Novel pyridyl nitrofuranyl isoxazolines show antibacterial activity against multiple drug resistant Staphylococcus species

Article abstract of DOI:10.1016/j.bmc.2017.05.037

A novel series of pyridyl nitrofuranyl isoxazolines were synthesized and evaluated for their antibacterial activity against multiple drug resistant (MDR) Staphylococcus strains. Compounds with piperazine linker between the pyridyl group and isoxazoline ri

Full text of DOI:10.1016/j.bmc.2017.05.037

Accepted Manuscript
Novel pyridyl nitrofuranyl isoxazolines show antibacterial activity against mul-
tiple drug resistant Staphylococcus species.
Pietro Picconi, Priya Prabaharan, Jennifer Auer, Stephanie Sandiford, Francesco
Cascio, Madiha Chowdhury, Charlotte Hind, Matthew E. Wand, J. Mark Sutton,
Khondaker M. Rahman
PII:
S0968-0896(17)30690-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.037
BMC 13755
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 March 2017
8 May 2017
17 May 2017
Please cite this article as: Picconi, P., Prabaharan, P., Auer, J., Sandiford, S., Cascio, F., Chowdhury, M., Hind, C.,
Wand, M.E., Sutton, J.M., Rahman, K.M., Novel pyridyl nitrofuranyl isoxazolines show antibacterial activity
against multiple drug resistant Staphylococcus species., Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.05.037
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Novel pyridyl nitrofuranyl isoxazolines show antibacterial activity
against multiple drug resistant Staphylococcus species.
Pietro Picconi1, Priya Prabaharan1, Jennifer Auer1, Stephanie Sandiford2, Francesco
Cascio1, Madiha Chowdhury1, Charlotte Hind2, Matthew E. Wand2, J. Mark Sutton2*
and Khondaker M. Rahman1*.
1Institute of Pharmaceutical Science, King’s College London, London SE1 1DB, UK.
2Public Health England, National Infections Service, Porton Down, Salisbury SP4 0JG,
Wiltshire, UK.
* Corresponding authors
E-mail: k.miraz.rahman@kcl.ac.uk (KMR) and mark.sutton@phe.gov.uk (JMS)
1

Abstract
A novel series of pyridyl nitrofuranyl isoxazolines were synthesized and evaluated for their
antibacterial activity against multiple drug resistant (MDR) Staphylococcus strains.
Compounds with piperazine linker between the pyridyl group and isoxazoline ring showed
better activity when compared to compounds without the piperazine linker. 3-pyridyl
nitrofuranyl isoxazoline with a piperazine linker was found to be more active than
corresponding 2-and 4-pyridyl analogues with MICs in the range of 4-32 µg/mL against
MDR Staphylococcus strains. The eukaryotic toxicity of the compounds was tested by MTT
assay and were found to be non-toxic against both non-tumour lung fibroblast WI-38 and
cervical cancer cell line HeLa. The most active pyridyl nitrofuranyl isoxazoline compound
showed improved activity against a panel Staphylococcus strains compared to nitrofuran
group containing antibiotic nitrofurantoin.
Key words: Antimicrobial Resistance, Nitrofuran Isoxazoline, MRSA, Medicinal
Chemistry, Antibacterial Activity, Structure Activity Relationship
2

1.0 Introduction
The development of novel antibacterial agents that are capable of killing resistant bacteria is
urgent due to the challenges posed by a number of, both Gram-positive and Gram-negative,
pathogens with multi- and sometimes pan-drug resistance [1-4]. An acronym, ESKAPEE has
been derived from the organisms recognised as the major threats (Enterococcus faecalis,
Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas
aeruginosa, Enterobacter spp and Escherichia coli) although there are a number of other
organisms that may become equally challenging to treat in the clinic [2-7]. The pipeline of
antibiotics is essentially empty and very few compounds are in the early stages of clinical trials
[2, 3, 8-10]. Of the Gram-positive pathogens, increasing resistance in S. aureus has become a
major clinical problem, particularly in the hospital environment, causing significant morbidity
and mortality in both healthy hosts and those with underlying comorbidities [11-13]. S. aureus is
found commensally on nasal passages, skin and mucous membranes. The emergence of resistant
strains of S. aureus has been reported since the mid-1900s. More recently MRSA has become the
number one cause of hospital-associated infections, and a large proportion of these infections are
caused by MRSA [14]. Approximately 150,000 infections are caused by MRSA in the Europe
and the mortality rate is estimated between 15-25%[15] . Therefore, development of novel
antimicrobial agents for the treatment of infections caused by MDR S. aureus is an urgent
priority.
Classical antimicrobial drug discovery involves in vitro screening for antimicrobial candidates,
Structure Activity Relationship (SAR) analysis, followed by in vivo testing for toxicity and
efficacy. However, the high cost of drug development, coupled with a relatively short window of
use to gain a return on investment for new antibiotics, have resulted in a waning interest in
3

antibiotic discovery among many pharmaceutical companies[16]. Lack of novel chemical
scaffolds with antibiotic activity has further compounded the problem[2], and no new classes of
antibiotics have been found since daptomycin, leading to a discovery void[17]. One of the key
approaches in discovering new antibacterial drugs is the modification of existing chemical
scaffolds, almost all the antibacterial drugs that reached the market after the 1960s are synthetic
derivatives of core-scaffolds discovered between 1930 and 1950. Modifications and
introductions of novel chemical moieties on the core-scaffold resulted in new generations of
molecules typically characterized by greater potency, a broader spectrum of activity and
capability of avoiding the mechanism of resistance[2]. In a bid to identify a new chemical
scaffold with activity against MDR Gram-positive bacteria, we explored nitrofuran-isoxazoline
scaffold that has been previously reported for notable activity against Mycobacterium
tuberculosis[18-21]. Incorporation of a pyridyl side chain with a piperazine spacer has resulted in
compounds (Fig 1a) with significant activity against MDR Staphylococcus species including
MRSA strains. Interestingly, the position of the nitrogen atom in the pyridine ring appears to be
very important for antibacterial activity with 3-pyridyl analogue showed the most notable
activity followed by the 2-pyridyl analogue while the 4-pyridyl analogue is either inactive or
active only at a very high concentration. Incorporation of electron withdrawing or donating
groups in the terminal pyridine ring retained activity of the 3-pyridyl compounds. The piperazine
spacer appeared to have a notable effect on the antimicrobial efficacy as compounds without the
piperazine spacers (Fig 1b) were found to be less active compared to their counterparts with
piperazine spacers, except the 4-pyridyl analogues which showed some gain of activity after the
removal of the piperazine spacer. The activity range of the eight compounds synthesized
4

provides SAR information about this modified chemical scaffold and its antimicrobial profile
that can be further explored to develop more potent analogues.
Fig 1. Structures of isomeric pyridyl nitrofuran isoxazolines with a piperazine spacer, 8a-e,
and without a piperazine spacer, 10a-c.
2.0 Materials and Methods
2.1 Chemistry
All solvents and reagents for the synthesis were obtained from commercial available sources
including Sigma-Aldrich, Fisher Scientific, Fluorochem and Alfa Aesar. Thin-layer-
chromatography (TLC) analysis was performed on silica gel plates (E. Merck silica gel 60 F254
plates) and visualized by ultra-violet (UV) radiation at 254 nm. Flash chromatography for the
purification of compound was performed with silica gel as a stationary phase (Merck 60, 230-
400 mesh). 1H and 13C nuclear magnetic resonance (NMR) analyses were performed on a Bruker
Spectrospin 400 Hz spectrometer. LC-MS analyses were performed on a Waters Alliance 2695
system, eluting in gradient with a flow rate of 0.5 mL/min according to the condition reported
5

herein in Table S1: The analyses were performed on a Monolithic C18 50 X 4.60 mm column by
Phenomenex. UV detection was performed on a Diode Array Detector. Mass spectra were
registered in both ESI+ and ESI- mode.
Synthesis of novel pyridyl nitrofuranyl isoxazolines with piperazine spacer, 8a-e
Synthesis of tert-butyl 4-(4-vinylphenyl)piperazine-1-carboxylate (3)
A mixture of 1 (1.06 g, 5.80 mmol, 1 eq.), NaOtBu (2.4 eq.), PdCl2[P(o-Tol)3]2 (0.06 eq.) and 2
(2 eq.) dissolved in toluene (50 mL) was left at reflux at 100°C with stirring in N2 atmosphere for
3 hours. The crude of reaction was filtered on Celite eluting with DCM and then concentrated
under reduced pressure. Purification by column chromatography on silica gel (mobile phase: 9/1,
1
v/v, hexane/EtOAc) provided 3 (1.19 g, 73%) as a yellow solid. H NMR (400 MHz, CDCl3 )
7.35 (d, J = 8.56 Hz, 2H), 6.89 (d, J = 8.56 Hz, 2H), 6.67 (dd, J = 10.83, 17.63 Hz, 1H), 5.63
(d, J = 17.63 Hz, 1H), 5.13 (d, J = 10.83 Hz, 1H), 3.52 - 3.65 (m, 4H), 3.09 - 3.21 (m, 4H), 1.53
(s, 9H).
13C NMR (100 MHz, CDCl3) 28.43, 31.44, 36.51, 49.14, 79.95, 111.22, 116.27, 127.13,
129.66, 136.23, 150.80, 154.72, 162.55. m/z (+EI) calc. for C17H24N2O2 (M)+ 288.30 found
289.10 ([M]+H)+.
Synthesis of N-hydroxy-5-nitrofuran-2-carbimidoyl chloride (5)
5-nitro-2-furaldoxime 4 (1.5 g, 10 mmol, 1 eq.) was dissolved in a solution of HCl 0.5M (1.1 eq.,
916 µl) in DMF (10.55 mL) and subsequently oxone (1.1 eq.) was added. The reaction mixture
was left under magnetic stirrer at room temperature until TLC showed total consumption of the
starting material. The reaction mixture was diluted with water (100 mL) and extracted with
6

EtOAc (3 x 40 mL). The organic layer was then washed with HCl 0.5 M aqueous solution (100
mL) and brine (100 mL). The collected organic phase was dried on MgSO4, filtered and
concentrated under reduced pressure providing 5 (1.87 g, yield was assumed 100%) as a yellow
solid.
Synthesis of tert-butyl 4-(4-(3-(5-nitrofuran-2-yl)-4,5-dihydroisoxazol-5-
yl)phenyl)piperazine-1-carboxylate (6)
Triethylamine (1.20 eq) was added to a solution of 3 (500 mg, 1.70 mmol 1 eq.) and 5 (1.20 eq.)
in CHCl3 (5 mL). The reaction mixture was left stirring in N2 atmosphere for 20 hours until TLC
showed total consumption of the starting material. At that point CHCl3 (11 mL) was added to the
reaction mixture and the organic phase was washed with brine (4 x 10 mL) and then dried on
MgSO4, filtered and concentrated under reduced pressure. The obtained crude product was
purified by column chromatography on silica gel (mobile phase: 7:3, v/v, diethyl ether-hexane)
provided 6 (558.60 mg 74.3%) as a deep red solid.
1H NMR (400 MHz, CDCl3) 7.29 (d, J = 3.78 Hz, 1H), 7.16 (d, J = 8.56 Hz, 2H), 6.90 (d, J =
4.03 Hz, 1H), 6.82 (d, J = 8.56 Hz, 2H), 5.64 (dd, J = 8.94 Hz, 10.95 Hz, 1H), 3.64 (dd, J =
11.08 Hz, 17.12 Hz, 1H), 3.41 - 3.53 (m, 4H), 3.27 (dd, J = 8.81 Hz, 17.12 Hz, 1H), 2.94 - 3.10
(m, 4H), 1.39 (s, 9H).
13C NMR (100 MHz, CDCl3) 28.43, 41.23, 49.01, 80.02, 83.99, 112.48, 113.15, 116.48,
127.21, 130.17, 147.50, 147.85, 151.60, 154.70. m/z (+EI) calc. for C22H26N4O6 (M)+ 442.4
found 443.1 ([M]+H)+.
7

2.2 General procedure for synthesis of final products (8a-e)
Concentrated hydrochloric acid solution (14 mL) was added to a solution of 6 (430 mg, 0.97
mmol, 1 eq.) in MeOH (15 mL) and the reaction mixture was left under magnetic stirrer at room
temperature for 15 minutes until TLC showed total removal of the protecting group. The solution
was then concentrated under reduced pressure to provide 7 (348 mg, 100%) as white solid.
Finally, the corresponding bromomethyl pyridine hydrobromide or chlorometyl pyridine
hydrochloride derivatives or bromomethyl 4-substitited pyridine derivatives (1.5 eq.) was added
to a solution of 7 (from 61 mg to 177 mg, 1 eq.) in DMF (8 mL) in the presence of K2CO3 (3
eq.). The reaction mixture was stirred at 50°C under N2 atmosphere for 16 hours. The reaction
was quenched by addition of water (20 mL) and subsequently extracted with EtOAc (3 x 20 mL).
The collected organic phases were washed with brine (3X15 mL), dried on MgSO4, filtered and
concentrated under reduced pressure. The crude product was purified by column chromatography
on silica gel (mobile phase: from 98:2, v/v, DCM-MeOH to 95:5, v/v, DCM-MeOH) obtaining
the final products 8a-e.
3-(5-nitrofuran-2-yl)-5-(4-(4-(pyridin-2-ylmethyl)piperazin-1-yl)phenyl)-4,5-
dihydroisoxazole (8a).
Obtained 68 mg (98%), as a dark yellow solid. 1H NMR (400 MHz, CDCl3) 8.57 (d, J = 4.78
Hz, 1H), 7.59 - 7.71 (m, 1H), 7.43 (d, J = 7.81 Hz, 1H), 7.37 (d, J = 3.78 Hz, 1H), 7.12 - 7.25
(m, 3H), 7.00 (d, J = 4.03 Hz, 1H), 6.90 (d, J = 8.81 Hz, 2H), 5.72 (dd, J = 8.94 Hz, 10.95 Hz,
1H), 3.64 - 3.79 (m, 3H), 3.37 (dd, J = 8.94 Hz, 17.25 Hz, 1H), 3.14 - 3.28 (m, 4H), 2.58 - 2.71
(m, 4H).
8

13C NMR (100 MHz, CDCl3) 41.15, 48.67, 53.14, 64.55, 84.12, 112.44, 113.15, 115.91,
122.23, 123.40, 127.16, 129.44, 136.51, 147.56, 147.85, 149.40, 151.68. HRMS (EI, m/z): calc.
for C23H24N5O4 ([M]+H)+ 434.1823 found 434.1819.
3-(5-nitrofuran-2-yl)-5-(4-(4-(pyridin-3-ylmethyl)piperazin-1-yl)phenyl)-4,5-
dihydroisoxazole (8b).
Obtained 44 mg (33.3%) as a yellow solid. 1H NMR (400 MHz, CDCl3)  8.51 (s, 1H), 8.46 (d,
J = 4.78 Hz, 1H), 7.65 (d, J = 7.81 Hz, 1H), 7.31 (d, J = 3.78 Hz, 1H), 7.12 - 7.25 (m, 3H), 6.94
(d, J = 3.78 Hz, 1H), 6.83 (d, J = 8.56 Hz, 2H), 5.66 (dd, J = 8.94 Hz, 10.95 Hz, 1H), 3.66 (dd, J
= 11.08, 17.12 Hz, 1H), 3.50 (s, 2H), 3.30 (dd, J = 9.06 Hz, 17.12 Hz, 1H), 3.04 - 3.20 (m, 4H),
2.42 - 2.59 (m, 4H).
13C NMR (100 MHz, CDCl3) 41.17, 48.71, 52.90, 60.17, 84.09, 112.45, 113.16, 115.95,
123.42, 127.18, 129.56, 133.32, 136.79, 147.54, 147.85, 148.81, 150.51, 151.60. HRMS (EI,
m/z): calc. for C23H24N5O4 ([M]+H)+ 434.1823 found 434.1863.
3-(5-nitrofuran-2-yl)-5-(4-(4-(pyridin-4-ylmethyl)piperazin-1-yl)phenyl)-4,5-
dihydroisoxazole (8c).
1
Obtained 104 mg (51%) as a yellow solid. H NMR (400 MHz, CDCl3)  8.51 - 8.61 (m, 2H),
7.39 (d, J = 3.78 Hz, 1H), 7.31 (d, J = 5.54 Hz, 2H), 7.23 - 7.27 (m, J = 8.81 Hz, 2H), 7.02 (d, J
= 3.78 Hz, 1H), 6.86 - 6.95 (m, J = 8.56 Hz, 2H), 5.75 (dd, J = 9.06 Hz, 11.08 Hz, 1H), 3.74 (dd,
9

J = 11.20 Hz, 17.25 Hz, 1H), 3.57 (s, 2H), 3.39 (dd, J = 8.94 Hz, 17.25 Hz, 1H), 3.19 - 3.28 (m,
4H), 2.57 - 2.67 (m, 4H).
13C NMR (100 MHz, CDCl3) 41.15, 48.72, 53.04, 61.67, 84.08, 112.52, 113.16, 115.91,
123.88, 127.19, 129.58, 147.34, 147.51, 147.83, 149.87, 151.58, 152.10. HRMS (EI, m/z): calc.
for C23H24N5O4 ([M]+H)+ 434.1823 found 434.1820.
5-(4-(4-((6-fluoropyridin-3-yl)methyl)piperazin-1-yl)phenyl)-3-(5-nitrofuran-2-yl)-4,5-
dihydroisoxazole (8d).
Obtained 11 mg (10.6%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ: 8.16 (d, J = 1.89 Hz,
1H), 7.83 (td, J = 7.96 Hz, 1.95 Hz, 1H), 7.40 (d, J = 3.78 Hz, 1H), 7.25 (d, J = 8.69 Hz, 2H),
7.03 (d, J = 3.90, 1H), 6.87 – 6.97 (m, 3H), 5.75 (dd, J = 11.02 Hz, 9.00 Hz, 1H), 3.75 (dd, J =
17.12 Hz, 11.08 Hz, 1H), 3.57 (s, 2 H), 3.39 (dd, J = 17.18 Hz, 8.88 Hz, 1H), 3.18 – 3.26 (m,
4H), 2.56 – 2.66 (m, 4H). 13C NMR (100 MHz, CDCl3) δ: 41.13, 48.62, 52.76, 59.06, 84.02,
109.15, 109.53, 112.44, 113.13, 115.94, 127.14, 129.63, 141.99, 142.07, 147.47, 147.73, 147.81,
147.86, 151.47, 161.91.
5-(4-(4-((6-methoxypyridin-3-yl)methyl)piperazin-1-yl)phenyl)-3-(5-nitrofuran-2-yl)-4,5-
dihydroisoxazole (8e)
Obtained 37 mg (34.6%) as a yellow solid. Obtained 37 mg (34.6%) as a yellow solid.
1H NMR (400 MHz, CDCl3) δ: 8.08 (d, J =2.14 Hz, 1H), 7.58-7.67 (m, 1H), 7.40 (d, J = 3.90
Hz, 1H), 7.25 (d, J = 8.69 Hz, 2H), 7.04 (d, J = 3.78 Hz, 1H), 6.91 (d, J = 8.81 Hz, 2H), 6.74 (d,
J = 8.44 Hz, 1H), 5.75 (dd, J = 11.02 Hz, 9.00 Hz, 1H), 3.94 (s, 3H), 3.75 (dd, J = 17.25 Hz,
10

11.08 Hz, 1H), 3.51 (br. s., 2H), 3.40 (dd, J = 17.25 Hz, 8.94 Hz, 1H), 3.22 (br. s., 4H), 2.60 (br.
s., 4H). 13C NMR (100 MHz, CDCl3) δ: 41.11, 48.60, 52.65, 53.41, 59.47, 84.04, 110.69, 112.41,
113.11, 115.87, 125.65, 127.11, 129.45, 139.87, 147.08, 147.48, 147.80, 151.55, 152.03, 163.64.
Synthesis of novel pyridyl nitrofuranyl isoxazolines without the piperazine spacer, 10a-c
Synthesis of 5-(4-bromophenyl)-3-(5-nitrofuran-2-yl)-4,5-dihydroisoxazole (9).
To a solution of 4-bromostyrene (400 mg, 2.18 mmol, 1 eq.) in CHCl3 (10 mL), 0.36 mL (1.20
eq) trimethylamine and 5 (500 mg, 2.62 mmol, 1.20 eq) were added. The reaction mixture was
stirred overnight under N2 atmosphere. The organic phase was extracted with CHCl3 (3 x 10
mL), washed with brine and dried over MgSO4, filtered and concentrated under reduced
pressure. The obtained crude product was purified by column chromatography on silica gel
(mobile phase: 1:1, v/v, diethyl ether-hexane) provided 9 (515 mg 70%) as a cream coloured
solid.
1H NMR (400 MHz, CDCl3)  7.52 (d, J = 8.31 Hz, 2H), 7.39 (d, J = 3.78 Hz, 1H), 7.24 (d, J =
8.56 Hz, 2H), 7.04 (d, J = 4.03 Hz, 1H), 5.79 (dd, J = 8.18 Hz, 11.20 Hz, 1H), 3.84 (dd, J = 11.33
Hz, 17.12 Hz, 1H), 3.35 (dd, J = 8.18 Hz, 17.25 Hz, 1H).
13C NMR (100 MHz, CDCl3)  147.58, 146.85, 138.44, 131.98, 127.40, 122.59, 112.98, 112.81,
82.91, 41.70, 29.61. m/z (+EI) calc. for C13H9BrN2O4 (M)+ 335.9 found 336.9 ([M]+H)+.
Synthesis of 3-(5-nitrofuran-2-yl)-5-(4-(pyridin-2-yl)phenyl)-4,5-dihydroisoxazole (10a).
Tetrakis(triphenylphosphine)palladium(0) (69.2 mg, 0.06 mmol, 0.08 eq.), K2CO3 (290.20 mg,
2.10 mmol, 3 eq.) and pyridine-2-boronic acid (129.04 mg, 1.05 mmol, 1.50 eq.) were added to a
11

solution of 9 (262.95 mg, 0.70 mmol, 1 eq.) in dioxane (5 mL) and water (0.50 mL). The
reaction mixture was stirred at reflux under an N2 atmosphere for 72 hrs. The reaction was not
completed, but it was decided to work up the reaction mixture to prevent formation of further
impurities. The reaction was filtered on a Celite pad, with the solvent evaporating under reduced
pressure and the crude reaction mixture dissolving in ethyl acetate (10 mL). This solution was
washed with water and the aqueous layer was extracted with ethyl acetate (3 x 10 mL). The
combined organic layer was dried over MgSO4 and evaporated in vacuo. The obtained crude
product was then purified by column chromatography on silica gel (mobile phase: from 100%
DCM to 95/5, v/v, DCM/acetone), obtaining the final product 10a as a yellow-red solid.
1H NMR (400 MHz, CDCl3) 8.74 (d, J = 5.54 Hz, 1H), 8.04 (d, J = 8.31 Hz, 2H), 7.73 - 7.83
(m, 2H), 7.49 (d, J = 8.31 Hz, 2H), 7.41 (d, J = 3.78 Hz, 1H), 7.30 (d, J = 6.04 Hz, 1H), 7.07 (d, J
= 3.78 Hz, 1H), 5.91 (dd, J = 8.31 Hz, 11.33 Hz, 1H), 3.88 (dd, J = 11.08 Hz, 17.12 Hz, 1H),
3.45 (dd, J = 8.56 Hz, 17.12 Hz, 1H). HRMS (EI, m/z): calc. for C18H13N3O4 (M)+ 336.0979
found 336.0979([M]+H)+.
2.3 General procedure for the synthesis of pyridyl isoxazolines without the piperazine linker
(10b-c)
[Pd2(dba)3] (48.9 mg, 0.053 mmol, 0.067 eq.), Tricyclohexylphosphine (131.24 mg, 0.47 mmol,
0.6 eq.), K3PO4 (496.71 mg, 2.34 mmol, 3 eq.) and the desired pyridine boronic acid (143.80
mg, 1.17 mmol, 1.5 eq.) were added to a solution of 9 (262.95 mg, 0.70 mmol, 1 eq.) in dioxane
(5 mL) and water (0.50 mL). The reaction mixture was stirred at reflux under N2 atmosphere for
20h at which point LC-MS monitoring showed complete consumption of the starting material.
The reaction was then filtered on a Celite pad, the solvent evaporated under reduced pressure and
the evaporated crude reaction mixture was dissolved in ethyl acetate (10 mL). This solution was
12

washed with water and the aqueous layer was extracted with ethyl acetate (3 x 10 mL). The
combined organic layer was dried over MgSO4 and evaporated in vacuo. The obtained crude
product was then purified by column chromatography on silica gel (mobile phase: from 100%
DCM to 95/5, v/v, DCM/acetone) obtaining the final products 10b-c.
3-(5-nitrofuran-2-yl)-5-(4-(pyridin-3-yl)phenyl)-4,5-dihydroisoxazole (10b).
Obtained 24 mg as a yellow solid. 1H NMR (400 MHz, CDCl3) 8.85 (d, J = 2.27 Hz, 1H), 8.62
(dd, J = 1.51 Hz, 4.78 Hz, 1H), 7.89 (td, J = 1.92 Hz, 7.99 Hz, 1H), 7.62 (d, J = 8.31 Hz, 2H),
7.49 (d, J = 8.31 Hz, 2H), 7.35 - 7.44 (m, 2H), 7.07 (d, J = 4.03 Hz, 1H), 5.90 (dd, J = 8.31 Hz,
11.33 Hz, 1H), 3.88 (dd, J = 11.20 Hz, 17.25 Hz, 1H), 3.45 (dd, J = 8.31 Hz, 17.12 Hz, 1H).
13C NMR (100 MHz, CDCl3)  148.97, 148.42, 148.04, 147.41, 139.73, 138.57, 136.24, 134.78,
128.02, 126.89, 123.98, 113.34, 113.05, 83.66, 42.14, 30.00. HRMS (EI, m/z): calc. for
C18H13N3O4 (M)+ 336.0979 found 336.0976 ([M]+H)+.
3-(5-nitrofuran-2-yl)-5-(4-(pyridin-4-yl)phenyl)-4,5-dihydroisoxazole (10c).
Obtained 17.40 mg as a yellow glassy solid. 1H NMR (400 MHz, CDCl3)  8.68 (d, J = 5.54 Hz,
2H), 7.68 (d, J = 8.31 Hz, 2H), 7.46 - 7.55 (m, 4H), 7.41 (d, J = 3.78 Hz, 1H), 7.08 (d, J = 3.78
Hz, 1H), 5.91 (dd, J = 8.18 Hz, 11.20 Hz, 1H), 3.89 (dd, J = 11.33 Hz, 17.12 Hz, 1H), 3.45 (dd, J
= 8.06 Hz, 17.12 Hz, 1H). HRMS (EI, m/z): calc. for C18H13N3O4 (M)+ 335.3 found 336.0977
([M]+H)+. 13C NMR (100 MHz, CDCl3) 150.44, 148.03, 147.35, 140.79, 138.83, 127.93,
126.89, 121.95, 113.34, 113.08, 83.54, 42.19, 30.00. HRMS (EI, m/z): calc. for C18H13N3O4
(M)+ 336.0979 found 336.0977 ([M]+H)+.
13

2.3 Toxicity evaluation of the compounds against eukaryotic cells
2.3.1 Cell Culture
HeLa (human cervical cancer) cell line and non-tumour WI38 cell line was obtained from the
American Type Culture Collection. The HeLa cell-line was maintained in monolayer culture in
75 cm2 flasks (TPP, Switzerland) under a humidified 5% CO2 atmosphere at 37°C. The HeLa
cell line was maintained in Dulbecco’s Modified Eagles Media (DMEM; Invitrogen)
supplemented with foetal bovine serum (10% v/v; Invitrogen), L-glutamine (2 mM; Invitrogen),
non-essential amino acids (1x; Invitrogen) and Penicillin-Streptomycin (1% v/v, Invitrogen). For
WI 38 cell line, Dulbecco’s MEM, supplemented with L-glutamine (2mM; Invitrogen), non-
essential amino acids (1x; Invitrogen), penicillin-streptomycin (1% v/v, Invitrogen) and foetal
bovine serum (15%, Biosera UK) was used. For seeding, cells were counted using a Neubauer
haemocytometer (Assistant, Germany) by microscopy (Nikon, USA) on a non-adherent
suspension of cells that were washed in PBS, trypsinised, centrifuged at 4°C at 4000 rpm for 5
min and re-suspended in fresh medium.
2.3.2 MTT Assay
The cells were grown in normal cell culture conditions at 37 ºC under a 5% CO2 humidified
atmosphere using appropriate medium. The cell count was adjusted to 105 cells/mL and 10,000
cells were added per well. The cells were incubated for 24 hours and 1 μl of the appropriate
ligand concentrations were added to the wells in triplicates. After 96 hours of continuous
exposure to each compound, the cytotoxicity was determined using the 3-(4,5-Dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Lancaster Synthesis Ltd, UK) colorimetric
14

assay.[34 Absorbance was quantified by spectrophotometry at λ = 570 nm (Envision Plate
Reader, PerkinElmer, USA). IC50 values were calculated by a dose-response analysis using the
Prism GraphPad Prism® software.
2.4 Microbiological Evaluation of the Compounds
2.4.1 Determination of minimum inhibitory concentration
MICs were determined using the broth microdilution method as outlined in the CLSI guidelines
[22]. Cell growth in Muller Hinton Broth (MHB) was determined by measuring optical density
(600 nm) using a FLUOstar Omega microplate reader (BMG Labtech). The MIC was determined
as the lowest concentration of drug at which growth was below an optical density of 0.10 at 600
nm after 20 hours growth. Tests were conducted in triplicate.
2.4.2 Determination of minimum bactericidal concentration
To determine the minimum bactericidal concentration (MBC), 10ul was taken from all wells of
the MIC plate at and above the MIC and spotted onto a tryptic soy agar plate. The agar plates
were incubated at 37 C for 24 hours and the MBC was defined as the concentration at which no
colonies were seen. The ratio MBC/MIC that was used to evaluate if the compound is
bactericidal (MBC/MIC = 1 or 2) or bacteriostatic (MBC/MIC = 4 or 16).
2.4.3 Time kill assays.
Flasks of MHB were inoculated with test organism at a concentration ~106 cfu/mL in a total
volume of 50 mL. The antimicrobial agents were then added at a concentration of 4 x MIC and
incubated at 37 C in a shaking incubator at 200 rpm. Samples (0.10 mL) were taken from each
15

sample every hour for 8 hours and at 24 hours following inoculation. The effect on bacterial
growth was determined using a modification of the Miles Misra dilution methods to estimate
viable counts.
3.0 Results
3.1 Chemistry
The synthetic scheme for the pyridyl nitrofuranyl isoxazoline compounds with piperazine spacer
(8a-e) is shown in Fig 2. First, the piperazine spacer was installed on p-bromo styrene by a
palladium-catalyzed aromatic amination reaction[23] on 1 with N-Boc piperazine in 73% yield.
Next, the 5-nitro-2-furaldoxime 4 was converted to the corresponding hydroximoyl chloride 5
using Oxone and HCl[24] in DMF which upon treatment with olefin 3, underwent a [3 + 2]
regioselective cycloaddition[25] under inert condition to give the reference Boc-protected
isoxazoline 6 in 74.3% yield. The Boc-deprotection of 6 was carried out in a methanolic solution
of HCl in room temperature in quantitative yield. Finally, the free amine 7 was treated with 2-
and 3-bromomethyl pyridine, 4-chloromethyl pyridine, 5-(bromomethyl)-2-fluoropyridine, 5-
(bromomethyl)-2-methoxypyridine, in the presence of K2CO3 to afford 8a, 8b 8c, 8d and 8e
with between 98%, 51%, 33%, 10.6% and 34.6% yield, respectively. Compounds 8a-c were
initially synthesized and evaluated for their antimicrobial activity. 8d and 8e are structural
analogues of 8b with an electron withdrawing F and electron donating methoxy group at 2-
position of the pyridine ring. The aim of this modification was to explore the role of electronic
environment in the terminal pyridyl group on the antimicrobial activity of this class of
compounds.
16

Fig 2. Synthesis of pyridyl nitrofuranyl isoxazolines with piperazine spacer 8a-e. Reagent
and conditions: a) PdCl2[P(o-Tol)3]2, NaOtBu, toluene, reflux, 3 hrs; b) Oxone, HCl 0.5M
aqueous solution, DMF, r.t., 24 hrs; c) Et3N, chloroform, r.t., 20 hrs; d) HCl 37% aqueous
solution, MeOH, r.t., 2 hrs; e) K2CO3, DMF, 50 oC, overnight.
To explore the role of the piperazine spacer between the phenyl ring and the terminal pyridyl
ring, three more compounds were designed in which the piperazine spacer was removed and the
phenyl ring was directly coupled to the pyridyl ring using Suzuki coupling. These compounds
(10a-c) were synthesized according to synthetic scheme reported in Fig 3. First, the 5-nitro-2-
17

furaldoxime 4 was converted to the corresponding hydroximoyl chloride 5 using Oxone and
HCl[24] in DMF which underwent a [3 + 2] regioselective cycloaddition[25] with p-bromo
styrene under inert condition to afford the Suzuki substrate 9 in 70% yield. Finally, the Suzuki
cross-coupling reactions between 9 and the pyridyl-3-boronic acid and pyridyl-4-boronic acid
were carried out using Pd2(dba)3 as a catalyst under inert condition in the presence of
tricyclohexylphosphine and K3PO4 to afford 10b, and 10c. The Suzuki reaction between 9 and
pyridyl-2-boronic acid did not proceed under same condition, and was carried out using tetrakis
palladium as the catalyst with a modest 10% yield to afford 10a.
Fig 3. Synthesis of pyridyl nitrofuranyl isoxazolines without the piperazine spacer 10a-c.
Reagent and conditions: a) Oxone, HCl 0.5M aqueous solution, DMF, r.t., 24 hrs; b)
bromostyrene, Et3N, chloroform, r.t., 20 hrs; c) [Pd2(dba)3], K3PO4, P(Cy)3, Dioxane, H2O
100 °C, d) Pd(PPh3)4, K2CO3, Dioxane/H2O, 100 °C
18

3.2 Microbiological Evaluation
3.2.1 MIC and MBC Determination
The antibacterial evaluation of compound 6, 8a—c and 10a-c were carried out using the broth
microdilution method as outlined in the CLSI guidelines[22]. Initially, the compounds were
tested against a panel of ESKAPEE pathogens consisting of three Gram-positive Staphylococcus
and six Gram-negative bacteria with a variety of antibiotic resistance mechanisms. The reference
isoxazoline 6, was found to be broadly inactive against both Gram-positive and Gram-negative
panel while newly synthesized pyridyl nitrofuranyl isoxazolines with piperazine spacer 8a-b
showed a wide range of activity against the Gram-positive panel with MICs between 16 to >128
µg/mL (Table 1), and 8c showed activity similar to that of 6 (Table 1). None of the compounds
showed any activity against the Gram-negative pathogens tested (Klebsiella pneumoniae strains
NCTC 13368 and M6[26], Acinetobacter baumannii strains ATCC BAA-1710 and ATCC
17978, Pseudomonas aeruginosa strains PAO1 and NCTC 13437; MIC values all >128 µg/mL;
results not shown). The initial Gram-positive panel included; methicillin sensitive (MSSA) S.
aureus MSSA4144, and methicillin resistant S. aureus (MRSA) isolates representative of
EMRSA-15 and EMRSA-16[27].
Table 1. Activity of 8a-e against the initial Staphylococcus panel.
MIC (µg/ml)
Nitrofur
- antoin
16
Vancomyc Amoxicil
Cipro
Bacteria
6
8a
8b
8c
8d
8e
in
lin
32
32
MSSA
9144
32
16
16
16
0.125
0.5
0.1-2
128
16
128
32
16
16
EMRSA
-15
>128
>128
32
32
16
32
>128
128
32
32
2
2
128
EMRSA
-16
>128
19

Compound 8b gave MIC values of 16-64 µg/mL for MSSA/MRSA strains. 8b showed
comparable activity against the MSSA 9144 and EMRSA 15 strains compared to the nitrofuran
group containing control antibiotic nitrofurantoin (MIC 16 µg/mL) which is expected to share a
similar mechanism of action (i.e., nitrocompound toxicity). This prompted us to synthesize two
additional analogues of 8b with weakly electron withdrawing F containing 8d and electron
donating methoxy group containing 8e. 8d showed an improvement in MIC value against
EMRSA-16 (MIC 16 µg/mL) compared to 8b, comparable activity against MSSA9144 but
notably lost activity against EMRSA-15. Compound 8e also showed a similar profile except an
improvement against EMRSA-16 wasn’t observed for this analogue. The MIC values of the
shortened pyridyl nitrofuranyl isoxazolines without the piperazine spacer are reported in Table
2. Compound 10a was found to be inactive against the Staphylococcus panel. Compound 10b
showed activity against all three Staphylococcus strains with MIC values between 16 to 128
µg/mL. Compound 10c also showed activity against all three Staphylococcus strains but the MIC
values were slightly higher (MIC range 64 to 128 µg/mL) than 10b. It appeared that the removal
of the piperazine spacer had a detrimental effect on the MIC values of 8a and 8b, but had a small
positive effect on the MIC values of 8c. Compound 10b maintained comparable activity against
methicillin resistant Staphylococcus EMRSA 16 compared to nitrofurantoin.
20

Table 2. Activity of 10a-c against the initial Staphylococcus panel.
MIC (µg/ml)
Bacteria
MSSA
9144
10a
10b
10c Nitrofurantoin
Cipro
Vancomycin Amoxicillin
>128
32
64
16
0.125
0.5
0.1-2
EMRSA -
15
>128
>128
64
16
128
16
32
32
2
2
128
64
16
>128
EMRSA-16
Compounds 8a and 8b were tested for their minimum bactericidal concentration (MBC) and the
ratio of MBC to MIC was calculated to determine the bactericidal vs bacteriostatic mode of
action the compounds. Both 8a and 8b showed MBC of ≥128 µg/mL against the MSSA 9144
strain with an MBC to MIC ratio of 8 suggesting a bacteriostatic mode of action as compounds
are usually regarded bactericidal if the MBC is no more than four times MIC. Both compounds
showed MBC values of >128 µg/mL against the EMRSA strains with MBC to MIC ratio of
greater than 4 in each cases.
Compounds 8a and 8b were tested against an extended panel of S. aureus and S. epidermidis
strains from a range of different lineages (Table 3). Both 8a and 8b showed good activity against
the extended Staphylococcus panel with MICs ranging from 8 to 32 µg/mL for 8a and 4-16
µg/mL for 8b. Both compound 8a and 8b showed either improved or comparable activities
(MICs 4-16 µg/mL) against the extended Staphylococcus panel compared to nitrofurantoin
(MICs 16 µg/mL). The levels of activity against different strains were unaffected by molecular
mechanisms which mediate resistance to ciprofloxacin and amoxicillin in Staphylococcus spp
(e.g. strains EMRSA-15 and EMRSA-16).
21

Table 3. Activity of 8a and 8b against extended Enterococcus and Staphylococcus panels.
MIC (µg/ml)
Nitrofurantoin
Vancomycin Amoxicillin
Cipro
0.125-
0.5
Bacteria
8a
8b
4
128
S. epidermidis
8-16
4-8
16
ATCC 35984
2
2
1
>128
0.25-0.5
>128
S. aureus A1988
16
8
8
16
16
0.25
0.5
S. aureus BGW541
8-16
MRSA NCTC
12493
8
4-8
16
16
0.5
2
2
0.25
0.25
S. aureus 13142
8-16
8-16
0.25
0.25-
0.5
S. aureus 6571 PVL
16-32
16
16
16
16
16
2
0.25-0.5
0.125-
0.25
S. aureus 8325 PVL
3.2.2 Time Kill Assay
Compound 8b was selected for time kill analysis against the EMRSA-15 strain to determine
whether 8b was bactericidal or bacteriostatic. 8b appeared to be bacteriostatic against with
activity against EMRSA-15 comparable to that of ciprofloxacin which was used as a control
(Fig4). There was no evidence of resistant colonies being selected for during the time-kill assays,
but there were viable cells which survived the treatment process with 8b for EMRSA-15 with no
significant reduction in the viable count between approximately 5 hours post treatment and the
end of the time course at 24 hours (Fig 4). The data suggests a bacteriostatic mode of action
which is consistent with the MBC to MIC ratio reported in Table 3.
22

Fig 4. Time kill analysis of compound 8b against Staphylococcus strain EMRSA-15.
EMRSA-15 was treated with 8b or ciprofloxacin at 4X MIC for 24 hours at 37C. Results are
the average of at least three replicate experiments and error bars show the standard error of the
mean.
3.2.3 Cytotoxicity Screening
Finally, the compounds were tested for their cytotoxic potential against the cervical cancer cell
line HeLa using the MTT assay[28] and the non-tumour lung fibroblast cell line WI-38 and the
results are shown in Fig 5 and S1Table. Doxorubicin was used as a positive control in the MTT
assay for comparison purpose. Doxorubicin showed notable cytotoxicity with an IC50 of 71 nM
(Fig 5), but none of the compounds demonstrated a measurable IC50 at the concentrations tested
23

and this lack of toxicity against the mammalian cells was encouraging from the anti-infective
point of view as this chemical scaffold offers selective toxicity against prokaryotic cells.
Fig 5. MTT cell-viability assay profile in HeLa and WI-38 cells treated with 8b, 10b and
control compound doxorubicin. HeLa and WI-38 cells were treated with selected compounds
for 96 hours. Results are the average of at least three replicate experiments.
4.0 Discussion
Eight new pyridyl nitrofuranyl isoxazolines were successfully synthesized using solution phase
linear chemistry. The compounds were derived from a reported molecule that has shown
antitubercular activity but was found to be broadly inactive against both Gram-positive and
Gram-negative bacteria. Compounds generated by modifying the terminal phenyl ring and
heterocyclic spacer (8a-b, d, e, 10a-c) showed significant activity against MDR Gram-positive
pathogens and no notable toxicity against eukaryotic cell lines tested. Although the presence of
the piperazine spacer wasn’t crucial for activity of these compounds against Gram-positive
24

bacteria, compounds with piperazine spacer showed slightly better activity compared to
analogues without the piperazine spacer. The position of the N-atom within the terminal pyridine
ring played a crucial role in conferring activity to these molecules and 3-pyridyl analogues (8b
and 10b) were significantly more potent compared to 2-pyridyl and 4-pyridyl analogues for both
sets of compounds (i.e., compounds with and without piperazine spacer). Electron donating and
electron withdrawing substituents in the terminal pyridine ring maintained or enhanced activity
against MSSA-9144 and EMRSA-16 strain but a loss of activity against EMRSA-15 strain was
observed in both cases. This SAR information will be helpful in designing future analogues of
this chemical class. The incorporation of the nitrofuran ring suggests that the compounds may be
active via nitrocompound toxicity, akin to that mediated by nitrofurantoin and benzothiazinone-
based compounds[29].
Compound 8b showed good activity against a wider panel of multiple drug-resistant
Staphylococcus strains. Many of the strains in the extended panel are multidrug resistant and
have a range of diverse resistance mechanisms. For example, strain A1988 is resistant to
trimethoprim, muciprocin, oxacillin, amoxicillin, gentamicin and kanamycin. No significant
difference in sensitivity was observed for strains 6571 and 8325 which express Panton Valentin
Leukocidin (PVL), which is a virulence factor associated with aggressive infection in healthy
individuals. Compound 8b showed similar activity (MIC 4-16 µg/mL) against most members of
the extended panel.
The increased susceptibility of Gram-positive strains compared to Gram-negative bacteria
mirrors other recent investigations of nitrofuran-containing compounds, such as N-
25

acylhydrazone[30, 31], which have been investigated for the control of nosocomial pathogens.
The reduced efficacy against Gram-negative pathogens probably reflects the greater barrier
functions of the bacterial membrane, but may also be influenced by cellular metabolism related
to nitrocompound activation and/or toxicity. Given the likely mechanisms of action of the
pyridyl nitrofuranyl isoxazolines being investigated here, we would not have expected any of the
existing resistance mechanisms in the Gram-positive strain panel to mediate cross-resistance to
the new compounds. Indeed there was no difference in the MIC levels (defined here as being
less than 4-fold difference in MIC) determined for strains resistant or sensitive to amoxicillin,
vancomycin or ciprofloxacin, respectively. Hence the compounds are equally active against
isolates which are resistant to key front-line antibiotics. The MICs of the pyridyl nitrofuranyl
isoxazolines are consistent with those of other novel nitrofuran-containing compounds tested
against Gram-positive pathogens[30, 31]. The absolute MIC values for drug resistant strains that
were susceptible against 8b were consistently lower for compound 8b (typically 4-16 µg/mL)
than for the control antibiotic nitrofurantoin which may have a similar mechanism of action (i.e.,
nitrocompound toxicity) and shares structural similarity. Nitrofurantoin is used routinely in the
United Kingdom and elsewhere for the treatment of urinary tract infections and the improved
activity of 8b compared to nitrofurantoin is both encouraging and significant from a drug-
discovery point of view as there is significant opportunity to make medicinal chemistry
modifications on the scaffold to develop more potent antibacterial agents. It should be noted that
the MIC values of 8b were higher than that for ciprofloxacin (0.125-2 µg/mL), vancomycin (0.5-
2 µg/mL) or amoxicillin (0.25-2 µg/mL), Given the different mode of action of these antibiotics,
this is unsurprising and does not necessarily preclude the development of pyridyl nitrofuranyl
isoxazolines as candidate antibiotics, especially given their low eukaryotic cell toxicity. Like
26

nitrofurantoin, there are several antibiotics which are used routinely in the clinic and where the
MIC for susceptible strains is higher than that observed for compound 8b. The European
Committee on Antimicrobial Susceptibility Testing (EUCAST) define resistance to
nitrofurantoin as being an MIC of 64 µg/mL and above, for Staphylococcus spp. In contrast,
S.aureus strains with an MIC of 2 µg/mL are defined as being resistant to ciprofloxacin. There is
significant opportunity to make medicinal chemistry modifications on the scaffold, to develop
more potent antibacterial agents.
Although this chemical scaffold showed a relatively narrow spectrum of activity, there is
increasing support within the clinical community for this since they do not have wide ranging
deleterious effects on the patients’ normal flora. Narrow spectrum agents can eliminate
resistance pressure on large sections of the microbiome, as these agents will be inactive against
them, and thereby play a pivotal role in minimising spread or resistance[32]. The number of
pipeline drugs presently undergoing clinical trials against MRSA is not particularly
encouraging[33] and, although the pipeline is not completely empty, it is important to exploit
existing as well as new chemical scaffolds to develop drug leads that can be optimized into
clinical candidates.
5.0 Conclusion
Concern over antibiotic resistance is growing, and novel classes of antibiotics are needed. We
have successfully modified a chemical scaffold that has previously shown activity against
Mycobacterium species, but was inactive against MDR Gram-positive pathogens, and introduced
notable activity against MRSA strains. It was possible to obtain limited SAR information from
27

the compound set and this information can be useful to generate future compounds of this type.
The pyridyl nitrofuranyl isoxazoline 8b showed improved activity against a wider panel of drug-
resistant and sensitive Staphylococcus strains compared to nitrofurantoin which like these
compounds shares nitrofuran group and may have a similar mechanism of action. The lack of
mammalian toxicity and the opportunity to make medicinal-chemistry modifications make
pyridyl nitrofuranyl isoxazoline scaffold an interesting starting point to develop more potent
antibacterial agents against MDR Gram-positive pathogens.
Acknowledgments: The authors would like to thank Public Health England [Grant reference no.
JGALAQR, 2015] and King’s Health School Studentships [Grant reference JGA3804, 2014] for
funding the research.
28

References
[1] C.A. Arias, B.E. Murray, Antibiotic-Resistant Bugs in the 21st Century -- A Clinical Super-
Challenge, New England Journal of Medicine, 360 (2009) 439-443.
[2] M.A. Fischbach, C.T. Walsh, Antibiotics for Emerging Pathogens, Science, 325 (2009) 1089-
1093.
[3] K. Lewis, Platforms for antibiotic discovery, Nature Reviews Drug Discovery, 12 (2013)
371-387.
[4] L.L. Maragakis, T.M. Perl, Acinetobacter baumannii: Epidemiology, antimicrobial
resistance, and treatment options, Clinical Infectious Diseases, 46 (2008) 1254-1263.
[5] K.K. Kumarasamy, M.A. Toleman, T.R. Walsh, J. Bagaria, F. Butt, R. Balakrishnan, U.
Chaudhary, M. Doumith, C.G. Giske, S. Irfan, Emergence of a new antibiotic resistance
mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study,
The Lancet infectious diseases, 10 (2010) 597-602.
[6] M.L. Cristina, A.M. Spagnolo, P. Orlando, F. Perdelli, The role of the environment in the
spread of emerging pathogens in at-risk hospital wards, Reviews in Medical Microbiology, 24
(2013) 104-112.
[7] D.E. Karageorgopoulos, M.E. Falagas, Current control and treatment of multidrug-resistant
Acinetobacter baumannii infections, Lancet Infectious Diseases, 8 (2008) 751-762.
[8] C. Nathan, Fresh Approaches to Anti-Infective Therapies, Science Translational Medicine, 4
(2012) 140sr142.
[9] A. MacGowan, M. Albur, Frontline antibiotic therapy, Clinical Medicine, 13 (2013) 263-268.
[10] A.M. Coates, G. Halls, Antibiotics in Phase II and III Clinical Trials, in: A.R.M. Coates
(Ed.) Antibiotic Resistance, Springer Berlin Heidelberg2012, pp. 167-183.
[11] L.M. de Almeida, M.R.E. de Araújo, M.F. Iwasaki, A.G. Sacramento, D. Rocha, L.P. da
Silva, M. Pavez, A.C. de Brito, L.C.S. Ito, A.C. Gales, N. Lincopan, J.L.M. Sampaio, E.M.
Mamizuka, Linezolid resistance in vancomycin-resistant Enterococcus faecalis and
Enterococcus faecium isolates in a Brazilian hospital, Antimicrobial Agents and Chemotherapy,
58 (2014) 2993-2994.
[12] B.P. Howden, J.K. Davies, P.D.R. Johnson, T.P. Stinear, M.L. Grayson, Reduced
vancomycin susceptibility in Staphylococcus aureus, including vancomycin-intermediate and
29