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amine of the previous amino acid in presence of NaCNBH 3 . After this reductive amination,a cyclisation was performed with TBTU/ DIEA to obtain the 4-amino-indolo[2,3-c]azepin-3-one structure [5]. After this, normal peptide synthesis was continued, followed by HFcleavage and by semi-prep reversed phase HPLC purification.Enzyme assay:Catalytic activity was measured by determining the rate of Leu-pNA cleavage in membranehomogenates of HEK293 cells transient transfected with human IRAP or AP-N in thepresence of different concentrations of compound. [6] Results are reported in Table 1.Table 1. Enzyme activity inhibited by Ang IV analogues in membranes of transfected HEK293 cellsCodeCompoundHEK 293 +IRAPpKi + SDHEK 293 +AP-NpKi + SDAng IV H-Val-Tyr-Ile-His-Pro-Phe 7.25 + 0.14 6.08 + 0.02AL11 H-(R)-β 2 -hVal-Tyr-Ile-His-Pro-β 3 -hPhe 7.56 + 0.21 5.23 + 0.04AL40 H-(R)-β 2 -hVal-Tyr-Ile-Aia-Gly-Phe 8.07 + 0.05 6.10 + 0.61IVDE73 H-(R)-β 2 -hVal-Tyr-Ile-Aia-Ala-Phe 7.56 + 0.18IVDE74 H-(R)-β 2 -hVal-Tyr-Ile-Aia-DAla-Phe 7.81 + 0.18IVDE75 H-(R)-β 2 -hVal-Tyr-Ile-Aia-Phe 7.37 + 0.11 5.20 + 0.03IVDE76 H-(R)-β 2 -hVal-Tyr-Ile-Aia-Pra-Phe 7.59 + 0.11 5.29 + 0.46IVDE77 H-(R)-β 2 -hVal-Tyr-Ile-Aia-Nva-Phe 5.80 + 0.24Enzyme stability study:The stability of the peptides was tested in human plasma at 37+1°C and incubated forvarious time intervals. The reaction was stopped by addition to CH 3 CN to precipitate theserum proteins. The supernatant was analyzed by RP-LC/MS.AngIV is first metabolised between Val 1 and Tyr 2 and subsequently between Tyr 2 andIle 3 . The half-life time is 1h 40min. All Aia-containing analogues are stable to enzymaticdegradation in these conditions.ConclusionsA rapid synthesis using chiral HPLC resolution of (R)-β 2 -h-Val was developed.Incorporation of the Aia constraint during solid phase synthesis provided AL-40 analogswithout problems. All new analogues are in the same potency range as the previouslysynthesized AL-11 and AL-40, except IVDE77. Deletion of Gly (analog IVDE75) did notreduce potency. All analogues containing (R)-β 2 -h-Val are stable in human plasma.AcknowledgmentsWe thank the 'The Fund for Scientific Research – Flanders' (FWO, Vlaanderen) and the VUB forfinancial support.References1. Chai, S.Y., et al. Cell Mol. Life Sci. 61, 2728-2737 (2004).2. Wright, J.W., et al. Prog. Neurobiol. 84, 157-181 (2008).3. Wright, J.W., et al. Fontiers in Neuroendocrinology 16, 23-52 (1995).4. Lukaszuk, A., et al. J. Med. Chem. 51, 2291-2296 (2008).5. Feytens, D., et al. J. Pept. Sci. 15, 16-22, (2009).6. Lukaszuk, A., et al. J. Med. Chem. 52, 6512-5618 (2009).139