Phenazine antibiotic inspired discovery of potent bromophenazine antibacterial agents against Staphylococcus aureus and Staphylococcus epidermidis

Article abstract of DOI:10.1039/c3ob42416b

Nearly all clinically used antibiotics have been (1) discovered from microorganisms (2) using phenotype screens to identify inhibitors of bacterial growth. The effectiveness of these antibiotics is attributed to their endogenous roles as bacterial warfare agents against competing microorganisms. Unfortunately, every class of clinically used antibiotic has been met with drug resistant bacteria. In fact, the emergence of resistant bacterial infections coupled to the dismal pipeline of new antibacterial agents has resulted in a global health care crisis. There is an urgent need for innovative antibacterial strategies and treatment options to effectively combat drug resistant bacterial pathogens. Here, we describe the implementation of a Pseudomonas competition strategy, using redox-active phenazines, to identify novel antibacterial leads against Staphylococcus aureus and Staphylococcus epidermidis. In this report, we describe the chemical synthesis and evaluation of a diverse 27-membered phenazine library. Using this microbial warfare inspired approach, we have identified several bromophenazines with potent antibacterial activities against S. aureus and S. epidermidis. The most potent bromophenazine analogue from this focused library demonstrated a minimum inhibitory concentration (MIC) of 0.78-1.56 μM, or 0.31-0.62 μg mL^-1, against S. aureus and S. epidermidis and proved to be 32- to 64-fold more potent than the phenazine antibiotic pyocyanin in head-to-head MIC experiments. In addition to the discovery of potent antibacterial agents against S. aureus and S. epidermidis, we also report a detailed structure-activity relationship for this class of bromophenazine small molecules.

Full text of DOI:10.1039/c3ob42416b

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Biomolecular Chemistry
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Phenazine antibiotic inspired discovery of potent
bromophenazine antibacterial agents against
Staphylococcus aureus and Staphylococcus
epidermidis†
Cite this: Org. Biomol. Chem., 2014,
12, 881
Received 3rd December 2013,
Accepted 17th December 2013
DOI: 10.1039/c3ob42416b
Nicholas V. Borrero, Fang Bai, Cristian Perez, Benjamin Q. Duong, James R. Rocca,
Shouguang Jin and Robert W. Huigens III*
www.rsc.org/obc
Nearly all clinically used antibiotics have been (1) discovered from development of drug resistant bacteria.1,2 Despite the growing
microorganisms (2) using phenotype screens to identify inhibitors need for new antibacterial agents, many pharmaceutical com-
of bacterial growth. The effectiveness of these antibiotics is attrib- panies have abandoned their antibacterial discovery programs
uted to their endogenous roles as bacterial warfare agents against as the anticipated success with target-based, high-throughput
competing microorganisms. Unfortunately, every class of clinically screening (HTS) campaigns has yet to be realized.2–5 The
used antibiotic has been met with drug resistant bacteria. In fact, health care emergency that has resulted from drug resistant
the emergence of resistant bacterial infections coupled to the bacterial infections has been gaining momentum over the past
dismal pipeline of new antibacterial agents has resulted in a global four decades as only two new classes of antibiotics have been
health care crisis. There is an urgent need for innovative anti- introduced into the clinic.2,4 Innovative antibacterial strategies
bacterial strategies and treatment options to effectively combat drug are desperately needed to meet the biomedical challenge of
resistant bacterial pathogens. Here, we describe the implemen- resistant bacterial infections.
tation of a Pseudomonas competition strategy, using redox-active
It is without question that microorganisms produce potent
phenazines, to identify novel antibacterial leads against Staphylo- antibiotics as agents of microbial warfare and competition. As
coccus aureus and Staphylococcus epidermidis. In this report, we a result, the large majority of our antibiotic arsenal is based
describe the chemical synthesis and evaluation of
a diverse on such natural products discovered in the antibiotic golden
27-membered phenazine library. Using this microbial warfare inspired era between the 1940s and 1960s (i.e., penicillin, streptomycin,
approach, we have identified several bromophenazines with potent erythromycin, tetracycline, vancomycin) or their synthetic
antibacterial activities against S. aureus and S. epidermidis. The derivatives.2 In fact, very few clinically useful treatment options
most potent bromophenazine analogue from this focused library for bacterial infections have been developed from purely syn-
demonstrated
a
minimum inhibitory concentration (MIC) of thetic origins (i.e., sulfonamides, quinolones, oxazolidinones).
Considering that many past successes in antibiotic discov-
0.78–1.56 µM, or 0.31–0.62 µg mL−1, against S. aureus and S. epi-
dermidis and proved to be 32- to 64-fold more potent than the ery have been grounded on bacterial warfare agents/strategies
phenazine antibiotic pyocyanin in head-to-head MIC experiments. from microorganisms, it stands to reason that future anti-
In addition to the discovery of potent antibacterial agents against bacterial treatments will also depend on the discovery and
S. aureus and S. epidermidis, we also report a detailed structure– implementation of innovative microbial-inspired antibacterial
activity relationship for this class of bromophenazine small molecules. strategies. One such strategy that we have become interested in
is the use of redox-active phenazine antibiotics by Pseudomonas
during competition with other bacteria and fungi through
the formation of reactive oxygen species (ROS).6,7 One example
Introduction
of this competition is in young cystic fibrosis (CF) patients.7
Many times, individuals with CF first develop Staphylococcus
aureus lung infections when they are young. As the CF patient
ages, Pseudomonas aeruginosa co-infects the lung and success-
fully competes against S. aureus for this niche using redox-
active phenazine antibiotics.
The emergence of multidrug resistant bacterial infections has
lead to a serious global crisis. Every class of antibiotic that has
been introduced into the clinic has been met with the
University of Florida, Gainesville, Florida 32610, USA. E-mail: rhuigens@cop.ufl.edu;
We have initiated a research program focused on the discov-
ery of novel antibacterial agents against S. aureus and
S. epidermidis inspired by phenazine antibiotics (Fig. 1,
Tel: +1-352-273-7718
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3ob42416b
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Scheme 1 Synthesis of 13 diverse phenazines for screening against
S. aureus and S. epidermidis. (a) H2O, sunlight, 50%; (b) H2O, sunlight;
NaOH, 37%; (c) 1.2 eq. NBS, PhMe, 21%; (d) 2.2 eq. NBS, PhMe, 99%; (e)
2-bromo-3-nitrobenzoic acid, CuCl, Cu°, N-ethylmorpholine, 2,3-buta-
nediol, 36–73% (6 analogues); (f) NaBH4, NaOEt, 7–54% (6 analogues);
(g) SOCl2, PhMe; NH3 (aq.), 53%; (h) DPPA, THF–TEA; H2O, 61%; (i) H2O2,
AcOH, 58%.
Fig. 1 Initial library of 5 phenazine natural products and 8 synthetic
phenazines in head-to-head MIC experiments against Staphylococcus
aureus.
naturally occurring phenazines: pyocyanin 1, 1-hydroxyphena-
zine 2, phenazine-1-carboxylic acid (PCA) 3, phenazine-1-
carboxamide (PCN) 4, and 2-bromo-1-hydroxyphenazine 5. In
addition to naturally occurring phenazine antibiotics, we also
synthesized eight non-natural phenazine small molecules
(compounds 6–13) for our initial screen against S. aureus
and S. epidermidis (Scheme 1). Phenazines 1, 2, 5 and 11 were
synthesized from phenazine methosulfate using previously
reported synthetic protocols.12–14 In addition, several electroni-
cally diverse PCA analogues (compounds 3, 8–10, 12, 13) were
synthesized using a previously described route.15 PCA 3 was
diversified via amidation reaction to the naturally occurring
phenazine PCN 4 using thionyl chloride followed directly
by treatment with aqueous ammonia. Curtius rearrangement
of PCA 3 readily afforded 1-aminophenazine 6 while oxidation
of 3 with hydrogen peroxide yielded PCA N-oxide 7.
This initial phenazine library was screened against
S. aureus ATCC 25923 and S. epidermidis ATCC 12228 according
to the Clinical and Laboratory Standards Institute (CLSI)
recommendations for microdilution MIC experiments.16 For
these MIC experiments, a concentration range of 0.1–100 µM
was used for phenazine/antibiotic compound made from
eleven 2-fold dilutions in 96-well microtiter plates. From the
initial screen of 13 phenazines, we identified two phenazine
small molecules that demonstrated potent growth inhibition
phenazines 1–5). S. aureus is a human pathogen that is notor-
ious for life-threatening drug resistant infections in hospitals
and the community.8 In fact, in the United States alone there
are more annual deaths from methicillin-resistant Staphylo-
coccus aureus (MRSA) related infections than AIDS.9 Staphylo-
coccus epidermidis is also a pathogen of great importance as
it is particularly prevalent in persistent catheter related
infections.10
Here we describe the synthesis and evaluation of electroni-
cally diverse phenazine natural products and synthetic ana-
logues against S. aureus and S. epidermidis. Reduction
potential and redox-cycling capabilities of the phenazine are
electronically influenced by functional group substitutions on
the phenazine heterocycle.6,11 We hypothesized that an electro-
nically diverse library of phenazine small molecules would
serve as a fruitful starting point for the discovery of promising
lead antibacterial agents against S. aureus and S. epidermidis
based on a ROS-based competition strategy model employed
by Pseudomonas aeruginosa.
Results and discussion
We first set out to synthesize a focused library of electronically
diverse phenazines. We were able to rapidly synthesize five
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Table 1 Minimum inhibitory concentration (MIC) results for phenazines 1–27 and kanamycin using a microdilution protocol in 96-well plates
S. aureus (ATCC 25923)
S. epidermidis (ATCC 12228)
P. aeruginosa (PAO1)
MIC (µM)
Phenazine
MIC (µM)
MIC (µg mL−1
)
MIC (µM)
MIC (µg mL−1
)
Pyocyanin 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
50
10.6
50
10.6
>19.7
>22.5
>22.4
1.72
>19.6
>24.1
>25.5
30.3
13.0
0.28–0.55
>31.5
>25.3
—
—
—
—
—
—
—
0.31
0.66
0.72
—
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
—
>100
>100
>100
6.25
>100
>100
>100
100
>19.7
>22.5
>22.4
1.72
>19.6
>24.1
>25.5
30.3
>100
>100
>100
6.25
>100
>100
>100
100
50
0.78–1.56
>100
>100
—
—
—
—
—
—
—
0.78
1.56
1.56
—
50
1.56
13.0
0.55
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.78–1.56
1.56
>31.5
>25.3
>27.4
>30.2
>43.2
>35.4
>38.3
>28.9
>27.5
0.31–0.62
0.66
—
—
—
1.56
1.56
0.72
0.74
25
26
27
3.13
1.68
3.13
—
—
1.68
—
—
—
—
—
—
>100
>100
1.56–6.25
>46.5
>36.9
0.76–3.03
Kanamycin
0.78–1.56
0.38–0.76
MIC experiments were carried out in duplicate, several active compounds were assayed up to 5 times during these investigations; — is designated
for compounds that were not tested against a particular bacterium.
activity against S. aureus and S. epidermidis. 2-Bromo-1-hydro-
xyphenazine 5 demonstrated impressive antibacterial activity
with MIC values of 6.25 µM (1.72 µg mL−1) against both
S. aureus and S. epidermidis while 2,4-dibromo-1-hydroxy-
phenazine 11 had improved potency against both Staphylococcus
strains (MICs of 1.56 µM/0.55 µg mL−1 against S. aureus;
0.78–1.56 µM/0.28–0.55 µg mL−1 against S. epidermidis; see
Table 1). To our surprise, bromophenazine 11 was found to be
32-fold more potent than the phenazine antibiotic pyocyanin 1
in head-to-head MIC experiments against S. aureus and
S. epidermidis (Fig. 1). Kanamycin (aminoglycoside antibiotic)
was used as
a positive control against S. aureus and
S. epidermidis.
The potent antibacterial activity of bromophenazines 5 and
11 against S. aureus and S. epidermidis prompted us to syn-
thesize a second small, yet diverse collection of bromophena-
zine small molecules (compounds 14–20; Scheme 2). This set
of bromophenazine compounds was
a combination of
Scheme 2 Synthesis of
7 diverse bromophenazines for screening
designed compounds to probe various structural elements of
compound 11 to establish an SAR (compounds 16, 17, 19, 20)
in addition to evaluating unrelated bromophenazines (com-
pounds 14, 15, 18) against S. aureus. With this series of bromo-
phenazines, we were primarily interested to know the impact
against S. aureus. (a) DPPA, THF–TEA; H2O, 69% for 14; (b) 2.1 eq. NBS,
PhMe, 8% for 20, 62% for 17; (c) SOCl2, PhMe; NH3 (aq.) 98%; (d)
2-bromo-3-nitrobenzoic acid, CuCl, Cu°, N-ethylmorpholine, 2,3-
butanediol, 29%; (e) NaBH4, NaOH, 32%; (f) 1 eq. NBS, PhMe–MeCN,
89%; (g) BBr3, CH2Cl2, 27%.
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of substituting the hydroxyl group in 11 for an amine group
(compounds 16 and 17) since the synthetic route we used to
make 1-amino-2,4-dibromophenazines would be ideal for the
synthesis of a more structurally diverse bromophenazine small
molecule library.
Phenazine 9 was converted into amine 14 through Curtius
rearrangement. The carboxylic acid of 9 was also transformed
to the corresponding primary amide in 15 using thionyl chlor-
ide followed directly by treatment with aqueous ammonia. The
1-aminophenazines
6 and 14 were dibrominated using
Fig. 2 Lead bromophenazines 11 and 21 in head-to-head evaluation in
MIC experiments against S. aureus.
N-bromosuccinimide to yield bromophenazines 16 and 17. 2,5-
Dibromoaniline was converted to 6,9-dibromophenazine-1-car-
boxylic acid 18 using the 2-step protocol to make PCA ana-
logues (Jourdan–Ullmann coupling, followed by reductive
ring closure with sodium borohydride). Finally, 1-methoxy-
phenazine was brominated in the 4-position to make 19,
which was demethylated to make 20 using a known route.14
All bromophenazines 14–20 were evaluated against S. aureus
in MIC experiments and found to be inactive as growth
inhibitors at the highest concentrations tested (100 µM).
Although we were surprised at this result, this small set of
compounds was useful in establishing a detailed structure–
activity relationship (SAR) for this class of bromophenazines
(Scheme 3).
Phenazines 1–20 were then screened for growth inhibition
activity against P. aeruginosa strain PAO1. In MIC experiments,
none of these phenazines demonstrated growth inhibition
against PAO1 at the highest concentration tested (100 µM). It
is well established that Pseudomonas is resistant to pyocyanin-
induced death at very high concentrations.17 The lack of
growth inhibition against PAO1 is supportive of phenazines
5 and 11 demonstrating potent antibacterial activity against
Bromophenazines 21–27 were evaluated against S. aureus
and bromophenazine 21 demonstrated the most potent activity
(Fig. 2) with an MIC of 0.78–1.56 µM (0.31–0.62 µg mL−1
against S. aureus and an MIC of 0.78 µM (0.31 µg mL−1
)
)
against S. epidermidis. The potent growth inhibition demon-
strated by 21 corresponds to a 32- to 64-fold increase in
potency against S. aureus and S. epidermidis when compared
head-to-head against pyocyanin 1. Bromophenazines 22, 23
and 24 also demonstrated impressive growth inhibition activi-
ties against S. aureus and S. epidermidis (24 not tested against
S. epidermidis) reporting MIC values of 1.56 µM while bromo-
phenazine 25 had an MIC of 3.13 µM against S. aureus and S.
epidermidis. Interestingly, bromophenazines 26 and 27 were
completely inactive against S. aureus at 100 µM in MIC exper-
iments. Bromophenazines 11 and 21 were found to be more
potent or equipotent to kanamycin in head-to-head MIC exper-
iments while the naturally occurring bromophenazine
5
demonstrated slightly lower potency than kanamycin against
S. aureus and S. epidermidis (Table 1).
S. aureus and S. epidermidis through
a ROS-generating
mechanism.
We then functionalized the phenolic hydroxyl group of bromo-
phenazine 11. We synthesized a small collection of diverse
ester analogues 21–26 by condensing bromophenazine 11 with
various acid chlorides. Additionally, we synthesized the corres-
ponding methyl ether 27 by refluxing bromophenazine 11 with
methyl iodide in acetone.14
Scheme 3 Synthesis of 7 diverse ester/ether bromophenazine ana-
logues for screening against S. aureus. (a) acid chloride, TEA, CH2Cl2,
76–94% (6 analogues); (b) K2CO3, acetone; iodomethane, 29%.
Fig. 3 Detailed structure–activity relationship (SAR) for this novel class
of antibacterial agents against S. aureus.
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If we use bromophenazine 11 (MIC 1.56 µM) as a starting compounds were prepared for biological evaluation as 10 mM
point to compare other analogs against, a detailed SAR DMSO stock solutions and were stored at room temperature in
emerges from our investigations against S. aureus (Fig. 3). the absence of light. DMSO served as our vehicle and negative
The loss of the bromine atom at the 4-position of 11 results in control in each microdilution MIC assay. DMSO was serially
a 4-fold reduction in potency (phenazine 5; MIC 6.25 µM). diluted at the same concentration as the phenazine com-
Substituting an amine group (compound 17; MIC >100 µM) for pounds with a top concentration of 1% v/v. Bacterial strains
the hydroxyl of 11 leads to a loss in activity against S. aureus. used during these investigations were S. aureus (ATCC 25923),
Ester analogues of 11 can either enhance potency (compound S. epidermidis (ATCC 12228) and P. aeruginosa (PAO1).
21 MIC 0.78–1.56 µM) or abolish growth inhibition (compound
26; MIC >100 µM), but most ester moieties evaluated during
our investigation are well tolerated and potent against
S. aureus (compounds 22–25 had MICs of 1.56–3.13 µM).
Acknowledgements
The methyl ether 27 demonstrated no growth inhibition
The authors gratefully acknowledge research support from
against S. aureus in MIC experiments at the highest
the University of Florida (UF College of Pharmacy and Division
concentration tested (100 µM). We can also note that the loss
of Sponsored Research). We would like to acknowledge
of a bromine atom at the 2-position of the phenazine
the National Science Foundation through the National High
ring (compounds 16 and 19; not shown in Fig. 3) results in
Magnetic Field Laboratory, which supported our NMR studies,
the complete loss of activity against S. aureus (MIC >100 µM).
in part, at the Advanced Magnetic Resonance Imaging and
This SAR will be beneficial in guiding future analogue develop-
Spectroscopy (AMRIS) Facility in the McKnight Brain Institute
ment pertaining to this class of bromophenazine small
of the University of Florida. High-resolution mass spectra
molecules.
(HRMS) were obtained from the Mass Spectrometry facility
(Chemistry Department) at the University of Florida. We would
like to thank Professor Hendrik Luesch for helpful discussions
regarding various aspects of this project and Nicholas Paciaroni
Conclusions
for synthesis assistance.
We have discovered a class of bromophenazine antibacterial
agents that demonstrate potent growth inhibition against
S. aureus and S. epidermidis inspired by a microbial warfare
strategy. These bromophenazines originated from a focused
library of 13 diverse phenazine compounds, including five
Notes and references
naturally occurring “phenazine antibiotics” that was evaluated
against S. aureus and S. epidermidis in MIC assays. These find-
ings are indeed timely as novel compounds that are potent
antibacterial against S. aureus and S. epidermidis are of
great importance as these pathogens are notorious for their
drug-resistant infections in humans.
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Experimental section
1
Synthetic procedures, H and 13C NMR spectra and HRMS are
reported in the ESI.†
Antibiotic susceptibility tests (MIC assay protocol)
The minimum inhibitory concentration (MIC) for each phena-
zine was determined by the broth microdilution method as
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886 | Org. Biomol. Chem., 2014, 12, 881–886
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