Design, synthesis and biological evaluation of oxazolidinone-quinolone hybrids

Article abstract of DOI:10.1016/S0968-0896(03)00083-X

Oxazolidinone-quinolone hybrids that combine the pharmacophores of a quinolone and an oxazolidinone were synthesised and shown to be active against a variety of resistant and susceptible Gram-positive and fastidious Gram-negative organisms. The best compo

Full text of DOI:10.1016/S0968-0896(03)00083-X

Bioorganic & Medicinal Chemistry 11 (2003) 2313–2319
Design, Synthesis and Biological Evaluation of
Oxazolidinone–Quinolone Hybrids
Christian Hubschwerlen,* Jean-Luc Specklin, Christine Sigwalt, Susanne Schroeder
and Hans H. Locher
Morphochem AG, Schwarzwaldallee 215, CH-4058 Basel, Switzerland
Received 22 October 2002; accepted 31 January 2003
Abstract—Oxazolidinone–quinolone hybrids that combine the pharmacophores of a quinolone and an oxazolidinone were synthe-
sised and shown to be active against a variety of resistant and susceptible Gram-positive and fastidious Gram-negative organisms.
The best compounds in this series overcome all types of resistance in relevant clinical Gram-positive pathogens. The nature of the
spacer greatly influences the antibacterial activity. The dual mode of action could be demonstrated for compounds having a
piperazinyl spacer. Antibacterial activity was higher at acidic pH.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
Several SAR studies on the oxazolidinones and quino-
The growing incidence of bacterial resistance to anti-
biotics represents a serious medical and socio-econom-
ical problem. The situation is getting particularly critical
in hospital settings where Gram-positive pathogens like
staphyloccocci and enteroccoci are becoming multi-
resistant to b-lactam, quinolone and glycopeptide anti-
biotics.1 Resistance to linezolid 1a (LZD; Chart 1), the
recently marketed oxazolidinone antibiotic, has already
been observed in clinical isolates of Staphylococcus aur-
eus and Enterococcus sp.2,3 Therefore, there is an urgent
need for new classes of antibiotics that are more potent
and less prone to resistance development than the cur-
rently marketed antibacterials. Besides the exploitation
of new targets there is an other approach consisting of
combining two pharmacophores in one molecule. These
two pharmacophores, by addressing the active site of
two different targets, offer the possibility to overcome
the current resistance and in addition to reduce the
appearance of new resistant strains. A similar strategy
was already applied to sulfanilyl–fluoroquinolone and
trimethoprime–fluoroquinolone hybrids.4,5 In the first
class, this principle could not be demonstrated since
sulfonamide activity was not retained.6 In contrast, the
second class clearly addressed the two targets DNA
gyrase and dihydrofolate reductase and both resistances
could be overcome in vitro to some extent.
lones have demonstrated a high tolerance for structural
variation at the 4- respectively 7-positions of the phenyl
ring.7,8
As eperezolid 1b and ciprofloxacin 2 (CIP) possess both
a piperazine substituent, it was appealing to merge these
two molecules through this common substituent. The
present paper describes the synthesis and the biological
evaluation of such oxazolidinone–quinolone hybrids 3
where the two pharmacophores were linked through a
spacer found either in quinolones or oxazolidinones.
*Corresponding author. Tel.: +41-61695-2103; fax: +41-61695-2122;
e-mail: christian.hubschwerlen@morphochem.ch
Chart 1.
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00083-X

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C. Hubschwerlen et al. / Bioorg. Med. Chem. 11 (2003) 2313–2319
Chemistry
rolidinone. This reaction was found to be sluggish with
quinolones 5 (Y=CH) and required the activation of
the quinolone moiety by the prior formation of a
boron complex.11 In the case of the naphthyridones 5
(Y=N), the boron activation was found to be trouble-
some at the stage of the hydrolysis. Alternatively, the
naphthyridone intermediates 5 were treated with tri-
ethylchlorosilane in presence of triethylamine at 100 ꢀC.
In general the yields were higher in the naphthyridone
series (Table 1).
The synthesis of the oxazolidinone–quinolone hybrids 3
is described in Scheme 1. The appropriate oxazolidinone
intermediates 4 containing a terminal basic nitrogen in
the Q substituent at the 4-position of the aromatic
ring were prepared following the synthetic strategy
adopted for the synthesis of eperezolid, starting from
3,4-difluoronitrobenzene or 2-chloro-5-nitropyridine
respectively (Scheme 2).9
The 7-chloro or -fluoroquinolone building blocks 5,
when not commercially available, were synthesised
using published procedures.10 The key coupling reaction
was performed either under thermal condition or under
microwave irradiation in the presence of an organic
base like DABCO, N,N-dimethyl-p-toluidine or triethyl-
amine in a polar solvent like DMSO or 1-methyl-2-pyr-
Results and Discussion
The oxazolidinone–quinolones hybrids 3 were tested for
in vitro antibacterial activity against a panel of well-
characterized susceptible and resistant Gram-positive
and -negative bacteria. Data for selected strains are
Scheme 1. Strategy for the assembly of the oxazolidinone-quinolone hybrids 3.
Scheme 2. (a) (1) 3(R)-1-Allyloxycarbonyl-3-amino-pyrrolidine, Et3N; (2) PdCl2(PPh3)2, AcOH, Bu3SnH; (3) BOC2O; (b) (1) H2, Pd/C; (2) ZCl,
NaHCO3; (c) n-BuLi, R-(À)-glycidyl butyrate; (d) (1) MsCl, TEA; (2) NaN3; (e) (1) H2, Pd/C; (2) Ac2O, AcOH; (3) Et3SiH, TFA; (f) 5a (Y=CH;
R2=cyclopropyl), DABCO.
Table 1. Yield of preparation of the oxazolidinone–quinolone hybrids 3
R1
X
Q
Y
R2
Yield (%)
3a
3b
3c
3d
3e
3f
COMe
COMe
COMe
C(=S)OMe
COMe
COMe
CF
CF
N
CF
CF
CF
CF
CF
Piperazinyl
Piperazinyl
Piperazinyl
Piperazinyl
Piperazinyl
CH
N
N
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
26
77
47
23
21
30
5
N
C–CH2–N(Me)
3-Amino-azetidinyl
3(R)-Amino-pyrrolidinyl
3(R)-Amino-pyrrolidinyl
N
CH
N
Cyclopropyl
Cyclopropyl
Cyclopropyl
3g
3h
COMe
COMe
5

C. Hubschwerlen et al. / Bioorg. Med. Chem. 11 (2003) 2313–2319
2315
Table 2. In vitro antibacterial activity of oxazolidinone-quinolone hybrids 3 against selected bacteria (MICs in mg/mL)
Compd
Bacterial straina
Sau1
Sau2
QRSA
Sau3
LZD-res.
Efs1
Efs2
LZD-res.
Efm1
VRE
Spn1
Spn2
QR
Hin
Mca
Eco
1a (LZD)
2
0.5
0.25
0.125
4
0.25
0.5
0.06
<0.03
<0.03
1
32
0.25
0.125
1
0.125
0.125
0.25
1
64
0.5
4
2
1
64
1
4
4
>32
16
2
>32
0.5
0.25
>32
0.25
0.5
8
16
8
0.5
0.5
0.5
32
8
<0.03
4
1
8
16
4
>64
2 (CIP)
3a
3b
3c
3d
3e
3f
3g
<0.03
0.5
0.25
64
0.25
1
0.125
0.06
<0.03
<0.03
32
32
0.125
0.125
2
0.25
0.125
0.5
0.25
0.125
0.125
0.125
0.125
0.06
0.5
0.25
0.25
0.25
0.25
0.25
0.25
2
8
>32
>32
8
0.06
<0.03
0.06
>64
>64
>64
2
8
8
0.5
0.125
0.06
0.06
0.06
<0.03
0.06
0.125
0.5
0.5
3h
1
0.06
<0.06
aSau1, S. aureus ATCC 29213; Sau2, S. aureus MRSA, quinolone-resistant; Sau3, S. aureus linezolid-resistant; Efs1, Enterococcus faecalis ATCC
29212; Efs2, E. faecalis linezolid-resistant; Efm1, E. faecium vancomycin- and quinolone-resistant; Spn1, Streptococcus pneumoniae ATCC 49619;
Spn2, S. pneumoniae quinolone-resistant; Hin, Haemophilus influenzae 11; Mca, Moraxella catarrhalis ATCC 8176; Eco, Escherichi coli ATCC 25922.
reported as minimum inhibitory concentration (MIC)
expressed in mg/mL (Table 2). For comparison, CIP (2)
and LZD (1a) were employed as reference drugs. As can
be deduced from these data, all of the synthesised com-
pounds exhibited potent antibacterial activity. This
activity can be modulated through the nature of the
spacer and the two pharmacophores.
Despite this general trend, representatives of both new
classes are superior to their respective comparators and
even to the ad hoc combination of both (see below).
Selected compounds were tested against 20strains each
of recent clinical isolates of methicillin- and quinolone-
resistant S. aureus (MRSA, QRSA), vancomycin-resis-
tant enterococci (VRE), and penicillin-resistant S.
pneumoniae (PRSP). The MIC90% (MIC that inhibits
ꢁ90% of all strains) for MRSA were 0.5, 0.5, 2, 2, >32
and 2 mg/mL for compounds 3a, 3b, 3g, LZD, CIP, and
the 1:1 combination of LZD and CIP, respectively. For
VRE the MIC90% values were 0.125, 0.25, 8, 2, >32,
and 4 mg/mL respectively, and for PRSP strains 0.125,
0.25, 0.25, 1, 4, and 1 mg/mL respectively. This data
illustrates the superior activity of 3a and 3b against
these multi-resistant organisms.
Compounds 3a–e which possess a piperazinyl linker
displayed an antibacterial spectrum similar to linezolid
by overcoming quinolone resistance in S. aureus (strain
Sau2), Streptococcus pneumoniae (strain Spn2) and
enteroccocci (strain Efm1). On the other hand, activity
against linezolid-resistant strains was somewhat lower
than towards wildtype strains, but nevertheless much
better than linezolid. The nature of the oxazolidinone
pharmacophore also plays an important role and the
same general trend was observed as in the oxazolidinone
series (i.e. 3dꢁ3b>3c).
In order to confirm this dual mode of action, we mea-
sured for compounds 3a–b, g both the inhibition of the
protein synthesis in an in vitro transcription/translation
assay and the inhibition of the enzymes that are targeted
by the quinolones, that is DNA gyrase and topoisome-
rase IV (Table 3). The enzyme data confirmed the
observed antibacterial activity with compounds 3a,b
showing a strong protein synthesis inhibition and com-
pound 3g a strong DNA gyrase and topoisomerase IV
inhibition. Moreover, compounds 3a,b also displayed
activity on topoisomerase IV and to a lesser extent on
the DNA gyrase indicating the dual mode of action of
these molecules. This difference of behaviour between
3a,b and 3g can best be explained by the different
molecular shape induced by the nature of the different
spacers. This may result in a preferred binding of the
In contrast, compounds 3f–h which contain an amino
pyrrolidinyl or amino azetidinyl spacer have a more
pronounced quinolone type antibacterial spectrum (i.e.,
potent activity against Gram-negative bacteria and
reduced activity against quinolone-resistant bacteria,
especially enteroccoccal strains like Efm1).
Table 3. Activity of oxazolidinone–quinolone hybrids 3 against
DNA gyrase, topoisomerase IV, and in an in vitro transcription/
translation assay
Compd
DNA gyrasea
Topo IVb
Inhib. of protein synthesisc
IC50 (mM)
IC50 (mM)
IC50 (mM)
Table 4. pH-dependent antibacterial activity (S. aureus Sau2)
3a
3b
3g
LZD
CIP
0
2.0810
5010
0.2
NT
0.5
2.8
1
Compd
MIC (mg/mL) at pH
>20
4.1
>20
NT
5
6.4
0.5
6.8
7.4
7.8
LZD
CIP
3a
3b
3g
0.5
32
0.125
0.06
0.25
1
16
0.25
0.25
0.5
1
16
0.5
0.5
1
NT, not tested.
aSupercoiling assay with E. coli gyrase.
bTopoisomerase IV relaxation assay.
cIn vitro transcription/translation assay with E. coli S30Extract System
(Promega).
32
0.03
0.03
0.125

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C. Hubschwerlen et al. / Bioorg. Med. Chem. 11 (2003) 2313–2319
molecules in one or the other active site of the two tar-
gets. Compounds 3a,b seem to be able to address mul-
tiple targets.
mL; 31.5 mmol) in AcOEt (100 mL) was stirred at reflux
for 20h. The reaction mixture was then worked up. The
residue was crystallized from Et2O–hexane to yield 6
(5.76 g; 18.6 mmol; 59%) as a yellow powder. NMR:
1.09–2.24 (m, 2H), 3.29–3.72 (m, 4H), 4.21–4.28 (m,
1H), 4.52 (d, J=1.5, 2H), 5.15–5.32 (m, 2H), 5.87–5.99
(m, 1H), 6.94 (t, J=9, 1H), 7.19 (d, J=6, 1H), 7.9–7.99
(m, 2H).
In contrast to LZD and CIP, the activity of compounds
3a,b is dependent on extracellular pH, that is MICs were
lower at acidic pH (Table 4). This could be explained by
an increased intrabacterial drug concentration when the
outside pH is lower than the intracellular pH (‘alka-
line trapping’).6 This property may contribute to
potent antibacterial activity and may be beneficial for
the treatment of infections involving acidic environ-
ments as, for example, found in abscesses or inflamed
tissues.12
(3R)-3-(2-Fluoro-4-nitro-phenylamino)-pyrrolidine-1-car-
boxylic acid tert-butyl ester (7). To a solution of 6 (5.76
g; 18.6 mmol) in THF (60mL) were added
PdCl2(PPh3)2 (0.13 g; 0.186 mmol), AcOH (12.12 mL;
37.2 mmol) and tributyltin hydride (49.87 mL; 37.2
mmol). The reaction was stirred at rt for 1 h. The sus-
pension was then diluted with Et2O (100 mL) and the
solid, collected by filtration, was washed with Et2O and
hexane and then dried. The solid was resuspended in
THF (10mL) and treated with Boc 2O (4.87g; 30mmol).
After stirring at rt for 3 h, the reaction mixture was
worked up and the residue was crystallized from Et2O–
hexane to afford 7 (4.15 g; 12.6 mmol; 68%) as a yellow
powder. NMR: 1.25 (s, 9H), 1.75–2.07 (m, 2H), 3.07–3.5
(m, 4H), 4.05–4.1 (m, 1H), 6.77–6.83 (t, J=9, 1H), 7.01
(d, J=5.7, 1H), 7.77–7.85 (m, 2H).
Conclusion
A
series of oxazolidinone-quinolone hybrid anti-
bacterials has been discovered. Their in vitro anti-
bacterial activity encompasses a large variety of clinically
relevant susceptible as well as resistant (i.e. MRSA,
QRSA, VRE and PRSP) Gram-positive and fastidious
Gram-negative bacteria. Various spacers are tolerated
and through their 3-D orientation influence the nature of
the mode of inhibition and the resulting antibacterial
spectrum. The most active compound 3b was 4–16 fold
more active than LZD and overcomes all types of resis-
tance in relevant clinical Gram-positive pathogens.13
(3R)-3-[Benzyloxycarbonyl-(4-benzyloxycarbonylamino-
2-fluoro-phenyl)-amino]-pyrrolidine-1-carboxylic
tert-butyl ester and (3R)-3-[(4-benzyloxycarbonylamino-
2-fluoro-phenyl)-amino]-pyrrolidine-1-carboxylic acid
acid
tert-butyl ester (8a and 8b). A solution of 7 (4 g; 12.29
mmol) in AcOEt (100 mL) and MeOH (50 mL) was
hydrogenated over Pd/C 10% (1 g). At the end of the
reaction, the catalyst was filtered off and the filtrate was
evaporated to dryness. The residue was dissolved in
AcMe (100 mL) and treated at 0 ꢀC with benzyl-
chloroformate (3.63 mL; 25.8 mmol) in presence of a
satd. solution of NaHCO3 (25 mL). The reaction was
stirred overnight at rt. The solvent was evaporated and
the aq layer was extracted twice with AcOEt and
worked up. The residue was chromatographed
(AcOEt–hexane 1:1) to give an unseparable equimolar
mixture of 8a and 8b (6.03 g; 10.8 mmol; 99%) as a
reddish oil. MS: 562.4 (MÀH)À, 428.4 (MÀH)À.
Method ESIÀ. This material was carried on without
further characterization.
Experimental
General
Proton (1H NMR) magnetic resonance spectra were
recorded in DMSO-d6 on a Bruker dpx 300 spectro-
meter. Chemical shifts (d) are given in ppm and refer to
TMS as internal standard and coupling constants are
given in Hz. Mass spectra were recorded on a HP1100
LC/API150EX with an ESI source and a single quad
analyser. Microwave experiments were performed using
Coherent SynthesisTM technology on a SmithCreatorTM
workstation (Personal Chemistry AB, Sweden). Flash
column chromatography was performed with Silica 32-
63 60A silica gel (Brunschwig). The term ‘worked up’
refers to the following sequence of operations: the aq
layer was extracted twice with AcOEt, the resulting
organic layer was then washed with H2O and brine,
dried over anhydrous magnesium sulfate, filtered and
evaporated to dryness. 7-Chloro-1-cyclopropyl-6-fluoro-
1,4-dihydro-4-oxo-[1,8]naphthyridine-3-carboxylic acid
and 7-chloro-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-
quinoline-3-carboxylic acid were purchased from com-
mercial sources. Ciprofloxacin and linezolid were syn-
thesised in our laboratories.
(3R)-3-{Benzyloxycarbonyl-[2-fluoro-4-{(5R)-5-hydroxy-
methyl-2-oxo-oxazolidin-3-yl}-phenyl]-amino}-pyrrolidine-
1-carboxylic acid tert-butyl ester and (3R)-3-{[2-fluoro-
4-{(5R)-5-hydroxymethyl-2-oxo-oxazolidin-3-yl}-phenyl]-
amino}-pyrrolidine-1-carboxylic acid tert-butyl ester (9a
and 9b). n-BuLi (1.6 M solution in n-hexane; 7.62 mL;
12.2 mmol) was added dropwise at À78 ꢀC to a crude
solution of 8a and 8b (6.02 g; 10.8 mmol) in THF (40
mL). The mixture was stirred at À78 ꢀC for 10min and
warmed up to reach 0 ꢀC. R-(À)-Glycidyl butyrate (2.11
g; 14.6 mmol) was added dropwise and the reaction was
allowed to reach rt. After stirring overnight, the reac-
tion was worked up. The residue was crystallized from
AcOEt-hexane to provide an unseparable equimolar
mixture of 9a and 9b (3.36 g; 6.48 mmol; 60%) of off-
white solid. MS: 530.3 (M+H)+, 396.1 (M+H)+,
Synthesis of the oxazolidinone building blocks 4
(3R)-3-(2-Fluoro-4-nitro-phenylamino)-pyrrolidine-1-car-
boxylic acid allyl ester (6). A solution of 3,4-difluoroni-
trobenzene (5.01 g; 31.5 mmol), (3R)-1-allyloxycarbonyl-
3-amino-pyrrolidine14 (5.1 g; 30mmol) and Et N (6.27
3

C. Hubschwerlen et al. / Bioorg. Med. Chem. 11 (2003) 2313–2319
2317
Method ESI+. This material was carried on without
further characterization.
(d, J=3, 1H), 8.31 (m, 1H). MS: 320.1 (M+H)+,
Method ESI+.
(3R)-3-{[4-{(5R)-5-Azidomethyl-2-oxo-oxazolidin-3-yl}-
2-fluoro-phenyl]-benzyloxycarbonyl-amino}-pyrrolidine-
1-carboxylic acid tert-butyl ester and (3R)-3-{[4-{(5R)-5-
Azidomethyl-2-oxo-oxazolidin-3-yl}-2-fluoro-phenyl]-
amino}-pyrrolidine-1-carboxylic acid tert-butyl ester
(10a and 10b). Methanesulfonylchloride (0.805 mL; 10.8
mmol) was added dropwise at 0 ꢀC to a crude solution of
9a and 9b (3.36 g; 10.8 mmol) and Et3N (2.05 mL; 10.8
mmol) in CH2Cl2 (40mL). The reaction was stirred at rt
for 1 h. The reaction was diluted with H2O and worked
up. The residue was dissolved in DMF (10mL), and
NaN3 (1.38 g; 10.8 mmol) was added. After stirring at
80 ꢀC for 20h, the reaction mixture was concentrated to
dryness and the residue was worked up to afford an unse-
parable equimolar mixture of 10a and 10b (4.07 g; 10.7
mmol; 99%) as an amorphous off-white material. MS:
555.5 (M+H)+, 421.3 (M+H)+, Method ESI+. The
latter was carried on without further characterization.
Thioacetamide {[(5S)-3-[3-fluoro-(1-piperazinyl) phenyl]-
2-oxo-5-oxalidinyl]methyl}-carbamothioic acid methyl
ester (4d; R1=C(=S)OMe, X=CF, Q=piperazinyl).
This compound was prepared in 99% from 4[4-[(5S)-5-
aminomethyl-2-oxo-oxazolidin-3-yl]-2-fluoro-phenyl]-
piperazine-1-carboxylic acid tert butyl ester17 according
to ref 18. NMR: 3.66 (s, 8H), 3.37 (m, 1H), 3.69–3.77
(m, 3H), 3.88 (s, 3H), 4.16 (t, J=9, 1H), 4.90(m, 1H),
7.12 (t, J=9, 1H), 7.23 (dd, J=9.5 and 2.5, 1H), 7.51
(dd, J=15 and 2.5, 1H), 9.57 (t, J=4.8, 1H). MS: 369.1
(M+H)+, Method ESI+.
N-{(5S)-3-[4-(azetidin-3-ylamino)-3-fluoro-phenyl]-2-oxo-
oxazolidin-5-ylmethyl}-acetamide (4f; R1=Ac, X=CF,
Q=3-amino-azetidinyl). This compound was prepared
according to the procedure reported for 4g starting from
3,4-difluoronitrobenzene and 1-diphenylmethyl-3-azeti-
dinamine in an 14% overall yield.16 NMR: 1.87 (s, 3H),
3.2–3.5 (m, 6H), 3.64–3.71 (m, 2H), 3.92–4.4 (m, 2H),
4.17 (m, 1H), 5.74–5.78 (m, 1H), 6.57–6.7 (m, 1H), 7.0
(d, J=9, 1H), 7.1–7.5 (m, 1H), 8.31 (m, 1H). MS: 322.34
(M+H)+, Method ESI+.
(3R)3-{4-[(5S)-5-(Acetylaminomethyl)-2-oxo-oxazolidin-
3-yl]-2-fluoro-phenylamino}-pyrrolidine-1-carboxylic acid
tert-butyl ester (11). A solution of 10a and 10b (4.2 g;
7.3 mmol) in AcOEt (50mL) was hydrogenated over
Pd/C 10% (400 mg). At the end of the reaction, the
catalyst was filtered off and the filtrate was evaporated
to dryness. The residue was dissolved in AcOH (5 mL)
and Ac2O (2 mL) was added. After stirring at rt for 2 h,
the solvents were evaporated and the residue was
worked up to yield 11 (3.1 g; 7.3 mmol) as a crude
amorphous off-white material. MS: 437.5 (M+H)+,
Method ESI+. This mixture was carried on without
further characterization.
Preparation of the oxazolidinone-quinolone hybrids 3
7-(4-{4-[(5S)-5-(Acetylamino-methyl)-2-oxo-oxazolidin-
3-yl]-2-fluoro-phenyl}-piperazin-1-yl)-1-cyclopropyl-6-
fluoro-4-oxo-1,4-dihydro-quinoline -3-carboxylic acid
(3a). A mixture of 7-chloro-1-cyclopropyl-6-fluoro-4-
oxo-1,4-dihydro-quinoline-3-carboxylate boron diace-
tate11 5a (0.103 g; 0.25 mmol), N-[{(5S)-3[3-fluoro-4-(1-
piperazinyl)phenyl]-2-oxo-5-oxazolidinyl}methyl]-acet-
amide9 4a (0.1 g; 0.3 mmol) and N,N-dimethyl-p-tolui-
dine (0.054 mL; 0.375 mmol) in 1-methyl-2-
pyrrolidinone (0.5 mL) was stirred at 120 ꢀC for 12 h.
The reaction mixture was poured into water and the
resulting crystals were collected by filtration and puri-
fied by chromatography on silica (CH2Cl2–MeOH 19:1)
to afford 3a (0.38 g; 0.06 mmol; 26%) as a beige solid.
Mp 315–320 ꢀC (dec). NMR: 1.32 (m, 2H), 1.44 (m,
2H), 1.95 (s, 3H), 3.34 (m, 4H), 3.52 (t, J=5.8, 3H), 3.60
(m, 4H), 3.83 (dd, J=7 and 8, 1H), 3.96 (m, 1H), 4.21
(t, J=8, 1H), 7.24–7.34 (m, 2H), 7.61–7.67 (dd, J=5
and 2, 1H), 7.75–7.77 (d, J=8, 1H), 8.06 (d, J=15, 1H),
8.35 (t, J=6, 1H), 8.79 (s, 1H), 15.3 (s, 1H). MS: 582.4
(M+H)+; 580.4 (MÀH)À. Anal. calcd for
C29H29F2N5O6 (0.45 H2O): C, 59.89; H, 5.03; N, 12.04;
H2O, 1.37. Found: C, 59.49; H, 5.04; N, 11.84, H2O,
1.38.
N-{(5S)-3-[3-Fluoro-4-{(3R)-pyrrolidin-3-ylamino}-
phenyl] - 2 - oxo - oxazolidin - 5 - ylmethyl] - acetamide (4g;
R1=Ac, X=CF, Q=3(R)-aminopyrrolidinyl). A solu-
tion of triethylsilane (0.93 mL; 7.3 mmol) and 11 (3.1g,
7.3 mmol) in CH2Cl2 (20mL) was treated with TFA
(20mL). After stirring at rt for 20 h, the volatiles were
evaporated and the residue was dissolved in H2O and
neutralized with a satd NaHCO3 solution. After evap-
oration in vacuo, the residue was taken up in a CH2Cl2–
MeOH (1:1) mixture. This suspension was treated with
Fuller’s earth, filtered and the filtrate evaporated to yield
4g (2.1 g; 6.2 mmol; 85%) as an off-white amorphous
solid. NMR: 1.87 (s, 3H), 2.21 (m, 1H), 3.16 (dd, J=12
and 2, 1H), 3.36 (m, 6H), 3.70(dd, J=6 and 9, 1H),
4.18 (m, 1H), 4.71 (m, 1H), 5.64 (d, J=5, 1H), 6.83 (t,
J=9, 1H), 7.12 (d, J=7, 1H), 7.42 (dd, J=14 and 2,
1H), 8.29 (t, J=6, 1H), 9.00 (s, 1H). MS: 337.6
(M+H)+, Method ESI+.
7-(4-{[(5S)-5-(Acetylamino-methyl)-2-oxo-oxazolidin-3-
yl]-2-fluoro-phenyl}-piperazin-1-yl)-1-cyclopropyl-6-fluoro
-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic acid
(3b). A suspension of 7-chloro-1-cyclopropyl-6-fluoro-
1,4-dihydro-4-oxo-[1,8]naphthyridine-3-carboxylic acid
5b (0.1 g; 0.35 mmol), 4a (0.13 g; 0.39 mmol), Et3N
(0.119 g; 1.17 mmol) and ClSi(Me)3 (0.085 g; 0.78
mmol) in DMSO (2 mL) was heated to 150 ꢀC in a
microwave oven for 10min. After evaporation under
reduced pressure, the residue was taken up in H2O, fil-
N-[(5S)-2-Oxo-3-(6-piperazin-1-yl-pyridin-3-yl)-oxazoli-
din-5ylmethyl]-acetamide (4c; R1=Ac, X=N, Q=pipera-
zinyl). This compound was prepared according to the
procedure reported for 4g starting with 4-(5-amino-pyri-
din-2-yl)-piperazine-1-carboxylic acid tert-butyl ester in
10% overall yield.15 NMR: 1.80(s, 3H), 2.81 (m, 3H),
3.46 (m, 5H), 3.62 (dd, J=6 and 10, 1H), 4.09 (t, J=9,
1H), 4.71 (d, J=9, 1H), 7.8 (dd, J=9 and 3, 1H), 8.21

2318
C. Hubschwerlen et al. / Bioorg. Med. Chem. 11 (2003) 2313–2319
tered and the solid was chromatographed (CH2Cl2-
MeOH 9:1). The fractions were collected and evapo-
rated. The residue was crystallized from MeCN to
afford 3b (0.154 g; 0.27 mmol; 77%) as a pale yellow
solid. NMR: 1.11 (m, 2H), 1.21 (m, 2H), 1.83 (s,
3H), 3.17 (m, 4H), 3.41 (t, J=5.8, 3H), 3.71 (m, 2H),
4.01–4.11 (m, 5H), 4.71 (m, 1H), 7.11 (t, J=8, 1H),
7.18 (dd, J=7 and 2, 1H), 7.51 (dd, J=8 and 2,
1H), 8.05 (d, J=13, 1H), 8.24 (t, J=6, 1H), 8.59 (s,
1H), 15.1 (s, 1H). MS: 583.3 (M+H)+, 581.6 (M+H)À
Method ESI+, ESIÀ. Anal. calcd for C28H28F2N6O6: C,
56.94; H, 4.93; N, 14.23. Found: C, 57.01; H, 4.97; N,
14.04, H2O, 1.38.
6-oxo-2,3-dihydro-6H-1-oxa-3,3a-diaza-phenalene-5-car-
boxylic acid (3e). A solution of 12 (0.044 g; 0.32 mmol)
in a mixture of 37% aq. HCl (1mL) and AcOH (1 mL)
was heated to 80 ꢀC for 20h. The solvents were eva-
porated. The residue was dissolved in a MeOH–
CH2Cl2 (1:1; 5 mL) mixture and treated with Et3N (0.5
mL). After evaporation to dryness, the residue was
taken up in AcOH (1 mL) and treated with Ac2O (1
mL). The reaction was left to proceed for 20h, and the
solvents were evaporated in vacuo. The residue was
purified by preparative HPLC (H2O–MeCN gradient)
to provide 3e (0.009 g; 0.06 mmol; 21%) as an off-white
solid. NMR: 1.84 (s, 3H), 3.02 (s, 3H), 3.11 (m, 4H),
3.50(m, 4H), 3.7 (dd, J=8 and 8.6, 1H), 4.09 (t, J=8,
1H), 4.71 (m, 1H), 5.3 (b, 1H), 7.13 (t, J=8, 1H), 7.20
(dd, J=7 and 2, 1H), 7.51 (dd, J=8 and 2, 1H), 7.63 (d,
J=13, 1H), 8.25 (t, J=6, 1H), 8.78 (s, 1H), 15.1 (s, 1H).
MS: 599.2 (M+H)+, 597.7 (MÀH)À, Method ESI+,
ESIÀ.
7-(4-{5-[(5S)-5-(Acetylamino-methyl)-2-oxo-oxazolidin-
3-yl]-pyridin-2-yl}-1-piperazin-1-yl)-1-cyclopropyl-6-
fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic
acid (3c). This compound was prepared according to
the procedure reported for 3b starting from 4c and 5b.
NMR: 1.2-1.4 (m, 4H), 1.85 (s, 3H), 3.45 (m, 6H), 3.61
(m, 6H), 3.71 (m, 1H), 4.1 (t, J=8, 1H), 4.67 (m, 1H),
7.00 (d, J=9, 1H), 7.63 (d, J=8, 1H), 7.83 (d, J=9,
1H), 7.93 (d, J=14, 1H), 8.25 (s, 2H), 8.67 (s, 1H), 15.1
(s, 1H). MS: 565.8 (M+H)+ Method ESI+. Anal. calcd
for C28H28F2N6O6 (0.5 H2O): C, 56.44; H, 5.09; N,
17.06; H2O, 1.56. Found: C, 56.59; H, 5.08; N, 16.89,
H2O, 1.56.
7-(3-{4-[5(S)-5-(Acetylamino-methyl)-2-oxo-oxazolidin-
3-yl]-2-fluoro-phenylamino}-azetidin-1-yl)-1-cyclopropyl-
6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-car-
boxylic acid (3f). This compound was prepared accord-
ing to the procedure reported for 3b starting from 4f
and 5b. NMR: 1.11–1.24 (m, 4H), 1.87 (s, 3H), 3.44 (t,
J=5, 2H), 3.68–3.76 (m, 2H), 4.09 (t, J=8, 1H), 4.32 (b,
2H), 4.51–4.57 (m, 1H), 4.67–4.77 (m, 3H), 6.24 (d,
J=7, 1H), 6.74 (t, J=9, 1H), 7.14 (d, J=9, 1H), 7.50
1-Cyclopropyl-6-fluoro-7-(4-{2-fluoro-4-[(5S)-5-(meth-
oxythiocarbonylamino-methyl)-2-oxo-oxazolidin-3-yl]-
phenyl}-piperazin-1-yl)-4-oxo-1,4-dihydro-[1,8]-naphthyri-
dine-3-carboxylic acid (3d). This compound was pre-
pared according to the procedure reported for 3b
starting from 4d and 5b. NMR: 1.11–1.27 (m, 5H), 3.22
(m, 4H), 3.80(m, 4H), 3.91 (s, 3H), 4.1 (m, 4H), 4.95
(m, 1H), 7.13–7.24 (m, 2H), 7.56 (dd, J=15 and 3,
1H), 8.12 (d, J=15, 1H), 8.66 (s, 1H), 9.54 (t, J=6,
1H), 15.23 (s, 1H). MS: 615.2 (M+H)+ Method ESI+.
Anal. calcd for C28H28F2N6O6S: C, 54.72; H, 4.59; N,
13.67; S, 5.22. Found: C, 54.41; H, 4.76; N, 13.33; S,
5.0.4.
(dd, J=14 and 2, 1H), 8.04 (d, J=11, 1H), 8.27 (t, J=6,
+
1H), 8.60(s, 1H), 15.41 (s, 1H). MS: 569.5 (M+H)
Method ESI+.
,
7-[(3R)-3-{4-[(5S)-5-(Acetylamino-methyl)-2-oxo-oxazoli-
din-3-yl]-2-fluoro-phenylamino}-pyrrolidin-1-yl]-1cyclo-
propyl-6-fluoro-4-oxo-1,4-dihydro-quinoline-1-carboxylic
acid (3g). A solution of 5a (0.204 g; 0.5 mmol), 4g (0.052
g; 0.75 mmol) and DABCO# (0.112 g; 1 mmol) in
DMSO (5 mL) was stirred for 50h at 120 ꢀC. After
evaporation under reduced pressure, the residue was
resuspended in EtOH (10mL), treated with Et N (0.1
3
mL) and further stirred at rt for 20h. The mixture was
diluted with H2O (20mL). The solid was collected by
filtration and crystallized in MeOH–EtOH–CH2Cl2 to
afford 3g (0.024 g; 0.027 mmol; 5.5%) as a beige pow-
der. NMR: 0.9 (t, J=8, 1H), 1.00 (m, 2H), 1.15 (d,
J=4, 1H), 1.6 (s, 3H), 1.96 (m, 1H), 2.15 (m, 1H), 3.15
(m, 1H), 3.22–3.68 (m, 6H), 3.9 (m, 1H), 4.15 (m, 1H),
4.5 (m, 1H), 5.3 (d, J=7, 1H), 6.73 (t, J=7, 1H), 6.95
(m, 2H), 7.15 (dd, J=15 and 2, 1H), 7.68 (d, J=15,
1H), 8.08 (t, J=6, 1 h), 8.42 (s, 1H). MS: 582.4
(M+H)+, Method ESI+.
9-(4-{4-[(5S)-5-(Acetylamino-methyl)-2-oxo-oxazolidin-
3-yl]-2-fluoro-phenyl}-piperazin-1-yl)-8-fluoro-3-methyl-
6-oxo-2,3-dihydro-6H-1-oxa-3,3a-diaza-phenalene-5-car-
boxylic acid ethyl ester (12). A solution of 8,9-
difluoro-3-methyl-6-oxo-2,3-dihydro-6H-1-oxa-3,3a-diaza-
phenalene-5-carboxylic acid ethyl ester10 (0.1 g; 0.32
mmol) and 4a (0.216 g; 0.64 mmol) in a mixture of pyri-
dine (1 mL) and DMSO (1 mL) was heated to 120 ꢀC
for 20h. The solvents were evaporated under reduced
pressure and the residue was taken up in H2O. The solid
was collected by filtration and purified by chromato-
graphy (CH2Cl2–MeOH 9:1) to yield 12 (0.044 g; 0.07
mmol; 22%) as an off-white solid. NMR: 1.4 (t, J=8,
3H), 2.0(s, 3H), 3.15 (s, 3H), 326 (m, 4H), 3.30(d, J=6,
2H), 3.61 (m, 5H), 3.74 (dd, J=8 and 9, 1H), 5.2 (m,
2H), 5.38 (m, 2H), 5.65 (m, 1H), 7.23–7.40(m, 2H), 7.61
(d, J=15, 1H), 7.65 (d, J=15, 1H), 8.4 (t, J=6, 1H),
8.62 (s, 1H). MS: 627.7 (M+H)+, Method ESI+.
7-[(3R)-3-{4-[(5S)-5-(Acetylamino-methyl)-2-oxo-oxazoli-
din-3-yl]-2-fluoro-phenylamino}-pyrrolidin-1-yl]-1-cyclo-
propyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]-naphthyridine-3-
carboxylic acid (3h). This compound was prepared
according to the procedure reported for 3g starting from
4g and 5b. NMR: 1.0–1.3 (m, 7H), 1.84 (s, 3H), 2.07 (m,
1H), 3.4 (t, J=5, 1H), 3.70(m, 1H), 3.8–4.0(m, 2H),
4.07 (t, J=9, 1H), 4.30–4.5 (m, 3H), 4.66 (m, 1H), 5.17
(d, J=6, 1H), 6.87 (t, J=12, 1H), 7.07 (d, J=8, 1H),
7.40(dd, J=14 and 2, 1H), 8.00 (d, J=14, 1H), 8.02 (t,
9-(4-{4-[(5S)-5-(Acetylamino-methyl)-2-oxo-oxazolidin-
3-yl]-2-fluoro-phenyl}-piperazin-1-yl)-8-fluoro-3-methyl-

C. Hubschwerlen et al. / Bioorg. Med. Chem. 11 (2003) 2313–2319
2319
J=6, 1 h), 8.58 (s, 1H), 15.4 (s, 1H). MS: 583.2
(M+H)+, Method ESI+.
References and Notes
1. Centers for Disease Control and Prevention MMWR Morb
Mortal Wkly. Rpt. 1999, 47, 1.
2. Tsiodras, S.; Gold, H. S.; Sakoulas, G.; Eliopoulos, G. M.;
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3. Bassetti, M.; Farrel, P. A.; Allan, D. A.; Dembry, L. M.;
Opal, J. E. Abstracts of Papers, 41st Interscience Conference
on Antimicrobial Agents and Chemotherapy, Chicago, IL,
USA, Sept. 22–25, 2001; American Society for Microbiology:
Washington, DC, 2001; Poster K-1193.
Microbiogical methods
Minimal inhibitory concentrations (MICs) were deter-
mined by a microdilution method following NCCLS
guidelines, except that IsoSensitest broth (Oxoid;
Basingstoke, UK) was used.19 The medium was supple-
mented with 3% lysed horse blood for fastidious
organisms. Serial dilutions in 96-well plates were pre-
pared with the help of a Biomek 2000 robot. The pH of
the test medium was 7.2–7.3, unless otherwise stated.
The bacterial strains used were from the Morphochem
collection. Clinical isolates were originally obtained
from the Kantonspital Basel, Switzerland, and from
other European and US hospitals.
4. Allemandi, D. A.; Alovero, F. L.; Manzo, R. H. J. Anti-
microb. Chemother. 1994, 34, 261.
5. Guerry, P.; Hartman, P. G.; Locher, H. H.; Jolidon, S.;
Broger, C.; Oefner, C.; Gmuender, H.; Then, R. L. Abstract of
Papers, 11th International Symposium Chemistry and Biology
of Pteridines and Folates, June 15–20, 1997, Berchtesgaden,
Germany.
6. Alovero, F.; Nieto, M.; Mazzieri, M. R.; Then, R.; Manzo,
R. H. Antimicrob. Agents Chemother. 1998, 42, 1495.
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In vitro transcription/translation assay
Inhibition of cell free protein synthesis was determined
with a coupled transcription/translation assay (Escher-
ichia coli S30Extract System, Promega, Madison, WI,
USA) using the plasmid pBestLuc as DNA template.20
After pre-incubation for 10min, the reactions were
initiated by adding 0.1 mg of plasmid DNA and incu-
bated at 37 ꢀC for 35 min. Then, the reaction was stop-
ped by cooling in ice, 15 mL was added to 15 mL
luciferase substrate (Bright Glo, Promega, Madison,
WI, USA), and luminescence was quantified in a Tecan
Spectrafluor Plus plate reader.
12. Konig, C.; Simmen, H. P.; Blaser, J. Eur. J. Microbiol.
Infect. Dis. 1993, 12, 519.
13. At the completion of our work, we have been made aware
of a recently published patent of Pharmacia describing analo-
gue compounds: Gordeev, M. F.; Patel, D.; Barbachyn, M. R.;
Gage, P. WO 0259116; Chem. Abstr. 2002, 137, 125148. Part
of our work has been presented recently: Locher, H. H.;
Specklin, J.-L.; Borer, Y.; Brohammer, S.; Schroeder, S.;
Sigwalt, C.; Hubschwerlen, C. Abstract of Papers, 42nd Inter-
science Conference on Antimicrobial Agents and Chemotherapy,
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Microbiology: Washington, DC, 2002; Poster F-1317.
14. Moriarty, K. J.; Shimshock, Y.; Ahmed, G.; Wu, J.; Wen,
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Topoisomerase assays
Inhibition studies on E. coli DNA gyrase and topo-
isomerase IV were performed by published proce-
dures.21,22 Compounds were tested initially at a wide
range of concentrations, which were then fine-tuned to
determine exact IC50s and confirm the results. Com-
pounds were serially diluted in water, re-heating gently
where required, to solubilize them, and added to the
reaction before the addition of the enzyme. Briefly,
DNA gyrase activity was measured in a supercoiling
assay using relaxed pBR322 DNA and supercoiled
pBR322 DNA was used for the topoisomerase IV
relaxation assay. The IC50 for gyrase supercoiling was
visually assessed as the concentration of compound,
which led to a 50% reduction of the supercoiled band
and a spread of topoisomers above. The IC50 for
relaxation was determined visually, as being the com-
pound concentration at which the relaxed band was
reduced by 50% and a supercoiled band with topoi-
somers appeared.
15. Dodic, N. WO 01/96327, 2001; Chem. Abstr. 2001, 136,
53765.
16. Arimoto, M.; Hayano, T.; Soga, T.; Yoshioka, T.;
Tagawa, H.; Furukawa, M. J. Antibiot. 1986, 39, 1243.
17. Kado, N.; Tokuyama, R.; Tsubouchi, M.; Tomita, Y. WO
2000 027830, 2000; Chem. Abstr. 2000, 132, 334453.
18. Tokuyama, R.; Takahasi, Y.; Tomita, Y.; Tsubouchi, M.;
Iwasaki, N.; Kado, N.; Okezaki, E.; Nagata, O. Chem. Pharm.
Bull. 2001, 49, 361.
19. National Committee for Clinical Laboratory Standards.
Methods for Dilution Antimicrobial SusceptibilityTests for
Bacteria that Grow Aerobically, 4th ed.; Approved standard:
NCCLS Document M7-A4; National Committee for Clinical
Laboratory Satandars: Villanova, PA, USA, 1997.
20. Kung, P.-P.; Casper, M. D.; Cook, K. L.; Wilson-Lingardo,
L.; Risen, L.; Vickers, T. A.; Ranken, R.; Blyn, L. B.; Wyatt,
J. R.; Cook, P. D.; Ecker, D. J. J. Med. Chem. 1999, 42, 4705.
21. Reece, R. J.; Maxwell, A. J. Biol. Chem. 1989, 264, 19648.
22. Peng, H.; Marians, K. J. J. Biol. Chem. 1993, 268, 24481.
Acknowledgements
Inhibition studies on DNA gyrase and topoisomerase IV
were kindly performed by A. Maxwell and A. Howells,
John Innes Enterprises, Ltd, Norwich, UK. We acknowl-
edge excellent technical assistance of Y. Borer and S.
Brohammer in performing microbiological testing.