Identification of a novel oxazolidinone (U-100480) with potent antimycobacterial activity.

Article abstract of DOI:10.1021/jm950956y

During the course of our investigations in the oxazolidinone antibacterial agent area, we have identified a subclass with especially potent in vitro activity against mycobacteria. The salient structural feature of these oxazolidinone analogues, 6 (U-10048

Full text of DOI:10.1021/jm950956y

680
J . Med. Chem. 1996, 39, 680-685
Id en tifica tion of a Novel Oxa zolid in on e (U-100480) w ith P oten t
An tim ycoba cter ia l Activity
Michael R. Barbachyn,*,† Douglas K. Hutchinson,† Steven J . Brickner,† Michael H. Cynamon,‡
J ames O. Kilburn,§ Sally P. Klemens,‡ Suzanne E. Glickman,§ Kevin C. Grega,† Susan K. Hendges,†
Dana S. Toops,† Charles W. Ford,† and Gary E. Zurenko†
Upjohn Laboratories, The Upjohn Company, Kalamazoo, Michigan 49001, Veterans Affairs Medical Center and State
University of New York Health Science Center, Syracuse, New York 13210, and Centers for Disease Control,
Atlanta, Georgia 30333
Received December 22, 1995X
During the course of our investigations in the oxazolidinone antibacterial agent area, we have
identified a subclass with especially potent in vitro activity against mycobacteria. The salient
structural feature of these oxazolidinone analogues, 6 (U-100480), 7 (U-101603), and 8 (U-
101244), is their appended thiomorpholine moiety. The rational design, synthesis, and
evaluation of the in vitro antimycobacterial activity of these analogues is described. Potent
activity against a screening strain of Mycobacterium tuberculosis was demonstrated by 6 and
7 (minimum inhibitory concentrations or MIC’s e0.125 µg/mL). Oxazolidinones 6 and 8 exhibit
MIC90 values of 0.50 µg/mL or less against a panel of organisms consisting of five drug-sensitive
and five multidrug-resistant strains of M. tuberculosis, with 6 being the most active congener.
Potent in vitro activity against other mycobacterial species was also demonstrated by 6. For
example, 6 exhibited excellent in vitro activity against multiple clinical isolates of Mycobac-
terium avium complex (MIC’s ) 0.5-4 µg/mL). Orally administered 6 displays in vivo efficacy
against M. tuberculosis and M. avium similar to that of clinical comparators isoniazid and
azithromycin, respectively. Consideration of these factors, along with a favorable pharmaco-
kinetic and chronic toxicity profile in rats, suggests that 6 (U-100480) is a promising
antimycobacterial agent.
In tr od u ction
aureus (MRSA) and Staphylococcus epidermidis (MRSE),
as well as the enterococci.5 DuP 721 has also been
reported to exhibit good activity against M. tuberculo-
sis.6 However, in drug safety studies conducted at The
Upjohn Co., it was shown that (()-DuP 721 exhibited
lethal toxicity in rats when dosed orally at 100 mg/kg
b.i.d. for 30 days.7 Therefore, the preparation of novel
oxazolidinones with potent antimycobacterial activity,
but without the liability of toxicity, remained a viable
goal.
Mycobacteria are ubiquitous organisms that are
becoming increasingly important pathogens. The re-
surgence of reported cases of tuberculosis, along with
the recent emergence of multidrug-resistant strains of
Mycobacterium tuberculosis, has refocused attention on
this disease.1 Perhaps the most important factor in the
reemergence of tuberculosis is the human immunode-
ficiency virus (HIV) pandemic. Tuberculosis prevalence
is high among patients infected with HIV.1 Dissemi-
nated Mycobacterium avium complex infections are also
closely associated with HIV-infected individuals.2 Such
infections contribute substantially to morbidity and
mortality and are difficult to control with current
treatment regimens. Therefore, an urgent need exists
for the development of new antimycobacterial agents
with a unique mechanism of action.
The oxazolidinones, exemplified by DuP 721 (1), are
a relatively new class of orally active, totally synthetic
antibacterial agents discovered by workers at DuPont.3
Preliminary inquiries into the mechanism of action of
the oxazolidinones have revealed that they are bacterial
protein synthesis inhibitors, with inhibition uniquely
occurring at an early event in the initiation phase of
protein synthesis.4 Their spectrum of activity encom-
passes anaerobic organisms and Gram-positive aerobic
bacteria, including methicillin-resistant Staphylococcus
Intensive studies at The Upjohn Co. have resulted in
novel and potent oxazolidinone antibacterial agents
incorporating a substituted piperazine moiety (see
generic structure 2).8 In contemplating alternative
analogues of 2, we were attracted to congeners contain-
ing various piperazine surrogates. One facet of this
structure-activity relationship effort focused on thio-
morpholine-derived compounds of generic structure 3
as potential targets. In this paper we describe the
preparation of selected examples of 3 and report the
remarkable in vitro activity manifested by these com-
pounds against M. tuberculosis, the causative agent of
tuberculosis, along with M. avium and other mycobac-
terial species.
† The Upjohn Co.
‡ Veterans Affairs Medical Center and State University of New York
Health Science Center.
§ Centers for Disease Control.
0022-2623/96/1839-0680$12.00/0 © 1996 American Chemical Society

Novel Oxazolidinone with Antimycobacterial Activity
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3 681
Sch em e 1
Ch em istr y
At the outset, our initial focus was on examples of 3
wherein at least one of the R2 and R3 groups was
fluorine. The often remarkable potentiating effect of one
or two fluorine atoms flanking the p-phenyl substituent
has been described previously.8-10 Much of the meth-
odology used to prepare these compounds closely paral-
lels that utilized for their piperazine- and morpholine-
substituted progenitors (see Scheme 1).11 To this end,
thiomorpholine and 3,4-difluoronitrobenzene were re-
acted to generate the adduct 4. Intermediate 4 was then
converted in three steps to the (R)-5-(hydroxymethyl)-
oxazolidinone 5 in 82% overall yield. Elaboration of the
C-5 hydroxymethyl moiety of 5 to the corresponding
acetamidomethyl subunit was accomplished as de-
scribed in the preceding paper, with the exception that
the intermediate C-5 azidomethyl group was reduced
by a reaction sequence involving treatment with tri-
phenylphosphine (THF) and then hydrolysis of the
resultant intermediate iminophosphorane.12 The prod-
uct amine was generally not purified but rather acety-
lated and the acetamide purified by recrystallization or
trituration to give the targeted oxazolidinone analogue
6 (U-100480) in 77-80% yield from 5.
Sch em e 2a
Thioether linkages are amenable to further elabora-
tion. We wanted to prepare the sulfur oxidation prod-
ucts of 6 to probe the antibacterial activity of such
derivatives and also to have potential metabolites
available. The metabolic oxidation of sulfides to sul-
foxides is a facile and generally reversible process. In
contrast, the sulfone oxidation state appears to be a
metabolic dead end.13 The chemical oxidation of sulfides
to sulfoxides and sulfones under a variety of conditions
is well precedented.14 As shown in Scheme 2, we were
pleased to find that 6 could be readily oxidized to the
corresponding sulfoxide 7 (U-101603) in 95% yield,
employing sodium metaperiodate under conditions de-
scribed by Leonard and J ohnson.15 The corresponding
sulfone 8 (U-101244) was prepared in 85% isolated yield
by treating 6 with catalytic osmium tetraoxide in the
presence of N-methylmorpholine N-oxide.16
Resu lts a n d Discu ssion
Oxazolidinones 6 and 7 were submitted for a prelimi-
nary evaluation of their in vitro activity against M.
tuberculosis17 (Table 1). As shown in the table, both
analogues were exceptionally active against a screening
strain of M. tuberculosis, being comparable to or more
active than the clinical comparator isoniazid. Com-
pounds 6 and 8 were tested against a panel of 10 M.
tuberculosis isolates (Table 2). Test isolates included
five “drug-sensitive” and five “drug-resistant” strains.
The resistant isolates’ phenotypes covered resistance to
the common antitubercular drugs isoniazid, streptomy-
a
Reagents: (a) NaIO4, MeOH, H2O; (b) catalytic OsO4, NMO,
acetone, H2O.
cin, rifampin, ethambutol, and ethionamide (see Table
3 for antibiograms of resistant M. tuberculosis isolates).
Oxazolidinone 6 exhibited a MIC range of 0.03-0.50 µg/

682 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3
Barbachyn et al.
ing.20 Compound 6 was well absorbed after oral ad-
ministration, although significant first-pass metabolism
to the sulfoxide 7 and, to a much lesser extent, the
sulfone 8 was observed. In a gratifying result, the
combined plasma concentrations of 6 and/or 7 were
found to be quite high and persistent; both 7 and 8
exhibit in vitro activity against M. tuberculosis similar
to that of 6.
Orally administered 6 had an acceptable safety profile
in rats when dosed at 50 mg/kg b.i.d. for 29 days.7 This
dosing regimen was very well tolerated, and drug-
related findings were considered to be of minor toxico-
logical relevance.
In summary, oxazolidinone 6 and its metabolites
exhibit potent in vitro activity against M. tuberculosis;
strains resistant to conventional antituberculosis drugs
were not cross-resistant to these compounds.17 The in
vitro activity of 6 extends to other mycobacterial species,
including M. avium complex, an opportunistic pathogen
associated with the acquired immune deficiency syn-
drome (AIDS). In general, oxazolidinone 6 was compa-
rable to or significantly more active in vitro than the
clinical comparator azithromycin against nontubercu-
losis species of mycobacteria. In mouse models, orally
administered 6 has shown efficacy similar to that of
isoniazid and azithromycin against M. tuberculosis and
M. avium, respectively.18,19 Oxazolidinone 6 exhibits
favorable pharmacokinetics in rats after oral dosing20
and was well tolerated in a chronic toxicity study.7
Consideration of the above findings suggests that
further studies probing the utility of 6 (U-100480) as a
general antimycobacterial agent are warranted.
Ta ble 1. In Vitro Activity of Selected Oxazolidinone Analogues
against M. tuberculosis H37Rv
MIC (µg/mL)a
organism
6
7
isoniazid
0.2
M. tuberculosis
e0.125
e0.125
a
For minimum inhibitory concentration (MIC) determinations
in Tables 1-3, the compounds were incorporated into 7H10 agar
medium (Difco Laboratories, Detroit, MI) at concentrations of 2.0,
0.50, 0.125, and 0.03 µg/mL. The M. tuberculosis test organisms
were grown in 7H9 medium (Difco) containing 0.05% Tween 80.
After 7 days of incubation at 37 °C, the broths were adjusted to
the turbidity of a 1.0 McFarland standard; the organisms were
then diluted 10-fold in sterile water containing 0.10% Tween 80.
The resultant bacterial suspensions were spotted onto the drug-
supplemented 7H10 plates. After a 21 day cultivation at 37 °C,
the growth of the organisms was scored. The MIC was defined
as the lowest concentration of drug that completely inhibited
growth of the organism.
Ta ble 2. In Vitro Activity of Selected Oxazolidinone Analogues
against Multiple Strains of M. tuberculosis
no. of strains inhibited at
concentration (µg/mL)
compd
organism groupa
0.03
1
0.125
0.50
2
1
6
S
R
S
R
2
3
1
2
2
4
4
8
a
S: group of five “drug-sensitive” isolates. R: group of five
“drug-resistant” isolates.
mL and was the most active oxazolidinone tested.
Interestingly, organisms resistant to numerous antitu-
bercular agents were not cross-resistant with 6 and 8.
Good in vitro activity was also observed for 6 against
other mycobacterial species (Table 4). Generally, 6 was
comparable to or significantly more active than the
current clinical benchmark azithromycin. Analogue 6
exhibited potent in vitro activity against multiple
isolates of M. avium complex, with a MIC50 of 2 µg/mL
and a MIC90 of 4 µg/mL (Table 5).
Oxazolidinone 6 has been tested in a M. tuberculosis
mouse infection model.18 The degree of efficacy seen for
orally administered 6 (mean cfu reduction in spleens
and lungs of 3.1 and 4.9 log units, respectively) was
comparable to that observed for the clinical standard
isoniazid (mean cfu reduction in spleens and lungs of
3.3 and 4.7 log units, respectively), although 6 was dosed
at a higher level (100 versus 25 mg/kg, administered
orally once daily, 5 days/week for 4 weeks). In a
comparative dose-response study in beige mice, 6 was
found to have activity against M. avium complex similar
to that of azithromycin (both drugs administered orally
at a level of 100 mg/kg once daily, 5 days/week for 4
weeks).19
Exp er im en ta l Section
Gen er a l. For general experimental techniques utilized, see
the preceding paper.11
4-(2-F lu or o-4-n itr op h en yl)th iom or p h olin e (4). A solu-
tion of 3,4-difluoronitrobenzene (68.703 g, 47.81 mL, 0.432 mol)
and N,N-diisopropylethylamine (83.724 g, 112.84 mL, 0.648
mol) in dry CH3CN (900 mL) was treated with thiomorpholine
(53.475 g, 0.518 mol) at ambient temperature and the reaction
mixture heated to gentle reflux for 24 h. At this point, the
reaction was determined to be complete by TLC (15% EtOAc/
hexane, short wave UV). The reaction mixture was cooled to
ambient temperature and concentrated by rotary evaporation
to a dark orange-red liquid which was diluted with EtOAc and
transferred to a separatory funnel. The organic layer was
washed with 1 N HCl until the washings were acidic and then
washed with saturated aqueous NaHCO3 and brine. The
organic layer was dried (Na2SO4), filtered, and concentrated
in vacuo to give 104.290 g (99%) of the title compound as an
orange crystalline solid: mp 59.5-60.5 °C; IR (mull) 1604,
1494, 1334, 1325, 1255, 1229, 1193, 1078, 806 cm-1; 1H NMR
(300 MHz, CDCl3) δ 7.98 (ddd, 1H, J ) 1.0, 2.6, 9.0 Hz), 7.91
(dd, 1H, J ) 2.6, 12.9 Hz), 6.93 (t, 1H, J ) 8.8 Hz), 3.60-3.56
(m, 4H), 2.82-2.79 (m, 4H); MS m/ z (rel intensity) 242 (M+,
92), 227 (16), 195 (30), 168 (100), 138 (19), 121 (16), 95 (13),
A preliminary evaluation of the pharmacokinetic
behavior and metabolism of 6 in the rat was encourag-
Ta ble 3. Antibiograms of Resistant M. tuberculosis Isolates
antibiotic and test concentration (µg/mL)
CDCa
isolate no.
organism
group no.
SMb
2.0
SM
10.0
INHc
0.2
INH
1.0
INH
5.0
RIFd
1.0
THAe
10.0
EMBf
5.0
91-2218
91-2219
91-2225
91-2227
91-2230
Rg
R
R
R
R
R
S
R
R
R
R
S
R
S
R
R
R
R
R
R
R
R
R
R
Sh
S
R
R
S
S
R
S
S
S
S
S
S
S
R
S
R
R
R
R
R
a
b
d
CDC: Centers for Disease Control. SM: streptomycin. c INH: isoniazid. RIF: rifampin. e THA: ethionamide. f EMB: ethambutol.
g
h
R: resistant. S: susceptible.

Novel Oxazolidinone with Antimycobacterial Activity
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3 683
Ta ble 4. In Vitro Activity of Oxazolidinone 6 (U-100480)
against Selected Mycobacteria
glycidyl butyrate (8.626 g, 8.474 mL, 59.83 mmol) dropwise
over 2 min. The reaction mixture was stirred for an additional
30 min at -78 °C, and then the cooling bath was removed.
The reaction mixture was allowed to warm to ambient tem-
perature overnight. Saturated aqueous NH4Cl (50 mL) was
added to the reaction mixture. The reaction mixture was
transferred with EtOAc washings to a separatory funnel
containing additional saturated aqueous NH4Cl and extracted
with EtOAc. The combined organic extracts were washed with
brine, dried over anhydrous Na2SO4, filtered, and concentrated
by rotary evaporation until a precipitate was noted. Hexane
was added at this point, and the solids were filtered off (hexane
wash of filter cake) and dried in vacuo to give 16.725 g (91%)
of the title oxazolidinone as a white solid: mp 136-137 °C;
[R]D -50° (c 1.0, EtOAc); IR (mull) 1731, 1707, 1517, 1431,
1423, 1324, 1228, 1218, 1198, 1154, 1098, 804 cm-1; 1H NMR
(400 MHz, CDCl3) δ 7.43 (dd, 1H, J ) 2.6, 14.0 Hz), 7.13 (dd,
1H, J ) 1.6, 7.8 Hz), 6.95 (dd, 1H, J ) 9.1, 9.1 Hz), 4.78-4.70
(m, 1H), 4.03-3.92 (m, 3H), 3.76 (dd, 1H, J ) 3.9, 12.6 Hz),
3.31-3.28 (m, 4H), 2.83-2.80 (m, 4H); MS m/ z (rel intensity)
312 (M+, 100), 238 (83), 164 (12), 151 (20), 149 (18), 138 (11),
135 (10); HRMS calcd for C14H17N2O3FS 312.0944, found
312.0942. Anal. (C14H17N2O3FS) C, H, N.
MIC (µg/mL)a
organism
6
azithromycin
4
M. avium complex (MAC)
ATCC 49601b
MAC 101c
4
0.5
2
4
4
M. simiae
M. xenopi
2
1
M. malmoense
M. fortuitum
2
8
16
>64
a
The minimum inhibitory concentrations (MICs) in Tables 4
and 5 were determined by broth dilution method using 7H11 broth,
pH 6.6, supplemented with 10% oleic acid-albumin-dextrose-
catalase enrichment (Difco Laboratories, Detroit, MI). MAC
ATCC 49601 was obtained as a clinical isolate from a patient with
AIDS at the State University of New York Health Science Center,
Syracuse. c MAC 101 (serotype 1) was kindly provided by Lowell
S. Young, Kuzell Institute for Arthritis and Infectious Diseases,
Pacific Presbyterian Medical Center, San Francisco, CA.
b
Ta ble 5. In Vitro Activity of Oxazolidinone 6 (U-100480)
against Multiple Clinical Isolates of M. avium Complex
(R)-[3-[3-F lu or o-4-(4-th iom or p h olin yl)p h en yl]-2-oxo-5-
oxa zolid in yl]m eth yl Meth a n esu lfon a te. A solution of (R)-
[3-[3-fluoro-4-(4-thiomorpholinyl)phenyl]-2-oxo-5-oxazolidinyl]-
methanol (5; 9.530 g, 30.51 mmol) in dry CH2Cl2 (250 mL) was
cooled with an ice bath to ca. 5 °C and treated with Et3N (4.631
g, 6.38 mL) and methanesulfonyl chloride (3.669 g, 2.48 mL).
After 2 h at this temperature, TLC analysis (10% MeOH/
CHCl3) revealed the reaction to be complete. The reaction
mixture was transferred to a separatory funnel (CH2Cl2 wash).
The organic layer was washed with H2O, saturated aqueous
NaHCO3, and brine. The organic layer was then dried (Na2-
SO4), filtered, and concentrated in vacuo (no heating) to a
white solid (11.780 g, 99%): mp 152-153 °C; [a]D -52° (c 0.98,
CHCl3); IR (mull) 1749, 1522, 1451, 1425, 1421, 1367, 1342,
MIC (µg/mL)b
species (no. of isolates)a
range
50%
2
90%
4
M. avium complex (20)
0.5-4
a
MAC isolates 1408 and 3404 were kindly provided by Leonid
B. Heifets, Mycobacteriology Clinical Reference Laboratory, Na-
tional J ewish Center for Immunology and Respiratory Medicine,
Denver, CO. The remaining MAC isolates were obtained from
patients with AIDS and disseminated MAC infections at the State
University of New York Health Science Center, Syracuse, NY.
b
50% and 90%, MICs at which 50% and 90% of the isolates are
inhibited, respectively.
1
1335, 1223, 1216, 1201, 1175, 990 cm-1; H NMR (300 MHz,
46 (17); HRMS calcd for C10H11N2O2FS 242.0522, found
242.0525. Anal. (C10H11N2O2FS) C, H, N.
CDCl3) δ 7.42 (dd, J ) 2.6, 13.9 Hz, 1H), 7.10 (ddd, J ) 1.2,
2.6, 8.8 Hz, 1H), 6.96 (dd, J ) 9.0, 9.0 Hz, 1H), 4.96-4.87 (m,
1H), 4.50 (dd, J ) 3.7, 11.7 Hz, 1H), 4.42 (dd, J ) 4.2, 11.7
Hz, 1H), 4.12 (dd, J ) 9.1, 9.1 Hz, 1H), 3.92 (dd, J ) 6.1, 9.1
Hz, 1H), 3.33-3.27 (m, 4H), 3.10 (s, 3H), 2.84-2.79 (m, 4H);
MS m/ z (rel intensity) 390 (M+, 100), 375 (1), 343 (5), 316 (64);
HRMS calcd for C15H19FN2O5S2 390.0719, found 390.0721.
Anal. (C15H19FN2O5S2) C, H, N.
4-[2-F lu or o-4-[(b e n zyloxyca r b on yl)a m in o]p h e n yl]-
th iom or p h olin e. A solution of 4-(2-fluoro-4-nitrophenyl)-
thiomorpholine (4; 1.89 g, 7.81 mmol) in 20% H2O/THF (75
mL) was treated with W-2 Raney nickel (400 mg). The
reaction mixture was thoroughly purged with N2 and then
shaken under 40 psi of H2. After 2 h, the reduction was
complete by TLC analysis (30% EtOAc/hexane). The pale
yellow reaction solution was decanted from the catalyst, and
the catalyst was rinsed several times with solvent. The
combined washings were immediately placed under an atmo-
sphere of N2, cooled with an ice bath, and then treated with
NaHCO3 (2.62 g, 31.2 mmol, 4 equiv) followed by benzyl
chloroformate (1.23 mL, 8.59 mmol, 1.10 equiv). After 0.5 h,
the reaction was concentrated under reduced pressure to a
dark tan solid. The crude material was dissolved in CH2Cl2,
washed with H2O and brine, dried (Na2SO4), filtered, and
concentrated under reduced pressure to a dark tan solid.
Purification by silica gel chromatography (100 g of silica gel,
eluted with 5-9% EtOAc/hexane) gave 2.42 g (90%) of the title
compound as a tan solid. Recrystallization of this lot from 30%
EtOAc/hexane (three crops) gave 2.24 g of the title product as
an off-white solid: mp 141-142 °C; IR (mull) 3308, 1696, 1535,
(R)-[3-[3-F lu or o-4-(4-th iom or p h olin yl)p h en yl]-2-oxo-5-
oxa zolid in yl]m eth yl p-Tolu en esu lfon a te. A solution of
(R)-[3-[3-fluoro-4-(4-thiomorpholinyl)phenyl]-2-oxo-5-oxazoli-
dinyl]methanol (5; 1.47 g, 4.69 mmol) in pyridine (15 mL) was
cooled to 0 °C (ice bath). p-Toluenesulfonyl chloride (1.34 g,
7.04 mmol) was added and the solution allowed to stir at 0 °C
under N2 for 7 h. At this point, the reaction mixture was
stoppered and stored in a freezer overnight (16 h). In the
morning, the reaction mixture showed only a trace of starting
material by TLC (5% MeOH/CHCl3, short wave UV). The
product was precipitated by quenching the reaction with ice
water (100 mL). The solid was isolated by suction filtration
and washed thoroughly with water. The title compound, 2.02
g (92%), was recovered as a white solid: mp 141 °C; [R]D -95°
(c 1.00, EtOAc); IR (mull) 1746, 1517, 1447, 1416, 1370, 1360,
1226, 1216, 1193, 1176, 1080, 972, 873 cm-1 1H NMR (300
;
1529, 1423, 1310, 1257, 1200, 1066, 893, 744, 696 cm-1 1H
;
MHz, CDCl3) δ 7.79 (d, 2H, J ) 8.3 Hz), 7.42-7.33 (obscured
dd, 1H), 7.36 (d, 2H, J ) 8.5 Hz), 7.06 (dd, 1H, J ) 2.5, 8.8
Hz), 6.98 (dd, 1H, J ) 8.8, 8.8 Hz), 4.87-4.78 (m, 1H), 4.27
(dd, 1H, J ) 4.0, 11.5 Hz), 4.22 (dd, 1H, J ) 4.6, 11.5 Hz),
4.06 (dd, 1H, J ) 9.1, 9.1 Hz), 3.86 (dd, 1H, J ) 5.9, 9.1 Hz),
3.34-3.28 (m, 4H), 2.87-2.80 (m, 4H), 2.46 (s, 3H); MS m/ z
(rel intensity) 466 (M+, 100), 392 (44), 176 (16), 164 (15), 150
(14), 148 (16), 91 (34); HRMS calcd for C21H23N2O5FS2 466.1032,
found 466.1036. Anal. (C21H23N2O5FS2) C, H, N.
(R)-[3-[3-F lu or o-4-(4-th iom or p h olin yl)p h en y]-2-oxo-5-
oxa zolid in yl]m eth yl Azid e. P r oced u r e 1: A solution of
(R)-[3-[3-fluoro-4-(4-thiomorpholinyl)phenyl]-2-oxo-5-oxazoli-
dinyl]methyl p-toluenesulfonate (2.00 g, 4.29 mmol) in dry
DMF (15 mL) was treated with solid NaN3 (1.67 g, 25.7 mmol)
NMR (400 MHz, CDCl3) δ 7.42-7.30 (m, 5H), 6.96 (apparent
d, J ) 8.7 Hz, 1H), 6.93-6.87 (m, 1H), 6.90 (dd, J ) 8.7 Hz,
1H), 6.59 (br s, 1H), 5.20 (s, 2H), 3.30-3.25 (m, 4H), 2.83-
2.79 (m, 4H); MS m/ z (rel intensity) 346 (M+, 49), 211 (73),
183 (13), 91 (100); HRMS calcd for C18H19N2O2FS 346.1151,
found 346.1157. Anal. (C18H19N2O2FS) C, H, N.
(R)-[3-[3-F lu or o-4-(4-th iom or p h olin yl)p h en yl]-2-oxo-5-
oxa zolid in yl]m eth a n ol (5). A solution of 4-[2-fluoro-4-
[(benzyloxycarbonyl)amino]phenyl]thiomorpholine (20.322 g,
o
58.66 mmol) in dry THF (250 mL) was cooled to -78 C (dry
ice/acetone bath) under N2. n-Butyllithium (37.40 mL of a 1.6
M solution in hexanes, 59.83 mmol) was added to the reaction
mixture over 10 min. The resultant light yellow solution was
stirred at -78 °C for 30 min and then treated with (R)-(-)-

684 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3
Barbachyn et al.
and heated to 65 °C (external temperature) for 6 h. The
reaction mixture was then allowed to cool to ambient temper-
ature, stirring over a weekend (158 h). At this point, the
reaction was found to be complete by TLC (6% CH3CN/CHCl3,
short wave UV). The reaction mixture was diluted with EtOAc
(300 mL) and then washed with water (3 × 250 mL). The
aqueous portions were back-extracted with more EtOAc (2 ×
150 mL). The EtOAc portions were combined, dried over Na2-
SO4, filtered, and concentrated under reduced pressure to give
1.447 g (100%) of the title compound as an off-white solid: mp
110-111 °C; [R]D -122° (c 1.00, EtOAc); IR (mull) 2101, 1736,
1520, 1449, 1421, 1361, 1241, 1230, 1196, 1126, 1040, 973
cm-1; 1H NMR (300 MHz, CDCl3) δ 7.42 (dd, 1H, J ) 2.5, 14.0
Hz), 7.12 (ddd, 1H, J ) 1.1, 2.6, 8.8 Hz), 6.96 (dd, 1H, J ) 9.0,
9.0 Hz), 4.83-4.74 (m, 1H), 4.05 (dd, 1H, J ) 8.9 Hz), 3.82
(dd, 1H, J ) 6.2, 8.9 Hz), 3.71 (dd, 1H, J ) 4.6, 13.2 Hz), 3.59
(dd, 1H, J ) 4.4, 13.2 Hz), 3.32-3.27 (m, 4H), 2.84-2.79 (m,
4H); MS m/ z (rel intensity) 337 (M+, 100), 263 (8), 224 (6),
191 (77), 164 (20), 150 (40), 135 (12); HRMS calcd for
C14H16N5O5FS 337.1009, found 337.1009. Anal. (C14H16N5O5-
FS) C, H, N.
(m, 4H), 2.84-2.79 (m, 4H), 2.02 (s, 3H); MS m/ z (rel intensity)
353 (M+, 100), 309 (31), 279 (5), 250 (17), 235 (14), 225 (20),
212 (7), 176 (19), 138 (18), 42 (28); HRMS calcd for C16H20N3O3-
FS 353.1209, found 353.1200. Anal. (C16H20N3O3FS) C, H,
N.
(S)-N-[[3-[3-Flu or o-4-(1-oxoth iom or ph olin -4-yl)ph en yl]-
2-oxo-5-oxa zolid in yl]m eth yl]a ceta m id e (7). A solution of
sodium metaperiodate (0.635 g, 2.97 mmol) in H2O (7 mL) was
cooled to 0 °C (ice bath). Solid (S)-N-[[3-[3-fluoro-4-(4-thio-
morpholinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]acetamide (6;
1.000 g, 2.83 mmol) was added and then MeOH (10 mL) to
increase solubility. The reaction mixture was stirred at 0 °C
for 3 h and then stoppered and placed in a refrigerator at 5
°C for several days. The reaction mixture was then filtered
through a medium-porosity sintered glass funnel (copious
CHCl3 wash of the filter cake). The filtrate was transferred
to a separatory funnel, and additional H2O was added. The
mixture was extracted with CHCl3. The combined organic
extracts were washed with brine, dried over anhydrous Na2-
SO4, filtered, and concentrated in vacuo to give a foamy solid.
Radial chromatography (silica gel, 400 µm thickness), eluting
with CHCl3, and then a gradient of 2-5% MeOH in CHCl3
afforded, after concentration of appropriate fractions, 0.995 g
(95%) of the title compound as a white solid: mp 159-161 °C;
[R]D -10° (c 0.68, CHCl3); IR (mull) 3283, 1750, 1727, 1652,
1559, 1520, 1451, 1432, 1337, 1234, 1222, 1196, 1051, 752
cm-1; 1H NMR (400 MHz, CDCl3) δ 7.50 (dd, 1H, J ) 2.4, 14.0
Hz), 7.09 (dd, 1H, J ) 2.4, 8.9 Hz), 7.05 (dd, 1H, J ) 8.8, 8.8
Hz), 6.09 (br t, 1H), 4.82-4.74 (m, 1H), 4.03 (dd, 1H, J ) 8.9,
8.9 Hz), 3.80-3.68 (m, 4H), 3.62 (dt, 1H, J ) 6.0, 14.7 Hz),
3.27 (dt, 2H, J ) 3.5, 13.3 Hz), 3.02-2.97 (m, 4H), 2.03 (s,
3H); MS m/ z (rel intensity) 369 (M+, 27), 325 (100), 308 (36),
306 (24), 262 (38), 234 (31), 165 (22), 151 (23); HRMS calcd
for C16H20N3O4FS 369.1158, found 369.1182. Karl-Fischer
titration: 2.0% H2O. Anal. (C20H16N3O4FS‚0.4H2O) C, H, N,
S.
P r oced u r e 2: A solution of (R)-[3-[3-fluoro-4-(4-thiomor-
pholinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl methanesulfonate
(23.860 g, 61.11 mmol) in dry DMF (300 mL) under N2 was
treated with solid NaN3 (19.867 g, 305.55 mmol) at ambient
temperature. The stirred slurry was then heated to 65 °C for
5 h, at which time TLC analysis (5% MeOH/CHCl3) revealed
the reaction to be complete. After cooling to ambient temper-
ature, the reaction mixture was transferred to a separatory
funnel with H2O (1000 mL) and EtOAc (500 mL). The mixture
was extracted with EtOAc. The combined EtOAc extracts were
thoroughly washed with H2O and brine, dried (Na2SO4),
filtered, and concentrated in vacuo to give 19.786 g (96%) of
the title azide as a pale yellow solid identical in all respects
with the compound obtained from the corresponding tosylate.
(S)-N-[[3-[3-Flu or o-4-(4-th iom or ph olin yl)ph en yl]-2-oxo-
5-oxa zolid in yl]m eth yl]a ceta m id e (6, U-100480). A solu-
tion of (R)-[3-[3-fluoro-4-(4-thiomorpholinyl)phenyl]-2-oxo-5-
oxazolidinyl]methyl azide (19.662 g, 58.28 mmol) in dry THF
(290 mL) was treated with triphenylphosphine (16.815 g, 64.11
mmol) over 10 min. After 2.0 h, TLC analysis (10% MeOH/
CHCl3) revealed the conversion to iminophosphorane was
complete. H2O (2.10 mL, 116.56 mmol) was added and the
reaction mixture heated to 40 °C (internal temperature) for 5
h and then allowed to cool to ambient temperature overnight.
At this point, TLC analysis (10% MeOH/CHCl3) indicated
incomplete hydrolysis of the iminophosphorane intermediate.
More H2O (8.40 mL) was added, and the reaction was heated
to 40 °C for 5 h. At this time, TLC indicated complete
conversion to the 5-(aminomethyl)oxazolidinone intermediate.
The reaction mixture was first concentrated by rotary evapo-
ration (benzene was added several times to azeotrope off the
H2O) and then under high vacuum to give the crude amine as
an off-white solid. This material was dissolved in CH2Cl2 (250
mL), treated with pyridine (46.099 g, 47.10 mL, 582.79 mmol)
and acetic anhydride (29.749 g, 27.49 mL, 291.40 mmol), and
then stirred overnight at ambient temperature. TLC analysis
(10% MeOH/CHCl3) showed complete conversion to 6. The
reaction mixture was diluted with CH2Cl2, transferred to a
separatory funnel, and then washed with 1 N HCl until the
washings were acidic. The organic layer was then washed
with saturated aqueous NaHCO3 and brine, dried over Na2-
SO4, filtered, and concentrated in vacuo to give crude 6 (U-
100480) as a cream-colored solid. The crude product was
triturated with hot CHCl3; most but not all of the solids
dissolved. After cooling to ambient temperature, the solids
were filtered off (cold CHCl3 wash) and dried in vacuo to
furnish 13.174 g of analytically pure title compound as a white
solid. A second crop of 3.478 g, also analytically pure, afforded
(S)-N-[[3-[3-F lu or o-4-(1,1-d ioxot h iom or p h olin -4-yl)-
p h en yl]-2-oxo-5-oxa zolid in yl]m eth yl]a ceta m id e (8).
A
slurry of (S)-N-[[3-[3-fluoro-4-(4-thiomorpholinyl)phenyl]-2-oxo-
5-oxazolidinyl]methyl]acetamide (6; 280 mg, 0.792 mmol) in
25% water/acetone solution (12 mL) was treated with solid
4-methylmorpholine N-oxide (278 mg, 2.38 mmol). Next,
osmium tetraoxide (0.037 mL of a 2.5 wt % solution in 2-methyl-
2-propanol, 15 mol %) was added dropwise. The reaction
mixture was allowed to stir for 16 h under N2 at room
temperature. At this time, the reaction was determined to be
complete by TLC (10% MeOH/CHCl3, short wave UV). The
reaction was quenched with saturated sodium bisulfite (15 mL)
and the mixture extracted into CH2Cl2 (4 × 25 mL). The
combined organic layers were then washed again with satu-
rated sodium bisulfite (100 mL) and once with brine (100 mL).
These aqueous portions were back-extracted with more CH2-
Cl2 (2 × 50 mL). All of the organic layers were combined, dried
over anhydrous Na2SO4, filtered, and then concentrated under
reduced pressure to yield an off-white solid. This solid was
adsorbed onto silica gel (2.5 g) and chromatographed on more
silica gel (30 g, packed with 10% CH3CN/CHCl3), eluting with
a gradient of 1-5% MeOH in 10% CH3CN/CHCl3, to give 224
mg (73%) of the title compound as a white solid: mp 202-203
°C; [R]D -20° (c 1.04, DMSO); IR (mull) 1744, 1652, 1644, 1519,
1442, 1424, 1322, 1231, 1219, 1193, 1128, 1048, 809, 749 cm-1
;
1H NMR (300 MHz, DMSO-d6) δ 8.25 (br t, 1H, J ) 5.5 Hz),
7.51 (dd, 1H, J ) 2.3, 15.7 Hz), 7.22 (dd, 1H, J ) 8.9, 8.9 Hz),
7.18 (dd, 1H, J ) 2.4, 8.9 Hz), 4.76-4.66 (m, 1H), 4.09 (dd,
1H, J ) 9.0, 9.0 Hz), 3.70 (dd, 1H, J ) 6.4, 9.0 Hz), 3.48-3.43
(m, 4H), 3.40 (“t”, 2H, J ) 5.5 Hz), 3.29-3.24 (m, 4H), 1.83 (s,
3H); MS m/ z (rel intensity) 385 (M+, 74), 341 (87), 282 (32),
257 (100), 163 (27), 85 (80), 56 (45), 42 (46); HRMS calcd for
C16H20N3O5FS 385.1108, found 385.1102. Anal. (C16H20N3O5-
FS‚0.4H2O) C, H, N.
o
a combined yield of 81%: mp 186.5-187.0 C; [R]D -8° (c 1.00,
CHCl3); IR (mull) 1749, 1746, 1641, 1656, 1518, 1448, 1419,
Ack n ow led gm en t. The authors acknowledge the
technical assistance of J ason Zamkoff in performing the
in vitro studies for the MAC isolates. Mark J . Ackland,
Robert A. Anstadt, Martin R. Howard, Iain J . Martin,
and Mary Lou Sedlock are thanked for the preliminary
1
1225, 1215, 1158, 1106, 1083, 867 cm-1; H NMR (300 MHz,
CDCl3) δ 7.42 (dd, 1H, J ) 2.6, 14.0 Hz), 7.06 (ddd, 1H, J )
1.0, 2.6, 8.8 Hz), 6.95 (dd, 1H, J ) 9.0, 9.0 Hz), 6.61 (br t, 1H,
J ) 6.0 Hz), 4.81-4.72 (m, 1H), 4.02 (dd, 1H, J ) 9.0, 9.0 Hz),
3.75 (dd, 1H, J ) 6.7, 9.1 Hz), 3.71-3.55 (m, 2H), 3.32-3.27

Novel Oxazolidinone with Antimycobacterial Activity
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3 685
and Structure-Activity Relationships of New Tropone-Substi-
tuted Oxazolidinone Antibacterial Agents. Abstracts of Papers;
35th Interscience Conference on Antimicrobial Agents and
Chemotherapy, San Francisco, CA, September 1995; American
Society for Microbiology: Washington, DC, 1995; Abstract No.
F206.
pharmacokinetic assessment of 6. The authors also
acknowledge the chronic toxicity testing of 6 conducted
by J ohn R. Palmer, Richard C. Piper, and Thomas F.
Platte. We are grateful to workers in the Structural,
Analytical and Medicinal Chemistry Unit of The Upjohn
Co. for elemental analyses, specific rotations, and IR and
mass spectral data.
(11) 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.; Zurenko, G. E.; Ford, C. W. Synthesis and
Antibacterial Activity of U-100592 and U-100766, Two Oxazo-
lidinone Antibacterial Agents for the Potential Treatment of
Multidrug-Resistant Gram-Positive Bacterial Infections. J . Med.
Chem. 1996, 39, 673-679.
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Primary Amines via a General Method Utilizing the Staudinger
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Organic Compounds; Damani, L. A., Ed.; Ellis Horwood Ltd.:
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