMMC-7721 and QGY-7701 was 0.91±0.16?M and2.23±0.16?M,respectively,which were alomost 20 times lower than the controls.Moreover,we revealed the structure-activity relationship of 58 Galaxamide analogs.Results indicated that Galaxamide analogs with phenylalanine substituent displayed better anticancer activity than other types of Galaxamide analogs.Galaxamide analogs with only one D-amino acid facilitated the formation of structure with single conformation and stronger selectivity.All the results indicated that anticancer activity of Galaxamide and its analogs greatly depended on their conformation.Furthermore,we elucidated the structure-activity relationship of those Galaxamide analogues.We revealed that analogue with Phe substituent group showed better anticancer activity than parent molecule Galaxamide.Number and spatial position of D-AA had great affect on the anticancer activity of analogues.Introduction of D-AA or^?N-Me-AA substituent could also significantly improved the inhibitory effects of analogues on cancer cells.Lastly,we investigated the anticancer mechanism of Galaxamide analogues on Hep G2through Annexin V-FITC/PI dual-staining method,PI staining method,Hoechst 33342 fluorescent staining method and western blotting technology.We revealed that,Analog-6 could cause the early apoptosis of Hep G2 cells by inhibiting their growth in the sub-G1 stage of the cell cycle and induce the chromatin condensation and fragmentation,which can be seen that 68%of Hep G2 cells inhibited in the sub-G1 stage.Moreover,a mitochondria-mediated pathway found to be involved in the apoptotic process of Analog-6 on Hep G2 cels.Our work not only expanded the research on Galaxamide and its analogues,but also the findings suggested that Galaxamide and its analogues are promising in further applications.Our work provided novel strategies for synthesis of Galaxamide analogues with better bio-reactive properties.