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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Synthesis, Characterization and Cytostatic Effect ofNew Pemetrexed-Peptide ConjugatesErika Orbán 1 , Zsanett Miklán 1 , Zoltán Bánóczi 1 , andFerenc Hudecz 1,21 Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös LorándUniversity, Budapest, H-1117, Hungary; 2 Department of Organic Chemistry, EötvösLoránd University, 1518, Budapest, 112 POB 32, H-1518, HungaryIntroductionPemetrexed is used as a folate-antimetabolite in the treatment of pleural mesothelioma andnon-small cell lung carcinoma. This drug inhibits at least three enzymes (thymidylatesynthase, dihydrofolate reductase and glycinamide-ribonucleotide formyl transferase)involved in purine- and pyrimidine synthesis. Pemetrexed (Pem) treatment could beaccompanied by various side-effects like nausea, vomiting, diarrhea, etc. Conjugation ofanticancer drug (e.g. daunomycin, methothrexate) with peptide carrier could decrease oreven eliminate side effects [1,2]. Oligoarginines are de novo designed cell penetratingpeptides capable to translocate covalently attached cargoes [2]. Oligoarginine and otheroligopeptides were conjugated with Pem in our group earlier [3]. It was observed thatpeptide IELLQAR used as selective inhibitor could inhibit the attachment of E-selectin tothe cell surface-carbohydrates and thus limit the risk of metastases formation [4].Results and DiscussionIn this study we report on the synthesis and characterization of novel Pem – peptideconjugates containing cell-penetrating octaarginine and/or E-selectin binding IELLQARpeptide and thioether bond between the moieties. The conjugates were characterized by RP-HPLC and ESI MS. The cytostatic effect of Pem derivatives and conjugates was studied onNCI-H358 non-small cell lung carcinoma and on HL-60 human leukemia cell lines.For the synthesis of these conjugates, we first prepared Pem dimethylester by thereaction between Pem disodium salt and thionyl chloride in methanol, based on publishedprocedure [5]. Pure Pem(OMe) 2 was obtained by crystallization from ethanol. In the nextstep ClAc-Pem(OMe) 2 was synthesized by the reaction of chloroacetic acid anhydride andPem(OMe) 2 in the presence of DIEA in DMF.C-terminally modified octaarginine and peptide IELLQAR as well as the conjugatepeptide, IELLQARGGCGGR 8 was prepared on solid phase by Fmoc-strategy using in situactive ester coupling strategy. Cysteine amino acid was incorporated into the sequence toprovide an appropriate site for thioether bond formation with the chloroacetyl group ofpemetrexed.Peptide conjugates of Pem(OMe) 2 were prepared by the reaction between chloroacetylgroup of Pem(OMe) 2 and thiol group of Cys, present in the peptide sequence, underalkaline conditions (pH 8). Finally, peptide conjugates of Pem were prepared by removingthe methylester protecting groups from the carboxylic groups of Pem. Removal of theprotecting groups is indispensable because these carboxylic groups are essential for thebiological activity of Pem, as demonstrated in in vitro experiments (Table 1). To removethe protecting groups three times molar excess of 0.1 M NaOH and acetone (1:1, v/v) wasused as cleavage mixture at 5°C for 60-90 min. The progress of the reaction was monitoredby analytical RP-HPLC and terminated by adding of 0. 1M HCl solution to the mixture.394