1. UFR Biomédicale des Saints-Pères Ecole Doctorale du Médicament Université René Descartes – Paris 5 UFR Biomédicale des Saints-Pères Ecole Doctorale du Médicament Strategic investigations for the design of a library of liposidomycins analogs, natural antibiotics dedicated to the MraY translocase Maryon GINISTY Direction : Pr. Yves Le Merrer Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques Direction : Dr. Daniel Mansuy - UMR 8601 – CNRS 45, rue des Saints-Pères - 75270 Paris Cedex 06- France

2. 2 ANTIBACTERIAL RESISTANCE : A MAJOR OBSTACLE FOR ANTIBIOTHERAPY  1940’s : development of penicillin and appearance of the concept of antibiotics « Agents with specific antibacterial action and with toxicity selectively directed against bacteria in low concentrations » ► Bacteriostatic effect (decrease or stop of bacterial growth) ► Bactericid effect (destruction of bacteria) ● Complexity and adaptability of bacterial world ► Therapeutic failure ► Development of a large number of antibiotics classified according to various criteria : site of action, origin, administation route, structure ⇒ Eight major families :  -lactams, aminosides, macrolides, sulfamides, poly- et glyco-peptides, cyclins, (fluoro)quinolons…

3. 3 ● Two types of resistance : ► Natural resistance (intrinsic property related to the bacterial genetic program) ► Acquired resistance (property resulting from genetic modifications of the bacterial cells) ● Five major mechanisms of resistance : - Overproduction of antibiotic target - Metabolic bypass of inhibited reaction - Inactivation of antibiotic by enzymatic modification - Modification of target eliminating or reducing antibiotic binding to target RESISTANT STRAINS AND MECHANISMS ⇒ Resistant strain : strain able to develop in the presence of an antibiotic concentration notably higher than that which inhibits development of other strains of same species antibiotic « modifying » enzyme modified antibiotic X modified receptor antibiotic receptor resistance gene pump - Decrease of cellular permeability to antibiotic

4. 4 ⇒ Four sites of action specific to procaryote bacterial cells - ribosomes responsible for protein synthesis - metabolism of nucleic acids ⇒ inhibition of DNA synthesis ⇒ inhibition of DNA transcription into messenger RNA - oxydoreduction (5-nitro-imidazoles) via formation of superoxides and nitro radicals responsible of irreversible damage on bacterial DNA - cell wall biosynthesis SITES OF ACTION OF ANTIBIOTICS AND POTENTIAL TARGETS Gram (-) cell Gram (+) cell lipopolysaccharide periplasm cytoplasmic membrane external membrane peptidoglycan cytoplasm mRNA ribosome DNA DNA-gyrase RNA-polymerase mRNA Bacterial wall BACTERIA aminoacid

5. 5 PERIPLASM ANTIBIOTICS AND BACTERIAL PEPTIDOGLYCAN BIOSYNTHESIS UDP-MurNAc-pentapeptide bacitracin vancomycin moenomycinpenicillin cephalosporin D -cycloserin fosfomycin tunicamycin muraymycin mureidomycin liposidomycin MraY CYTOPLASM UMP Pi UDP-GlcNAc UDP BacA MurG PBPs PBPs Lipid I Lipid II Acceptor Polymer Peptidoglycan Undecaprenyl-PP Undecaprenyl-P MEMBRANE

6. 6 INHIBITORS AND NATURAL SUBSTRATE OF MraY TRANSLOCASE

7. 7 STRUCTURE OF LIPOSIDOMYCINS S S S S

8. 8 SYNTHETIC APPROACHES DESCRIBED IN LITTERATURE ⇖ ⇖ ⇖  1,4-diazepan-2-one moiety Knapp et coll. Tetrahedron Lett. 1992, 33, 5485. Knapp et coll. J. Org. Chem. 2001, 66, 5822.

9. 9  Ribosyl-diazepanone Isono et coll. Heterocycles 1992, 34, 1147. SYNTHETIC APPROACHES DESCRIBED IN LITTERATURE

10. 10  Nucleosidyl-diazepanone Knapp et coll. Org. Lett. 2002, 4, 603. 1/ Oxidation 2/ NaN 3 1/ Ozonolysis 2/ Azide reduction 3/ Reductive amination SYNTHETIC APPROACHES DESCRIBED IN LITTERATURE

11. 11  Nucleosidyl-ribosyl-diazepanone Angew. Chem. Int. Ed. 2005, 44, 1854. SYNTHETIC APPROACHES DESCRIBED IN LITTERATURE

12. 12 STRUCTURE-ACTIVITY RELATIONSHIP : DEVELOPMENT OF A NEW PHARMACOPHORE MraY

13. 13 STRUCTURE-ACTIVITY RELATIONSHIP: DEVELOPMENT OF A NEW PHARMACOPHORE Target scaffold Pharmacophore structure Structure of natural molecules

14. 14 SCAFFOLD RETROSYNTHESIS N-ALKYLATION O-GLYCOSYLATION PEPTIDE COUPLING ⇒

15. 15 STRATEGIES TOWARDS SCAFFOLD SYNTHESIS GLYCOSYLATION N-ALKYLATION PEPTIDE COUPLING N-ALKYLATION PEPTIDE COUPLING GLYCOSYLATION

16. 16 ACCESS TO THE SCAFFOLD BY « CHAIN EXTENSION »  STEPS AND PRECURSORS ⇗ ⇘ STRATEGY 1 STRATEGY 2

17. 17 H+H+ R-OH acceptor X= Br, Cl, F, SR = OCOR, O 3 SR, OP(OR) 2, OPO 2 -OR'. R-OH ( acceptor) Substitution via activation of anomeric position Direct acid-catalyzed substitution  GLYCOSYLATION STEP X= Br, Cl, F ACCESS TO THE SCAFFOLD BY « CHAIN EXTENSION »

18. 18 ⇒ Tricky step : - for the formation of O-glycosidic derivatives, less known than that of N-glycosidic analogs - in the particular case of threonyl and serinyl acceptors ELABORATION OF O-GLYCOSYLATION STEP (1) Basic lability Acid lability

19. 19 ⇒ Success of the reaction and control of stereochemistry depending on three principal factors : - nature of glycosylation activator - nature of activation in anomeric position -nature of the C-2 substituent of the ribose controling  - or  -selective introduction of serine (anchimeric assistance) activator   ELABORATION OF O-GLYCOSYLATION STEP (2)

20. 20 R 1 = H R 2 = Bn Koenigs-Knorr method R 1 = Ac R 2 = Ac, Bz, Bn R 1, R 2 = H ACTIVATION IN ANOMERIC POSITION X = Cl SOCl 2, DCM, 0°C to RT X = Br TMS-Br, DCM, -40°C to RT. STRATEGY 1 DAST, THF, -30°C to RT, 1h. 95% (  = 99/1) STRATEGY 2

21. 21 1/ DOWEX 50W-H +, MeOH, 65°C, 17h. 2/ Ac 2 O, pyridine, RT, 2h. FORMATION OF PREFUNCTIONALIZED RIBOFURANOSIDES a : 1/ H 2 SO 4 (0,1N), 65°C, 4h; 2/ Me 2 C(OMe) 2, CSA, Me 2 CO, 50°C, 30 min.

22. 22 R= Boc, Cbz, FmocR= BocR= CbzR= Fmoc, Cbz SYNTHESIS OF L -SERINYL ACCEPTORS

23. 23  STRATEGY 1 (riboses not functionalized )  STRATEGIE 2 (prefunctionalized riboses) P1P1P1P1 P2P2P2P2YActivationX X = Br, Cl, F Z AgOTf, DCM, -15°C, 15 h.  : 100% 32Z 92 Boc0,369 Boc0,369 AcAcOAc TMSBr, DCM, -40°C à TA BrBzAcOBzBr BnAcOBnBr CMe 2 H N3N3N3N3 DAST, THF, -30°C à TA, 2h. F CMe 2 H N3N3N3N3F BnHOBnF SnCl 2 / AgClO 4Boc SnCl 2, AgClO 4, -15°C à TA, 48 à 72 h. 2,1564Fmoc1,7100 Boc1,244 SELECTION OF ACTIVATORS AND OPTIMIZATION OF GLYCOSYLATION CONDITIONS Ginisty M., Gravier-Pelletier C., Le Merrer Y., Tetrahedron: Asymmetry 2004, 15, 189-193. Ginisty M., Gravier-Pelletier C., Le Merrer Y., Tetrahedron: Asymmetry 2006, 17, 142-150.  Hg(CN) 2  AgClO 4  TMS-OTf  BF 3.OEt 2  AgOTf  SnCl 2 / AgClO 4 P3P3P3P3GlycosylationRatio  ) Yield (%)

24. 24 ⇗ ⇘ STRATEGY 1 STRATEGY 2 ACCESS TO THE SCAFFOLD BY « CHAIN EXTENSION »

25. 25 FUNCTIONALIZATION OF RIBOSYL MOIETY functionalization at C-2’ and C-3’ positions substitution of the 5’-OH function deprotection C-2’ and C-3’ protection substitution of the 5’-OH function P 1 = Ac, Bz, Bn P 2 = C(CH 3 ) 2 X R = Z 82% Boc -

26. 26 ⇗ ⇘ STRATEGY 1 STRATEGY 2 ACCESS TO THE SCAFFOLD BY « CHAIN EXTENSION »

27. 27 HCO 2 NH 4 Pd/C 10% MeOH, TA. AMINE DEPROTECTION  STRATEGY 1 H 2, Pd(OH) 2 / C, CH 3 CO 2 H, EtOH abs., RT, 24h. H 2, Pd(OH) 2 / C, CH 3 CO 2 H, EtOH abs., RT, 48h. H 2, Pd black, CH 3 CO 2 H, RT, 48h. R = Ac, Bz X  STRATEGY 2 Y= PhtN-, ZHN- X

28. 28  Powerful glycosylation conditions for the diastereoselective formation of serinyl-5’-amino-  -D- ribofuranoside derivatives ⇒ unfinished strategy because of difficult functionalization of the ribosyl moiety and amine deprotection. ⇒ unfinished strategy because of difficult functionalization of the ribosyl moiety and amine deprotection.  Perspective : ⇒ strategy 2 : glycosylation of 2,3-O-isopropyliden- D -ribofuranoside derivatives differently ⇒ strategy 2 : glycosylation of 2,3-O-isopropyliden- D -ribofuranoside derivatives differently N-protected, whose synthesis was already carried out.. N-protected, whose synthesis was already carried out.. ACCES TO THE SCAFFOLD BY « CHAIN EXTENSION » : CONCLUSION AND PERSPECTIVES Y = PhtN-, ZHN- X = activated group

29. 29 ACCESS TO THE SCAFFOLD BY DIRECT COUPLING GLYCOSYLATION N-ALKYLATION PEPTIDE COUPLING GLYCOSYLATION NH 2 PO amino-dihydroxy- butane HO Y

30. 30 CAG STRATEGY ACG STRATEGY ACCESS TO THE SCAFFOLD BY DIRECT COUPLING

31. 31 FORMATION OF N 1 -C 2 LINKAGE BY PEPTIDE COUPLING - FIRST STEP OF THE CAG STRATEGY -  SYNTHESIS OF AMINO-BUTANOL PRECURSORS 3,4-O-methylethyliden- L -threonine ethyl ester

32. 32 L -ascorbic acid 180° rotation introduction in C 3 position of the azido group introduction of the azido group in C 2 position P : protecting group R = OEt, H introduction of an electrophilic group in C 4 position Introduction of an electrophilic group in C 1 position   180° rotation FORMATION OF N 1 -C 2 LINKAGE BY PEPTIDE COUPLING - FIRST STEP OF THE CAG STRATEGY -  SYNTHESIS OF AMINO-BUTANOL PRECURSORS

33. 33  SYNTHESIS OF AMINO-BUTANOL PRECURSORS FORMATION OF N 1 -C 2 LINKAGE BY PEPTIDE COUPLING - FIRST STEP OF THE CAG STRATEGY -

34. 34 P1P1 P2P2 Coupling reagent FmocBn PyBOP 78 % HBTU 49 % ZtBuPyBOP 80 % BocBnPyBOP 99 % PEPTIDE COUPLING : SUBSTRATES AND PRODUCTS PEPTIDE COUPLING 100 % a : PyBOP, DIEA, CH 2 Cl 2 b : HBTU, DIEA, DMF a a a or b * 25 % of epimerization in C 2 position * COUPLING PRODUCTS YIELD

35. 35 CAG STRATEGY ACG STRATEGY ACCESS TO THE SCAFFOLD BY DIRECT COUPLING

36. 36 CAG STRATEGY ACG STRATEGY INTRAMOLECULAR PATHWAY INTERMOLECULAR PATHWAY ACCESS TO THE SCAFFOLD BY DIRECT COUPLING

37. 37 FORMATION OF N 4 -C 5 LINKAGE BY N-ALKYLATION INTRAMOLECULAR PATHWAY (CAG Strategy) INTERMOLECULAR PATHWAY (ACG Strategy)

38. 38 P1P1P1P1 P2P2P2P2 Opening conditions Opening conditionsYieldHH.HCl tBuOH, NaH, 100°C Cs 2 CO 3, DMF, 65°C Cs 2 CO 3, DMF, 110°C - BnTBDMS tBuOH, NaH, 100°C Cs 2 CO 3, DMF, 110°C MeOH, Et 3 N, 60°C Yb(OTf) 3, (Et 3 N), DCM, TA - tBu Bn Yb(OTf) 3, DCM, RT, 7 days 65 % FORMATION OF N 4 -C 5 LINKAGE BY N-ALKYLATION : NUCLEOPHILIC ATTACK OF AN ACTIVATED PRIMARY CARBON  INTERMOLECULAR PATHWAY P1P1P1P1 P2P2P2P2HH.HCl BnTBDMS tBu Bn

39. 39 piperidine, DMF, RT 65%  INTRAMOLECULAR PATHWAY deprotection X Acid conditions : Yb(OTf) 3, (Et 3 N), CH 2 Cl 2, RT, 6 days. LiNTf 2, CH 2 Cl 2, RT, 48h. Basic conditions : Cs 2 CO 3, DMF, RT to 110°C, 20h. tBuOH, NaH, 100°C. « Neutral » conditions : MeOH, RT, 15 days. iPrOH, RT, 18h. iPrOH, 50°C, 4 days. X epoxide opening FORMATION OF N 4 -C 5 LINKAGE BY N-ALKYLATION : NUCLEOPHILIC ATTACK OF AN ACTIVATED PRIMARY CARBON

40. 40 «  -stacking » interactions Primary carbon atom of epoxide ring Amine function involved in epoxide ring opening MOLECULAR MODELING OF AMINO-EPOXIDE «  -stacking » interactions

41. 41 N-ALKYLATION BY REDUCTIVE AMINATION reductive amination reductive amination functionalization of the diol moiety functionalization of the diol moiety peptide coupling  SYNTHESIS OF PRECURSORS INVOLVED IN REDUCTIVE AMINATION 1/ Aldehyde derivatives P= TBDPS

42. 42 Y AR 84 58 27 47 84 58 27 47 Y ZC l 84 95 Y TFA 76 83 FUNCTIONALIZATION OF THE DIOL MOIETY N-ALKYLATION BY REDUCTIVE AMINATION Aldehyde derivative R1R1R1R1Bn tBu Et reductive amination 2/ Serinyl derivatives reductive amination R = H R = Z R’ = H R’ = TBDPS Y N3 85 - 1/ Step 1 2/ NaBH 3 CN, EtOH abs., 18 h. 1/ Step 1 2/ NaBH 3 CN, EtOH abs., 18 h.  SYNTHESIS OF PRECURSORS INVOLVED IN REDUCTIVE AMINATION Step 1 : reductive amination DIEA, DCM, 4Ả molecular sieves, RT, 15h. Ti(OiPr) 4, DCM, RT, 3h Y TBDPS 94 90

43. 43 CAG STRATEGY ACG STRATEGY ACCESS TO THE SCAFFOLD BY DIRECT COUPLING

44. 44 R1R1R1R1 R2R2R2R2 R3R3R3R3 Azido reduction YieldDeprotectionYieldPeptideCouplingYieldBnZTBDPS H 2, Pd/C 10 %, MeOH, AcOEt, RT, 24h. DCC, HOBt, DCM, RT - EtHTBDPS 1 nBu 3 P, toluene, RT (3h) to reflux (5h) 2 TFA, THF, H 2 O, RT, 15h. 1 nBu 3 P, toluene, RT (3h) to reflux (5h) 2 TFA, THF, H 2 O, RT, 15h.- azido reduction FORMATION OF N 1 -C 2 LINKAGE BY PEPTIDE COUPLING deprotection peptide coupling deprotection AND 12 X X R1R1R1R1 R2R2R2R2 R3R3R3R3FormationBnZTBDPS Reductive amination EtHTBDPS tBu HH Epoxide ring opening tBu HTBDPS Reductive amination R 2 = H tBu HH HCO 2 NH 4, Pd/C, MeOH, RT, 20 min 51 TFA, DCM, RT, 20h. 100  DCC/ HOBt, DIEA, DCM/ DMF, RT, 24h.  PyBOP, DIEA, DCM, RT, 24h - tBu HTBDPS71100  EDCI/ HOBt, DIEA, DCM/ DMF, RT, 24h.  DCC, DIEA, DCM, RT. -

45. 45 « hydrophobic site »  -stacking interaction  -stacking interaction acid function involved in peptide coupling amine function involved in peptide coupling hydrophobic interactions MOLECULAR MODELING OF THE « COMPLEX AMINO-ACIDS » « COMPLEX AMINO-ACIDS » bis-O-silylated compound Mono-O-silylated compound  -stacking interaction

46. 46 X O-GLYCOSYLATION PEPTIDE COUPLING N-ALKYLATION ⇒ RING CLOSURE ? CONCLUSION AND PERSPECTIVES 1 5 2 3 4 6 7 ⇒ HOAt

47. 47 TOWARDS A NEW FAMILY OF POTENTIAL ANTIBIOTICS N- A lkylation of L -serine tert-butyl ester + Intramolecular peptide C oupling + O- G lycosylation of diazepanone heterocycle Ribosyl-diazepanone scaffold + R 1 / R 2 / R 3 Family of powerful MraY inhibitors ⇒ Biologic evaluation (Laboratoire des Enveloppes Bactériennes et Antibiotiques – Dr D. Blanot – Dr. D. Mengin-Lecreulx)