Synthesis of novel oxazolidinone antimicrobial agents

Article abstract of DOI:10.1016/j.bmc.2007.11.040

The oxazolidinone class of antimicrobials represents a promising advance in the fight against resistant Gram-positive bacterial infections. Four novel oxazolidinone antimicrobial compounds, each containing a benzodioxin ring system, have been prepared. The general synthesis of each compound begins with the construction of a benzodioxin ring system containing a nitro substituent that ultimately becomes the nitrogen of the oxazolidinone ring. Three of the compounds utilize high yielding 'click chemistry' in their final step. The antimicrobial activities of the new oxazolidinones have been measured and the MIC against Staphylococcus aureus for one of the antimicrobials was determined to be 2-3 μg/mL, which is comparable to the well-known oxazolidinone, linezolid.

Full text of DOI:10.1016/j.bmc.2007.11.040

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2651–2656
Synthesis of novel oxazolidinone antimicrobial agents
David C. Ebner,a Jeffrey C. Culhane,a Tyler N. Winkelman,a Mitchell D. Haustein,b
Jayna L. Dittyb and J. Thomas Ippolitia,*
aDepartment of Chemistry, University of St. Thomas, St. Paul, MN 55105, USA
bDepartment of Biology, University of St. Thomas, St. Paul, MN 55105, USA
Received 16 October 2007; revised 9 November 2007; accepted 13 November 2007
Available online 19 November 2007
Abstract—The oxazolidinone class of antimicrobials represents a promising advance in the fight against resistant Gram-positive bac-
terial infections. Four novel oxazolidinone antimicrobial compounds, each containing a benzodioxin ring system, have been pre-
pared. The general synthesis of each compound begins with the construction of a benzodioxin ring system containing a nitro
substituent that ultimately becomes the nitrogen of the oxazolidinone ring. Three of the compounds utilize high yielding ‘click chem-
istry’ in their final step. The antimicrobial activities of the new oxazolidinones have been measured and the MIC against Staphylo-
coccus aureus for one of the antimicrobials was determined to be 2–3 lg/mL, which is comparable to the well-known oxazolidinone,
linezolid.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
is blocked by Zyvox’s interaction with the 23S rRNA of
the 50S subunit.
Multi-drug resistance among Gram-positive bacterial
pathogens represents a serious challenge for health prac-
titioners in treating both nosocomial and community-
acquired infections.1 Several reports have shown that
antibacterial resistance is increasing at a rapid rate in
both community and hospital settings.2,3 This continued
rise in antibacterial resistance has called for antimicro-
bial agents to be developed that are effective against
resistant strains of bacteria. In April 2000, the Food
and Drug Administration approved the release of lin-
ezolid, 1, trademarked Zyvox (Fig. 1).4
Brickner et al. described the syntheses of Zyvox, 1, and
an analog, 2, in 1996.8 The common structural elements
in these analogs are the ortho electron donating groups,
a fluorine and an amino nitrogen on the aryl ring con-
nected to the nitrogen of the oxazolidinone ring, as well
as the stereochemistry at carbon 5 of the oxazolidinone
ring. It has also been reported that an acetamide group
connected to carbon 5 of the oxazolidinone ring is
required for high activity.9
Since the introduction of Zyvox there has been much re-
search directed toward the synthesis of oxazolidinone
antimicrobials.10,11 Wright has recently reviewed the dis-
covery of Zyvox and syntheses of new members of this
class.12
Zyvox, an antimicrobial oxazolidinone first marketed by
the Pharmacia Corporation, became the first approved
drug that was effective against antibacterial resistant
bacteria.5 Zyvox prevents initiation of protein synthesis
via a novel mechanism in Gram-positive bacteria includ-
ing Enterococcus strains, Staphylococcus strains, and
Streptococcus pneumoniae.6,7 Specifically, the fMet-
tRNA necessary for bacterial protein synthesis initiation
To explore the ability of conjugated oxygens as electron
donors to affect the antibacterial activity versus the
nitrogen and fluorine atoms of linezolid, we have de-
signed an oxazolidinone containing a benzodioxin moi-
ety. Others have been successful in attaching fused rings
to the phenyl group to yield molecules with desirable
activity. For example, oxazolidinones containing fused
tricyclic ring systems such as 3 and 4 have been synthe-
sized (Fig. 2).13 These compounds have MIC values less
than 0.125–1 lg/mL.
Keywords: Oxazolidinone; Benzodioxin; Triazole; Antimicrobial
agents.
*
Corresponding author. Tel.: +1 651 962 5582.; e-mail: jtippoliti@
stthomas.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.040

2652
D. C. Ebner et al. / Bioorg. Med. Chem. 16 (2008) 2651–2656
O
O
O
O
O
O
N
N
N
N
N
HO
F
F
HN
O
HN
O
1
2
Figure 1. Zyvox (1) and analog (2).
O
O
O
O
O
O
N
N
N
N
O
N
N
O
O
F
HN
H2N
HN
O
O
N
N
3
13
4
N
Figure 2. Oxazolidinones that demonstrate high antibacterial activity.
In addition, Phillips et al. have replaced the amide group
of linezolid with a triazole ring to give compound 13 and
found comparable antibacterial activity to linezolid.14
This result supports the idea that triazoles can act as
amide surrogates.15
accomplished in good yield (83%) starting with enantio-
merically pure (R)-(ꢀ)-glycidyl butyrate. Although the
enantiomeric excess for this reaction was not measured,
the identical reaction conditions used by Brickner et al.
were followed and the authors reported greater than
99.7% ee.8 Mesylation followed by substitution with
azide and then reduction of the azide gave amine 11 in
high overall yield. Acylation of the amine with acetic
anhydride proceeded smoothly to give the final product
5 in 83% yield. Purification of the product did not re-
quire chromatography and was accomplished readily
by washing with cold ethanol.
Herein we describe the synthesis and antibacterial activ-
ity of four novel oxazolidinones. The first compound (5)
contains a benzodioxin ring system with the benzodioxin
oxygens in the same positions as the fluorine and amino
nitrogen of linezolid (Fig. 3). We have also explored the
effect of replacing the amide functional group on com-
pound 5 with a substituted triazole ring by synthesizing
compounds 12a–12c.
Replacement of amide group in compound 5 with a tri-
azole was easily accomplished using Sharpless click
chemistry conditions on intermediate 10.16 Three novel
oxazolidinones containing triazole rings were synthe-
sized according to Scheme 2. Substituted alkynes reacted
readily with the azide 10 to give 12a with a nonpolar bu-
tyl group and 12b with a polar carboxylic acid group.
Synthesis of the unsubstituted triazole was not straight-
forward. Phillips et al. reacted azides with acetylene gas
under high pressure in a steel bomb. To avoid these
potentially hazardous conditions we used methodology
reported by Biagi et al. to form the unsubstituted tria-
zole ring.17 We first synthesized a triazole substituted
with a carboxylic acid group (12b). Decarboxylation
proceeded smoothly by refluxing in DMF to give com-
pound 12c in excellent yield.
2. Results and discussion
The synthesis of the new antimicrobial agent 5 was
accomplished in seven steps as shown in Scheme 1.
The benzodioxin ring was readily constructed by a dou-
ble nucleophilic aromatic substitution reaction using
catechol and 3,4-difluoronitrobenzene in 85% yield.
Reduction of the nitro group and formation of the ben-
zyl carbamate was carried out in the same reaction ves-
sel without isolation of the amine in an overall 74%
yield. The formation of the oxazolidinone ring was
2.1. In vitro antimicrobial testing
O
O
O
O
O
O
The MIC of the oxazolidinone derivative 5 for Gram-
positive Staphylococcus aureus ATCC 29213 was 2–
3 lg/mL. The determined MIC concentration is similar
in efficacy range compared to other published oxazolid-
inone derivative MIC values.7 The MICs of the oxazo-
lidinone derivatives 12a, 12b, and 12c for S. aureus
were greater than 50 lg/mL. Concentrations above
50 lg/mL were not tested of derivatives 12a, 12b, and
12c. None of the oxazolidinone derivatives were effective
against the Gram-negative Escherichia coli ATCC 25922
up to a concentration of 100 lg/mL. The 100 lg/mL
N
N
O
O
5
N
HN
N
R
N
O
12a - R= CH2CH2CH2CH3
12b - R= COOH
12c - R= H
Figure 3. Novel oxazolidinones.

D. C. Ebner et al. / Bioorg. Med. Chem. 16 (2008) 2651–2656
2653
O
O
d
O
O
O
O
F
F
OH
OH
b, c
NO2
a
O
+
N
H
O
O
NO2
7
6
O
O
O
O
O
e
f
O
O
O
g
N
N
O
N
O
O
O
9
8
10
O
O
HO
N3
S
O
CH3
O
O
O
O
O
h
O
N
N
O
O
O
NH
11
H2N
5
Scheme 1. Reagents: (a) K2CO3, DMF/toluene (80:20); (b) H2, 10% Pd/C, THF; (c) CbzCl; (d) LiN(Si(CH3)3)2 followed by addition of (R)-(ꢀ)-
glycidyl butyrate; (e) MeSO2Cl/NEt3/CH2Cl2; (f) NaN3/DMF; (g) H2, 10% Pd/C, THF; (h) acetic anhydride, pyridine.
O
O
O
O
O
O
O
O
i
j
N
N
O
O
O
N
O
10
N
N
N3
N
N
R
N
R
N
12c - R= H
12a - R= CH2CH2CH2CH3
12b - R= COOH
Scheme 2. Reagents and condition: (i) sodium ascorbate, CuSO4Æ5H2O, t-BuOH/H2O, for 12a: 1-hexyne, for 12b: propiolic acid; (j) from 12b, DMF,
reflux.
concentration was the highest concentration of the oxa-
zolidinone derivative that could be administered without
limiting the growth of E. coli due to the presence of
greater than 5% EtOH in the growth medium. The oxa-
zolidinone derivative was determined to have a bacterio-
static mode of action, as cells that were harvested from
post MIC incubations were viable after removal of the
antimicrobial.
crobial activity against S. aureus compared to linezolid,
which has been reported to have an MIC of 4 lg/mL. It
appears that the benzodioxin ring system does not alter
the activity of the oxazolidinone with an amide at-
tached. However, in contrast to the results of Phillips
et al., our compounds with triazole rings show no anti-
microbial activity, which suggests there is a link between
the benzodioxin moiety and the amide functionality for
activity of these oxazolidinones.
3. Conclusions
4. Experimental
4.1. Chemistry
Four novel oxazolidinones containing a benzodioxin
moiety have been synthesized. The antimicrobial activity
of each of these compounds has been measured. The
MIC against S. aureus was determined to be 2–3 lg/
mL for compound 5. This compound has similar antimi-
All reagents and solvents were purchased from Aldrich
Chemical Co. and used as received. H NMR and 13C
1

2654
D. C. Ebner et al. / Bioorg. Med. Chem. 16 (2008) 2651–2656
NMR data were collected at 300 MHz on a Bruker
NMR spectrometer.
134.9, 124.6, 124.3, 116.5, 116.5, 116.3, 113.2, 106.2,
73.2, 61.7, 46.1. HRMS(ESI) calcd for C16H13NO5 + Na,
exact 322.0686, found 322.0708.
4.1.1. 2-Nitrodibenzo[b,e][1,4]dioxine (6). Catechol
(20.0 g, 0.182 mol) and K2CO3 (50.2 g, 0.363 mol) were
stirred in a solution of 80:20 DMF:toluene (1000 mL).
The solution was treated by dropwise addition of 3,4-
difluoronitrobenzene (20.1 mL, 0.182 mol) and brought
to reflux for 2 h. The reaction mixture was cooled and
then poured over ice water (1500 mL). The resulting yel-
low precipitate was vacuum filtered and recrystallized
from MeOH. The procedures yielded 28.0 g (68%) of 6
4.1.4. ((2R)-4-(dibenzo[b,e][1,4]dioxin-7-yl)-tetrahydro-5-
oxofuran-2-yl)methyl methanesulfonate (9). 8 (2.68 g,
8.95 mmol) and NEt3 (2.19 mL, 15.7 mmol) in CH2Cl2
(250 mL) were cooled to 0 ꢁC and allowed to stir for
30 min.
Methanesulfonyl
chloride
(0.867 mL,
11.2 mmol) was added dropwise to the solution. The
reaction mixture was warmed to ambient temperature
while stirring for 24 h. The reaction mixture was diluted
over CH2Cl2, washed with water (3·) and brine, and
dried over Na2SO4. Concentration in vacuo yielded
1
as a yellow solid. H NMR (CDCl3, 300 MHz): d 6.93
(m, 5H), 7.74 (d, J = 2.44 Hz, 1H), 7.83 (dd,
J = 8.85 Hz, J0 = 2.75 Hz, 1H). 13C NMR (CDCl3,
300 MHz): d 147.4, 143.5, 141.9, 140.7, 140.7, 124.9,
124.6, 119.9, 116.5, 116.5, 116.3, 112.2. HRMS(ESI)
calcd for C12H7NO4 + Na, exact 252.0267; found
252.0274.
1
3.29 g (97%) of 9 as a white solid. H NMR (CDCl3,
300 MHz): d 3.11 (s, 3H), 3.91 (dd, J = 9.16 Hz,
J0 = 6.11 Hz, 1H), 4.11 (t, J = 9.16 Hz, 1H), 4.43 (dd,
J = 11.59 Hz,
J0 = 3.96 Hz,
1H),
4.50
(dd,
J = 11.59 Hz, J0 = 3.96 Hz, 1H), 4.91 (m, 1H), 6.87 (m,
5H), 6.98 (dd, J = 8.85 Hz, J0 = 2.74 Hz, 1H), 7.18 (d,
J = 2.75 Hz, 1H). 13C NMR (CDCl3, 300 MHz): d
153.5, 142.3, 141.8, 141.5, 138.9, 133.3, 124.0, 123.9,
116.4, 116.3, 116.3, 113.3, 107.3, 69.3, 67.9, 46.6, 37.8.
HRMS(ESI) calcd for C17H15NO7S + Na, exact
400.0461; found 400.0447.
4.1.2. Benzyl dibenzo[b,e][1,4]dioxin-7-ylcarbamate (7).
The nitro compound 6 (15.0 g, 65.4 mmol) was dissolved
in THF (300 mL) followed by the addition of 10% Pd on
carbon (1.0 g). Hydrogenation in a Parr Shaker was car-
ried out for 20 h. The reaction mixture was filtered
through Celite, treated with saturated NaHCO3 solu-
tion, and cooled to ꢀ20 ꢁC. After addition of benzyl
chloroformate (14.0 mL, 98.1 mmol) the reaction mix-
ture was allowed to warm to ambient temperature while
stirring for 20 h. The solution was concentrated to half
the volume on a rotary evaporator followed by dilution
with EtOAc. The solution was washed with water (4·)
and brine, dried over Na2SO4, and concentrated in va-
cuo. The crude product was washed with hot hexanes
4.1.5. (R)-5-(azidomethyl)-3-(dibenzo[b,e][1,4]dioxin-7-
yl)oxazolidin-2-one (10). 9 (3.25 g, 8.61 mmol) in DMF
(250 mL) was treated with sodium azide (5.60 g,
86.1 mmol) and heated to 60 ꢁC for 18 h. The reaction
mixture was diluted with EtOAc, washed with water
(5·) and brine, and dried with Na2SO4. Concentration
in vacuo yielded 2.77 g (99%) of 10 as a white solid.
1H NMR (CDCl3, 300 MHz): d 3.59 (dd, J = 13.12 Hz,
J0 = 4.27 Hz, 1H), 3.70 (dd, J = 13.42 Hz, J0 = 4.88 Hz,
1H), 3.81 (dd, J = 8.85 Hz, J0 = 6.1 Hz, 1H), 4.04 (t,
8.85 Hz, 1H), 4.77 (m, 1H), 6.87 (m, 5H), 6.99 (dd,
J = 8.85 Hz, J0 = 2.75 Hz, 1H), 7.18 (d, J = 2.75 Hz,
1H). 13C NMR (DMSO, 300 MHz): d 154.1, 141.6,
141.6, 141.3, 137.6, 134.8, 124.8, 124.7, 116.8, 113.7,
106.8, 71.5, 53.0, 47.2. HRMS(ESI) calcd for
C16H12N4O4 + Na, exact 347.0751, found 347.0742.
1
to yield 19.6 g (78%) of 7 as a beige solid. H NMR
(CDCl3, 300 MHz): d 5.19 (s, 2 H), 6.53 (br s, 0.5H),
6.84 (m, 7H), 7.38 (m, 5H). 13C NMR (CDCl3,
300 MHz): d 154.2, 142.2, 142.1, 141.7, 138.1, 135.9,
133.6, 128.6, 128.4, 128.3, 123.7, 116.4, 116.3, 116.3,
67. HRMS(ESI) calcd for C20H15NO4 + Na, exact
356.0893; found 356.0898.
4.1.3. (R)-3-(Dibenzo[b,e][1,4]dioxin-7-yl)-5-(hydroxy-
methyl)oxazolidin-2-one (8). A solution of compound 7
4.1.6. (S)-5-(aminomethyl)-3-(dibenzo[b,e][1,4]dioxin-7-
yl)oxazolidin-2-one (11). Azide 10 (2.75 g, 8.48 mmol)
was dissolved in THF (250 mL) followed by the addition
of 10% Pd on carbon (0.90 g). Reaction vessel was
charged with H2 and hydrogenation for 20 h ensued in
a Parr Shaker. The reaction mixture was filtered through
Celite and concentrated in vacuo to yield 2.50 g (99%) of
11 as a white solid. 1H NMR (CDCl3, 300 MHz): d 2.97
(dd, J = 14.19 Hz, J0 = 5.8 Hz, 1H), 3.11 (dd,
J = 13.73 Hz, J0 = 5.27 Hz, 1H), 3.80 (dd, J = 8.54 Hz,
J0 = 6.71 Hz, 1H), 4.00 (t, J = 8.44 Hz, 1H), 4.66 (m,
1H), 6.86 (m, 5H), 7.00 (dd, J = 8.85 Hz, J0 = 2.44 Hz,
1H), 7.20 (d, J = 2.75 Hz, 1H). 13C NMR (CDCl3,
300 MHz): d 154.6, 142.3, 142.0, 141.7, 138.5, 134.2,
124.0, 123.8, 116.4, 116.4, 113.1, 107.1, 73.8, 47.7,
45.0. HRMS(ESI) calcd for C16H14N2O4 + H, exact
299.1024, found 299.1026.
(3.85 g, 11.6 mmol) in THF (200 mL) was cooled to
1.0 M
ꢀ78 ꢁC.
lithium
bis(trimethylsilyl)amide
(12.8 mL, 12.8 mmol) was added dropwise to the solu-
tion and allowed to stir for 30 min. (R)-(ꢀ)-glycidyl
butyrate (1.64 mL, 11.6 mmol) was added dropwise to
the solution. The reaction mixture was warmed to ambi-
ent temperature while stirring for 24 h. The reaction
mixture was diluted with EtOAc, washed with water
and saturated NaCl solution (2·), and dried over
Na2SO4. Precipitation of product during in vacuo con-
centration yielded 2.75 g (79%) of 8 as a white solid.
1H NMR (DMF, 300 MHz):
d
3.56 (ddd,
J = 12.21 Hz, J0 = 9.15 Hz, J00 = 3.66 Hz, 1H), 3.67
(ddd, J = 12.21 Hz, J0 = 9.15 Hz, J00 = 3.66 Hz, 1H),
3.81 (dd, J = 8.85 Hz, J0 = 6.91 Hz, 1H), 4.02 (t,
J = 8.85 Hz,1H), 4.63 (m, 1H), 5.23 (t, J = 5.8 Hz, 1H),
6.85 (m, 5H), 6.99 (dd, J = 8.85 Hz, J0 = 2.44 Hz, 1H),
7.27 (d, J = 2.44 Hz, 1H). 13C NMR (DMSO,
300 MHz): d 154.434, 141.4, 141.235, 141.0, 137.1,
4.1.7. N-(((S)-3-(dibenzo[b,e][1,4]dioxin-7-yl)-2-oxooxaz-
olidin-5-yl)methyl)acetamide (5). Amine 11 (2.47 g,

D. C. Ebner et al. / Bioorg. Med. Chem. 16 (2008) 2651–2656
2655
8.28 mmol) in pyridine (300 mL) was treated with acetic
anhydride (0.94 mL, 9.94 mmol) and stirred for 21 h.
Concentration in vacuo yielded 2.35 g (83%) of crude
product 5 as a beige solid. Crude product 5 (2.00 g)
was washed with cold absolute EtOH while stirring for
20 min to yield 1.15 g (58%) of 5 as a white solid. mp
233.0–233.3 ꢁC. 1H NMR (CDCl3, 300 MHz): d 2.03
(s, 3H), 3.66 (m, 3H), 4.01 (t, J = 9.15 Hz, 1H), 4.76
(m, 1H), 6.01 (br s, 1H), 6.89 (m, 6H), 7.17 (d,
J = 2.44 Hz, 1H). 13C NMR (CDCl3, 300 MHz): d
171.0, 154.3, 142.4, 142.0, 141.7, 138.8, 133.8, 124.1,
123.9, 116.4, 116.4, 113.2, 107.2, 71.8, 47.7, 42.0, 23.2.
HRMS(ESI) calcd for C18H16N2O5 + Na, exact
363.0951, found 363.0951.
at 243 ꢁC. 1H NMR (DMSO, 300 MHz): d 3.86 (dd,
J = 9.17 Hz, J0 = 5.75 Hz, 1H), 4.20 (t, J = 8.80 Hz,
1H), 4.83 (d, J = 5.01 Hz, 2H), 5.12 (m, 1H), 7.00 (m,
6H), 7.21 (s, 1H), 7.77 (s, 1H), 8.18 (s, 1H). 13C NMR
(DMSO, 300 MHz): d 153.4, 141.2, 141.1, 140.8, 137.3,
134.2, 125.8, 124.5, 124.3, 116.4, 116.4, 113.6, 106.6,
70.7, 51.7, 47.1. HRMS(ESI) calcd for C18H14N4O4 + -
Na, exact 373.0907, found 373.0907.
5. General procedure for preparation of antimicrobial
drugs18
A 2.0 mg/mL stock solution of linezolid (Pharmacia
Corporation, New York) and the oxazolidinone deriva-
tive was prepared using 100% ethanol (EtOH) or 100%
dimethylsulfoxide (DMSO, for derivative 12c only be-
cause of insolubility in EtOH) as the solvent. Linezolid
was prepared by grinding Zyvox pills with a mortar
and pestle and placing in 100 mL of methylene chloride.
The Zyvox/CH2Cl2 solution was filtered and the CH2Cl2
was evaporated. The resultant white crystalline powder
was analyzed by TLC and NMR and shown to be pure
linezolid. This material was used to make the linezolid
stock solution.
4.1.8.
(R)-5-((4-butyl-1H-1,2,3-triazol-1-yl)methyl)-3-
(dibenzo[b,e][1,4]dioxin-8-yl)oxazolidin-2-one (12a). To
azide 10 (252.0 mg) in 20 mL H2O were added (+)-so-
dium ascorbate (82.3 mg) and copper(II) sulfate penta-
hydrate (7.4 mg). The solution was stirred and hexyne
(67.0 mg) was added. The reaction mixture was stirred
for 5 days under ambient conditions and then filtered.
After washing with water and 1.0 M HCl, 152.0 mg
1
(60.3%) of 12a was obtained. mp 215.8–216.2 ꢁC. H
NMR (DMSO, 300 MHz): d 0.85 (t, J = 7.34 Hz, 3H),
1.26 (m, 2H), 1.513 (m, 2H), 2.59 (t, J = 7.34 Hz, 2H),
3.82 (dd, J = 9.41 Hz, J0 = 5.38 Hz, 1H), 4.19 (t,
J = 9.17 Hz, 1H), 4.73 (d, J = 4.89 Hz, 2H), 5.09 (m,
1H), 6.99 (m, 6H), 7.19 (d, J = 1.47 Hz, 1H), 7.87 (s,
J = Hz, 1H). 13C NMR (DMSO, 300 MHz): d 153.4,
146.9, 141.2, 141.1, 140.8, 137.2, 134.2, 124.5, 124.3,
122.7, 116.4, 113.5, 106.5, 70.7, 51.8, 47.0, 31.1, 24.5,
21.5, 13.7. HRMS(ESI) calcd for C22H22N4O4 + H, ex-
act 407.1714, found 407.1721.
5.1. Strains, growth conditions, and minimum inhibitory
concentration (MIC)
Staphylococcus aureus ATCC 29213 and Escherichia coli
ATCC 25922 strains were maintained in Muller Hinton
broth (MHB; Oxoid, Ogdensburg, New York). Over-
night cultures incubated at 37 ꢁC for 24 h were used to
test antimicrobial efficacy. The MIC of Zyvox and the
oxazolidinone derivatives were determined as previously
described.18 The gradient of both Zyvox and oxazolidi-
none derivative 5 tested against S. aureus ranged from
final concentrations of 0.5 to 10.0 lg/mL. The gradient
of oxazolidinone derivatives 12a, 12b, and 12 c tested
against S. aureus ranged from final concentrations of
0.5 to 50.0 lg/mL. The range of antimicrobial concen-
tration used against E. coli was from 10 to 100 lg/mL.
Cultures exposed to the antimicrobials and either EtOH
or DMSO controls were statically incubated at 37 ꢁC for
24 h and subsequently visualized for microbial growth.
4.1.9. 1-(((R)-3-(dibenzo[b,e][1,4]dioxin-8-yl)-2-oxooxaz-
olidin-5-yl)methyl)-1H-1, 2, 3-triazole-4-carboxylic acid
(12b). To azide 10 (209.0 mg) in 10 mL t-BuOH were
added (+)-sodium ascorbate (76.4 mg) and copper(II)
sulfate pentahydrate (45.5 mg). The solution was stirred
and propiolic acid (80% in THF) (3 mL) was added. The
reaction was refluxed overnight under N2 pressure and
constant stirring. Reaction mixture was filtered resulting
in a purple solid. The purple solid was stirred in 0.5 M
HCl overnight, filtered, and washed with 50 mL H2O
and 2· 25 mL MeOH to yield 220.0 mg (87%) of 12b
1
as a light gray solid. H NMR (DMSO, 300 MHz): d
5.2. Determination of cell viability
3.90 (m, 1H), 4.22 (t, J = 8.19 Hz, 1H), 4.84 (d,
J = 4.40 Hz, 2H), 5.16 (m, 1H), 7.00 (m, 6H), 7.23 (s,
1H), 8.72 (s, 1H). 13C NMR (DMSO, 300 MHz): d
161.4, 153.3, 141.2, 141.1, 140.8, 139.6, 137.3, 134.2,
129.8, 124.5, 124.3, 116.4, 113.6, 106.6, 70.5, 52.1,
47.1. HRMS(ESI) calcd for C19H14N4O6 + Na, exact
417.0806, found 417.0803.
To determine if oxazolidinone derivative 5 had a bacte-
riostatic or bacteriocidal mode of action, cell viability
was determined after exposure to the oxazolidinone
derivative. Bacterial cultures exposed to oxazolidinone
derivative 5 concentrations around the determined
MIC and the EtOH control were selected. The 1.0 mL
MIC culture was placed into a 1.5 mL microfuge tube
and centrifuged for 2 min at 16,000g. The supernatant
was removed and the pellet was resuspended in 1 mL
of MHB medium to wash the cell pellet. The cell suspen-
sion was centrifuged again for 2 min at 16,000g, the
supernatant was removed, and a final 1 mL of MHB
medium was used to resuspend the cell pellet. Dilutions
were plated in duplicate on MHB agar (purchased from
4.1.10. (R)-5-((1H-1,2,3-triazol-1-yl)methyl)-3-(dibenzo[-
b,e][1,4]dioxin-8-yl)oxazolidin-2-one (12c). 12b (250.0 mg)
was added to 30 mL DMF. The solution was refluxed
for 3.25 h under N2 pressure and constant stirring. The
reaction mixture was cooled to room temperature and a
brown solid was precipitated into 150 mL H2O. The solid
was filtered and washed with 3· 25 mL EtOAc yielding
200.0 mg 12c (90.1%) as a light tan solid. Decomposes

2656
D. C. Ebner et al. / Bioorg. Med. Chem. 16 (2008) 2651–2656
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Oxoid) and incubated at 37 ꢁC for 24 h to determine cell
number and viability.
Supplementary data
10. Sbardella, G.; Mai, A.; Artico, M.; Loddo, R.; Setzu, M.
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