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As noted, eperezolid (2) was the first Upjohn oxazolidinone drug candidate, a piperazinyl monofluorophenyl-5-acetamidomethyl oxazolidinone with a hydroxyacetyl moiety on the piperazine distal nitrogen.45 While this is the identical group that had proved optimal in the 5'-indolinyl series (with U-97456), the sequence of events leading to the identification of eperezolid was somewhat more roundabout than may be obvious. At an early point in the piperazinyl project, the Hutchinson laboratory had installed the hydroxyacetyl group on the racemic, nonfluorinated piperazinyl phenyl oxazolidinone template. The resultant compound did not particularly distinguish itself from other analogs on the basis of its level of in vitro activity, and thus was not pursued further at that point. It was only later, after we had access to and exclusively worked with the optically active compounds (which provided a twofold increase in potency, as the 5-(R)-acetamidomethyl oxazolidinone enantiomers are inactive), and the monofluorophenyl template (which also elevated potency over the des-fluoro analog by twofold), that the beneficial properties of this moiety would become evident. One of the piperazinyl substitutions my laboratory had prepared was that having an acetoxyacetyl group on the distal nitrogen. Upon evaluation by our biology colleagues in the Ford laboratory, this compound particularly stood out, demonstrating excellent oral in vivo activity, with an ED50 considerably better than its in vitro MICs would have predicted. Working on a hypothesis that it was likely this acetoxyacetyl could be functioning as an oral ester prodrug (i.e., undergoing esterase-mediated cleavage of the acetate in vivo to release the true active component), we immediately targeted the synthesis of the presumed ester cleavage product. As surmised, this hydroxyacetyl piperazine fluorophenyl oxazolidinone (U-100592) proved to be of outstanding interest, and went on to become our first drug candidate, eperezolid.

The Zurenko laboratory demonstrated that eperezolid had excellent antibacterial potency against all of the MDR Gram-positive strains of interest, including vancomycin-resistant enterococci.53 Likewise, our colleagues in the Ford laboratory extensively evaluated this compound, and also determined that U-100592 had the properties we had been seeking for a drug candidate, with excellent oral efficacy in a number of models of sensitive and resistant Gram-positive infection.54 Oral eperezolid performed very well in a model of infection with vancomycin-resistant E. faecium (ED50 = 12.5 mgkg_ 1) in immunocompromised mice, was more active than vancomycin (dosed s.c.) against MRSA, and showed exceptional oral activity (ED50 = 2.0mgkg_ 1) against penicillin-and cephalosporin-resistant Streptococcus pneumoniae.54 Eperezolid dosed orally was very well tolerated in 30 day toxicology studies in the rat and dog; for both species, the no-observed-adverse-effect-level (NOAEL) was 25 mgkg _ 1 day _ 1.55 Upon the endorsement of eperezolid as our first drug candidate in May 1994, the early clinical development team charged with the planning and executing of Phase I clinical trials was formed, and chaired by Susan Speziale.

As our first oxazolidinone clinical candidate, eperezolid would come to be the compound with which a number of important MOA studies were conducted, in an effort to add to a more detailed understanding of where the oxazolidinones bind on the bacterial ribosome. While DuPont scientists reported that the oxazolidinones had a unique MOA, inhibiting an early event in bacterial protein synthesis,56 the details of the MOA were not fully understood. One particularly compelling aspect of the oxazolidinones was the difficulty with which resistant mutants could be raised in the laboratory. Using serial passages (20, over 7 weeks) of a sensitive S. aureus strain on a spiral gradient plater, Zurenko was able to raise a stable eperezolid-resistant mutant, which had utility in MOA studies.57 Keith Marotti, Jerry Buysse, Dean Shinabarger, and their colleagues Robert Murray, Alice Lin, Earline Melchior, Steve Swaney, Donna Dunyak, and William Demyan along with Alex Mankin (University of Chicago), determined that the resistance was associated with a mutation in the peptidyl transferase region of 23S ribosomal RNA.58 This was supported by their demonstration that eperezolid binds to the 50S ribosomal subunit at a site overlapping chloramphenicol and lincomycin.59'60 Some of the most revealing mechanistic work would come later from this group working together with Lisa Thomasco, Jerry Colca and Robert Gadwood.61 They prepared a radioactive photosensitive probe that was directly attached to eperezolid through its alcohol, to give 22, and then photolyzed this compound in growing S. aureus cells. They determined that 22 cross-linked to several components, most importantly tRNA and the universally conserved nucleotide at position A-2602 (E. coli numbering) in the peptidyl transferase center of 23S rRNA. This work extensively advanced the understanding of the location of the oxazolidinone binding site on the 50S bacterial ribosomal subunit, suggesting that it is located in the vicinity of the ribosomal peptidyl transferase center near the P site, where the peptide bond is constructed by the ribosome. Additional work conducted later included nuclear magnetic resonance (NMR) studies by Brian Stockman and Casey Zhou,62 who studied the binding of eperezolid to E. coli bacterial ribosomes, using 1H NMR transferred nuclear Overhauser enhancement, and demonstrated that it bound only to the 50S ribosomal subunit, not the 30S.

Within the chemistry team, upon having consolidated all of our medicinal chemistry efforts on the piperazinyl series, one of the concepts that came forth from one of many brainstorming group sessions (Hutchinson and Barbachyn) was to

8.13.10 The Identification of Linezolid (U-100766) and U-100480, a Potent Antimycobacterial also examine isosteres of the piperazine ring, notably the morpholine and thiomorpholine rings. Prosecution of this work in my laboratory led directly to the morpholinyl compound linezolid (1, U-100766, PNU-100766),45 and the interesting thiomorpholinyl oxazolidinone U-100480 (23, PNU-100480)63 was made in the Barbachyn laboratory. An interesting aspect, from a discovery time-line perspective, is that the very first samples of eperezolid and linezolid were synthesized within only 2 days of each other, in the spring of 1993.

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