1. Linezolid – a true small molecule antibiotic Alexei Pushechnikov, Ph.D. Disney group January 13, 2009

2. Outline Introduction Synthetic Approaches Activity Mode of Action Conclusions 2

3. Introduction 3 E.I. du Pont de Nemours & Co. Slee, A.M. et al 1987 DuPont’s SAR of the oxazolidinonepharmacophore: electron-withdrawing groups in the aryl para- position provided optimal activity importance of the N-aryl group additional substitutions at the aryl ortho- position or C-4 of the oxazolidinone ring had a detrimental or indifferent effect on the antibacterial activity C-5 (S)-configuration required for antibacterial activity optimal activity of a C-5 acetamidomethyl group Ford, C. W.; Zurenko, G. E.; Barbachyn, M. R. Cur. Drug Targets2001, 1, 181-199 Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010-2023 Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008,51, 1981-1990

4. Introduction 4 Barbachyn, M.R.; Brickner, S.J.; Hutchinson, D.K. WO95/07271 The Upjohn Company (Filed on April 1994) Brickner, S.J.; Hutchinson, D.K.; Barbachyn, M.R.; Manninen, P.R.; Ulanowicz, D.A.; Garmon, S.A.; Grega, K.C.; Hendges, S.K.; Toops, D.S.; Ford, C.W.; Zurenko, G.E. J. Med. Chem. 1996,39, 673

5. Introduction 5 Generalized testing scheme for oxazolidinones development Analogues In Vitro testing - Intrinsic activity - Activity against resistant strains - Spectrum of activity SAR In Vivo testing - Acceptable in vivo activity - Route of administration Pharmacokinetics/Toxicology - Useful blood levels - Frequency of dosing - Acceptable toxicity profile Further Biological Characterization - Mechanism of action - Pharmacodynamics - Tissue penetration Clinical Trials Registration Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010-2023

6. Introduction 6 Drug-like compounds (Lipinski’s rule) * Not more than 5 hydrogen bond donors : 1 * Not more than 10 hydrogen bond acceptors : 6 (8) * A molecular weight under 500 daltons : 337 * A partition coefficient logP less than 5 : 0.9 * Number of atoms from 20 to 70 : 44 * Number of rotatable bonds less than 10 : 5 * Polar surface area (PSA) less than 140 : 91 Computed Properties - Chem3D Properties Broker Linezolid (PNU-100766) Good solubility : 3.7 mg/mL in pH 7 phosphate buffer The oral bioavailability : 100% (rapid and complete absorption) The excretion : 20–30% of the dose found in the urine as the parent drug Has been approved by the FDA in 2000 under the trade name Zyvox Ford, C. W.; Zurenko, G. E.; Barbachyn, M. R. Cur. Drug Targets2001, 1, 181-199 Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010-2023 Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008,51, 1981-1990

7. Introduction 7

8. Introduction 8 Pharmacia&Upjohn’s revised SAR : electron-donating nitrogen atom well tolerated and often improves safety profile N-aryl group required for activity fluorination of phenyl ring often improves antibacterial activity/efficacy C-5 (S)-configuration necessary for antibacterial activity C-5 acetamidomethyl group essential for good activity

9. Synthetic approaches 9 Ford, C. W.; Zurenko, G. E.; Barbachyn, M. R. Cur. Drug Targets2001, 1, 181-199 Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010-2023 Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008,51, 1981-1990

10. Synthetic approaches 10 Ford, C. W.; Zurenko, G. E.; Barbachyn, M. R. Cur. Drug Targets2001, 1, 181-199 Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010-2023 Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008,51, 1981-1990

11. Synthetic approaches 11 Process scale synthesis Ford, C. W.; Zurenko, G. E.; Barbachyn, M. R. Cur. Drug Targets2001, 1, 181-199 Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010-2023 Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008,51, 1981-1990

12. Synthetic approaches 12 Lohray, B. B.; Baskaran, S.; Rao, B. S.; Reddy, B. Y.; Rao, I. N. Tetrahedron Lett. 1999, 40, 4855-4856

13. Activity 13 In vitro activities of linezolid and vancomycin, Minimum Inhibitory Concentration (mg/L) Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010-2023

14. Activity 14 In vivo activities of linezolid and vancomycin, Effective Dose (mg/kg) Linezolid – orally Vancomycin - subcutaneously Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010-2023

15. Activity 15 Activities against Mycobacterium tuberculosis In Vitro In Vivo Treatment was started 1 day after the mice received 7x106 viable mycobacteria. Cynamon,M.H.; Klemens,S.P.; Sharpe,C.A.; Chase,S. Antimicrob. Agents Chemother. 1999, 43,1189-1191

17. Despite the observed in vitro activity, linezolid was ineffective against the Moraxellacatarrhalisand H. influenzae, even at concentrations above the MIC

18. However, in the absence of cell membranes and cell walls the oxazolidinones were very active in inhibiting E. coli protein synthesis

19. Making the E. coli transmembrane pump nonfunctional made whole E. coli cells sensitive to linezolid both in vitro and in vivo

21. Activity 18 Linezolid behaves as a cidal drug in vivo although it is clearly static for staphylococci and enterococci in the test tube Linezolid is generally considered to be well tolerated in humans. Most common side effects in the clinical trials were (percent incidence) : diarrhea (2.8-11%) nausea (3.4-9.6%) headache (0.5-11.3%) With longer term usage of linezolid (>2 weeks), there is an association of reversible myelosuppression (anemia, thrombocytopenia, leukopenia, or pancytopenia) Linezolid is a weak, reversible, and nonselective inhibitor of Monoamine Oxidase. A risk of serotonin toxicity is anticipated with linezolid. Avoidance of large quantities of food with a high tyramine level (aged cheese, beer, or red wine) along with administering linezolid is suggested Lawrence, K.R.; Adra, M.; Gillman, P.K. Clin. Infect. Dis. 2006, 42, 1578-1583 Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008,51, 1981-1990

22. Mode of Action 19 Swaney,S.M.; Aoki,H.; Ganoza,M.C.; Shinabarger,D.L. Antimicrob. Agents Chemother. 1998, 42, 3251-3255 Clemett,D.; Markham,A. Drugs 2000, 59, 815-827

23. Mode of Action 20 [14C]Eperezolid binding to E. coli ribosomes. (A) Total ribosomes; (B) 50S subunits; (C) 30S subunits. ▓, total binding; █, specific binding. Lin,A.H.; Murray,R.W.; Vidmar,T.J.; Marotti,K.R. Antimicrob. Agents Chemother. 1997, 41, 2127-2131

24. Mode of Action 21 Binding to ribosomes. Eperezolid and linezolid bind to the 50S ribosomal subunit with Kd ~20 M Competition by various concentrations of unlabelled antibiotics. (A) [14C]eperezolid binding; (B) [14C]chloramphenicol binding. ●, eperezolid; ■, linezolid; ▲, chloramphenicol; ▼, lincomycin. Lin,A.H.; Murray,R.W.; Vidmar,T.J.; Marotti,K.R. Antimicrob. Agents Chemother. 1997, 41, 2127-2131

26. The resulting distorted site may prevent the correct positioning of the 30S initiation complex from forming the 70S initiation complex and hence inhibit translation initiation.Lincomycin Chloramphenicol Lin,A.H.; Murray,R.W.; Vidmar,T.J.; Marotti,K.R. Antimicrob. Agents Chemother. 1997, 41, 2127-2131 Thompson, J.; O’Connor, M.; Mills, J.A.; Dahlberg, A.E. J. Mol. Biol. 2002, 322, 273–279

28. E. coli linezolid resistance mutations are shown by arrows (thickness proportional to the level of linezolid resistance for each mutation).

29. Marked positions of nucleotide substitutions that confer linezolid resistance in H. halobium(boxed) and in S. aureusand E. faecalis(circled).Kloss,P.; Xiong,L.; Shinabarger,D.L.; Mankin, A.S. J. Mol. Biol. 1999, 294, 93-101 Xiong,L.; Kloss,P.; Douthwaite, S.; Andersen, N.M.; Swaney,S.; Shinabarger,D.L.; Mankin, A.S. J. Bacteriol. 2000, 182, 5325-5331

30. Mode of Action 24 A B Orientation of Linezolid at the Peptidyltransferase Center of the Ribosome. Model for the binding position of linezolid (Lnz, red) with respect to nucleotides (blue) at the E. coli PTC. Relative position of linezolid (red) compared to chloramphenicol (Cam, green). PTC nucleotides are shown as green surface representation. Leach, K.L., Swaney, S.M., Colca, J.R., McDonald, W.G., Blinn, J.R., Thomasco, L.M., Gadwood, R.C., Shinabarger, D., Xiong, L., Mankin, A.S. Mol. Cell2007, 26, 393-402 Wilson, D.N.; Nierhaus, K.H. Mol. Cell2007, 26, 460-462

31. Mode of Action 25 Superposition of the structure of linezolid (cyan) with the structures of A-site (orange) and P-site (green) substrate analogues bound to H50S Linezolid (cyan) and CCA-Phe (gold) binding to H50S. Linezolid molecule occupied the A-site and CCA-Phe occupied the P-site (PDB code 3CPW) Ippolito, J.A.; Kanyo, Z.F.; Wang, D.; Franceschi, F.J.; Moore, P.B.; Steitz, T.A.; Duffy, E.M. J. Med. Chem. 2008, 51, 3353-3356

32. Mode of Action 26 The binding site of oxazolidinones. Linezolid bound to the Deinococcusradiodurans50S ribosomal subunit. Oxazolidinones induce an A/O state recognized by LepA. Relative position of linezolid (red), P-tRNA (cyan), A-tRNA (pale green), LepA (maroon density and ribbon) and A/L-tRNA (blue). Wilson, D.N.; Schluenzen, F.; Harms, J.M.; Starosta, A.L.; Connell, S.R.; Fucini, P. Proc. Natl. Acad. Sci. USA 2008,105, 4673-4678

33. Mode of Action 27 Events during normal translation (A–D), compared with the effect of the linezolid (red) during translation (E–H). Wilson, D.N.; Schluenzen, F.; Harms, J.M.; Starosta, A.L.; Connell, S.R.; Fucini, P. Proc. Natl. Acad. Sci. USA 2008,105, 4673-4678

34. Conclusions 28 Linezolid has been approved in the U.S. for the treatment of nosocomial and communityacquired pneumonia caused by S. aureus(methicillin-susceptible or MRSA) or S. pneumoniae(penicillin-susceptible or multidrug-resistant strains) and vancomycin-resistant E. faecium(including concurrent bacteremias) for use in children and newborns against Gram-positive infections for treatment of complicated skin and skin-structure infections including those due to MRSA (including Gram-positive bacterial diabetic foot infections (MRSA) without concomitant osteomyelitis) the only approved agent for treatment of hospital-acquired MDR S.pneumoniaeinfectionsand is the first and only oral drug approved for the treatment of VRE infections. Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008,51, 1981-1990

35. Conclusions 29 Early 1993 - first synthesis April 1995 - entered phase I trials 1996 - initiated phase II studies January of 1998 - phase III trials began April 18, 2000 - approved by the FDA January of 2008 - has been used in an estimated 3 million patients Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008,51, 1981-1990