Synthesis of β-Turn and Pyridine Based Peptidomimetics David Blomberg Department of Chemistry, Organic Chemistry Umeå University Umeå 2007 Department of Chemistry, Organic Chemistry Umeå University SE-901 87 Umeå Copyright 2007 by David Blomberg ISBN 978-91-7264-305-5 Printed in Sweden by Intellecta DocuSys, Västra Frölunda, 2007 Contents 1. List of Papers.................................................................................................. 3 2. List of Abbreviations ..................................................................................... 5 3. Introduction .................................................................................................... 7 3.1 Structure and function of peptides and proteins.................................... 7 3.2 Peptidomimetics ...................................................................................... 9 3.2.1 Development of a peptidomimetic drug - Exanta ....................... 10 4. Objectives of the thesis ................................................................................ 12 5. Peptidomimetics of Leu-enkephalin ........................................................... 13 5.1 Biological action and conformation of Leu-enkephalin ..................... 13 5.2 Design of Leu-enkephalin peptidomimetics........................................ 14 5.3 Synthesis of peptidomimetics incorporated in Leu-enkephalin ......... 15 5.3.1 Synthesis of a ten membered β-turn mimetic.............................. 15 5.3.2 Conformational studies of the ten membered β-turn mimetic using NMR spectroscopy ....................................................................... 20 5.3.3 Synthesis of a seven membered β-turn mimetic on solid phase. 21 5.3.4 Synthesis of linear Leu-enkephalin analogues ............................ 22 5.4 Biological evaluation ............................................................................ 24 5.4.1 Opioid receptor binding assay ...................................................... 24 5.4.2 Binding to µ- and δ- opioid receptors .......................................... 25 5.5 Summary................................................................................................ 27 6. β-Strand peptidomimetics............................................................................ 29 6.1 β-Strands................................................................................................ 29 6.2 Design and retrosynthetic analysis of a β-strand mimetic.................. 30 6.3 Attachment of an N-terminal leucine analogue at position 4 of the pyridine ring................................................................................................. 32 6.4 Attachment of a C-terminal glycine analogue at position 2 of the pyridine ring................................................................................................. 34 6.4.1 Nucleophilic aromatic substitution............................................... 34 6.4.2 A reductive amination strategy..................................................... 40 6.4.3 Changing the substitution order and starting with the SNAr reaction .................................................................................................... 41 6.5 Completing the synthesis − A successful Boc strategy ...................... 42 1 6.6 Incorporation of a second chiral amino acid analogue and attempts to elongate the β-strand mimetic..................................................................... 45 6.6.1 Introducing a chiral amino acid analogue instead of glycine as C- terminus................................................................................................... 45 6.6.2 Attempts to elongate the β-strand mimetic.................................. 46 6.6.3 Conclusions.................................................................................... 49 6.7 Summary................................................................................................ 49 7. Thrombin inhibitors ..................................................................................... 51 7.1 Biological action of thrombin............................................................... 51 7.2 Structure based design .......................................................................... 53 7.3 Retrosynthetic analysis of the thrombin inhibitors ............................. 53 7.4 Synthesis of thrombin inhibitors .......................................................... 54 7.4.1 Attempts to obtain thrombin inhibitors via a Grignard exchange reaction followed by an SNAr reaction using substituted benzylamines .................................................................................................................. 54 7.4.2 A reductive amination approach................................................... 57 7.4.3 Conversion of the cyano group to the desired benzamidines ..... 58 7.5 Biological evaluation ............................................................................ 61 7.6 Crystal structure .................................................................................... 61 7.7 Summary................................................................................................ 62 8. Thrombin inhibitors with reduced basicity................................................. 64 8.1 Introduction............................................................................................ 64 8.2 Structure based design and retrosynthetic analysis............................. 64 8.3 Synthesis of thrombin inhibitors .......................................................... 65 8.3.1 Synthesis of Boc-protected alaninal and glycinal ....................... 65 8.3.2 A Grignard reaction and nucleophilic aromatic substitution with cyclic amines........................................................................................... 66 8.3.3 Completing the synthesis .............................................................. 67 8.4 Biological evaluation ............................................................................ 68 8.5 Summary................................................................................................ 68 9. Concluding remarks ..................................................................................... 70 10. Acknowledgement ..................................................................................... 73 11. References .................................................................................................. 75 Appendix........................................................................................................... 85 Experimental section for chapter 8........................................................ 85 2 1. List of Papers I David Blomberg, Mattias Hedenström, Paul Kreye, Ingmar Sethson, Kay Brickmann and Jan Kihlberg; Synthesis and conformational studies of a β-turn mimetic incorporated in Leu- enkephalin. J. Org. Chem., 2004, 69, 3500-3508. II David Blomberg, Paul Kreye, Kay Brickmann, Chris Fowler and Jan Kihlberg; Synthesis and biological evaluation of leucine enkephalin turn mimetics. Org. Biomol. Chem., 2006, 4, 416- 423. III David Blomberg, Kay Brickmann and Jan Kihlberg; Synthesis of a β-strand mimetic based on a pyridine scaffold. Tetrahedron, 2006, 62, 10937-10944. IV David Blomberg, Tomas Fex, Yafeng Xue, Kay Brickmann and Jan Kihlberg; Design, synthesis and biological evaluation of thrombin inhibititors based on a pyridine scaffold. Submitted. V David Blomberg, Tomas Fex, Kay Brickmann and Jan Kihlberg; Design, synthesis and biological evaluation of thrombin inhibitors lacking a strong basic functionality in P1. Manuscript. Reprinted with kind permission from the publishers. 3 4 2. List of Abbreviations Bn benzyl Boc tert-butoxycarbonyl BSA bovine serum albumin Cbz benzyloxycarbonyl DAMGO [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DCC dicyclohexyl carbodiimide DCE 1,2-dichloroethane DIBAL diisobutylaluminium hydride DIC N,N’-diisopropyl carbodiimid DIPEA diisopropylethylamine DMAP N,N-dimethylaminopyridine DPDPE [3H] [D-Pen2, D-Pen5]enkephalin DTI direct thrombin inhibitor EWG electron withdrawing group Fmoc 9-fluorenylmethyloxycarbonyl GPCR G protein-coupled receptor HATU O-(7-azabenzotriazole-1-yl)-N, N,N’N’- tetramethyluronium hexafluorophosphate HMDS hexamethyldisilazane HOAt 1-hydroxy-7-azabenzotriazole HOBt N-hydroxybenzotriazole K-selectride potassium tri-sec-butylborohydride LCMS liquid chromatography mass spectrometry LHRH luthenizing hormone releasing hormone N,O-DMHA N,O-dimethylhydroxylamine NMO N-methyl morpholine N-oxide NMR nuclear magnetic resonance NOE nuclear overhauser enhancement NOESY nuclear overhauser enhancement spectroscopy PAM 4-(Hydroxymethyl)phenylacetamidomethyl PMB p-methoxybenzyl chloride Q tetrabutylammonium rt room temperature 5 TBAF tetrabutylammonium fluoride TBDMS tert-butyldimethyl silyl TEA triethylamine TMP 2,4,6-trimethylpyridine TFA trifluoro acetic acid TFAA trifluoro acetic anhydride TFE 2,2,2-trifluoroethanol TMS trimethylsilyl Tris tris hydroxymethylaminoethane UHP urea hydrogen peroxide 3-D three dimensional 6 3. Introduction 3.1 Structure and function of peptides and proteins On a molecular level proteins are built up by small residues, amino acids, that are connected via amide bonds to form chains (Figure 3.1). Shorter amino acid sequences, usually containing 2−50 amino acid residues, are defined as peptides, while longer chains are defined as proteins. There are 20 naturally occurring