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Table 1. Antitumor activity in vivo (prostate cancer model, ACI rats)Peptide Dose (µg/kg) Inhibition of tumor growth (%) aII 100 9IV 10 67V 10 45Buserelin 100 63a 29th day of treatmentFor the synthesis of cytotoxic peptide conjugates we applied both stable amide bondformation by means of CMFU-ONp derivative and hydrazone ligation using CMFUhydrazide. In the first case solid-phase conjugation was the method of choice for histidinecontaining peptides due to the side reaction of Ser and Tyr O-acylation, observed during thesynthesis in solution.In order to optimize the hydrazone formation conditions we prepared model RGDanalogues containing reactive keto groups (Figure 1). Peptides (IX) and (X) were obtainedas a result of N-terminal acylation by levulinic or pyruvic acid. In accordance withliterature data it was shown that presence of ethanedithiol at the stage of final deprotectionlead to the formation of dithioketal as the main side product. Triisopropylsilane should alsobe excluded from the cleavage cocktail due to its ability to reduce the carbonyl group. Inthe case of pyruvoyl peptide X RP-HPLC analysis of final product gave a broad peak dueto the equilibrium between keto- and hydrate- forms [4].At the stage of hydrazone formation, surprisingly, levuloyl peptide IX did not interactwith CMFU hydrazide at all, both in water solution at different pH and in MeOH withcatalytic amount of TFA. In contrast, for pyruvoyl derivative X under the same conditions,reaction was completed in 24 hr. Investigation of stability towards acid hydrolysisunexpectedly revealed that analog XI is completely stable at pH 5 and substantiallyhydrolyzed only at pH 3. Thus hydrazone bond should be intact at acidic endosomal pHand the only chance of CMFU release is enzymatic cleavage.By using different 5-FU attachment technique we synthesised two GnRH analoguesVII and VIII and investigated their cytotoxic activity in vitro. The biological experimentswere done on GnRH receptor-positive human HepG2 cells. The comparison of cytotoxicproperties revealed that peptide VII has at least the same activity as free CMFU, whilecompound VIII is slightly less active. Considering the number of synthetic stages andsurprisingly high stability of corresponding hydrazone it seems that application of CMFUhydrazide has no evident advantages.Biological experiments in vivo on prostate cancer model (Table 1) showed thatcytotoxic GnRH conjugates significantly suppress tumor growth as compared to controlgroup and parent compounds. Moreover, peptides V and VI were efficient at the dose of 10μg/kg, while clinically applied anticancer GnRH analog buserelin has a similar effect onlyat the dose of 100 μg/kg. These data suggested the possibility of CMFU application for thesynthesis of cytotoxic peptide conjugates useful for the suppression of tumor growth invivo.References1. Putnam, D.A., Shiah, J.G., Kopecek, J. Biochem. Pharmacol. 52, 957-962 (1996).2. Tada, M. Bull. Chem. Soc. Jpn. 48, 3427-3428 (1975).3. Vlasov, G.P., Burov, S.V., Semko, T.V., Veselkina, O.S., Sepetov, N.F., Makusheva, V.P. Peptides1990 (Proceedings of the 21 th European Peptide Symposium), Escom, Leiden, 1991, p. 682.4. Katayama, H., et al. Tetrahedron Lett. 50, 818-821 (2009).311