Synthesis and antibacterial evaluation of isoxazolinyl oxazolidinones: Search for potent antibacterial

Article abstract of DOI:10.1016/j.bmcl.2009.04.133

A series of (5S) N-(3-{3-fluoro-4-[4-(3-aryl-4,5-dihydro-isoxazole-5-carbonyl)-piperazin-1-yl]-phenyl}-2-oxo-oxazolidin-5-ylmethyl)-acetamide(6a-o) were synthesized and their in vitro antibacterial activity against various resistant Gram-positive and Gram-negative bacteria were evaluated. Most of the synthesized compounds showed 2 to 10 fold lower MIC values compared to linezolid against Staphylococcus aureus ATCC 25923, ATCC 70069, ATCC 29213, Bacillus cereus MTCC 430, Enterococcus faecalis MTCC439, Klebsiella pneumoniae ATCC 27736, and Streptococcus pyogens.

Full text of DOI:10.1016/j.bmcl.2009.04.133

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3573–3576
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antibacterial evaluation of isoxazolinyl oxazolidinones:
Search for potent antibacterial q
Vandana Varshney a, Nripendra N. Mishra b, Praveen K. Shukla b, Devi P. Sahu a,
*
a Medicinal & Process Chemistry Division, Central Drug Research Institute, Lucknow 226 001, India
b Fermentation Technology Division, Central Drug Research Institute, Lucknow 226 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of (5S) N-(3-{3-fluoro-4-[4-(3-aryl-4,5-dihydro-isoxazole-5-carbonyl)-piperazin-1-yl]-phenyl}-
2-oxo-oxazolidin-5-ylmethyl)-acetamide(6a–o) were synthesized and their in vitro antibacterial activity
against various resistant Gram-positive and Gram-negative bacteria were evaluated. Most of the synthe-
sized compounds showed 2 to 10 fold lower MIC values compared to linezolid against Staphylococcus aur-
eus ATCC 25923, ATCC 70069, ATCC 29213, Bacillus cereus MTCC 430, Enterococcus faecalis MTCC439,
Klebsiella pneumoniae ATCC 27736, and Streptococcus pyogens.
Received 28 January 2009
Revised 16 April 2009
Accepted 29 April 2009
Available online 3 May 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Oxazolidinone
Isoxazoline
Antibacterial
Eperazolid
In spite of available potent antimicrobials for management of
infections, development of bacterial resistance to existing drugs
is a major concern in antibacterial therapy and necessitates contin-
uing research for new classes of antibacterial. Of particular concern
are severe infections caused by multi drug-resistant Gram-positive
pathogens, which result in high mortality rates especially in the
hospital settings. The individual organisms responsible include
methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-
resistant Enterococcus faecalis (VRE) and penicillin-resistant Strep-
tococcus pneumoniae. There are several antibiotics effective against
these organisms but these organisms easily acquire resistance
against them. Resistant development is inherent in the mode of ac-
tion of all antibiotics and hence posses challenges in the develop-
ment of new antimicrobial agents to overcome it. Some bacterial
strains have been found to be resistant to all currently available
antibacterial agents.1
Among the antibacterial, oxazolidinone covers the important
Gram-positive pathogens and show unique mechanism of action.
Linezolid2 (Fig. 1), the first commercially available oxazolidinone
shows activity against Gram-positive bacteria3 including multi-
drug-resistant bacteria. Linezolid is a bacteriostatic antibiotic that
exerts its action by binding to 50S subunit of bacterial ribosome,
blocking synthesis of bacterial proteins.4 Despite of its unique
mode of action resistance against linezolid was reported in early
2001.5
As a result, new antimicrobial agents are being developed and
old ones are reevaluated as potential alternatives for control of
infections due to multiple resistant Gram-positive microorganism.
The hybrid approach has been one of the alternate approaches fol-
lowed by several research groups6 to treat resistant bacterial
strains. Among oxazolidinone family, Eperazolid (Fig. 1) is another
lead molecule but it was not taken for further commercial develop-
ment due to its shorter half life7 and bone marrow toxicity.8 How-
ever it has been modified by various groups9 to enhance activity
and to reduce its toxicity level. Johnson10 and other research
groups9a reported synthesis and activity of isoxazolinyl oxazolidi-
none (2, Fig. 2). Barbachyn et al.11 replaced oxazolidinone ring by
isoxazoline ring (3, Fig. 2), these compounds are showing good
activity against resistant microorganisms.
These literature findings prompted us to synthesize hybrid ana-
logues of oxazolidinones and isoxazoline 6a–o (Fig. 3). We herein
report the synthesis of novel hybrid analogues of Eperazolid and
isoxazoline and their in vitro activity against Gram-positive and
Gram-negative bacteria resistant to various drugs in this Letter.
Key intermediate 1 was synthesized from 3,4-difluoro nitroben-
zene by reported protocol12 (Scheme 1).
The isoxazoline components were synthesized by following the
literature precedence. Conversion of aldehydes 2a–o to the oxime
3a–o by hydroxylamine hydrochloride followed by chlorination
with N-chlorosuccinimide produced the nitrile oxide precursor.13
Dehydrohalogenation of chloroxime with base in the presence of
dipolarophile ethylacrylate afforded exclusively the 5-substituted
cycloadduct (4a–o) in good yield with none of the regioisomeric
4-substituted product being observed by NMR.14 A doublet of dou-
q
CDRI Communication No. 7695.
* Corresponding author. Tel.: +91 522 2612415x4378; fax: +91 522 2623405.
E-mail addresses: dpsahuin@yahoo.com, dp_sahu@cdri.res.in (D.P. Sahu).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.133

3574
V. Varshney et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3573–3576
O
O
O
O
O
O
O
O
O
O
N
N
N
N
N
N
N
N
(a)
HO
NHCOCH3
NHCOCH3
NHCOCH3
O
N
O
H N
N
N
F
F
F
NHCOCH3
LINEZOLID
EPERAZOLID
R1
F
6a-o
1
Scheme 3. Reagents and conditions: (a) compounds 5a–o, EDCÁHCl, HOBt, Et3N,
0 °C, overnight.
R2
R1
O
N
R1
N
O
O
NHCOCH3
NHCOCH3
O
N
1.0–2.0 Â 107 cells/mL by matching with 0.5 McFarland standards.
The activity of compounds was determined as per NCCLS protocol
using Mueller Hinton broth (Becton Dickinson, USA) in 96-well tis-
sue culture plates. Proper growth control, drug control and the
negative control were adjusted onto the plate. Compounds were
R3
F
3
2
Figure 2.
dissolved in DMSO at a concentration of 1 mg/mL and 20
was added to each well of 96-well tissue culture plate having
180 L Mueller Hinton broth. From here the solution was serially
diluted resulting in twofold dilution of the test compounds in sub-
sequent wells. 100 L of Mc Farland matched bacterial suspension
was diluted in 10 ml of media and then 100 L of it was added in
each well and kept for incubation. The maximum concentration
of compounds tested was 50 g/mL. The micro-titer plates were
lL of this
O
N
O
R1
O
l
N
N
N
NHCOCH3
O
F
l
6a-6o
l
Figure 3. R1 = Substituted aryl/substituted heteroaryl.
l
incubated at 35 °C in a moist, dark chamber and MICs were re-
corded spectrophotometrically after 24 h using SOFT max Pro 4.3
Software (Molecular Devices, Sunnyvale, USA).
The analogues 6a–o of this series exhibited better activity than
linezolid against Gram-positive resistant strain but did not show
any activity against Escherichia coli (ATCC 9637) and Pseudomonas
aeruginosa (ATCC-BAA 427). Surprisingly, these compounds
showed excellent activity against a Gram-negative bacteria Klebsi-
ella pneumoniae (ATCC 27736) resistant to various drugs. Minimum
O
O
F
NO2
H N
N
N
NHCOCH3
F
F
1
Scheme 1.
inhibitory concentration MIC (
reported in Table 1.
Antibacterial activity evaluation largely depended on various
substitutions in aryl ring present at 3-position of isoxazoline ring.
Attempts were made to correlate antibacterial activity of these
compounds with changing substitution at aryl ring. Compounds (
lM) of these novel compounds are
blet for C-5 proton of isoxazoline appeared at d 5.10 (J = 10.6,
7.6 Hz) and signal of C-4 Hcis appeared at d 3.66 showed character-
istic geminal and cis coupling (J = 16.9, 7.6 Hz).The C-4Htrans
appearing at d 3.58 showed geminal and trans coupling constant
16.8 and 10.6 Hz, respectively. Isoxazoline ester 4a–o were hydro-
lyzed in the presence of LiOH to furnish compounds 5a–o (Scheme
2).
Compounds 5a–o were coupled with intermediate 1 mediated
by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochlo-
ride (EDCÁHCl) as coupling reagent at 0 °C in the presence of trieth-
ylamine to afford novel isoxazolinyl oxazolidinone analogues 6a–o
in good yields (Scheme 3).
This series of oxazolidinone antimicrobial agents was screened
against panel of susceptible and resistant bacteria. Compounds
were tested against both Gram-negative and Gram-positive strains
where linezolid and vancomycin were used as standard drug.
The bacterial strains were grown on nutrient agar at 37 °C. After
24 h of incubation, bacterial cells were suspended in normal saline
containing Tween 20 at 0.05% at a concentration of approximately
6a–o) demonstrated a range of MIC 0.0866–24.5580 lM against
resistant bacteria tested in this study. All compounds except 6a,
6m and 6n were more potent than linezolid. From the results in
Table 1 it is clear that unsubstituted phenyl ring at 3-position of
isoxazoline (6a) showed higher MIC values (lower activity) against
all bacterial strains tested. Substitution of chloro group either at 4
position or at 3,4 position of phenyl ring increased antibacterial
activity of compounds (6c and 6e) These compounds 6c and 6e
showed MIC of 0.1559–0.3499
eus ATCC 29213 with MIC of 2.879
tively. Compounds 6b and 6d with fluoro substitution at 3 and at
3,4 position respectively were less potent than compounds 6c
and 6e with chloro substitution suggesting electronegative substi-
tuent was not suitable for antibacterial activity.
lM against all strains except S. aur-
l
M and 2.7036 M, respec-
l
Compound 6h having CF3 group at meta position was proved to
be better than compound 6g having CF3 at para position against all
resistant strains evaluated. This indicated that substitution at meta
position enhanced the antibacterial activity.
Compound 6i was the most potent compound of series (6a–o)
showing MIC of 0.0866 lM against E. faecalis MTCC 439 suggesting
(a)
N
O
(b)
R1
N
OH
R1CHO
R1
3a-o
O
C2H5
2a-o
O
4a-o
(c)
electron withdrawing group at meta position enhanced the
activity.
Among the S. aureus resistant strains ATCC 25293, ATCC 70069,
ATCC 29213 compounds 6c (p-chloro), 6k (3,4-methylenedioxy)
were the most potent compound showing MIC 0.1657 and
N
O
R1
OH
O
5a-o
0.1627
tive compound among heterocyclic analogues with MIC of 0.3725–
1.5294 M. Thiophenyl analogues 6m and 6n were inferior to fura-
lM, respectively. Compound 6o (pyridyl) was the most ac-
Scheme 2. Reagents and conditions: (a) NH2OHÁHCl, NaOH, H2O; (b) NCS, ethyl-
acrylate, Et3N, 0 °C; (c) LiOH, THF:MeOH:H2O, (3:3:1).
l

V. Varshney et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3573–3576
3575
Table 1
MIC values (
l
M) for oxazolidinones 6a–o
O
N
O
R1
O
N
N
N
NHCOCH3
O
F
6a-6o
S.ab
Compounds
R1
S.a.a
S.a.c
B.c.d
E.f.e
K.pn.f
S.p.g
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
Phenyl
24.5580
0.7400
0.1657
0.3486
0.3292
0.3632
1.3518
0.6759
0.3429
0.3423
0.1627
0.7815
6.0582
12.1359
0.7647
4.6290
0.2624
24.5580
0.7400
0.3499
0.3486
0.3292
1.4913
1.3518
0.6759
0.7039
0.7027
0.3435
1.5631
24.2718
6.0583
1.5294
4.6290
4.2066
24.5580
0.7400
2.8729
0.7155
2.7036
2.9827
2.7036
2.7036
0.7039
0.7027
2.8209
6.2525
24.2718
24.2718
1.5294
9.2581
1.0499
24.5580
0.7400
0.3499
0.3486
0.1559
0.3632
1.3518
0.6759
0.1624
0.3423
0.3435
1.5631
6.0582
12.1359
0.7647
2.3145
0.1278
24.5580
0.7400
0.3499
0.3486
0.3292
0.7456
1.3518
0.3292
0.0866
0.3423
0.3435
0.7815
24.2718
12.1359
0.3725
9.2581
0.5249
24.5580
1.4800
0.1657
0.7155
0.3292
0.3633
2.7036
0.6759
0.7039
0.3423
0.1627
1.5631
6.0583
6.0583
0.7647
4.6290
0.2624
24.5580
0.7400
0.3490
0.3486
0.3292
0.7456
1.3518
0.6759
0.7039
0.7027
0.3435
0.7815
12.1359
12.1359
0.3725
4.6290
0.2624
4-Fluorophenyl
4-Chlorophenyl
3,4-Difluorophenyl
3,4-Dichlorophenyl
4-Tolyl
3-Trifluoromethyl phenyl
4-Trifluoromethyl phenyl
3-Nitrophenyl
3-Hydroxy-4-methoxyphenyl
3,4-Methylene dioxyphenyl
2-Furanyl
2-Thiophenyl
3-Thiophenyl
2-Pyridyl
Linezolid
Vancomycin
a
Staphylococcus aureus ATCC 25923 (floxacin and methicillin-resistant).
Staphylococcus aureus ATCC 70069 (methicillin-resistant).
Staphylococcus aureus ATCC 29213 (methicillin and vancomycin-resistant).
Bacillus cereus MTCC 430.
Enterococcus faecalis MTCC 439.
Klebsiella pneumoniae ATCC 27736.
Streptococcus pyogens.
b
c
d
e
f
g
nyl analogue 6l against all strains evaluated. All Compounds except
6a, 6m, and 6n of this series were more potent than linezolid
against Bacillus cereus MTCC 430. Compounds 6e and 6i were as
par with vancomycin against B. cereus MTCC 430 with MIC
Acknowledgements
We are thankful to the Director, CDRI, Lucknow, India for con-
stant encouragement of the drug development programme. We
also acknowledge the SAIF division, CDRI for providing spectro-
scopic data.
0.1559 lM and 0.1624 lM. Rest of the compounds was either equi-
potent or inferior to vancomycin against B. cereus. Among Staphy-
lococcus strains, all compounds except 6i, 6j, 6b and 6c were
more potent than vancomycin against S. aureus ATCC 29213 (resis-
tant to vancomycin) in similar comparison of the respective MIC
Supplementary data
values expressed in
were found to be more active than vancomycin against methicil-
lin-resistant S. aureus ATCC 70069.
lg/mL. All compounds except 6a, 6m and 6n
Supplementary data (the physical and spectral data of all syn-
thesized compounds) associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.04.133.
It is interesting to note that these compounds were also active
against Gram-negative bacterial strains. Generally oxazolidinones
are devoid of activity against Gram-negative bacteria but to our
great surprise these compounds showed promising activity against
K. pneumoniae ATCC 27736, a Gram-negative bacteria, resistant to
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