An azido-oxazolidinone antibiotic for live bacterial cell imaging and generation of antibiotic variants

Article abstract of DOI:10.1016/j.bmc.2014.05.054

An azide-functionalised analogue of the oxazolidinone antibiotic linezolid was synthesised and shown to retain antimicrobial activity. Using facile 'click' chemistry, this versatile intermediate can be further functionalised to explore antimicrobial structure-activity relationships or conjugated to fluorophores to generate fluorescent probes. Such probes can report bacteria and their location in a sample in real time. Modelling of the structures bound to the cognate 50S ribosome target demonstrates binding to the same site as linezolid is possible. The fluorescent probes were successfully used to image Gram-positive bacteria using confocal microscopy.

Full text of DOI:10.1016/j.bmc.2014.05.054

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
An azido-oxazolidinone antibiotic for live bacterial cell imaging
and generation of antibiotic variants
⇑
Wanida Phetsang, Mark A. T. Blaskovich , Mark S. Butler, Johnny X. Huang, Johannes Zuegg,
⇑
Sreeman K. Mamidyala, Soumya Ramu, Angela M. Kavanagh, Matthew A. Cooper
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
An azide-functionalised analogue of the oxazolidinone antibiotic linezolid was synthesised and shown to
retain antimicrobial activity. Using facile ‘click’ chemistry, this versatile intermediate can be further func-
tionalised to explore antimicrobial structure–activity relationships or conjugated to fluorophores to gen-
erate fluorescent probes. Such probes can report bacteria and their location in a sample in real time.
Modelling of the structures bound to the cognate 50S ribosome target demonstrates binding to the same
site as linezolid is possible. The fluorescent probes were successfully used to image Gram-positive bac-
teria using confocal microscopy.
Received 25 March 2014
Revised 16 May 2014
Accepted 22 May 2014
Available online xxxx
Keywords:
Antibiotics
Linezolid
Ó 2014 Published by Elsevier Ltd.
Fluorescent probes
Bacteria
Click chemistry
1. Introduction
(composed of 30S and 50S subunits), whereas mammalian ribo-
somes are 80S structures (composed of 40S and 60S subunits).10
Bacterial infection is a leading cause of death worldwide, and
antimicrobial resistance has become one of the most serious public
health concerns.1,2 It has proven very difficult to identify new anti-
biotics, especially those with activity against drug-resistant bacte-
ria, with very few new classes identified in the past 40 years.3,4 The
growing incidences of these drug-resistant ‘superbugs’ is fuelling a
resurgence in microbiology research focused on the identification
of new antibiotics, bacterial resistance mechanisms, new biological
targets, and mode of action (MoA) studies.
Antibiotics bind to bacterial ribosomes selectively because they dif-
fer from mammalian ribosomes. Oxazolidinone antibiotics bind to
the 50S subunit and block formation of the 70S initiation complex
by preventing assembly of the N-formyl-methionyl-tRNA–
ribosome–mRNA ternary complex.11,12 In addition, they interfere
with translocation of peptidyl-tRNA from the A site to the P site.13
Resistance to linezolid 1 is associated with modifications of its bind-
ing site on the ribosome.14 Resistance to oxazolidinones also
involves mutation of 23S rRNA, resulting in decreased binding
affinity.15
Linezolid 1 (ZyvoxÒ, ZyvoxidÒ, ZyvoxamÒ; Pfizer) was the first
clinically used example of a novel class of chemically synthesized
antimicrobial agents known as oxazolidinones (Fig. 1).5,6 Developed
in the 1990s and FDA approved in 2000, linezolid 1 is administered
orally or intravenously for the treatment of Gram-positive infec-
tions. It is effective against methicillin-resistant Staphylococcus aur-
eus (MRSA) and vancomycin resistant Enterococcus spp. (VRE).
Resistance was noted within a year after its clinical introduction7,8
and is still a concern.9 Linezolid 1 acts by inhibiting bacterial protein
synthesis by targeting the ribosome, one of the most common
antibiotictargets.10 Bacterialribosomesare knownas 70S ribosomes
A number of new oxazolidinone derivatives are in clinical devel-
opment (Fig. 1).4 Tedizolid phosphate 2a (Sivextro, torezolid phos-
phate, TR-701, DA-7218; Cubist/Trius Therapeutics) is a prodrug
that has completed phase-III trials and a New Drug Application
(NDA) has been filed in the US and Marketing Authorization Appli-
cation (MAA) for Europe, with FDA approval granted in June
2014.16–18 Dephosphorylation in vivo unmasks tedizolid 2b, which
is active against linezolid resistant strains.19 Cadazolid
3
(ACT-179811; Actelion Pharmaceuticals) is a quinolonyl-oxazolidi-
none chimeric antibiotic that recently started a phase-III trial for the
treatment of patients with Clostridium difficile infection (CDI).20,21
Four oxazolidinones have completed or are in phase-II trials.
Sutezolid 4 (PNU-100480, PF-02341272; Sequella/Pfizer) is a close
analogue of linezolid 1 that was optimized for activity against tuber-
culosis.22–26 Radezolid 5 (RX-1741; Melinta Therapeutics, formally
⇑
Corresponding authors. Tel.: +61 7 3346 2966 (M.A.T.B.), +61 7 3346 2044
(M.A.C.).
E-mail addresses: m.blaskovich@uq.edu.au (M.A.T. Blaskovich), m.cooper@uq.
edu.au (M.A. Cooper).
http://dx.doi.org/10.1016/j.bmc.2014.05.054
0968-0896/Ó 2014 Published by Elsevier Ltd.
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2
W. Phetsang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
F
O
vancomycin probe was used as an optical imaging tool for detect-
ing infecting bacteria in vivo.40 Vancomycin linked with
near-infrared (NIR) probe binds to Gram-positive bacteria, allow-
ing invasive infection to be detected by NIR optical imaging.
Boron-dipyrromethene (BODIPY)-labeled daptomycin (BDP-DAP)
a
O
N
O
N
N
H
N
1)
linezolid (
O
F
O
N
N
O
was used to determine a novel resistance mechanism of
N
N
OR
vancomycin-resistant enterococci (VRE) to cationic antimicrobial
peptides.41 From this study, VRE was shown to resist DAP-elicited
cell membrane damage by diverting the antibiotic away from its
principal target to other distinct cell membrane regions.
N
tedizolid phosphate: R = PO3H2 (2a)
2b
tedizolid: R = H (
)
O
F
The Cu-catalysed azide–alkyne cycloaddition (an azide–alkyne
version of the Huisgen 1,3-dipolar cycloaddition) is a useful reac-
tion for derivatising azide/alkyne substituents under mild condi-
tions.42–44 The ‘click’ generated triazole is very useful in
biological studies and medicinal chemistry due to the favourable
physicochemical properties of the triazole ring.45 Triazoles are sta-
ble to reductive and oxidative reactions and acidic and basic
hydrolysis reactions, under conditions where amides can be hydro-
lytically cleaved.46 Furthermore, the aromatic structure of triazoles
resists enzymatic degradation and also participates in hydrogen
O
OH
O
N
OH
N
O
N
F
3
cadazolid ( )
HO
F
O
N
O
O
H
N
S
N
O
4
sutezolid ( )
bond formations and p
-stacking interactions.47,48
O
F
O
In this study, we prepare an azide-functionalised oxazolidinone
antibiotic, and use it as a convenient intermediate in azide–alkyne
click reactions to rapidly generate modified analogues, including
fluorescent probes.
H
N
N
NH
O
radezolid (5)
HN
N
N
O
N
F
O
2. Materials and methods
O
N
N
O
N
HO
O
2.1. Synthesis of azide-derivatised linezolid
OH
F
posizolid (6)
O
F
Theintermediate linezolidderivative19(Scheme1)wassynthes-
ised as described in the literature,49 except that a tosylate was used
instead of a mesylate for the conversion of 15 to 16. In short, 3,
4-difluoronitrobenzene 10 was treated with piperazine, selectively
resulting in p-substituted nitrobenzene 11. Catalytic reduction of
11 and subsequent acylation of amine 12 with benzylchloroformate
gave protected carbamate 13. Reaction of 13 with (R)-glycidyl
butyrate in THF at ꢀ78 °C in the presence of n-BuLi provided 5-(R)-
hydroxymethyl-2-oxazolidinone 14. To form the desired oxazolidi-
none ring it was essential to use a lithium counter ion in the base
for regiochemical control. Sodium and potassium bases generate
5-hydroxymethyl-2-oxazolidinone as a major by-product.50 Treat-
ment of compound 14 with p-toluenesulfonyl chloride at 0 °C in
the present of triethylamine gave the tosylated product 15, which
was displaced by phthalimide to produce compound 16. Deprotec-
tion of phthalimide with methylamine generated the free amine
17 which was acetylated with acetic anhydride to provide 18.
Catalytic deprotection of the Cbz group with palladium on activated
carbon gave the intermediate linezolid derivative 19.
O
N
N
N
OH
F
O
MRX-I (7)
O
N
H
N
O
N
N
O
F
F
8
LCB01-0371 ( )
O
F
O
N
N
N
H
N
N3
9
O
Figure 1. Oxazolidinone antibiotics in the clinic and the azide-functionalised
linezolid analogue 9.
RibX) is similar in structure to tedizolid, and has activity against
both Gram-positive and some Gram-negatives bacteria.27–32 Posizo-
lid 6 (AZD5847, AZD2563; AstraZeneca) is another oxazolidinone
being assessed for treatment of tuberculosis, although it was origi-
nally developed as a broad spectrum Gram-positive antibiotic.33–35
MRX-I (MicuRx) 7 recently started Phase II trials in China.36,37
LCB01-0371 8 (LegoChem Biosciences)38 is currently being evalu-
ated in phase-I trials.
A linker 22 containing the azido group was synthesised in 2
steps from 3-bromo-1-propanol 20, which was treated with
sodium azide in THF/H2O. The reaction mixture was refluxed with
stirring for 16 h to yield 3-azido-1-propanol 21.51 Treatment of
with p-toluenesulfonyl chloride at 0 °C in the present of triethyl-
amine gave the tosylated product 22.
The development of new antibiotics and alternative strategies
to combat antibiotic-resistant bacteria is aided by an improved
understanding of antibiotic interactions with bacteria and bacterial
cellular complexity. Fluorescent imaging has been used to improve
our comprehension. For example, the peptidoglycan layer (PG)
plays an important role in cell wall structure. Vancomycin fluores-
cent probes binding to peptidoglycan precursors were used to stain
Bacillus subtilis, and indicated that although bacterial actin homo-
logues played an important role in cell shape determination, other
proteins controlled the spatial localization of the biosynthetic com-
plexes responsible for new PG synthesis.39 In another study a
Finally, the secondary amine contained in 19 was alkylated with
azide linker 22 to yield azide-derivatised linezolid analogue 9 in
good yield.
2.2. Synthesis of alkyne-derivatised fluorophores
Two fluorophores, 7-(dimethylamino)-coumarin-4-acetic acid
(DMACA, prepared according to literature procedures52,53) and 7-
nitrobenzofurazan (NBD, Sigma–Aldrich), were functionalized with
an alkyne substituent (Scheme 2) so that they could be coupled to
the linezolid–azide analogue 9 by click chemistry. DMACA 23 was
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W. Phetsang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
F
O
F
F
F
O
O
O
ii
i
iii
N
N
NH
F
NO2
HN
N
NO2
HN
N
NH2
10
11
12
13
F
O
F
O
F
O
O
iv
v
O
O
O
vi
Cbz
N
N
N
Cbz
N
N
N
Cbz
N
N
N
OH
OTs
N
O
14
15
16
O
O
F
F
O
F
O
O
vii
ix
viii
Cbz
OH
N
N
N
HN
N
N
H
N
H
N
O
Cbz
N
N
N
NH2
O
O
17
20
18
19
xii
x
xi
N3
N3
OTs
Br
OH
21
22
N3
F
O
O
N
N
N
H
N
O
9
Scheme 1. Synthesis of azide derivatised oxazolidinone antibiotic 9, following literature procedure49 for 19. Reagents and conditions: (i) piperazine, CH3CN, reflux; (ii) H2,
10% Pd/C, THF; (iii) benzyloxycarbonyl chloride (Cbz-Cl), Na2CO3, acetone/H2O; (iv) n-BuLi, THF, ꢀ78 °C, (R)-glycidyl butyrate; (v) TsCl, NEt3, CH2Cl2; (vi) potassium
phthalimide, CH3CN, H2O, reflux; (vii) aqueous MeNH2, EtOH, reflux; (viii) Ac2O, pyridine; (ix) H2, 10% Pd/C, MeOH/CH2Cl2; (x) NaN3, THF/H2O, reflux; (xi) TsCl, NEt3, CH2Cl2,
0 °C–rt; (xii) 22, NEt3, NaI, EtOH, reflux.
O
O
O
O
O
O
O
O
N
N
N
N
i
ii
OH
N
H
O2N
Cl
(25
O2N
NH
N
N
(23
24
26
DMACA
)
NBD-Cl
)
Scheme 2. Synthesis of alkyne-functionalised fluorophores. Reagents and conditions: (i) propargylamine, HATU, DIPEA, DMF rt; (ii) propargylamine, Cs2CO3, THF.
reacted with propargylamine in the presence of HATU as coupling
agent to give DMACA linked alkyne 24. NBD linked alkyne 26 was
prepared by a substitution reaction from NBD-Cl (4-chloro-7-nitro-
benzofurazan) 25 by an improved method based on that previously
reported in the literature.54 The use of Cs2CO3 in THF for the substi-
tutiongave improvedyields compared to aqueousNaHCO3 in MeOH.
2.4. MIC assays
Compounds were tested for antimicrobial activity against
twelve Gram-positive bacterial strains: Enterococcus faecalis
(VanA clinical isolate), Enterococcus faecium (MDR Van A ATCC
51559), Streptococcus pneumoniae (MDR ATCC 700677), Staphylo-
coccus aureus (MRSA ATCC 43300, MRSA clinical isolate, MRSA
daptomycin resistant clinical isolate, GISA NRS 1, GISA NRS 17,
VRSA NARSA VRS1, VRSA NARSA VRS4 and VRSA NARSA
VRS10). All experiments were performed in duplicate with vanco-
mycin, and linezolid used as positive controls (see Table 1). Posi-
tive growth control rows of bacteria and DMSO + bacteria as well
as a negative control row of only media were included for every
plate.
2.3. Click chemistry of azide-derivatised oxazolidinone
To demonstrate the potential of the azide-functionalised oxazo-
lidinone to generate analogues to explore antimicrobial SAR and
produce fluorescent probes, two different alkynes and the two
alkyne derivatised fluorophores were reacted with the azide. Lin-
ezolid azide analogue 9 was reacted with propargyl alcohol, phen-
ylacetylene, and alkyne fluorophores 24 and 26 in the presence of
copper sulfate and sodium ascorbate to provide the corresponding
triazole derivatives 27, 28, 29, and 30, respectively (Scheme 3). The
cycloaddition reactions of the alkyne-fluorophores and azide 9 at
room temperature were found to be very slow, hence were con-
ducted at 50 °C with completion after 16 h. All products were puri-
fied by reverse-phase HPLC.
2.5. Fluorescent properties of probes
The fluorescent spectra of the two oxazolidinone-fluorescent
probe conjugates 29 and 30 were measured (Fig. 2). Note that
the DMACA emission peak of 29 (kmax = 490 nm) overlaps with
the NBD absorption peak of 30 (kmax = 475 nM).
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W. Phetsang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
i
O
N3
F
Ph
or
CH2OH
O
N
N
N
H
N
or ii
9
24 or 26
O
N
N
O
N
F
O
N
N
N
O
H
N
N
27
N
HO
N
F
O
O
N
N
N
H
N
28
O
O
N
N
H
N
O
O
N
F
N
N
N
H
N
O
O
O
N
Figure 2. Excitation/emission of fluorophore probes 29 and 30.
29
O
O2N
slides were obtained using a confocal microscope (Zeiss LSM 510
META).
N
N
N
N
H
O
N
F
N
O
2.7. Docking studies of oxazolidinone derivatives
N
N
N
H
N
30
The derivatives were modelled into the crystal structure of
native Deinococcus radiodurans large ribosomal subunit (D50S)
bound with linezolid (Pdb: 3DLL55) using Schrödinger software
package and its Induced Fit Docking module (Induced Fit Docking
protocol 2013-2, Glide version 5.9, Prime version 3.2, Schrödinger,
LLC, New York, NY, 2013), to account for the reported structural
flexibility of the peptidyl transferase centre.
O
Scheme 3. ‘Click’ reaction to generate functionalized oxazolidinone antibiotics and
fluorescent probes. Reagents and conditions: (i) sodium ascorbate, CuSO4, MeOH, rt;
(ii) sodium ascorbate, CuSO4, MeOH, DMF, 50 °C.
2.6. Fluorescent imaging of bacteria
3. Results and discussion
The ability of the fluorescent probes to label bacteria was exam-
ined using log phase cultures of two S. aureus strains (an MRSA
clinical isolate and a VISA NARSA VRS3b) and an Enterococcus fae-
cium strain (MDR Van A ATCC 51559). Probes 29 and 30 were incu-
bated with the bacteria for one hour at a concentration of 64 lg/
mL, after which the bacteria were smeared onto glass slides. After
3.1. Design of azide-derivatised linezolid analogue
The oxazolidinone antibiotic linezolid 1 possesses 4 key struc-
tural elements; the A-, B-, and C-rings, and the C-5 position of the
A-ring (Fig. 3). Modification of linezolid’s A-ring, B-ring, and C-5
position influence its antimicrobial activity. Most modified oxazo-
lidinone antibiotics keep the B-ring to maintain potent activity.
fixation, washing and mounting steps, fluorescent images of the
Table 1
MIC (minimum inhibitory concentrations) of oxazolidinone derivatives against Gram-positive bacteria
Compound
MIC (lg/mL)
Staphylococcus aureus
Streptococcus
pneumoniae
VRSA NARSA MDR ATCC
Enterococcus Enterococcus
faecalis
Van A Clin.
Isol
faecium
Van A ATCC
51559
MRSA ATCC MRSA
DaptRs
GISA
GISA
VRSA
VRSA
NARSA
VRS4
43300
Clin. Isol. Clin. Isol.
NRS1
NRS 17. NARSA
VRS1
VRS10
700677
Vancomycin
Linezolid 1
19
1
1
8
2
1
1
2
2
2
1
2
2
8
1
4
2
4
1
4
2
>64
1
4
32
2
32
2
1
2
8
2
1
1
4
4
>64
2
8
>64
2
0.5
4
9 Lz-N3
2
4
27 Lz-tz-Ph
28 Lz-tz-CH2OH
29 Lz-tz-DMACA
30 Lz-tz-NBD
64
4
>64
16
32
2
64
8
>64
4
>64
8
16
2
>64
8
16
4
>64
16
16
4
64
16
64
8
>64
32
32
8
64
32
64
4
64
4
16
32
64
32
16
2
32
16
Clin. Isol. = clinical isolate; DaptRs = daptomycin resistant; MRSA = methicillin resistant S. aureus, GISA = glycopeptide insensitive S. aureus, MDR = multidrug resistant,
VRSA = vancomycin resistant S. aureus.
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W. Phetsang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
4-position is the
most tolerant of
variation with different
functional groups
Fluorophenyl moiety sits optimally
in 50S ribosomal subunit.
Change to other aromatic
ring positions affect activity
N-aryl group is required
Forms H-bond.
F
O
Alkyl groups
affect activity
due to steric or
conformational
effect
C
N
O
B
N
F
O
A
C-5
NH
Difluoro compound
has been found
to be promising
H
C
3
Figure 4. In silico docking studies of the azide-functionalised oxazolidinone 9
within the binding site of linezolid in ribosomal subunit 50S of D. radiodurans.
Crystal structure 3DLL shown as cartoon, with key nucleotides in green and
linezolid in yellow. Docked structure of 9 shown in grey with conformational
variations of nucleotide U2585 in orange.
(S)-configuration is critical
sp3 center is required
O
N atom possessing sp3-like
character can be used
Can be replaced
with heterocyclic ring
but large substituents
lose activity
S atom exhibits
slightly less potency
3.3. Fluorophore-derivatised oxazolidinone antibiotics
Figure 3. Known structure–activity relationships of oxazolidinone antibiotics.
Two different fluorophores, 7-(dimethylamino)-coumarin-4-
acetic acid (DMACA) and 7-nitrobenzofurazan (NBD) were selected
as initial fluorophores due to their low molecular size compared to
most other fluorophores (such as rhodamines and fluoresceins),
increasing both the potential for cellular penetration of the antibi-
otic-fluorophore probes, and the likelihood that they could be
accommodated within the linezolid binding site. They also have
differing fluorescence colours (blue and green, respectively), with
an emission/excitation overlap that may be useful for FRET studies.
The alkyne-derivatised fluorophores were readily coupled with the
azide-oxazolidinone 9 to generate fluorescent probes 29 and 30.
Incubation of these probes with Gram-positive bacteria resulted
in selective staining of the bacteria cells (Fig. 5), with the staining
pattern consistent with internalization when compared to other
fluorophore probes known to stain the cell surface (e.g., similar
to internal staining pattern of DAPI, 40,6-diamido-2-phenylindole,
which binds internal nucleic acids, but not like the membrane
binding dye FM4-64, N-(3-triethylammoniumpropyl)-4-(6-(4-
(diethylamino)phenyl)-hexa-trienyl)pyridinium dibromide60). The
probes labelled both S. aureus and E. faecium despite relatively poor
MIC values.
However, the 40-position of the C-ring was tolerant to alteration and
does not show significant loss of activity after replacement with dif-
ferent functional groups.12,56–58 This correlates with structural anal-
ysis of different ribosomal subunits in comparison with the binding
mode of linezolid55,59 in which the nucleotide U2585 at the peptid-
yltransferase centre shows considerable conformational flexibility,
and is able to accommodate large substituents at the 40-position of
linezolid. Therefore, an azide-derivatized linezolid 9 was designed
to explore the 40-position of the C-ring.
3.2. ‘Click’ chemistry and antimicrobial activity of conjugates
Four different alkynes were successfully employed for proof-of-
principle Cu-catalysed azide–alkyne cycloadditions with the azide-
derivatised oxazolidinone, generating the triazole-linked conju-
gates. The reactions proceeded in good yield to give the desired
products. As expected, the reaction was tolerant of a range of func-
tionalities on both the azide and alkyne components.
The linezolid/sutezolid analogue 19, replacing the morpholine
oxygen with a nitrogen, consistently lost approximately 4-fold
potency, with the exception of multidrug resistant E. faecium ATCC
51559, where potency was gained. Substitution with the azidopro-
pyl substituent, in the azide-functionalised linezolid intermediate
9, gave a compound that retained similar activity to linezolid against
4. Conclusion
In conclusion, we have demonstrated that a morpholine-to-
piperidine analogue of the oxazolidinone antibiotic linezolid can
be alkylated with azidopropane to produce an azide-modified
derivative that retains the antibacterial activity of linezolid. This
versatile intermediate can be applied to rapidly assess
structure–activity relationships using Cu-catalysed azide–alkyne
cycloadditions with substituted alkynes, and to easily conjugate
fluorophores with different physicochemical and photochemical
properties. The oxazolidinone-fluorophore probes successfully
labelled a number of different types of Gram-positive bacteria. This
approach allows for a rapid ‘mix and match’, with alternate fluoro-
phore colours readily attached to the derivatised antibiotic in a sin-
gle step. Future studies will examine the utility of these probes in
bacterial detection systems, and their ability to detect internal bac-
terial structures when combined with high resolution imaging,
such as 3D structured illumination microscopy (3D-SIM-Micros-
copy).61 The azide-modified oxazolidinone will also be combined
with other functionalized antibiotics to rapidly generate hybrid
antibiotics similar to cadazolid 3.
all strains tested (MIC 2 to 4 lg/mL). Triazole formation with phen-
ylacetylene or propargyl alcohol resulted in significantly different
activity, with the phenyl-substituted triazole 27 much less active
(MIC 32 to >64
2 to 32 g/mL). The linezolid-DMACA probe 29 lost most antimicro-
bial activity (MIC 32 to >64 g/mL) whereas the linezolid-NBDprobe
30 retained some activity (4 to 32 g/mL) against most strains
lg/mL) than the hydroxymethyl derivative 28 (MIC
l
l
l
tested. From the in silico docking studies the azide-functionalised
oxazolidinone 9 was able to fit into a similar position observed for
linezolid in the crystal structure of ribosomal subunit 50S of D. radio-
durans (Fig. 4).55 Due to the extended size of the moleculean induced
fit workflow had to be applied in order to accommodate the mole-
cule. As reported in other crystal structures, the nucleotide U2585
was able to adopt a variation of conformations, while the piperazine
azide moiety oriented itself along the P-site. The fact that some
activity was retained for the fluorescent probes was promising, as
it indicated that some compound was penetrating the bacterial
membrane, and for staining of live bacteria sub-MIC concentrations
could be used.
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W. Phetsang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 5. Labelling of Gram positive bacteria with fluorescent probes 29 and 30. Negative controls: bacteria only. Bars, 2 lm.
5.1.3. 3-Azidopropyl 4-methylbenzenesulfonate 22
5. Experimental
5.1. Chemistry
The mixture of 3-bromo-1-propanol 20 (1.0 g, 7.19 mmol) and
sodium azide (1.4 g, 21.58 mmol) in THF/H2O (20:5 mL) was stir-
red at 80 °C for 16 h. The reaction mixture was extracted with
CH2Cl2, dried over MgSO4, and concentrated under reduced pres-
sure to give compound 21 (0.59 g, 81%) as an oil which was used
for the next reaction without further purification.
5.1.1. General
All materials, unless otherwise noted, were obtained from
commercial suppliers and used without further purification.
Non-aqueous reactions were conducted under an inert atmo-
sphere of nitrogen. Reactions were monitored by thin layer chro-
matography (TLC). Analytical TLC was performed on Merck TLC
alumina sheets pre-coated with Silica Gel 60 F254, and com-
pounds were visualized using UV lamp and appropriate TLC
stains. Column chromatography was performed using silica gel
60 (0.063–0.200 mm), 70–230 mesh ASTM. Biotage Isolera and
Grace Reveleris chromatography systems were used for com-
pound purification. 1H (600 MHz) and 13C (125 MHz) NMR spec-
To the solution of 3-azidopropan-1-ol 21 (1.47 g, 14.55 mmol)
in CH2Cl2 was added p-toluenesulfonyl chloride (3.05 g,
16.01 mmol). The reaction mixture was stirred at rt overnight.
The reaction was checked by TLC for completion. The resulting res-
idue was diluted with water and extracted with CH2Cl2, dried over
MgSO4, and concentrated under reduced pressure. Purification by
column chromatography (silica gel, 50% EtOAc/Hexane) gave com-
pound 22 (2.58 g, 69%) as an oil. 1H NMR (600 MHz, CDCl3): d 7.76
(d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 4.07 (t, J = 5.8 Hz, 2H),
3.34 (t, J = 6.5 Hz, 2H), 2.42 (s, 3H), 1.85 (quin, J = 6.2 Hz, 2H); 13C
NMR (125 MHz, CDCl3): d 145.2, 132.8, 130.1, 128.0, 67.2, 47.4,
tra were obtained using
a Bruker Avance-600 spectrometer
equipped with a TXI cryoprobe. Chemical shifts are reported rel-
ative to the residual solvent signals in parts per million (d)
(CDCl3: 1H: d 7.27, 13C: d 77.2; CD3OD: 1H: d 3.30, 13C: d 49.5;
DMSO-d6: 1H: d 2.50, 13C: d 39.5). High resolution mass spec-
trometry (HRMS) was performed on a Bruker Micro TOF mass
spectrometer using (+)-ESI calibrated to NH4OAc.
28.6, 21.8; (+)-ESI-HRMS calc for
278.0575, found 278.0580.
C
10H13N3NaO3S [M+Na+]+:
5.1.4. (S)-N-((3-(4-(4-(3-Azidopropyl)piperazin-1-yl)-3-
fluorophenyl)-2-oxooxazolidin-5-yl)methyl)-acetamide 9
To the mixture of 19 (100 mg, 0.30 mmol), 22, and NaI
(10 mol %, 5 mg, 0.003 mmol) in EtOH (40 mL), NEt3 was added.
The mixture was stirred under reflux for 6 h and then concentrated
under reduced pressure. Purification by column chromatography
(Grace MPLC; C18 reverse phase with eluents 0.1% TFA in MeOH/
5.1.2. Synthesis of linezolid analogue 19
Linezolid analogue 19 was synthesized by a slight modification
(mesylate displacement replaced with tosylate) of a literature
method49 as shown in Scheme 1.
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W. Phetsang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
0.1% TFA in H2O) gave compound 9 (46 mg, 37%) as white solid. 1H
NMR (600 MHz, CD3OD): d 7.48 (dd, J = 14.7, 2.5 Hz, 1H), 7.16 (dd,
J = 8.8, 2.6 Hz, 1H), 7.05 (t, J = 9.4 Hz, 1H), 4.80 (m, 1H), 4.11 (t,
J = 9.0 Hz, 1H), 3.80–3.77 (m, 1H), 3.55 (d, J = 5.0 Hz, 2H), 3.40 (t,
J = 6.6 Hz, 2H), 3.10 (br s, 4H), 2.69 (s, 4H), 2.55 (t, J = 7.5 Hz, 2H),
1.96 (s, 3H), 1.85–1.80 (m, 2H); 13C NMR (125 MHz, CD3OD): d
174.2, 156.9 (d, J = 245.1 Hz), 156.8, 137.6 (d, J = 9.1 Hz), 135.1 (d,
J = 10.5 Hz), 120.5 (d, J = 3.9 Hz), 115.6 (d, J = 2.9 Hz), 108.6 (d,
J = 26.5 Hz), 73.6, 56.7, 54.4, 51.6 (d, J = 2.6 Hz, 2C), 50.7, 49.3,
43.3, 27.1, 22.6; (+)-ESI-HRMS calc for C19H27FN7O3 [M+H]+:
420.2159, found 420.2174.
106.6 (d, J = 26.0 Hz), 71.6, 53.0, 51.2, 47.4, 47.3, 46.9, 41.2, 24.3,
22.4; (+)-ESI-HRMS calc for C27H33FN7O3 [M+H]+: 522.2629, found
522.2627.
5.1.8. (S)-N-((3-(3-Fluoro-4-(4-(3-(4-(hydroxymethyl)-1H-1,2,3-
triazol-1-yl)propyl)piper-azin-1-yl)phenyl)-2-oxooxazolidin-5-
yl)methyl)acetamide 28
To a solution of 9 (24 mg, 0.057 mmol) and propargyl alcohol
(3.2 lL, 0.057 mmol) in MeOH (4 mL) was added a solution of
CuSO4 (5 mol %, 0.71 mg, 0.0029 mmol) and treated with aqueous
sodium ascorbate (10 mol %, 1.13 mg, 0.0057 mmol). The reaction
was stirred vigorously for 16 h. The reaction was concentrated
under reduced pressure. Purification by column chromatography
(Grace MPLC; C18 reverse phase with eluents 0.1% TFA in MeOH/
0.1% TFA in H2O) gave compound 28 (13.3 mg, 49%) as a white
solid. 1H NMR (600 MHz, DMSO-d6): d 8.24 (t, J = 5.8 Hz, 1H),
8.02 (s, 1H), 7.51 (dd, J = 14.7, 2.5 Hz, 1H), 7.20 (dd, J = 8.9,
2.2 Hz, 1H), 7.13 (t, J = 9.2 Hz, 1H), 4.73–4.69 (m, 1H), 4.53 (s,
2H), 4.45 (t, J = 6.8 Hz, 2H), 4.08 (t, J = 9.0 Hz, 1H), 3.71–3.69 (m,
1H), 3.59 (br s, 2H), 3.41–3.36 (m, 4H), 3.19 (br s, 4H), 3.01 (br s,
2H), 2.26, (br s, 2H), 1.83 (s, 3H); 13C NMR (125 MHz, CD3OD): d
174.3, 157.1 (d, J = 245.4 Hz), 156.7, 136.4 (d, J = 10.6 Hz), 135.7
(d, J = 9.5 Hz), 121.3 (d, J = 3.2 Hz), 115.6 (d, J = 2.9 Hz), 108.6 (d,
J = 26.3 Hz), 73.7, 56.5, 55.4, 53.6, 49.2, 48.4, 43.3, 25.9, 22.6; 13C
NMR (125 MHz, DMSO-d6): d 170.0, 154.5 (d, J = 244.3 Hz), 154.0,
148.1, 122.8, 119.92, 114.1 (d, J = 3.0 Hz), 106.6 (d, J = 26.0 Hz),
71.6, 55.1, 53.0, 51.2, 47.3, 46.6, 41.4, 22.4; (+)-ESI-HRMS calc for
5.1.5. 2-(7-(Dimethylamino)-2-oxo-2H-chromen-4-yl)-N-(prop-
2-yn-1-yl)acetamide 24
To a solution of the 2-(7-(dimethylamino)-2-oxo-2H-chromen-
4-yl)acetic acid 23 (0.3 g, 1.21 mmol) in DMF (5 mL) was added
HATU in DMF (5 mL) followed by DIPEA (386
lL), and propargyl-
amine (71 L, 1.1 mmol). The solution was stirred at rt overnight.
l
The reaction was evaporated under reduced pressure to remove
DMF. The residue was diluted with water and extracted with ethyl
acetate, dried over MgSO4, and concentrated under reduced pres-
sure. The crude compound was recrystallized in CH2Cl2. The solid
was filtrated and washed with CH2Cl2 to give pure compound 24
(0.149 g, 48%) as a green solid. 1H NMR (600 MHz, DMSO-d6): d
8.65 (t, J = 5.4 Hz, 1H), 7.52 (d, J = 9.0 Hz, 1H), 6.72 (dd, J = 9.1,
2.6 Hz, 1H), 6.55 (d, J = 2.6 Hz, 1H), 6.00 (s, 1H), 3.88–3.87 (m,
2H), 3.62 (s, 2H), 3.13 (t, J = 2.5 Hz, 1H), 3.01 (s, 6H); 13C NMR
(125 MHz, DMSO-d6): d 167.7, 160.7, 155.4, 152.9, 151.0, 126.0,
109.4, 109.1, 108.1, 97.5, 80.9, 73.3, 39.7, 38.4, 28.2; (+)-ESI-HRMS
calc for C32H32N4NaO6 [2M+Na]+: 591.2220, found 591.2190.
C
22H31FN7O4 [M+H]+: 476.2422, found 476.2419.
5.1.9. (S)-N-((1-(3-(4-(4-(5-(Acetamidomethyl)-2-oxooxazolidin-
3-yl)-2-fluorophenyl)piperazin-1-yl)propyl)-1H-1,2,3-triazol-4-
yl)methyl)-2-(7-(dimethylamino)-2-oxo-2H-chromen-4-yl)
acetamide 29
5.1.6. 7-Nitro-N-(prop-2-yn-1-yl)benzo[c][1,2,5]-oxadiazol-4-
amine 26
To
(300 mg, 1.5 mmol) in THF (10 mL) was added a solution of prop-
argyl amine (110 L, 1.65 mmol), Cs2CO3 (480 mg, 1.5 mmol). The
a
solution of 4-chloro-7-nitrobenzo[c][1,2,5]oxadiazole
To a solution of azide 9 (50 mg, 0.12 mmol) and DMACA fluoro-
phore 24 (34 mg 0.12 mmol) in DMF (4 mL) was added a solution
of CuSO4 (5 mol %, 1.5 mg, 0.006 mmol) and treated with aqueous
sodium ascorbate (10 mol %, 2.4 mg, 0.012 mmol). The reaction
was stirred vigorously at 50 °C for 16 h. Purification by column
chromatography (Grace MPLC; C18 reverse phase with eluents
0.1% TFA in ACN/0.1% TFA in H2O) gave compound 54 (61.5 mg,
79%) as a green solid. 1H NMR (600 MHz, DMSO-d6): d 8.76 (t,
J = 5.7 Hz, 1H), 8.24 (t, J = 5.9 Hz, 1H), 7.98 (s, 1H), 7.54–7.50 (m,
2H), 7.21 (dd, J = 8.8, 2.2 Hz, 1H), 7.14 (t, J = 9.4 Hz, 1H), 6.71 (dd,
J = 9.1, 2.5 Hz, 1H), 6.55 (d, J = 2.5 Hz, 1H), 5.99 (s, 1H), 4.73–4.69
(m, 1H), 4.44 (t, J = 6.9 Hz, 2H), 4.33 (d, J = 5.7 Hz, 2H), 4.08 (t,
J = 9.0 Hz, 1H), 3.71–2.20 (m, 11H), 3.19 (br s, 4H), 3.02 (s, 6H),
2.26 (quin, J = 6.9 Hz, 2H), 1.83 (s, 3H); 1H NMR (600 MHz, CD3OD):
d 7.79 (s, 1H), 7.53–7.51 (m, 2H), 7.17 (d, J = 8.8 Hz, 1H), 7.07 (t,
J = 9.2 Hz, 1H), 6.72–6.71 (m, 1H), 6.50 (s, 1H), 5.95 (s, 1H), 4.79–
4.75 (m, 1H), 4.53 (t, J = 6.6 Hz, 2H), 4.46 (s, 2H), 4.10 (t,
J = 9.1 Hz, 1H), 3.80–3.77 (m, 1H), 3.71 (s, 2H), 3.62 (br s, 2H),
3.55 (m, 4H), 3.26–3.23 (m, 4H), 3.11 (br s, 2H), 3.05 (s, 6H),
2.43–2.38 (m, 2H), 1.96 (s, 3H); 13C NMR (125 MHz, DMSO-d6): d
170.0, 167.8, 160.7, 154.6 (d, J = 246.2 Hz), 154.0, 152.8, 151.2,
144.6, 134.3 (d, J = 10.5 Hz), 133.7 (d, J = 9.0 Hz), 126.1, 123.1,
119.9 (d, J = 2.9 Hz), 114.1 (d, J = 2.4 Hz), 109.2, 109.0, 108.2,
106.6 (d, J = 26.0 Hz), 97.5, 71.6, 53.0, 51.2, 47.4, 47.3, 46.6, 41.4,
39.7, 38.5, 34.4, 24.3, 22.4; (+)-ESI-HRMS calc for C35H43FN9O6
[M+H]+: 704.3320, found 704.3309.
l
reaction mixture was stirred at 50 °C for 4 h. After completion of
the reaction, the reaction mixture was diluted with EtOAc
(50 mL), washed with H2O (30 mL), brine (30 mL). The organic
phase was separated, dried (MgSO4), and evaporated to give the
residue. The residue was purified by Si column chromatography
(petroleum ether/EtOAc, 7:3) to afford 26 (240 mg, 75%). 1H NMR
(600 MHz, CDCl3): d 8.54 (d, J = 8.4 Hz, 1H), 6.35 (d, J = 8.4 Hz,
1H), 6.32 (s, 1H, NH), 4.3 (dd, J = 2.4, 5.6 Hz, 2H), 2.44 (t,
J = 2.4 Hz, 1H).
5.1.7. (S)-N-((3-(3-Fluoro-4-(4-(3-(4-phenyl-1H-1,2,3-triazol-1-
yl)propyl)piperazin-1-yl)phenyl)-2-oxooxazolidin-5-
yl)methyl)acetamide 27
To a solution of 9 (18.9 mg, 0.045 mmol) and phenylacetylene
(5 lL, 0.045 mmol) in MeOH (4 mL) was added a solution of CuSO4
(5 mol %, 0.56 mg, 0.0023 mmol) and treated with aqueous sodium
ascorbate (10 mol %, 0.89 mg, 0.0045 mmol). The reaction was stir-
red vigorously for 16 h. The reaction was concentrated under
reduced pressure. Purification by column chromatography (Grace
MPLC; C18 reverse phase with eluents 0.1% TFA in MeOH/0.1%
TFA in H2O) gave compound 27 (5.66 mg, 24%) as a white solid.
1H NMR (600 MHz, DMSO-d6): d 8.62 (s, 1H), 8.24 (t, J = 5.8 Hz,
1H), 7.86–7.84 (m, 2H), 7.51 (dd, J = 14.7, 2.5 Hz, 1H), 7.48–7.45
(m, 2H), 7.35 (tt, J = 7.4, 1.2 Hz, 1H), 7.21 (dd, J = 8.8, 2.2 Hz, 1H),
7.14 (t, J = 9.4 Hz, 1H), 4.73–4.69 (m, 1H), 4.54 (t, J = 6.7 Hz, 2H),
4.08 (t, J = 9.0 Hz, 1H), 3.71–3.69 (m, 1H), 3.61 (br s, 2H), 3.45–
3.39 (m, 4H), 3.24 (br s, 4H), 3.01 (br s, 2H), 2.34 (br s, 2H), 1.83
5.1.10. (S)-N-((3-(3-Fluoro-4-(4-(3-(4-(((7-nitrobenzo[c]
[1,2,5]oxadiazol-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)
propyl)piperazin-1-yl)phenyl)-2-oxooxazolidin-5-yl)
methyl)acetamide 30
(s, 3H); 13C NMR (125 MHz, DMSO-d6):
d 170.0, 154.5 (d,
J = 243.6 Hz), 154.0, 146.4, 134.3 (d, J = 10.2 Hz), 130.7, 128.9,
127.9, 125.1, 121.6, 119.9 (d, J = 3.4 Hz), 114.1 (d, J = 3.1 Hz),
To a solution of azide 9 (50 mg, 0.12 mmol) and NBD fluoro-
phore 26 (26 mg 0.12 mmol) in DMF (4 mL) was added a solution
Please cite this article in press as: Phetsang, W.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.054

8
W. Phetsang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
of CuSO4 (5 mol %, 1.5 mg, 0.006 mmol) and treated with aqueous
sodium ascorbate (10 mol %, 2.4 mg, 0.012 mmol). The reaction
was stirred vigorously at 50 °C for 16 h. Purification by column
chromatography (Grace MPLC; C18 reverse phase with eluents
0.1% TFA in ACN/0.1% TFA in H2O) gave compound 54 (51.8 mg,
68%) as a orange solid. 1H NMR (600 MHz, DMSO-d6): d 9.88 (t,
J = 6.1 Hz, 1H), 8.53 (d, J = 8.9 Hz, 1H), 8.24 (t, J = 5.8 Hz, 1H), 8.16
(s, 1H), 7.51 (dd, J = 14.8, 2.3 Hz, 1H), 7.20 (dd, J = 8.8, 1.9 Hz,
1H), 7.12 (t, J = 8.9 Hz, 1H), 6.51 (d, J = 8.9 Hz, 1H), 4.78 (br s,
2H), 4.73–4.69 (m, 1H), 4.44 (t, J = 6.8 Hz, 2H), 4.08 (t, J = 9.0 Hz,
1H), 3.71–3.69 (m, 1H), 3.56 (br s, 2H), 3.40–3.35 (m, 4H), 3.19
(br s, 4H), 3.0 (br s, 2H), 2.24 (br s, 2H), 1.83 (s, 3H); 13C NMR
orientations were limited to poses similar to the orientation of lin-
ezolid in the crystal structure.
Acknowledgments
This research was supported financially by NHMRC Grants
APP631632, APP1026922 and AF511105. W.P. is supported by an
Australian Postgraduate Award (APA) PhD scholarship, M.A.B. and
M.S.B. by
a Wellcome Trust Seeding Drug Discovery Award
(094977/Z/10/Z), and M.A.C. by an NHMRC Principal Research Fel-
lowship (APP1059354). Microscopy was performed at the Austra-
lian Cancer Research Foundation (ACRF)/Institute for Molecular
Bioscience Cancer Biology Imaging Facility, which was established
with the support of the ACRF.
(125 MHz, DMSO-d6):
d 170.0, 154.5 (d, J = 243.3 Hz), 154.0,
145.7, 144.5, 144.0, 142.7, 137.7, 134.2, 123.6, 119.9, 114.0 (d,
J = 2.2 Hz), 106.5 (d, J = 26.0 Hz), 99.8, 71.6, 52.9, 52.0, 51.1, 47.2,
47.7, 41.3, 38.5, 24.2, 22.4; (+)-ESI-HRMS calc for C28H33FN11O6
[M+H]+: 638.2599, found 638.2602.
Supplementary data
Supplementary data (NMR spectra and HPLC chromatograms
for 9, 22, 24, and 27–30) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmc.2014.
05.054. These data include MOL files and InChiKeys of the most
important compounds described in this article.
5.2. Biology
5.2.1. Determination of MIC (minimum inhibitory
concentration)
MICs were determined by a two-fold serial broth microdilution
according to the recommendation of CLSI standards with an inoc-
ulum of 5 ꢁ 105 cfu/mL. The compounds along with standard anti-
biotics were serially diluted twofold across the wells of 96-well
non-binding surface plates (NBS, Corning). Standards ranged from
References and notes
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64–0.03
lg/mL, and the compounds from 128–0.06
lg/mL with
L per well. Gram-positive and Gram-negative
final volumes of 50
l
bacteria were cultured in Mueller Hinton broth (MHB) (Bacto lab-
oratories, Cat. No. 211443) at 37 °C overnight. A sample of each
culture was then diluted 40-fold in fresh MHB broth and incubated
at 37 °C for 2–3 h. The resultant mid-log phase cultures were
diluted to the final concentration of 5 ꢁ 105 cfu/mL, then 50
lL
was added to each well of the compound containing 96-well plates.
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5.2.2. Fluorescent microscopy of bacteria with fluorescent
probes
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5.3. Modelling
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The in silico docking was done using Schrödinger software
package and its Induced Fit Docking module (Induced Fit Docking
protocol 2013-2, Glide version 5.9, Prime version 3.2, Schrödinger,
LLC, New York, NY, 2013). For the docking studies the crystal struc-
ture of ribosomal subunit 50S of D. radiodurans bound with linezo-
lid (Pdb: 3DLL) was used.55 Default parameters for the induced fit
docking workflow were used, using the bound linezolid molecule
as ligand for the definition of the receptor site (box size of 40 Å
around the ligand) and refinement of the receptor/pose complex
including residues with 5 Å of the ligand. Ligands were prepared
using the default Ligand Prep workflow, using the protonation
state of the ligand at pH 7.0. The analysis of the different ligand
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