Anionic fluoroquinolones as antibacterials against biofilm-producing Pseudomonas aeruginosa

Article abstract of DOI:10.1016/j.bmcl.2016.01.012

Pseudomonas aeruginosa is a common biofilm-forming bacterial pathogen implicated in diseases of the lungs. The extracellular polymeric substances (EPS) of respiratory Pseudomonas biofilms are largely comprised of anionic molecules such as rhamnolipids and

Full text of DOI:10.1016/j.bmcl.2016.01.012

Accepted Manuscript
Anionic fluoroquinolones as antibacterials against biofilm-producing Pseudo-
monas aeruginosa
Timothy E. Long, Lexie C. Keding, Demetria D. Lewis, Michael I. Anstead, T.
Ryan Withers, Hongwei D. Yu
PII:
S0960-894X(16)30012-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.01.012
BMCL 23477
Reference:
Bioorganic & Medicinal Chemistry Letters
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22 December 2015
5 January 2016
6 January 2016
Please cite this article as: Long, T.E., Keding, L.C., Lewis, D.D., Anstead, M.I., Ryan Withers, T., Yu, H.D., Anionic
fluoroquinolones as antibacterials against biofilm-producing Pseudomonas aeruginosa, Bioorganic & Medicinal
Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.01.012
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Anionic fluoroquinolones as antibacterials against
biofilm-producing Pseudomonas aeruginosa
Timothy E. Long, Lexie C. Keding, Demetria D. Lewis,
Michael I. Anstead, T. Ryan Withers, and Hongwei D. Yu

1
Bioorganic & Medicinal Chemistry Letters
Anionic fluoroquinolones as antibacterials against biofilm-producing
Pseudomonas aeruginosa
Timothy E. Longa,b, Lexie C. Kedingb, Demetria D. Lewisa, Michael I. Ansteadd, T. Ryan Witherse, and
Hongwei D. Yub,c,e
a Department of Pharmaceutical Science and Research, School of Pharmacy, Marshall University, Huntington, WV 25755
b Departments of Biochemistry and Microbiology, and cPediatrics, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25755
dDepartment of Pediatrics, College of Medicine, University of Kentucky, Lexington, KY 40506
eProgenesis Technologies, LLC, Huntington WV 25755
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Pseudomonas aeruginosa is a common biofilm-forming bacterial pathogen implicated in
diseases of the lungs. The extracellular polymeric substances (EPS) of respiratory Pseudomonas
biofilms are largely comprised of anionic molecules such as rhamnolipids and alginate that
promote a mucoid phenotype. In this paper, we examine the ability of negatively-charged
fluoroquinolones to transverse the EPS and inhibit the growth of mucoid P. aeruginosa. Anionic
fluoroquinolones were further compared with standard antibiotics via a novel microdiffusion
assay to evaluate drug penetration through pseudomonal alginate and respiratory mucus from a
patient with cystic fibrosis.
Available online
Keywords:
fluoroquinolone
Pseudomonas
mucoid
biofilm
antibacterial
2009 Elsevier Ltd. All rights reserved.
Pseudomonas aeruginosa is an opportunistic bacterium that is
frequently implicated in burn wound, bladder, and lower
respiratory tract infections [1]. The Gram-negative aerobe is also
the primary etiology of chronic pulmonary infections in cystic
fibrosis (CF) due to its ability to evade host defenses within the
accumulated mucus lining the airway of the lungs. When
Pseudomonas colonizes the lower respiratory tract, neutrophils
infiltrate the lungs and release reactive oxygen species (ROS) to
suppress the infection. Exposure to ROS is thought to cause
DNA-level mutations and activation of gene products that further
protects P. aeruginosa from the immune system [2]. Among
these are the enzymes and proteins involved in the biosynthesis
and secretion of rhamnolipids [3] and alginate [4], two
components of the extracellular polymeric substance (EPS) found
in Pseudomonas biofilms of CF airways.
this premise, we set forth to evaluate anionic fluoroquinolones as
growth inhibitors of biofilm-producing P. aeruginosa and their
ability to penetrate the alginate component of Pseudomonas EPS
and CF respiratory mucus.
Like the sulfated and carboxylated components of mucus
located in the lower respiratory tract, rhamnolipids and alginate
possess an overall negative charged (Figure 1) [5]. In CF, the
anionic environment of the lungs is amplified by DNA and F-
actin released from necrotic neutrophils. The physiochemical
properties of this dense complex mixture of negatively charged
biomolecules consequentially serves as a barrier against cationic
and zwitterionic antimicrobials [6]. It was rationalized from here
that antibiotics carrying a net -1 or -2 charge would more readily
transverse these barriers and reach the bacteria entrenched within
Figure 1. Structures of (a) rhamnolipids and (b) alginate in biofilm-producing
Pseudomonas aeruginosa.
The initial series of anionic fluoroquinolones explored were
derived from FDA-approved fluoroquinolones 1a-5a (Figure 2)
due to the lack of ionic interactions with the EPS and mucus. On
———
 Corresponding author. Tel.: +1-304-696-7393; fax: +1-304-696-7309; e-mail: longt@marshall.edu

2
including ciprofloxacin (1a), a first-line treatment for urinary
tract infections due to P. aeruginosa. The initial design of the
anionic fluoroquinolones was to acetylate the C-7 amino residue
of the quinolone ring to preclude zwitterion formation at
physiological pH. The reactions were performed in
dichloromethane with acetic anhydride (1.5 equiv.), Et3N (1.3-2.6
equiv.), and DMAP (0.1 equiv.) to afford the N-acetyl
fluoroquinolones 1b-5b. The derivatives and parent compounds
were then evaluated for antipseudomonal activity against the
reference non-mucoid P. aeruginosa PAO1 and its mucoid
derivative, PAO581. The genomes of both bacterial strains have
been sequenced [7,8] with the mutation causing the stable
production of EPS alginate previously defined [9].
to compare the ability of each analog to inhibit bacterial viability
on regular growth and mucoidy-promoting agar media,
respectively (Table 1). The compounds were evaluated as the K+
salts using 10 mm cellulose discs impregnated with 10 M of
drug. Following overnight air incubation at 37 °C, the plates were
examined for zones of growth inhibition and it was discovered
that acetylation of the C-7 amino residue negated the inhibitory
activity for most of the derivatives against both PAO variants. In
contrast, the unmodified fluoroquinolones proved to be active
displaying moderate to strong growth inhibitory effects with a
zone diameter range of 10-38 mm.
As expected, ciprofloxacin demonstrated the greatest
antipseudomonal activity and its N-acetylated form 1b was the
lone derivative in the series to inhibit growth. To further explore
impact of lipophilicity on the activity, the N-formyl (1c, Figure
2) [10] and 10 additional N-acyl derivatives (1d-1m, Figure 3) of
ciprofloxacin [11,12] were prepared for microbiological
evaluation. The compounds were once again tested at 10 M as
K+ salts and with the exception of the N-formyl analog 1c, each
was found to have no inhibitory effects on Pseudomonas growth.
It was concluded from the data that the anionic character and
lipophilicity (clogP 1.96 – 3.98) furnished to analogs 1b-1m of
ciprofloxacin (clogP 1.63) had adversely affected antimicrobial
activity. These findings further suggested that an ionizable group
at the C-7 position of the quinolone ring may be a pre-requisite
for transport and/or gyrase binding in Pseudomonas as noted for
the reduced efficacy of pefloxacin (6, Table 1).
Figure 3: N-Acyl fluoroquinolones 1d-1m.
A second series of ciprofloxacin analogs modified with an
ionizable carboxylate substituent on the C-7 piperazine ring were
subsequently prepared (Scheme 1) and evaluated. Compounds
1n-1p were synthesized by heating ciprofloxacin (1 equiv.) with
their respective cyclic anhydrides (1 equiv.) overnight in DMSO
[13]. Activity comparisons were made with the polycationic
aminoglycoside tobramycin and the carboxypenicillin ticarcillin,
which like the analogs carries a net -2 charge.
Figure 2: N-Protio fluoroquinolones and N-substituted derivatives 1-6.
Table 1: Zones of growth inhibition (mm) against non-mucoid PAO1 and
mucoid PAO581 for fluoroquinolones 1-6.a
PAO1
LB
30
29
23
24
25
18
10
18
0
PAO1
PIA
24
21
12
10
17
12
0
PAO581
PAO581
strain
media
ciprofloxacin (1a)
gatifloxacin (2a)
lomefloxacin (3a)
moxifloxacin (4a)
pazufloxacin (5a)
pefloxacin (6)
LB
38
31
29
31
30
19
0
21
0
0
0
0
PIA
26
23
15
14
20
14
13
9
0
0
0
0
1b
1c
2b
3b
4b
5b
(20)c
0
0
0
0
0
0
0
a Compounds were tested as K+ salts at 10 M concentration in water.
b Pseudomonas isolation agar (PIA) is supplemented with 5% glycerol as a C source for
mucoid production. Luria-Bertani (LB) agar lacks glycerol to promote the mucoid
phenotype.
c Partial inhibition.
Scheme 1: Synthesis of carboxyfluoroquinlones 1n-1p.
The N-acetyl fluoroquinolones 1b-5b were tested by the
In this series, the dianionic N-phthalyl derivative 1p was the
conventional disc diffusion (Kirby Bauer) assay on LB and PIA
only compound found to effectively inhibit the growth of

3
Pseudomonas (Table 2). This was a surprising finding on
comparison with the inactive N-benzoyl analog 1i. Moreover, a
53-58% reduction in zone size was observed for bacteria grown
on the mucoidy-promoting PIA media in contrast to 20-32% for
ciprofloxacin. When compared with tobramycin, the N-phthalyl
analog exhibited similar activity while ticarcillin was found to be
inactive suggesting that the PAO strains used in the study were
harboring the genes for one or more penicillin-degrading -
lactamase (e.g., AmpC) [14].
ciprofloxacin and tobramycin. Of note, the MIC results (i.e.,
ciprofloxacin ≈ tobramycin > 1c = 1p) did not correlate to the
zone data (i.e., ciprofloxacin > 1p > tobramycin > 1c) performed
on solid media. It is believed that the smaller zone sizes exhibited
for the charged fluoroquinolones and tobramycin was a
consequence of truncated diffusion through the uncharged agar
media. These results prompted us to examine the influence of
charge on the ability the antibiotics to penetrate through the
alginate component of biofilm Pseudomonas EPS and CF
respiratory mucus.
Table 2: Zones of growth inhibitions (mm) for fluoroquinolones 1n-1p.a
Table 4: Minimum inhibitory concentration (MIC) and minimum bactericidal
PAO1
LB
0
PAO1
PIA
0
PAO581
LB
0
PAO581
PIA
0
strain
media
1n
1o
1p
concentration (MBC) for select compounds.a
PAO1
MIC
3.125
3.125
1.56
PAO1
MBC
6.25
6.25
6.25
PAO581
MIC
3.125
3.125
1.56
PAO581
MBC
6.25
12.5
6.25
strain
0
0
0
0
23
18
0
11
13
0
31
20
0
13
17
0
1c
1p
tobramycin
ticarcillin
ciprofloxacin
tobramycin
a Compounds were tested as K+ salts at 10 M concentrations in water.
3.125
1.56
0.78
6.25
a Compounds were tested as K+ salts in Luria-Bertani broth. Values reported in M.
With these results, an additional series of carboxy-
fluoroquinolones were evaluated. The acetic and butyric acid
derivatives 1v and 1w were prepared by acid hydrolysis of their
parent methyl esters 1q and 1r, respectively (Scheme 2) [15].
Additional N-alkyl derivatives 1s-1u were synthesized from their
respective alkyl bromides for comparison with the analogs and
the N-methyl fluoroquinolone, pefloxacin 6 (Figure 2). The
antipseudomonal activity for this series was found to be mostly
limited to N-alkylated analogs 1r and 1s (Table 3). On
comparison with pefloxacin, the methyl ester derivative 1r
displayed similar activity against mucoid PAO581.
To probe the penetrating ability of charged antibiotics through
solid media, a novel microdiffusion assay was developed using
centrifuge filter columns containing 1% PAO581 alginate [16] or
CF sputum in Noble agar. As depicted in Figure 4, the filter
columns containing the semi-permeable barriers were inserted
into a 1.5 mL centrifuge tube containing a 5 x 105 inoculum of P.
aeruginosa and 50 µL of 25 µM antibiotic was applied to the
agar surface. The tubes were sealed and incubated overnight at 37
°C with shaking. The penetrating ability was assessed by
calculating the inhibition of antibiotic activity from the OD600
measurements and Equation 1 with Noble agar, an uncharged
medium, representing 100% drug diffusion into the broth culture.
Scheme 2: Synthesis of N-alkyl fluoroquinolones 1q-1w.
Table 3: Zones of growth inhibitions (mm) for fluoroquinolones 1q-1w.a
PAO1
LB
0
PAO1
PIA
0
PAO581
LB
0
PAO581
PIA
0
strain
media
1q
Figure 4: Microdiffusion assay design.
16
23
0
0
0
6
0
0
0
0
0
12
17
13
0
13
0
9
0
0
0
0
0
14
1r
1s
1t
1u
1v
1w
Table 5: Inhibition of antibiotic activity using PAO581 in the microdiffusion
assay.
strain
1c
1p
ciprofloxacin
tobramycin
PAO581 alginate
CF sputum
0.89
0.61
0.80
0.97
0.98
0.49
-0.33
0.0
0
18
0
19
pefloxacin (6)
a Compounds were tested as K+ salts at 10 M concentrations in water.
n = 0 (drug bioavailability not affected)
n = 0 < to 1 (drug bioavailability decreased)
n < 0 (drug bioavailability increased)
Minimum inhibitory and bactericidal concentrations were
subsequently determined by broth microdilution for the two most
active fluoroquinolones from the study, the N-formyl and N-
phthalyl substituted ciprofloxacin analogs 1c and 1p, respectively
(Table 4). The carboxy-fluoroquinolone 1p was found to be
inferior to both ciprofloxacin and tobramycin in liquid culture.
Likewise, the N-formyl analog 1c proved to be two fold less
effective at inhibiting the pseudomonal growth compared to
The microdiffusion assay revealed that Pseudomonas alginate
and the components in CF sputum likely modulate antibiotic
penetration (Table 5). The data suggests that the negatively
charged sugars in alginate (i.e., mannuronate, guluronate) reduces
the EPS permeation of both charged and zwitterionic antibiotics
through Pseudomonas biofilms. Based on the preliminary

4
findings from this assay, the anionic fluoroquinolones 1c and 1p
were less hindered by the alginate barrier compared to
ciprofloxacin and tobramycin. Conversely, the constituents found
in CF patient sputum did not appear to affect the penetration of
these standard antibiotics. Follow up studies using sputum from
different CF patient populations will be performed to further
corroborate these findings.
responsibility of the authors and does not necessarily represent
the official views of the NIH.
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Figure 5: Respiratory mucus and EPS of Pseudomonas biofilms reduce the
bioavailability of inhaled and systemic antibiotics.
Acknowledgements
This research was supported in part by the Marshall University
School of Pharmacy FRS Grant Program and the Office of
Experiential Learning for financial support. Special thanks is also
given to NASA West Virginia EPSCoR grant program, the
National Center for Research Resources, the National Center for
Advancing Translational Sciences, and National Institutes of
Health (NIH), through Grant UL1TR000117. HDY is supported
by NIH P20GM103434 to the West Virginia IDeA Network for
Biomedical Research Excellence, and is the co-founder of
Progenesis Technologies, LLC. The content is solely the