Antibacterial oxazolidinones possessing a novel C-5 side chain. (5R)-trans-3-[3-fluoro-4-(1-oxotetrahydrothiopyran-4-yl)phenyl] -2-oxooxazolidine-5-carboxylic acid amide (PF-00422602), a new lead compound

Article abstract of DOI:10.1021/jm070708p

Oxazolidinones possessing a C-5 carboxamide functionality (reverse amides) represent a new series of compounds that block bacterial protein synthesis. These reverse amides also exhibited less potency against monoamine oxidase (MAO) enzymes and thus posses

Full text of DOI:10.1021/jm070708p

5886
J. Med. Chem. 2007, 50, 5886-5889
Antibacterial Oxazolidinones Possessing a Novel
C-5 Side Chain. (5R)-trans-3-[3-Fluoro-4-
(1-oxotetrahydrothiopyran-4-yl)phenyl]-2-
oxooxazolidine-5-carboxylic Acid Amide
(PF-00422602), a New Lead Compound
Toni-Jo Poel, Richard C. Thomas, Wade J. Adams,
Paul A. Aristoff, Michael R. Barbachyn, Frederick E. Boyer,
Joan Brieland, Roger Brideau, Joanne Brodfuehrer,
Alan P. Brown, Allison L. Choy, Michael Dermyer,
Michael Dority, Charles W. Ford, Robert C. Gadwood,
Debra Hanna, Cai Hongliang, Michael D. Huband,
Christopher Huber, Rose Kelly, Ji-Young Kim,
Joseph P. Martin, Jr., Paul J. Pagano, Daniel Ross,
Laura Skerlos, Mark C. Sulavik, Tong Zhu,
Figure 1. Structures.
pneumonia caused by S. aureus or Streptococcus pneumoniae,
and VRE infections.
Gary E. Zurenko, and J. V. N. Vara Prasad*
Generally, these antibacterial oxazolidinones possess a C-5
aminomethyl functionalized as the amide, urethane, urea, or
thiourea (2).4-6 On the basis of the structures of known revers-
ible and competitive inhibitors (e.g., toloxatone 3, befloxatone
4) of monoamine oxidase A (MAO-A),7-9 it is not surprising
that linezolid has safety considerations in its label regarding
MAO inhibition. In the literature, linezolid is shown to be a
weak reversible inhibitor of MAO. In the literature, other side
chains such as -CH2-heterocycle (5) are reported to address
the MAO inhibition issue.10 Herein we report a novel C-5 side
chain for these oxazolidinones, namely, -CONH2 [termed
“reverse amide” to differentiate from the orginal methylamide
(-CH2NHCOR) functionality]. Analogues bearing a C-5 reverse
amide are of lower molecular weight and easily accessible via
total synthesis. This structural modification affords analogues
with the reduction in MAO inhibition and a reduction in
myelotoxicity, which can be a significant side effect with
linezolid when used over the long term (>21 days).11
As shown in Scheme 1, the reverse amides described herein
were synthesized from the corresponding anilines (6) or car-
bamates (7) by treating with n-butyl (R)-glycidate in the presence
of a Lewis acid or potassium (R)-glycidate.12 The intermediates
8 and 9 thus obtained were further manipulated to afford the
final products 10. The carbamate used to prepare 11 was
described in the synthesis of 1.13 Compound 13 was prepared
by coupling the 5-(R)-3-[4-trimethylstannyl-3-fluorophenyl]-2-
oxo-5-oxazolidinonecarboxamide with 3,6-dihyro-2H-pyran-4-
yltrifluoromethanesulfonic acid ester in the presence of palla-
dium,14,15 followed by reduction of the double bond. The amine
used to prepare 14 was synthesized in a stereodivergent fashion
as detailed previously.16 A similar procedure was adopted to
synthesize the aniline precursor to difluoro analogue 15.
These oxazolidinone analogues were tested against a panel
of Gram-positive and fastidious Gram-negative bacteria. Mini-
mum inhibitory concentration (MIC, µg/mL) values were
determined by microbroth technology.17 Selected analogues were
also tested against levofloxacin and oxacillin resistant S. aureus
(LORSA), levofloxacin, pencillin resistant S. pneumoniae
(LPRSP), and vancomycin resistant Enterococcus faecalis and
Enterococcus faecium (VREF) strains for the determination of
MIC90. The Escherichia coli in vitro transcription and translation
assay (TnT), providing an assessment of protein synthesis
inhibition in a cell-free format, was performed in 96-well
microtiter plates using a luciferase reporter system.18,19 MAO
inhibition was measured using recombinant enzymes and a
Pfizer Global Research and DeVelopment, Michigan Laboratories,
2800 Plymouth Road, Ann Arbor, Michigan 48105
ReceiVed June 18, 2007
Abstract: Oxazolidinones possessing a C-5 carboxamide functionality
(reverse amides) represent a new series of compounds that block
bacterial protein synthesis. These reverse amides also exhibited less
potency against monoamine oxidase (MAO) enzymes and thus possess
less potential for the side effects associated with MAO inhibition. The
title compound (14) showed reduced in vivo myelotoxicity compared
to linezolid in a 14-day safety study in rats, potent in vivo efficacy in
murine systemic infection models, and excellent pharmacokinetic
properties.
Oxazolidinones, as exemplified by (S)-N-((3-fluoro-4-mor-
pholinophenyl)-2-oxaoxazolidin-5-yl)methyl)acetamide (linezo-
lid, 1), are a novel, completely synthetic class of antibacterial
agents that possess potent activity against Gram-positive
bacteria. Herein is reported a series of oxazolidinones bearing
a novel C-5 side chain (reverse amide) replacement for the
typical substituted aminomethyl side chain that provides a safety
advantage over earlier oxazolidinones. Structure-activity studies
in this novel series eventually led to the discovery of 14 (PF-
00422602), which possesses good antibacterial efficacy in
animal models and good pharmacokinetic properties in multiple
species.
The continual emergence of multidrug-resistant Gram-positive
bacterial pathogens including methicillin-resistant Staphylo-
coccus aureus (MRSAa), Staphylococcus epidermidis (MRSE),
and vancomycin-resistant enterococci (VRE) creates a need for
novel antimicrobial agents.1,2 Linezolid 1, the first member of
a new class of synthetic antibacterials (oxazolidinone family)
approved by the FDA, possesses Gram-positive activity, includ-
ing activity versus the above-mentioned resistant strains.3 This
drug was proven to be effective in treating skin and soft tissue
infections caused by S. aureus or S. pyogenes, nosocomial
* To whom correspondence should be addressed. Phone: 734-622-2866.
Fax: 734-622-2265. E-mail: Josyulav@gmail.com.
a Abbreviations: MAO, monoamine oxidase; MRSA, methicillin-resistant
Staphylococcus aureus; MRSE, methicillin-resistant Staphylococcus epi-
dermidis; VRE, vancomycin-resistant enterococci; FDA, Food and Drug
Administration; MIC, minimum inhibitory concentration; LORSA, levof-
loxacin and oxacillin resistant Staphylococcus aureus; LPRSP, levofloxacin
and pencillin resistant Streptococcus pneumoniae; VREF, vancomycin
resistant Enterococcus faecalis; TnT, transcription and translation assay;
PEG, polyethylene glycol; PK, pharmacokinetic; CYP, cytochrome P;
NOAEL, no adverse effect level.
10.1021/jm070708p CCC: $37.00 © 2007 American Chemical Society
Published on Web 11/08/2007

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 5887
Scheme 1a
a Reagents and conditions: (i) n-butyllithium, -78 °C, potassium (R)-glycidate; (ii) oxalylchloride, room temp; (iii) ammonia, 0-25 °C; (iv) n-butyl
(R)-glycidate, lithium triflate, 60 °C; (v) 1,1′-carbonyldimidazole, room temp; (vi) ammonia, room temp.
Table 1. Enzymatic Activity (IC50), Minimum Inhibitory Concentrations (MIC, µg/mL), and Human Monoamine Oxidase Activity (IC50) of
Oxazolidinones (MIC Values in Bold Are MIC90 Values for the Denoted Organism
a Staphylococcus aureus UC-76 SA-1. b Streptococcus pneumoniae SV1 SP-3. c Enterococcus faecalis MGH-2 EF-1. d Enterococcus faecium.
chromogenic substrate as described in the literature.20 The
activities of the oxazolidinone analogues are summarized in
Table 1, and the values for linezolid are included for comparison.
Reverse amides 13-17 demonstrated significant inhibition
of TnT activity and had potent Gram-positive antibacterial
activities. However, 11 and 12 are less active compared to
linezolid (Table 1). In general, the activities of the reverse
amides were somewhat similar to or slightly less than those of
the corresponding C-5 acetamide analogues. Among the reverse
amide analogues, oxazolidinones possessing difluorophenyl or
monofluorophenyl groups showed better antibacterial activities
than their des-fluoro congeners.21 Against the fastidious Gram-
negative strains Haemophilus influenzae and Moraxella
catarrhalis, these reverse amides (11-17) showed MIC values
of >8 µg/mL. Reverse amides 12 and 14-16 were also tested
for their MIC90 values; the strains include drug-resistant S.
aureus, S. pneumoniae, E. faecalis, and E. faecium (VREF) as
described above, and results are shown in bold in Table 1. More
interestingly, reverse amide analogues (11 and 14-17) showed
reduced human MAO-A inhibition compared to linezolid. This
suggests that these compounds have much less potential than
linezolid to alter neurotransmitter homeostasis and/or cause
adverse drug interactions with compounds whose clearance is
MAO-mediated (e.g., sympathomimetic amines). Time kill
kinetics indicate that oxazolidinone 14 is bacteriostatic versus
staphylococci and enterococci at 48 h following continuous drug
exposure at (2-16)× the MIC. In contrast, continuous exposure
of 14 at (2-16)× the MIC results in eradication of S.
pneumoniae at 48 h. These results are similar to those of
linezolid. Selected carboxamide analogues were tested for in
vivo efficacy in mouse systemic S. aureus and S. pyogenes
infection models via oral dosing. These analogues showed

5888 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Letters
Table 2. Antibacterial Efficacies (PD50) of Selected Reverse Amides in
ceived vehicle or 14 orally (gavage) at 200, 600, or 900 (mg/
kg)/day for 14 days. All animals at 900 (mg/kg)/day were
euthanized early in moribund condition. The 600 (mg/kg)/day
dose produced toxicity and hematology and bone marrow
changes, and 200 (mg/kg)/day was the no-adverse effect level
(NOAEL). Hematology changes at 600 (mg/kg)/day included
8% increases in red blood cells, hemoglobin, and hematocrit
(likely hemoconcentration) and decreases in reticulocytes (62%).
The bone marrow myeloid/erythroid (M:E) ratio was increased
4.5-fold at 600 (mg/kg)/day. Compound 14 plasma AUC(0-
24) values were 223 and 619 µg‚h/mL at 200 (NOAEL) and
600 (mg/kg)/day, respectively. These results were compared to
a 14-day oral toxicology study of linezolid in rats. Female rats
were dosed by gavage with linezolid at 40, 200, or 1000 (mg/
kg)/day. Lethality occurred at 1000 (mg/kg)/day, and 40 (mg/
kg)/day was the NOAEL. Hematology and bone marrow
changes at 200 (mg/kg)/day included decreases of 9-12% in
red blood cells, hemoglobin, and hematocrit, a 97% decrease
in reticulocytes, and a 8.4-fold increase in M:E ratio. Linezolid
AUC(0-24) values were 105 and 666 µg‚h/mL at 40 (NOAEL)
and 200 (mg/kg)/day, respectively. On the basis of these in vivo
data, it was concluded that 14 was at least 2× less myelotoxic
than linezolid because of the higher plasma drug levels at the
NOAEL and decreased hematology and bone marrow changes
at higher exposures.
a Mouse Systemic Infection Modela
MIC
(µg/mL)
PD50
compd
strain
(mg/kg)b
1
1
1
S. aureus (UC-76)
4
4
4
4
4
2
4
4
2
2
4
3.1-8.0
6.25
5
8.22
6.7
1.25
1.7
<5
1.1
S. aureus (SA-1417, MRSA)
S. pyogenes (C-203)
S. aureus (UC-76)
S. aureus (UC-76)
S. aureus (UC-76)
S. aureus (SA-1417, MRSA)
S. pyogenes (C-203)
S. aureus (UC-76)
S. aureus (UC-76)
S. aureus (UC-76)
11
12
14
14
14
15
16
17
2.1
15
a Female CD-1 mice were challenged with 100 × LD50 of bacterial
inoculum by intraperitoneal injection, and therapy was initiated immediately
by administering the drug by gavage at 2× increasing doses. Mortality of
mice was monitored after 7 days of the infection. b PD50 is the dose at which
there is 50% mortality.
Table 3. PK Properties of Selected Oxazolidinones in Sprague-Dawley
Male Ratsa
parameter
14
15
16
C
max (µg/mL), po
15.9
109
15.6
6.2
5.8
83
4.69
15.40
12.8
0.83
2.24
65
1.88
8.62
12.9
1.11
1.58
40
AUC (µg‚h/mL), po
CL ((mL/min)/kg), iv
V
dss (L/kg), iv
1/2 (h), po
F (%)
t
In summary, we report the synthesis and in vitro and in vivo
activity of novel C-5 carboxamide oxazolidinones. These
analogues are low in molecular weight and easy to synthesize,
as demonstrated by the scale-up of reverse amide 14 in
multigram quantities. This subclass of oxazolidinones showed
antibacterial activities similar to those of linezolid but with a
decreased monoamine oxidase inhibitory potency. These com-
pounds also showed excellent PK properties and were low risk
for drug-drug interactions. Results of in vitro time kill studies
demonstrate that, like linezolid, 14 is bacteriostatic for staphy-
lococci and enterococci but is bactericidal for streptococci.
Importantly, 14 also showed a reduction in myelotoxicity
compared to linezolid.
a For po: dose of 20 mg/kg, formulation of 5% PEG 2000/95% methyl
cellulose (0.5%). For iv, dose of 10 mg/kg, formulation of 5% DMA/95%
50 mM Tris base.
Table 4. PK Properties of Compound 14 in Mouse and Dogsa
parameter
mouse
dog
C
max (µg/mL), po
12.3
48.3
25.2
3.3
17.9
76.2
5.7
0.7
6.2
AUC (µg*hr/mL), po
CL ((mL/min)/kg), iv
V
dss (L/kg), iv
1/2 (h), po
F (%)
t
1.5
64
a For mouse: po dose of 20 mg/kg, iv dose of 10 mg/kg. For dog: po
dose of 40 mg/kg, formulation of 5% PEG 2000/95% methyl cellulose
(0.5%); iv dose of 4 mg/kg, formulation of 5% DMA/95% 50 mM Tris
base.
Supporting Information Available: Experimental procedures
and characterization data for all compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
efficacies comparable to linezolid (Table 2), demonstrating their
potential as antibacterial agents.
References
Selected oxazolidinones (14-16) were evaluated for their
pharmacokinetic (PK) properties by dosing Sprague-Dawley rats
via intravenous and oral administration. The results are shown
in Table 3. Since 14 showed better PK properties compared to
15 and 16, it was evaluated further in mouse and dog, and the
results are shown in Table 4. Oxazolidinone 14 showed low
plasma clearance and good oral bioavailability in rats and dogs.
Pooled human liver microsomes were incubated with CYP
isozymes, selective markers of metabolism, in the presence of
varying concentrations of the drug to determine the potential
of the drug to inhibit their metabolic activity.22 Compound 14
also had low potential for drug-drug interactions, as this
compound showed very little inhibition (IC50 > 900 µM) against
CYP 3A4, CYP 2C9, CYP 2C19, CYP 1A2, and CYP 2D6.
Because of the excellent profile of oxazolidinone 14, this
compound was evaluated in an exploratory toxicity study in
rats to evaluate myelotoxic potential. Toxicology studies of
linezolid were previously performed in rats and myelotoxicity
was produced, providing an animal model of this clinically
relevant toxicity. Female Sprague-Dawley rats (5/group) re-
(1) Levy, S. B.; Marshall, B. Antibacterial resistance worldwide: causes,
challenges and responses. Nat. Med. 2004, 10, S122-S129.
(2) Payne, D. J.; Gwynn, M. N.; Holmes, D. J.; Pompliano, D. L. Drugs
for bad bugs: confronting the challenges of antibacterial discovery.
Nat. ReV. Drug DiscoVery 2006, 1-12.
(3) Moellering, R. C., Jr. Linezolid: the first oxazolidinone antimicrobial.
Drugs Drug Ther. 2003, 138, 135-142.
(4) Barbachyn, M. R.; Ford, C. W. Oxazolidinone structure-activity
relationships leading to linezolid. Angew. Chem., Int. Ed. 2003, 42,
2010-2023.
(5) Hutchinson, D. K. Recent advances in oxazolidinone antibacterial
agent research. Expert Opin. Ther. Pat. 2004, 14, 1309-1328.
(6) Hutchinson, D. K. Oxazolidinone antibacterial agents: a critical
review. Curr. Top. Med. Chem. 2003, 3, 1021-1042.
(7) Mai, A.; Artico, M.; Valente, S.; Sbardella, G.; Turini, P.; Befani,
O.; Vedova, L. D.; Agostinelli, E. 3-(1H-Pyrrol-2-yl)-2-oxazolidi-
nones as novel monoamine oxidase type A inhibitors. Med. Chem.
2005, 1, 117-124 and references therein.
(8) Keung, W. M. Monoamine oxidase inhibitors. Expert Opin. Ther.
Pat. 2002, 12, 1813-1829.
(9) Wouters, J.; Moureau, F.; Evrard, G.; Kownig, J. U.-J.; Jegham, S.;
George, P.; Durant, F. A reversible monoamine oxidase A inhibitor,
befloxatone: structural approach of its mechanism of action. Bioorg.
Med. Chem. 1999, 7, 1683-1693.

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 5889
(10) Reck, F.; Zhou, F.; Girardot, M.; Kern, G.; Eyermann, C. J.; Hales,
N. J.; Ramsay, R. R.; Gravestock, M. B. Identification of 4-substituted
1,2,3-triazoles as novel oxazolidinone antibacterial agents with
reduced activity against monoamine oxidase A. J. Med. Chem. 2005,
48, 499-506.
(17) Methods for Antimicrobial Susceptibility Testing of Anaerobic
Bacteria, 6th ed.; NCCLS Document M11-A6; Clinical and Labora-
tory Standards Institute: Wayne, PA; Volume 24, Issue 2.
(18) Murray, R. W.; Melchoir, E. P.; Hagadorn, J. C.; Marotti, K. R.
Staphylococcus aureus cell extract transcription-translation assay:
firefly luciferase reporter system for evaluating protein translation
inhibitors. Antimicrob. Agents Chemother. 2001, 45, 1900-1904.
(19) Zubay, G. In vitro synthesis of protein in microbial systems. Annu.
ReV. Genet. 1973, 7, 267-287.
(20) Flaherty, P.; Castignoli, K.; Wang, X. Y.; Castignoli, N., Jr. Synthesis
and selective monoamide oxidase B-inhibiting properties of 1-methyl-
1,23,6-tetrahydropyrid-4-yl carbamate derivatives: potential prodrugs
of (R)- and (S)-nordeprenyl. J. Med. Chem. 1996, 39, 4756-4761.
(21) Data not shown.
(22) Incubations were performed in duplicate with 0.03-0.2 mg/mL
human liver microsomes, substrate (30 µM phenacetin for CYP 1A2,
7.5 µM diclofenac for CYP 2C9, 50 µM phenacetin for CYP 2C19,
10 µM dextromethorphan for CYP 2D6, 50 µM phenacetin, 50 µM
midazolam, 1.5 µM felodipine for CYP 3A4), 1 mM NADPH, and
in the presence of a positive control (furafylline for CYP1A2,
sulfaphenazole for CYP 2C9, ticlopidine for CYP 2C19, quinidine
for CYP 2D6, ketoneconazole and midazolam for CYP 3A4) or drug
in 50 mM potassium phosphate buffer at pH 7.4. The concentrations
of drug were 0, 1, 3, 7.5, 15, 40, 100, 500, and 1000 µM. The
reactions were quenched with 500 µL of 100 ng/mL acetonitrile (ice-
cold) after 7-30 min. The samples were analyzed by LC/MS after
centrifugation.
(11) Kutler, D. J.; Trillotson. Hematologic effects of antimicrobials: focus
on the oxazolidinone linezolid. Pharmacotheraphy 2001, 21, 1010-
1013.
(12) Roush, W. R.; Brown, B. B. Enantioselective synthesis of 2-alkyl-
5-methylene-1,3-dioxolan-4-ones and exo-selective Diels-Alder
reactions with cyclopentadiene. J. Org. Chem. 1992, 57, 3380-
3387.
(13) 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. Synthesis and antibac-
terial activity of U-100592 and U-100766, two oxazolidinone
antibacterial agents for the potential treatment of multidrug-resistant
Gram-positive bacterial infections. J. Med. Chem. 1996, 39, 673-
679.
(14) Farina, V.; Krishnan, B. Large rate accelerations in the Stille reaction
with tris-2-furylphosphine and triphenylarsine as palladium ligands:
mechanistic and synthetic implications. J. Am. Chem. Soc. 1991, 113,
9585-9595 and references therein.
(15) Stille, J. K. The palladium-catalyzed cross-coupling reactions of
organotin reagents with organic electrophiles. Angew. Chem., Int.
Ed. Engl. 1986, 25, 508-524.
(16) Gage, J. R.; Perrault, W. R.; Poel, T.-J.; Thomas, R. C. Stereo-
divergent synthesis of sulfoxide-containing oxazolidinone antibiotics.
Tetrahedron Lett. 2000, 41, 4301-4305.
JM070708P