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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, 2010Amidine Neighbouring-Group Effect on the Stability of B9870, aHighly Potent Anti-Cancer Bradykinin B1/B2Antagonist Peptide DimerLajos Gera 1,3 , Richard Duke 2,3 , Daniel C. Chan 2,3 , Paul A. Bunn, Jr. 2,3 ,Robert S. Hodges 1 , John M. Stewart 1,3 , and James Blodgett 41 Department of Biochemistry and Molecular Genetics, University of Colorado Denver,Aurora, CO, 80045, U.S.A.; 2 Cancer Center, University of Colorado Denver, Aurora,CO, 80045, U.S.A.; 3 ApopLogic Pharmaceuticals, Inc., Aurora, CO, 80010, U.S.A.;4 Indianapolis, IN, 46236, U.S.A.IntroductionBradykinin (BK) is a biologically active nonapeptide that plays an important role in manyphysiological processes, such as cardiovascular diseases, septic shock, pain, chronic andacute inflammation, and cancer. The first full-chain, highly enzyme-resistant BK antagonistwhich showed high affinity at both B1 and B2 receptors, B9430 (DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg, where Hyp is trans-4-hydroxyproline, Igl is -(2-indanyl)glycineand Oic is octahydroindole-2-carboxylic acid), was developed more than a decade ago [1].Since BK is an autocrine growth factor for many types of cancer, notably of lung andprostate, it was logical to try the potent BK antagonist B9430 as a growth inhibitor forcancer. Disappointingly, the B9430 potent BK antagonist peptide monomer was almostineffective against cancer but the N-terminal suberimidyl-crosslinked B9430 dimer, knownas B9870 (later also named B201, CU201, NSC 710295, and Breceptin), was found to bean extremely potent anti-cancer agent. The synthesis, stability, and the anti-cancer activityof B9870 will be discussed in this article.Results and DiscussionThe synthesis of the monomer peptide B9430 was accomplished via standard solid-phasemethodology on Merrifield resin using Boc-chemistry. The dimerization of B9430 wasindependently conceived and carried out by Dr. Gera [2] (notwithstanding other claims [3])and involved the formation of the amidine-structure dimer with dimethyl suberimidate.This approach to the dimerization was based on Gera's previous work involving amidinechemistry [4]. The dimerization of B9430 with thio-suberimidates increases the yieldsignificantly (Gera, et al., unpublished results). B10346 [Sub-(B9430) 2 , Sub: suberyl), thebis-amide analog of dimer B9870, was prepared with the dimerization on the resin withsuberyl dichloride. The peptides were purified by preparative RP-HPLC and characterizedby analytical RP-HPLC, TLC, mass spectroscopy and amino acid analysis. BOP-HOBt wasused for the coupling of amino acids. The stability study was carried out by RP-HPLC andthe degradation products were characterized by LC/MS. The stability of B9870 (CU201)has also been studied at the National Cancer Institute [5]. The bis-amidine dimer, B9870 ishighly stable when stored frozen as a dry powder. It is also highly stable when reconstitutedin physiological saline and stored in the refrigerator (4ºC; 5% loss at 4 weeks). Thecompound undergoes degradation in saline solution buffered with phosphate (pH 7.4) toB10660 (DArg-Suim-B9430, Suim: suberimidyl; an alternative, isomeric ring-openedproduct is also possible but B10660 appears more likely) and B9598 (des-DArg 0 -B9430)via amidine group participation according to the proposed mechanism (Figure 1). Otherdegradation products also observed were the hemi-amide dimer (B10658), bis-amide dimer(B10346) and 7-mer monomer peptide (B9924, Hyp-Gly-Igl-Ser-DIgl-Oic-Arg), the latterpossibly formed via N-terminal diketopiperazine formation from B9598 by a mechanismfor which precedents exist in the literature [6]. B10346 bis-amide peptide is stable underphysiological conditions, where B9870 undergoes degradation. Our potent BK antagonistswere routinely screened [7] for anti-cancer activity. The compounds, including themetabolism-resistant, highly potent BK antagonist B9430, blocked Ca 2+ mobilization bybradykinin in vitro but did not show significant growth inhibition of lung or prostate cancercells (Table 1). However, B9870, the suberimidyl dimer of B9430 monomer BK antagonistwas found to have high anti-cancer activity in vitro and in vivo in nude mouse xenografts[8]. Concerning the pH-sensitive N-terminal amidine structure, its inclusion in B9870 was308