Piperazinyl oxazolidinone antibacterial agents containing a pyridine, diazene, or triazene heteroaromatic ring

Article abstract of DOI:10.1021/jm980274l

Oxazolidinones are a novel class of synthetic antibacterial agents active against gram-positive organisms including methicillin-resistant Staphylococcus aureus as well as selected anaerobic organisms. Important representatives of this class include the mo

Full text of DOI:10.1021/jm980274l

J . Med. Chem. 1998, 41, 3727-3735
3727
P ip er a zin yl Oxa zolid in on e An tiba cter ia l Agen ts Con ta in in g a P yr id in e,
Dia zen e, or Tr ia zen e Heter oa r om a tic Rin g
J ohn A. Tucker,* Debra A. Allwine, Kevin C. Grega, Michael R. Barbachyn,1 J ennifer L. Klock,
J enifer L. Adamski, Steven J . Brickner,2 Douglas K. Hutchinson, Charles W. Ford, Gary E. Zurenko,
Robert A. Conradi, Phillip S. Burton, and Randy M. J ensen
Discovery Research, Pharmacia & Upjohn, 7000 Portage Road, Kalamazoo, Michigan 49001
Received May 4, 1998
Oxazolidinones are a novel class of synthetic antibacterial agents active against gram-positive
organisms including methicillin-resistant Staphylococcus aureus as well as selected anaerobic
organisms. Important representatives of this class include the morpholine derivative linezolid
2, which is currently in phase III clinical trials, and the piperazine derivative eperezolid 3. As
part of an investigation of the structure-activity relationships of structurally related
oxazolidinones, we have prepared and evaluated the antibacterial properties of a series of
piperazinyl oxazolidinones in which the distal nitrogen of the piperazinyl ring is substituted
with a six-membered heteroaromatic ring. Compounds having MIC values e 2 µg/mL vs
selected gram-positive pathogens were discovered among each of the pyridine, pyridazine, and
pyrimidine structural classes. Among these the cyanopyridine 17, the pyridazines 25 and 26,
and the pyrimidine 31 exhibited in vivo potency vs S. aureus comparable to that of linezolid.
In tr od u ction
selection studies demonstrated that eperezolid- and
linezolid-resistant mutants exist with a frequency of
<10-9 among selected staphylococcal species.15 Serial
passage studies using drug gradient plates performed
with these oxazolidinones failed to find evidence for the
rapid development of resistance.17 Mechanism of action
studies demonstrated that eperezolid binds specifically
to the 50s ribosomal subunit and prevents formation of
a functional initiation complex.19,20 The utility of lin-
ezolid in the treatment of gram-positive infections is
currently being examined in phase III clinical trials.
Previous reports have described the design and
synthesis of a series of oxazolidinone antibacterial
agents related to linezolid by replacement of the mor-
pholine ring with an alkyl-, acyl, or sulfonyl-substituted
piperazinyl ring.13 In the present work we describe the
synthesis and antibacterial activity of a related family
of compounds A in which the distal nitrogen of the
Oxazolidinones are a new class of synthetic antibacte-
rial agents with activity against anaerobic and gram-
positive aerobic bacteria.3 This class was discovered
through broad screening and is exemplified by the
erstwhile clinical candidate DuP 721 (1).4-6 Early
studies of DuP 721 revealed a number of attractive
features, including activity against problematic resis-
tant pathogens, a lack of cross resistance with existing
antimicrobial agents, oral activity in animal models of
human infection, and a unique mechanism of action
involving inhibition of a very early stage of protein
synthesis. Despite these attractive features the devel-
opment of DuP 721 was terminated.7
The potential of this new antibacterial class stimu-
lated an exploratory chemical analogue program in our
discovery research laboratories. Two oxazolidinone
analogues, linezolid 2 (PNU-100766) and eperezolid 3
(PNU-100592), emerged as lead compounds with the
piperazinyl ring is substituted with a six-member het-
eroaromatic ring.
best overall combination of positive attributes.8-13 Lin-
ezolid and eperezolid exhibit useful levels of activity
against staphylococci (including methicillin-resistant
Staphylococcus aureus [MRSA] and methicillin-resistant
Staphylococcus epidermis [MRSE]), enterococci (includ-
ing vancomycin-resistant strains), and pneumococci
(including penicillin-resistant strains).14-18 Single-step
Resu lts a n d Discu ssion
Ch em istr y. The first compound of this series was
prepared from the commercially available piperazine
derivative 4. The chemistry involved in building up the
oxazolidinone ring and its acetamidomethyl substitu-
S0022-2623(98)00274-X CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/21/1998

3728 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Sch em e 1. Synthesis of Oxazoidinone 9
Tucker et al.
ents has been described previously.9-13 These methods
were readily applied to the preparation of compound 9
as shown in Scheme 1.
prepared by catalytic hydrogenation of 42 and 34,
respectively.
The in vitro and in vivo antibacterial activity exhib-
ited by 9 encouraged us to develop a synthetic route
which would more readily lend itself to the rapid
exploration of the structure-activity relationship (SAR)
of related compounds. In particular it seemed likely
that at least some analogues A might readily be
prepared in a single step by nucleophilic aromatic
substitution reactions of haloaromatics with the readily
available piperazine 10.8 In the event, a wide variety
Str u ctu r e-Activity Rela tion sh ip s. In vitro and
in vivo antibacterial assays were performed following
previously described methods.8 The results of these
studies are summarized in Table 1. Analogues having
in vitro activity comparable to eperezolid were discov-
ered among each of the pyridine, pyridazine, pyrazine,
and pyrimidine structural classes. In the pyridine and
pyrimidine classes the in vitro potency is relatively
independent of whether the heteroaromatic ring is
attached to the rest of the molecule through its 2- or
4-position (compounds 9 and 20; compounds 31 and 33).
These results suggest that the binding of these ana-
logues does not involve strong hydrogen bonds between
the binding site and the heteroaromatic ring nitrogens.
The effect on in vitro activity of substituents on the
heteroaromatic ring is in most cases modest and follows
similar trends in each of the heterocyclic classes.
Within each heterocyclic class, the unsubstituted het-
erocycle(s) (e.g., 9, 20, 25, 31, 33, and 36) exhibits
activity roughly similar to that of eperezolid. Attach-
ment of a single halogen atom (12, 13, 34, and 37),
methyl group (15, 19, 21, and 26), or cyano group (16,
17, and 18) in place of a hydrogen has little effect on
potency. The presence of multiple halogen substituents
exerts either a modest (14 and 30) or a strongly negative
(22 and 23) effect. The activity difference between the
active trihalogenated analogue 14 and the inactive
tetrahalogenated analogues 22 and 23 may arise in part
of analogues A in which Ar is a six-membered het-
eroaromatic ring were readily prepared by this method
using reaction temperatures between 0 and 135 °C.
Dimethylpropyleneurea (DMPU) and ethoxyethanol
were especially useful solvents for these reactions. In
some cases the use of dimethylformamide (DMF) led to
the production of a side product which was difficult to
separate from the desired product. With the exception
of 9, 25, and 33, all of the target compounds of the
present work were prepared by nucleophilic aromatic
substitution reactions. Compounds 25 and 33 were

Piperazinyl Oxazolidinone Antibacterial Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3729
Ta ble 1. In Vitro and in Vivo Antibacterial Activity of N-Heteroaryl Piperazinyl Oxazolidinones

3730 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Ta ble 1 (Continued)
Tucker et al.
a
Strains: S.a.-1 ) Staphylococcus aureus UC 9213 (methicillin-susceptible); S.a.-2 ) S. aureus UC 6685 (methicillin-resistant); S.e. )
Staphylococcus epidermis UC 12084 (methicillin-resistant); E.f. ) Enterococcus faecalis UC 9217; S.p. ) Streptococcus pneumoniae UC
b
9912. PO administration; 95% confidence limits are -50% and +100% of the nominal value. c Positive control is eperezolid administered
d
PO unless otherwise indicated. Eperezolid and linezolid MIC and ED50 values taken from refs 8 and 14. e Positive control is vancomycin
administered sc.
from conformational differences, as 22 and 23 are
unique among the compounds of this report in that their
heteroaromatic rings bear substituents in both postions
ortho to the point of attachment to the piperazine ring.
Hydrophilic substituents such as amino (compounds 35
and 39) and acetamido (29) were associated with
reduced potency.
ezolid. Several other compounds which exhibited prom-
ising in vitro activity gave disappointing results in vivo.
One interesting trend is that all five of the compounds
tested in vivo that have a halogen substituent attached
to the heteroaromatic ring gave ED50 values at least
2-fold and in several cases greater than 6-fold higher
than that of eperezolid.
Representative compounds having in vitro activity
versus S. aureus were tested for in vivo activity against
this organism in a lethal systemic mouse model. The
ED50 values from this screen correspond the amount of
drug required (mg/kg bodyweight/dose) to reduce mor-
tality by 50%. These data are presented in Table 1
along with ED50 values obtained in the same trial for
eperezolid as a positive control. The pyridine 17, the
pyridazines 25 and 26, and the pyrimidine 31 each
exhibited in vivo potency comparable to that of eper-
In view of the known propensity of halogen substit-
uents on heteroaromatic rings to undergo facile nucleo-
philic aromatic substitution reactions, a series of ex-
periments was performed to determine whether the
disappointing in vivo activities of representative halo-
genated analogues 13, 34, and 30 were due to facile
glutathione conjugation or some other cause. No reac-
tion was observed when compounds 13 and 34 were
heated for several hours at 40 or 80 °C with glutathione
(GSH) in pH 10.4 carbonate buffer. In contrast, two new

Piperazinyl Oxazolidinone Antibacterial Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3731
Ta ble 2. Permeability Assessment of Oxazolidinones A across
a Monolayer of Caco-2 Cells in the Presence and Absence of the
GST Inhibitor Ethacrynic Acid
compounds were observed by HPLC when the dichlo-
ropyridazine 30 was treated with glutathione in carbon-
ate buffer at 40 °C, and a corresponding decrease in the
area of the peak corresponding to 30 was observed.
Additional experiments were designed to assess the
susceptibility of these compounds to enzymatic glu-
tathionylation and to evaluate the effect of the halogen
substituent on membrane permeability.
The Caco-2 cell line derives from a human colon
adenocarcinoma. These cells spontaneously differenti-
ate in culture, producing monolayers of cells displaying
morphological and biochemical characteristics of normal
intestinal absorptive cells.21 For this reason, Caco-2 cell
monolayers are frequently used to model intestinal
absorption and transport.22 Because these cells contain
relatively high levels of GSH23 and glutathione S-
transferase (GST) enzymes24 and exhibit little or no
cytochrome P-450 activity, they also provide a useful
tool for assessing susceptibility to enzymatic GSH
conjugation.25
Compounds 13, 34, 30, and their analogues 9, 33, and
25 which lack halogen substitution on the heteroaro-
matic ring were assayed for permeability across a
Caco-2 cell monolayer using a previously described
procedure.26,27 Each compound was combined with
phosphate-buffered saline to a target concentration of
40 µM. After sonication, the solutions were equilibrated
to 25 °C and filtered. These donor solutions were used
to determine permeability coefficients for transport
across Caco-2 cell monolayers into a buffered receiver
solution. The susceptibility of each compound to glu-
tathione conjugation was assayed by performing each
measurement in the presence and absence of the broad
spectrum GST inhibitor ethacrynic acid (EA). Mass
recovery was monitored by summing the total drug in
the donor compartment at the end of the experiment
with the total drug recovered from the receiver solu-
tions. The ratio of this amount to the initial donor
amount corresponds to the mass recovery.
a
Abbreviations: No. ) compound number, EA ) ethacrynic
acid, Pe ) permeability coefficient (units of 10-6 cm/s), M.R. ) mass
recovery.
can also present a barrier to oral absorption by reducing
the lumenal to serosal concentration gradient.29 Taken
in sum, these studies suggest that the diminished in
vivo efficacy of the halogenated compounds of this report
relative to their unhalogenated counterparts can be
explained by either poor solubility or metabolic poten-
tial.
Con clu sion s. Thirty-one new piperazinyl oxazoli-
dinone antibacterial agents substituted with a six-
member heteroaromatic ring on the distal piperazine
nitrogen were prepared and examined for in vitro and
in vivo antibacterial activity. Several of these com-
pounds exhibit in vitro and in vivo activity vs S. aureus
comparable to that of eperezolid. A series of experi-
ments designed to elucidate the factors responsible for
the unexpectedly poor in vivo activity of orally dosed
analogues bearing halogen substituents on the het-
eroaromatic ring was performed. These experiments
suggest that the negative impact of halogen substituents
may be related to facile glutathione conjugation or to
the reduced solubility of the resulting analogues.
Permeabilities and mass recoveries for the six com-
pounds in the presence and absence of EA are recorded
in Table 2. Mass recovery was close to 100% for all
compounds except 13 and 30. The latter compound was
clearly metabolized in transit as indicated by the nearly
complete restoration of mass recovery and the 2-fold
increase in apparent permeability caused by the GST
inhibitor EA. In addition, the same adduct peaks were
observed as were seen when the compound was treated
with GSH in aqueous base. Compound 13 displayed no
evidence of adduct formation and neither mass recovery
nor apparent permeability was affected by EA. The
relatively high lipophilicity and low solution concentra-
tion of 13 (necessitated by poor solubility) may have
resulted in a significant fraction of the compound being
retained by the cells.28
Exp er im en ta l Section
All of the compounds displayed moderate to high
permeability in Caco-2 monolayers, suggesting that
intestinal permeability is not likely to be a significant
barrier to oral absorption. Intestinal metabolism may
be a problem only for 30 which undergoes facile glu-
tathionylation. In contrast to their counterparts 9 and
33 which lack halogen substitution on the heteroaro-
matic ring, both 13 and 34 were significantly less
soluble than 40 µM. Low solubility or slow dissolution
Gen er a l Meth od s. 1H NMR spectra were recorded at 300
or 400 MHz; chemical shifts are reported relative to tetra-
methylsilane as an internal standard at 0.00 ppm. Infrared
spectroscopy was performed on mineral oil mulls. Combustion
analyses were performed for each novel compound and agree
within (0.4% of the calculated theoretical value. All reactions
were performed in oven-dried glassware which was flushed
with nitrogen prior to cooling. DMF, DMPU, ethoxyethanol,
triethylamine, and ethyldiisopropylamine were dried over

3732 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Tucker et al.
activated 3 Å molecular sieves for 24 h before use. Other
solvents and reagents were used as supplied. Both the in vitro
and in vivo biological screens have been described in previous
publications.8
of 2-chloro-3-cyanopyridine, and 0.300 mL (1.7 mmol) of
diisopropylethylamine was added 20 mL of EtOH. The solu-
tion was transferred to a sealed tube and heated to 110 °C for
7 days. After cooling to 25 °C the white solid precipitate was
collected by filtration to yield 75.3 mg (28%) of the title
compound: mp 190-192 °C; MS (EI) m/z (rel intensity) 438
(M+, 46). Anal. (C22H23FN6O3‚0.3H2O) C, H, N.
Gen er a l P r oced u r e A. The appropriate pyridine (1.1
mmol), 10 (1.2 mmol), and diisopropylethylamine (2.1 mmol)
were stirred in n-butanol (10 mL) for 2-3 days at 100-110
°C. Upon cooling, a precipitate formed and was collected by
filtration.
3-F lu o r o -4-[4-(2-p y r id y l)p ip e r a zin -1-y l]n it r o b e n -
zen e, 6. A stirred solution of 1-(2-pyridyl)piperazine (5.00 g,
32.8 mmol), diisopropylethylamine (6.81 mL, 39.4 mmol), and
3,4-difluoronitrobenzene (3.99 mL, 36.1 mmol) in CH3CN (300
mL) was stirred at 25 °C. After 2 days, the solvent was
removed under reduced pressure. The crude yellow solid was
dissolved in CH2Cl2, washed with H2O followed by brine, dried
(Na2SO4), filtered, and concentrated under reduced pressure.
The crude orange solid was triturated with Et2O to give 6.63
g (67%) of product as a orange solid: mp 149.5-150 °C; MS
(EI) m/z (rel intensity) 302 (M+, 15). Anal. (C15H15FN4O2) C,
H, N.
N-Ben zyloxycar bon yl-3-flu or o-4-[4-(2-pyr idyl)piper azin -
1-yl]a n ilin e, 7. A vigorously stirred solution of 3-fluoro-4-
[4-(2-pyridyl)piperazin-1-yl]nitrobenzene 6 (4.00 g, 13.2 mmol)
in 15% aqueous THF (300 mL) was charged with 10% Pd-C
(500 mg). The resulting mixture was placed under an atmo-
sphere of H2. After 9 h, the mixture was cooled to 0 °C and
treated with NaHCO3 (4.44 g, 52.8 mmol) followed by benzyl-
oxycarbonyl chloride (2.27 mL, 15.8 mmol). After 1 h, the
solvent was removed under reduced pressure. The remaining
aqueous suspension was extracted with EtOAc. Purification
by silica gel chromatography (5-20% EtOAc/hexanes) gave
3.57 g (67%) of the title compound as a pale yellow solid: mp
168-169.5 °C; MS (EI) m/z (rel intensity) 406 (M+, 8). Anal.
(C23H23FN4O2) C, H, N.
(R)-[3-[3-F lu or o-4-(4-(2-p yr id yl)p ip er a zin -1-yl)p h en yl]-
2-oxo-5-oxa zolid in yl]m eth a n ol, 8. A stirred solution of
N-benzyloxycarbonyl-3-fluoro-4-[4-(2-pyridyl)piperazin-1-yl]-
aniline 7 (3.24 g, 8.00 mmol) in anhydrous THF (80 mL) was
placed under an atmosphere of N2 and cooled to -78 °C. The
cold solution was treated with n-BuLi (5.05 mL, 8.09 mmol).
After 20 min, the solution was treated with R-glycidyl butyrate
(1.14 mL, 8.09 mmol) and then allowed to warm to 25 °C. After
2 h, the mixture was treated with saturated aqueous am-
monium chloride solution (5 mL) and concentrated under
reduced pressure. The remaining material was dissolved in
CH2Cl2, washed with H2O followed by brine, dried (Na2SO4),
filtered, and concentrated under reduced pressure to an amber
solid. Purification by recrystallization from CHCl3/Et2O (4:1)
gave 2.57 g (86%) of the title compound as an off-white solid:
mp 157-161 °C; HRMS (EI) (C19H21FN4O3) calcd 372.1598,
found 372.1593. Anal. (C19H21FN4O3) C, H, N.
(S)-N-[[3-[3-Flu or o-4-(4-(2-p yr id yl)p ip er a zin -1-yl)p h en -
yl]-2-oxo-5-oxa zolid in yl]m eth yl]a ceta m id e 9. A solution
of (R)-[3-[3-fluoro-4-(4-(2-pyridyl)piperazin-1-yl)phenyl]-2-oxo-
5-oxazolidinyl]methanol 8 (1.02 g, 2.74 mmol) in dry CH2Cl2
(55 mL) was cooled to 0 °C. The solution was treated with
Et3N (420 µL, 3.02 mmol) followed by methanesulfonyl chloride
(234 µL, 3.02 mmol). After 0.5 h, the solution was washed
with H2O followed by brine, dried (Na2SO4), filtered, and
concentrated under reduced pressure to give a yellow solid
(1.19 g): mp 165.5-166.5 °C; MS (EI) m/z (rel intensity) 450
(M+, 8).
(S)-N-[(3-{4-[4-(5-Cya n o-2-p yr id in yl)-1-p ip er a zin yl]-3-
flu or op h e n yl}-2-oxo-1,3-oxa zolid in -5-yl)m e t h yl]a ce t -
a m id e, 17. According to procedure A, 2-chloro-5-carboni-
trilepyridine was heated to 110 °C for 2 days in n-butanol.
The title compound was recrystallized from 10 mL of 1-pro-
panol to give 75.0 mg (28%) of a yellow solid: mp 175-177
°C. MS (EI) m/z (rel intensity) 438 (M+, 41). Anal. (C22H23
FN6O3‚1.1H2O) C, H, N.
-
(S)-N-[(3-{4-[4-(3-Cya n o-4,6-d im eth yl-2-p yr id in yl)-1-p i-
p er a zin yl]-3-flu or op h en yl}-2-oxo-1,3-oxa zolid in -5-yl)-
m eth yl]a ceta m id e, 19. According to procedure A, 2-chloro-
4,6-dimethyl-3-carbonitrilepyridine was heated to 110 °C for
2 days. The precipitate was recrystallized from EtOH to give
61.2 mg (12%) of the title compound: mp 221.5-222 °C.
HRMS (EI) (C24H27FN6O3) calcd 466.2129, found 466.2117.
Anal. (C24H27FN6O3‚0.5H2O) C, H, N.
(S)-N-[[3-[3-F lu or o-4-(4-(2,3,5,6-tetr a ch lor o-4-p yr id yl)-
p ip er a zin -1-yl)p h en yl]-2-oxo-5-oxa zolid in yl]m eth yl]a cet-
a m id e, 23. This compound was made using pentachloropyr-
idine according to procedure A except that 1-propanol was the
solvent. The reaction mixture was refluxed at 100 °C for 3
days. The crude product was then suspended in 50 mL of
EtOAc, and the mixture was stirred for 24 h. After filtration
this process was repeated in 20 mL of EtOAc for 2 days to
yield 83.5 mg (15%) of the desired product as a solid: mp 219-
220 °C; MS (EI) m/z (rel intensity) 549 (M+, 16). Anal.
(C21H20Cl4FN5O3‚0.75H2O) C, H, N.
(S)-N-[(3-{4-[4-(6-Br om o-2-p yr id in yl)-1-p ip er a zin yl]-
3-flu or op h en yl}-2-oxo-1,3-oxa zolid n -5-yl)m et h yl]a cet -
a m id e, 13. This compound was prepared by applying proce-
dure A to 2,6-dibromopyridine in refluxing ethoxyethanol as
the solvent with a reaction time of 26 h. The product was
purified by silica gel chromatography (3.5% MeOH in CH2-
Cl2). The title compound was obtained as 230 mg (29%) of a
yellow-green powder: mp 173-175 °C; MS (ESI+): m/z 492
(M + H+), 494 (M + H+ + 2). Anal. (C21H23BrFN5O3) C, H,
N.
(S)-N-[[3-[3-F lu or o-4-(4-(2,3,5,6-tetr a flu or o-4-p yr id yl)-
p ip er a zin -1-yl)p h en yl]-2-oxo-5-oxa zolid in yl]m eth yl]a cet-
a m id e, 22. To a mixture of 200 mg (0.54 mmol) of 10 and
0.187 mL (1.07 mmol) of diisopropylethylamine was added 10
mL of EtOH. The suspension was cooled to 0 °C. Pentafluo-
ropyridine (0.059 mL, 0.536 mmol) was added to 5 mL of
EtOH, and the resulting solution was cooled to 0 °C. The
suspension of 10, diisopropylethylamine, and EtOH was added
to the cold pentafluoropyridine solution. This mixture was
stirred in an ice bath for 1.5 h. The mixture was allowed to
stir at room temperature for 24 h, during which a white
precipitate formed and was collected to give the desired
product: mp 205-206 °C; MS (EI) m/z (rel intensity) 485 (M+,
99). Anal. (C21H20F5N5O3) C, H, N.
A mixture of this solid (870 mg, 1.93 mmol) in 1:1:1 THF/
2-propanol/14 M aqueous ammonium hydroxide solution (12
mL) was heated in a heavy walled sealed tube to 95 °C for 16
h. After this time the solvent was removed under reduced
pressure. The remaining crude solid was dissolved in CH2Cl2
(40 mL), treated with pyridine (328 mL, 4.05 mmol) followed
by Ac2O (201 µL, 2.12 mmol), and allowed to stir at 25 °C for
0.5 h. Then the reaction solution was washed with H2O
followed by brine, dried (Na2SO4), filtered, and concentrated
under reduced pressure. Purification by silica gel chromatog-
raphy (1.5-3.5% MeOH/CHCl3) gave 476 mg (60%) of the title
compound as a off-white solid: mp 190-192 °C; MS (EI) m/z
(rel intensity) 413 (M+, 11). Anal. (C21H24FN5O3) C, H, N.
(S)-N-[[3-[3-F lu or o-4-(4-(6-m eth yl-2-p yr id yl)p ip er a zin -
1-yl)p h en yl]-2-oxo-5-oxa zolid in yl]m eth yl]a ceta m id e, 15.
To 1.6 mL of ethoxyethanol were added 0.300 g (0.80 mmol)
of 10, 0.410 g (3.69 mmol) of 2-fluoro-6-methylpyridine, and
0.31 mL (1.78 mmol) of diisopropylethylamine. The mixture
was refluxed for 27 h. A light brown solid precipitated upon
cooling. It was recrystallized from butanol to yield 100 mg
(11%) of the title compound: MS (ES+) m/z 428.2 (M + H).
Anal. (C22H26FN5O3‚0.5H2O) C, H, N.
(S)-N-[[3-[3-F lu or o-4-(4-(3-cya n o-2-p yr id yl)p ip er a zin -
1-yl)p h en yl]-2-oxo-5-oxa zolid in yl]m eth yl]a ceta m id e 18.
To a mixture of 200 mg (0.54 mmol) of 10, 84.6 mg (0.61 mmol)
(S)-N-[[3-[3-Flu or o-4-(4-(4-p yr id yl)p ip er a zin -1-yl)p h en -
yl]-2-oxo-5-oxazolidin yl]m eth yl]acetam ide, 20. A mixture

Piperazinyl Oxazolidinone Antibacterial Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3733
of 3.2 mL of ethoxyethanol, 0.600 g (1.61 mmol) of 10, 0.266 g
(1.77 mmol) of 4-chloropyridine hydrochloride, and 0.925 mL
(5.31 mmol) of diisopropylethylamine was refluxed for 21 h.
The mixture was partitioned between excess aqueous potas-
sium carbonate solution and a 1:1 mixture of EtOAc and EtOH.
The organic layer was dried (MgSO4) and filtered. After the
solution stood for 3 days at 25 °C, the product precipitated
from this solution and it was recovered by filtration. The
product was purified by silica gel chromatography eluting with
8% ammonia-saturated EtOH in CH2Cl2 to give 127 mg (53%
yield): MS (ES+) m/z 414 (M + H)+. Anal. (C22H24FN5O3) C,
H, N.
(S)-N-[[3-[3-F lu or o-4-(4-(2-m eth yl-4-p yr id yl)p ip er a zin -
1-yl)p h en yl]-2-oxo-5-oxa zolid in yl]m eth yl]a ceta m id e, 21.
A mixture of 1.0 g (2.68 mmol) of 10, 0.408 g (3.21 mmol) of
4-chloro-2-methylpyridine, and 1.40 mL (8.04 mmol) of Hunig’s
base was refluxed for 1 h in 6 mL of ethoxyethanol. The
product precipitated out of solution upon adddition of 10 mL
of H2O. The solid was collected by filtration, washed with H2O,
and dried to give the title compound: mp 188-189 °C; MS
(ESI-) for m/z 426.2 (M-H)-. Anal. (C22H26FN5O3‚H2O) C,
H, N.
filtered, and the solvent was evaporated at reduced pressure.
The residue was recrystallized from 2-propanol to give the title
compound as 0.20 g (33%) of a white solid: mp 197-198 °C;
MS (EI) m/z (rel intensity) 414 (M+, 48). Anal. (C20H23FN6O3)
C, H, N.
(S)-N-[[3-[3-F lu or o-4-[4-(4-p yr id a zin yl)]-1-p ip er a zin yl]-
p h en yl-2-oxo-5-oxa zolid in yl]m et h yl]a cet a m id e, 33.
A
suspension of 34 (150 mg) in 15 mL of 2:1 absolute EtOH/
EtOAc was agitated and treated with 0.3 mL of 3.0 N
hydrochloric acid. Approximately 50 mg of 5% palladium on
carbon was added to the resulting clear solution, and the
mixture was agitated under an atmosphere of 50 psi hydrogen
gas for 3 days. The catalyst was removed by filtration, and
the solvent was evaporated at reduced pressure. The residue
was partitioned between 20 mL of EtOAc and 20 mL of 1 M
dipotassium hydrogen phosphate solution. The solvent was
evaporated at reduced pressure. The residue was chromato-
graphed on 10 g of silica gel eluting with 93:7 CH2Cl2/absolute
EtOH. The white solid thus obtained was washed with
hexanes and dried to give 52 mg (36%) of the title compound:
mp 145-147 °C; HRMS (EI) (C20H23FN6O3) calcd 414.1816,
found 414.1808. Anal. (C20H23FN6O3‚0.15C6H14‚0.75H2O) C,
H, N. The presence of 0.15 equiv of hexane, not readily
removed by heating under vacuum, was confirmed by 1H NMR.
Gen er a l P r oced u r e B. A mixture of triethylamine (5
mmol), 10 (1.4 mmol), and the appropriate aryl halide (1.5
mmol) was stirred in DMF (5-12 mL) overnight. The mixture
was partitioned between EtOAc and H2O. The organic phase
was dried (MgSO4), and the solvent was evaporated at reduced
pressure.
(S)-N-[(3-{4-[4-(2-Ch lor o-4-p yr im id in yl)-1-p ip er a zin yl]-
3-flu or op h en yl}-2-oxo-1,3-oxa zolid in -5-yl)m et h yl]a cet -
a m id e, 34. According to procedure B, 2,4-dichloropyrimidine
was stirred in DMF for 16 h at 25 °C. The residue was
triturated with toluene and dried in a stream of air to give
0.499 g (83%) of the title compound as a white powder: mp
189-191 °C; MS (EI) m/z (rel intensity) 448 (M+, 6). Anal.
(C20H22ClFN6O3‚0.15H2O) C, H, N.
(S)-N-[(3-{4-[4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-1-piperazinyl]-
3-flu or op h en yl}-2-oxo-1,3-oxa zolid in -5-yl)m et h yl]a cet -
am ide, 38. Procedure B was applied to 2-chloro-4,6-dimethoxy-
1,3,5-triazine. The crude product was recrystallized from
EtOAc and hexanes to give 0.419 g (65%) of the title compound
as a white solid: mp 206.5-207 °C; HRMS (EI) (C21H26FN7O5)
calcd 475.1979, found 475.1987. Anal. (C21H26FN7O5) C, H,
N.
(S)-N-[(3-{4-[4-(4-Chloro-6-propoxy-1,3,5-triazin-2-yl)-1-pipera-
zinyl]-3-fluorophenyl}-2-oxo-1,3-oxazolidin-5-yl)methyl]acetamide,
40. Procedure B was applied to 2,4-dichloro-6-n-propoxy-1,3,5-
triazine. The crude product was purified by silica gel chro-
matography (3-5% MeOH/CH2Cl2) to give 0.298 g (41%) of
material. This material was recrystallized from EtOAc to give
0.143 g (20%) of the title compound as a white solid: HRMS
(EI) (C22H27ClFN7O4) calcd 507.1797, found, 507.1797. Anal.
(C22H27ClFN7O4) C, H, N.
(S)-N-[[3-[3-F lu or o-4-[4-(3-p yr id a zin yl)]-1-p ip er a zin yl]-
p h en yl-2-oxo-5-oxa zolid in yl]m eth yl]a ceta m id e, 25. Pro-
cedure B was applied to 3,6-dichloropyridazine in 15 mL of
70 °C DMF using a reaction time of 7 days. The crude product
was purified by silica gel chromatography (3-10% MeOH/CH2-
Cl2) to give a light yellow solid. The combined product from
two such preparations was hydrogenated in 1:1 MeOH/EtOAc
using palladium black as a catalyst at 1 atm hydrogen
pressure. The crude material was purified on a preparative
TLC plate (4-8% MeOH/CH2Cl2) to give 0.114 g (54%) of the
desired material as a cream solid: mp 207-208 °C; HRMS
(EI) (C20H23FN6O3) calcd 414.1815, found 414.1818. Anal.
(C20H23FN6O3‚0.5H2O) C, H, N.
(S)-N-[[3-[3-F lu or o-4-(4-(2-q u in olin yl)p ip er a zin -1-yl)-
p h en yl]-2-oxo-5-oxa zolid in yl]m eth yl]a ceta m id e, 24.
A
mixture of 1.0 g (2.86 mmol) of 10, 0.53 g (3.21 mmol) of
4-chloroquinoline, and 1.40 mL (8.04 mmol) of Hunig’s base
was refluxed for 1 h in 6 mL of ethoxyethanol. The reaction
mixture was partitioned between EtOAc and 3 N NaOH.
During this procedure, a precipitate formed and was collected
by filtration. The solid was washed with H2O followed by
EtOAc and dried. The solid was recrystallized from H2O to
give 0.538 g of the title compound as a tan powder: mp 138-
141 °C; HRMS (FAB) (C25H26FN5O3+H1) calcd 464.2098, found
464.2105. Anal. (C25H26FN5O3‚0.6H2O) C, H, N.
(S)-N-[(3-{4-[4-[(4-Cya n o-2-p yr id in yl)-1-p ip er a zin yl]-3-
flu or op h e n yl}-2-oxo-1,3-oxa zolid in -5-yl)m e t h yl]a ce t -
a m id e, 16. A mixture of 5.4 mL of ethoxyethanol, 0.600 g
(1.61 mmol) of 10, 0.245 g (1.81 mmol) of 2-chloropyridine-4-
carbonitrile, and 0.620 mL (3.56 mmol) of diisopropylethyl-
amine was refluxed for 21 h. Upon cooling, brown crystals
formed. The crystals were washed with two portions of
butanol to yield 351 mg (50%) of the title compound as a tan
powder: mp > 200 °C; MS (ES+) m/z 461.3 (M + Na). Anal.
(C22H23FN6O3‚0.2H2O) C, H, N.
(S )-N -[[3-[3-F lu or o-4-(4-(3,5,6-t r iflu or o-2-p yr id yl)-
p ip er a zin -1-yl)p h en yl]-2-oxo-5-oxa zolid in yl]m eth yl]a cet-
a m id e, 14. A mixture of 5.4 mL of EtOH, 0.600 g (1.61 mmol)
of 10, 0.267 g (1.77 mmol) of 2,3,5,6-tetrafluoropyridine, and
0.620 mL (3.56 mmol) of diisopropylethylamine was refluxed
for 48 h. Upon cooling, a light brown solid formed. The solid
was collected by filtration to yield 450 mg (60%) of the title
compound as a tan powder: mp 176-178°C;HRMS (C21H21F4N5O3)
calcd 467.1581, found 467.1599. Anal. (C21H21F4N5O3‚0.5H2O)
C, H, N.
(S)-N-[[3-[3-F lu or o-4-(4-(6-flu or o-2-p yr id yl)p ip er a zin -
1-yl)p h en yl]-2-oxo-5-oxa zolid in yl]m eth yl]a ceta m id e, 12.
A mixture of 5.4 mL of ethoxyethanol, 0.60 g (1.61 mmol) of
10, 0.204 g (1.77 mmol) of 2,6-difluoropyridine, and 0.620 mL
(3.56 mmol) of diisopropylethylamine was refluxed for 16 h. A
light brown solid precipitated upon cooling. The solid was
collected by filtration and recrystallized from EtOH. The title
compound was isolated as 0.23 g (33% yield) of a tan solid:
mp 183-185 °C; MS (ES+) m/z 432.3 (M + H). Anal.
(C21H23F2N5O3‚0.2H2O) C, H, N.
(S)-N-[[3-[3-Flu or o-4-[4-(2-p yr im id in yl)]-1-p ip er a zin yl]-
p h en yl-2-oxo-5-oxa zolid in yl]m et h yl]a cet a m id e, 31.
A
solution of 0.50 g (1.34 mmol) of 10, 0.153 g (1.34 mmol) of
2-chloropyrimidine, and 0.55 mL (4.0 mmol) of triethylamine
in 5.0 mL of absolute EtOH was refluxed for 5.5 h and then
stirred 21 h at 25 °C. The mixture was then partitioned
between 50 mL of CH2Cl2 and 50 mL of a saturated aqueous
solution of sodium bicarbonate. The solvent was evaporated
at reduced pressure, and the residue was dissolved in 50 mL
of CH2Cl2 and treated with 15 g of silica gel. The mixture was
(S )-N -[[3-[3-F lu o r o -4-[4-[2-[4-(t r i flu o r o m e t h y l)-
pyr im idin yl]]]-1-piper azin yl]ph en yl-2-oxo-5-oxazolidin yl]-
m eth yl]a ceta m id e, 32. A mixture of 0.50 g (1.34 mmol) of
10 and 0.37 mL (2.68 mmol) of triethylamine in 5 mL of DMF
was treated with 0.16 mL (1.34 mmol) of 2-chloro-4-(trifluo-
romethyl)pyrimidine. The mixture was stirred at 25 °C for 4

3734 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Tucker et al.
days, and then it was poured into 50 mL of distilled H2O. The
precipitate was collected by filtration and recrystallized from
95% aqueous EtOH to give 0.42 g (65%) of the title com-
pound: mp 189-192 °C (dec); MS (EI) m/z (rel intensity) 482
(M+, 73). Anal. (C21H22F4N6O3) C, H, N.
the 5-position: HRMS (EI) (C20H21Cl2FN6O3) calcd 482.1036,
found 482.1032. Anal. (C20H21Cl2FN6O3‚0.5H2O) C, H, N, Cl.
Hydrogenation of this substance gave a compound which is
isomeric to 25 (1H NMR, MS).
(S)-N-[(3-{4-[4-(6-Ch lor o-4,5-d im eth yl-3-p yr id a zin yl)-1-
p ip er a zin yl]-3-flu or op h en yl}-2-oxo-1,3-oxa zolid in -5-yl)-
m eth yl]a ceta m id e, 28. According to procedure C, a DMPU
solution of 3,6-dichloro-4,5-dimethylpyridazine and 10 was
stirred at 60 °C for 3 days and at 110 °C for 2 days. The
reaction yielded 0.148 g (21%) of the desired material as a tan
solid: mp 253-254 °C; HRMS (EI) (C22H26ClFN6O3) calcd
476.1739, found 476.1732. Anal. (C22H26ClFN6O3) C, H, N,
Cl.
(S)-N-[[3-[3-F lu or o-4-[4-(2-p yr a zin yl)]-1-p ip er a zin yl]-
p h en yl-2-oxo-5-oxa zolid in yl]m eth yl]a ceta m id e, 36. Ac-
cording to procedure C, a DMPU solution of 10 with chloro-
pyrazine was stirred at 60 °C for 3 days and at 110 °C for 1
day. The reaction yielded 0.360 g (52%) of the desired material
as a yellow solid: mp 193-194.5 °C; HRMS (C20H23FN6O3)
calcd 414.1815, found 414.1816. Anal. (C20H23FN6O3‚0.4H2O)
C, H, N.
(S)-N-[(3-{4-[4-(6-Ch lor o-2-p yr a zin yl)-1-p ip er a zin yl]-3-
flu or op h e n yl}-2-oxo-1,3-oxa zolid in -5-yl)m e t h yl]a ce t -
a m id e, 37. A DMPU solution of 10 with 2,6-dichloropyrazine
was heated at 100 °C overnight. The reaction yielded 0.174 g
(27%) of the desired material as a light orange solid: mp 160-
164 °C; HRMS (EI) (C20H22ClFN6O3) calcd 448.1426, found
448.1418. Anal. (C20H22ClFN6O3‚0.4H2O) C, H, N, Cl.
(S)-N-[[3-[3-Fluoro-4-[4-[3-(6-methylpyridazinyl)]]-1-piper-
azin yl]ph en yl-2-oxo-5-oxazolidin yl]m eth yl]acetam ide, 26.
According to procedure C, a DMPU solution of 10 with
3-chloro-6-methylpyridazine was stirred 2 days at 90 °C. This
material was further purified on a preparative TLC plate (4%
MeOH/CH2Cl2) to give 0.060 g (10%) of the title compound as
a light tan solid: mp 237-238 °C; HRMS (EI) (C21H25FN6O3)
calcd 428.1972, found 428.1954. Anal. (C21H25FN6O3) C, H,
N.
(S)-N-[(3-{4-[4-(4,6-Dia m in o-1,3,5-tr ia zin -2-yl)-1-p ip er -
a zin yl]-3-flu or op h en yl}-2-oxo-1,3-oxa zolid in -5-yl)m eth yl]-
a cet a m id e h yd r och lor id e, 39. A mixture of 0.50 g (1.34
mmol) of 10, 0.195 g (1.34 mmol) of 4,6-diamino-2-chlorotria-
zene, 0.276 mL (2.0 mmol) of triethylamine, and 15 mL of 95:5
EtOH/H2O was refluxed for 48 h. The white solid precipitate
that formed upon cooling was collected by filtration. The solid
was dissolved in a warm mixture of 25 mL of 95:5 EtOH/H2O
and treated with 8 mL of 1.0 N hydrogen chloride in Et2O.
The solvent was evaporated at reduced pressure. The residue
was recrystallized from absolute EtOH to give 116 mg (19%)
of the title compound as a pale yellow solid: mp 172-174 °C;
MS (FAB) m/z (rel intensity) 446 (M + H, 99). Anal. (C19H24
FN9O3‚HCl‚1.5H2O) C, H, N.
-
(S)-N-[(3-{4-[4-(2,6-Dia m in o-4-p yr im id in yl)-1-p ip er a zi-
n yl]-3-flu or op h en yl}-2-oxo-1,3-oxa zolid in -5-yl)m et h yl]-
a ceta m id e, 35. A mixture of 0.30 g (0.80 mmol) of 10, 0.116
g (0.80 mmol) of 2,6-diamino-4-chloropyrimidine, 2.0 mL of
ethylene glycol, and 0.52 mL (3.0 mmol) of diisopropylethyl-
amine was stirred at 100 °C for 18 h. The mixture was cooled
and then partitioned between 3.0 N aqueous sodium hydroxide
solution and EtOAc, and the organic phase was dried (MgSO4).
The volume of the organic phase was reduced to 2 mL by
evaporation at reduced pressure. The resulting solution was
diluted with 18 mL of diethyl ether. The tan solid precipitate
was collected by filtration to give 83 mg (23%) of the title
compound: mp 215 °C (dec); MS (EI) m/z (rel intensity) 444
(M+, 1). Anal. (C20H25FN8O3‚0.5H2O) C, H, N.
Gen er a l P r oced u r e C. The remaining compounds were
made using this general procedure: A solution of triethylamine
(5 mmol), 10 (1.5 mmol), and the aryl halide (1.8 mmol) in
DMF (9 mL) or DMPU (3 mL) was stirred 1-7 days at 40-
110 °C. The solvent was removed via bulb-to-bulb distillation,
and the crude material was purified by silica gel chromatog-
raphy (3% MeOH/CH2Cl2).
(S)-N-{[3-(4-{4-[5-(Acetylam in o)-6-ch lor o-3-pyr idazin yl]-
1-p ip er a zin yl}-3-flu or op h en yl)-2-oxo-1,3-oxa zolid in -5-
yl]m eth yl}a ceta m id e, 29. According to procedure C, 4-acet-
amido-3,6-dichloropyridazine stirred with 10 in DMF at 55 °C
for 7 days. The product was then recrystallized from MeOH
to give 0.190 g (27%) of cream-colored amorphous solid: IR
(mull, cm-1) 3350 (m), 3259 (m), 3206 (m), 3171 (m), 3143 (m),
3126 (m), 3104 (m), 3091 (m), 3063 (m), 3044 (m), 1743 (sh),
1734 (s), 1712 (m), 1653 (s), 1543 (s), 1531 (s), 1516 (s), 1483
(s), 1224 (s); HRMS (FAB) (C22H25ClFN7O4 + H+) calcd
506.1718, found 506.1715. Anal. (C22H25ClFN7O4) C, H, N,
Cl.
(S)-N-[(3-{4-[4-(6-Ch lor o-5-m eth yl-3-p yr id a zin yl)-1-p i-
p er a zin yl]-3-flu or op h en yl}-2-oxo-1,3-oxa zolid in -5-yl)-
m eth yl]a ceta m id e, 27. According to procedure C, a DMF
solution 3,6-dichloro-4-methylpyridazine was heated with 10
at 55-95 °C for 7 days. Trituration with MeOH gave 0.135 g
(S)-N-[(3-{4-[4-(3-Acetyl-2-m eth ylim id a zo[1,2-b]p yr id -
a zin -6-yl)-1-p ip er a zin yl]-3-flu or op h en yl}-2-oxo-1,3-ox-
a zolid in -5-yl)m eth yl]a ceta m id e, 41. According to proce-
dure C, a DMPU solution of 10 and 3-acetyl-6-chloro-2-methyl-
imidazo[1,2-b]pyridazine30 was stirred at 100 °C for 4 days.
The material was further purified by preparative TLC (4-8%
MeOH/CH2Cl2) to give 0.101 g (26%) of the desired material
as a light yellow solid: mp 217-218 °C; HRMS (FAB) (C25H28
FN7O4 + H+) calcd 510.2265, found 510.2264. Anal. (C25H28
FN7O4‚0.5H2O) C, H, N.
-
-
Ca co-2 P er m ea bility Stu d ies. Solid test compounds were
weighed out and combined with phosphate-buffered saline
(containing 15 mM HEPES, 0.1% glucose, pH 7.2) to a target
concentration of 40 µM. After sonication the solutions were
equilibrated at 25 °C and filtered. Half of each solution was
supplemented with 50 µM EA. These donor solutions, with
and without EA, were used to determine permeability coef-
ficients across Caco-2 cell monolayers. The cell monolayers
were prepared and absorptive permeability was determined
as previously described.26,27 Briefly, 1.5 mL of solution was
placed on top of buffer-washed confluent Caco-2 monolayers
which had been grown on Costar Transwell filters. The
Transwell filters were placed in six well tissue culture plates
containing 2.5 mL buffer per well and were moved at regular
time intervals to wells containing fresh buffer. The appear-
ance of the test compound in these receiver solutions as a
function of time was used to calculate permeability coefficients.
Mass recovery was monitored by summing total drug in the
donor solution at the end of the experiment with total drug
recovered from receiver solutions. The ratio of this amount
to the initial donor solution amount yields percent mass
recovery.
(19%) of pure material as a white solid: HRMS (FAB) (C21H24
ClFN6O3 + H+) calcd 463.1660, found 463.1667. Anal. (C21H24
ClFN6O3) C, H, N, Cl.
-
-
(S)-N-[(3-{4-[4-(5,6-Dich lor o-4-p yr id a zin yl)-1-p ip er a zi-
n yl]-3-flu or op h en yl}-2-oxo-1,3-oxa zolid in -5-yl)m et h yl]-
a ceta m id e, 30. According to procedure C, 3,4,5-trichloropy-
ridazine and 10 were stirred at 40 °C overnight. The reaction
yielded 0.139 g (21%) of the title compound as a light tan
solid: mp 132-134 °C; 1H and 13C chemical shift assignments
were made using 2D heteronuclear one-bond (HMQC) and
multiple-bond (HMBC) NMR experiments conducted at 500
MHz (1H). Identification of the regioisomer was accomplished
using single-frequency irradiation nuclear Overhauser effect
(NOE) difference experiments conducted at 500 MHz. A strong
NOE observed between four downfield methylene protons on
the piperazine ring and the single proton of the pyridazine
ring shows that attachment of the pyridazine ring occurs at
A gradient HPLC system was used to determine solution
concentrations of the test compounds. A BDS-Hypersil-C18
column (150 × 4.6 mm2) from Keystone Scientific was used
with a gradient ranging from 10 to 45% acetonitrile. Peaks
were detected by UV absorbance at 254 nm.

Piperazinyl Oxazolidinone Antibacterial Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3735
C. W. Synthesis and Antibacterial Activity of [6,5,5] and [6,6,5]
Tricyclic Fused Oxazolidinones. Bioorg. Med. Chem. Lett. 1998,
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Ack n ow led gm en t. The authors thank J ohn W.
Allison, Ronda D. Schaadt, and Betty Yagi for the in
vitro data; J udith C. Hamel and Douglas Stapert for
the in vivo data. The personnel of Pharmacia and
Upjohn’s Structural, Analytical, and Medicinal Chem-
istry unit are thanked for elemental analysis and mass
spectral data.
(13) Hutchinson, D. K.; Barbachyn, M. R.; Brickner, S. J .; Buysse,
J . M.; Demyan, W.; Ford, C. W.; Garmon, S. A.; Glickman, S.
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Su p p or tin g In for m a tion Ava ila ble: NMR spectral data
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Ordering information is given on any current masthead page.
Refer en ces
(1) Current address: Bristol-Meyers-Squibb Pharmaceutical Re-
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(2) Current address: Central Research, Pfizer, Inc., Groton, CT
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