Synthetically Tuning the 2-Position of Halogenated Quinolines: Optimizing Antibacterial and Biofilm Eradication Activities via Alkylation and Reductive Amination Pathways

Article abstract of DOI:10.1002/chem.201600926

Agents capable of eradicating bacterial biofilms are of great importance to human health as biofilm-associated infections are tolerant to our current antibiotic therapies. We have recently discovered that halogenated quinoline (HQ) small molecules are: 1) capable of eradicating methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE) and vancomycin-resistant Enterococcus faecium (VRE) biofilms, and 2) synthetic tuning of the 2-position of the HQ scaffold has a significant impact on antibacterial and antibiofilm activities. Here, we report the chemical synthesis and biological evaluation of 39 HQ analogues that have a high degree of structural diversity at the 2-position. We identified diverse analogues that are alkylated and aminated at the 2-position of the HQ scaffold and demonstrate potent antibacterial (MIC≤0.39 μm) and biofilm eradication (MBEC 1.0–93.8 μm) activities against drug-resistant Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecium strains while demonstrating <5 % haemolysis activity against human red blood cells (RBCs) at 200 μm. In addition, these HQs demonstrated low cytotoxicity against HeLa cells. Halogenated quinolines are a promising class of antibiofilm agents against Gram-positive pathogens that could lead to useful treatments against persistent bacterial infections.

Full text of DOI:10.1002/chem.201600926

DOI: 10.1002/chem.201600926
Full Paper
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Medicinal Chemistry |Hot Paper|
Synthetically Tuning the 2-Position of Halogenated Quinolines:
Optimizing Antibacterial and Biofilm Eradication Activities via
Alkylation and Reductive Amination Pathways
Akash Basak,[a] Yasmeen Abouelhassan,[b] Verrill M. Norwood IV,[b] Fang Bai,[c]
Minh Thu Nguyen,[b] Shouguang Jin,[c] and Robert W. Huigens, III*[a, b]
We dedicate this work to the memory of the late Professor Alan Katritzky
Abstract: Agents capable of eradicating bacterial biofilms
are of great importance to human health as biofilm-associat-
ed infections are tolerant to our current antibiotic therapies.
We have recently discovered that halogenated quinoline
(HQ) small molecules are: 1) capable of eradicating methicil-
lin-resistant Staphylococcus aureus (MRSA), methicillin-resist-
ant Staphylococcus epidermidis (MRSE) and vancomycin-re-
sistant Enterococcus faecium (VRE) biofilms, and 2) synthetic
tuning of the 2-position of the HQ scaffold has a significant
impact on antibacterial and antibiofilm activities. Here, we
report the chemical synthesis and biological evaluation of 39
HQ analogues that have a high degree of structural diversity
at the 2-position. We identified diverse analogues that are al-
kylated and aminated at the 2-position of the HQ scaffold
and demonstrate potent antibacterial (MICꢀ0.39 mm) and
biofilm eradication (MBEC 1.0–93.8 mm) activities against
drug-resistant Staphylococcus aureus, Staphylococcus epider-
midis and Enterococcus faecium strains while demonstrating
<5% haemolysis activity against human red blood cells
(RBCs) at 200 mm. In addition, these HQs demonstrated low
cytotoxicity against HeLa cells. Halogenated quinolines are
a promising class of antibiofilm agents against Gram-positive
pathogens that could lead to useful treatments against per-
sistent bacterial infections.
Introduction
Small molecules capable of eradicating free-floating planktonic
bacteria and surface-attached bacterial biofilms serve as prom-
ising therapeutic leads as persistent, biofilm-associated bacteri-
al infections are innately tolerant to our current arsenal of anti-
biotics.[1–3] Bacteria exist in two distinct lifestyles, which in-
clude: 1) rapidly-dividing, free-floating (planktonic) bacteria,
and 2) surface-attached communities of specialized, non-repli-
cating persister cells encased within an extracellular matrix of
biomolecules (i.e., biofilm; Figure 1).[1,2,4] Bacterial biofilms play
a significant role in human health as it is estimated that 17 mil-
lion new biofilm infections will result in >500000 deaths in
the United States each year.[2,5]
Figure 1. Illustration of the dynamic “biofilm cycle” including free-floating
planktonic and surface-attached bacteria. Halogenated quinolines eradicate
both planktonic and persistent biofilm-associated bacteria.
[a] A. Basak, Prof. R. W. Huigens, III
Since penicillin’s discovery in 1928, each new class of antibi-
otic has been discovered as bacterial growth inhibitors that
either kill or inhibit the proliferation of planktonic cells. As
a consequence, our antibiotics operate through growth-depen-
dent mechanisms and are rendered ineffective against non-
replicating, persistent biofilms.[1,2,6] Several classes of biofilm in-
hibitors and dispersal agents have been discovered and al-
though these are promising agents, they typically operate
through the perturbation of bacterial signaling processes (i.e.,
quorum sensing)[3,7,8] and do not kill persister cells housed
within biofilms.
Department of Chemistry, University of Florida
1600 SW Archer Road, Gainesville, FL 32610 (USA)
E-mail: rhuigens@cop.ufl.edu
[b] Y. Abouelhassan, V. M. Norwood IV, M. T. Nguyen, Prof. R. W. Huigens, III
Department of Medicinal Chemistry, Center for Natural Products
Drug Discovery and Development (CNPD3), University of Florida
1345 Center Drive, Gainesville, FL 32610 (USA)
[c] Dr. F. Bai, Prof. S. Jin
Department of Molecular Genetics and Microbiology
University of Florida, Gainesville, FL 32610 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201600926.
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In order to eradicate persister cells that live within biofilms,
new small molecules are needed that operate through growth-
independent mechanisms. The most prominent class of bio-
film-eradicating agents are antimicrobial peptide mimics (i.e.,
quaternary ammonium cations[9,10]); however, other small mol-
ecules have demonstrated biofilm eradication, including: dicat-
ionic porphyrins (i.e., XF-73)[11], carbonyl cyanide m-chlorophe-
nylhydrazone (CCCP),[12] N-acetyl cysteine (NAC),[13] mitomycin
C[14] and halogenated phenazines.[15,16] In addition, the co-treat-
ment of ADEP4 (acyldepsipeptide antibiotic and ClpP activator)
and rifampin successfully eradicated a methicillin-resistant
Staphylococcus aureus (MRSA) biofilm infection using a mouse
model.[17]
Results and Discussion
Chemical synthesis of wave 1 HQ analogues
With HQ-1 demonstrating potent antibacterial (minimum in-
hibitory concentration or MIC 0.78 mm) and biofilm eradication
(minimum biofilm eradication concentration or MBEC 250 mm)
activities against MRSA,[20] our initial goal was to synthesize
a series of HQ analogues that possessed diverse alkylated
products at the 2-position of the HQ scaffold (A-HQs). To this
end, we treated 1 with 1.1 equivalents of lithium diisoproplya-
mine (LDA) to generate the corresponding carbanion at the
methyl group of the 2-position, followed by subsequent addi-
tion of an alkyl halide to serve as an electrophile in an SN2 re-
action (Scheme 1A). This alkylation reaction proceeded in 20–
45% yield to generate 6 diverse alkylated products 2a–f. Meth-
ylation with methyl iodide gave a 20% yield of the bismethyl
product 2b, which was unanticipated; however, we carried
this analogue forward to 2-isopropyl A-HQ-2 (Scheme 1).
Following the alkylation step, 8-methoxyquinolines 2a–f were
demethylated using hydrobromic acid in acetic acid to afford
the corresponding 8-hydroxyquinolines in 65–91% yield. Final
halogenation with N-bromosuccinimide (NBS) or N-iodosuc-
cinimide (NIS) yielded A-HQ-1 through A-HQ-7 in 40–75%
yield.
Recently, we reported a series of five halogenated quinolines
with potent biofilm-eradicating activities against MRSA, MRSE
(methicillin-resistant Staphylococcus epidermidis) and VRE (van-
comycin-resistant Enterococcus faecium).[18] These HQs were dis-
covered through a reductive amination reaction at the 2-posi-
tion of the HQ scaffold, which plays a critical role in antibacte-
rial activities. Here, we describe a full account of our investiga-
tions of HQ analogues that have been diversified at the 2-posi-
tion of the HQ scaffold, including: analogue design, chemical
synthesis, biological evaluation and structure-activity relation-
ship analysis.
In our previous studies, we observed that the 2-position of
HQ scaffold plays a significant role on the corresponding anti-
bacterial activities on HQ analogues.[19] Broxyquinoline (Brox-Q;
Figure 2) has a hydrogen atom in the 2-position compared to
HQ-1, which has a methyl group in the 2-position of the HQ
scaffold. This single methyl group difference enhances the anti-
bacterial activity of HQ-1 16-fold against staphylococcal patho-
gens while eliminating antibacterial activity against the Gram-
negative pathogen Acinetobacter baumannii 128-fold com-
pared to Brox-Q. In addition, we found that HQ-1 is capable of
eradicating MRSA biofilms.[18,20] These observations motivated
us to design multiple synthetic routes to achieve rapid and
highly diverse analogues at the 2-position of the HQ scaffold
for evaluation in antibacterial and biofilm eradication assays
against drug-resistant strains of major human pathogens.
In addition to 2-alkylated HQ analogues, we designed an al-
ternative route to incorporate a diverse series of amines and
anilines at the 2-position of the HQ scaffold through reductive
amination. HQ-2 (Scheme 1B), which was synthesized by the
NBS bromination of 8-hydroxyquinoline-2-carbaldehyde, was
used as a key building block for analogue synthesis through
a divergent reductive amination reaction. Initial attempts to
carry out reductive amination on HQ-2 were unsuccessful de-
spite extensive scouting of solvents (i.e., toluene, acetonitrile,
methanol), temperatures (i.e., room temperature, reflux), reac-
tion times (i.e., hours to multiple days) and catalysts (i.e., acid,
no acid). Upon close examination of these reaction conditions,
we encountered problems with initial imine formation which
led to no or unacceptable yields of reductive amination. How-
ever, we found that changing the solvent to 1,2-dichloro-
ethane (1,2-DCE) allowed the desired reductive amination to
proceed smoothly at room temperature. We condensed HQ-2
with a diverse panel of amines and anilines for 15 min to 1 h
before adding sodium triacetoxyborohydride (NaBH(OAc)3) to
afford eleven reductive amination HQ analogues (RA-HQ-
1 through RA-HQ-11) in 40–85% yield. Aliphatic amines (4 ex-
amples; 45–85% yield) and anilines (7 examples; 43–77%
yield) demonstrated near identical efficiencies using this reduc-
tive amination route (Scheme 1B).
Commercially available or easily synthesized 8-hydroxyqui-
nolines with different substitution at the 2-position were bro-
minated or iodinated to yield a diverse collection of HQs (see
Scheme 1C for structures; see the Supporting Information for
synthesis details). Several of these analogues were used to
probe diverse electronic properties at the 2-position of the HQ
scaffold, including: HQ-2/HQ-3 (aldehyde), HQ-7 (carboxylic
acid), HQ-8 (nitrile), HQ-9 (amine) and HQ-10 (amidine). HQ-4,
Figure 2. Preliminary findings that the 2-position of the HQ scaffold controls
antibacterial properties led to the discovery of HQ biofilm-eradicating
agents.
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Scheme 1. Synthetic routes involving: A) alkylation, and B) reductive amination pathways for the diversification of the 2-position of the HQ scaffold. C) Initial
HQ library synthesized for biological evaluation in antibacterial and biofilm eradication assays.
HQ-5 and HQ-6 possessed alcohol functional groups, comple-
menting analogues the alkylated series.
Staphylococcus aureus (methicillin-sensitive; ATCC 29213),
Staphylococcus epidermidis (methicillin-sensitive; ATCC 12228)
and Enterococcus faecium (ATCC 700221; vancomycin-resistant,
VRE) to identify potent antibacterial agents in microdilution
MIC assays (MICꢀ3.13 mm; full summary in the Supporting In-
formation Table S1) before advancing the most active antibac-
terial agents to biofilm eradication studies using Calgary Bio-
Biological investigations of wave 1 HQ analogues
With this panel of 29 HQ analogues (including Brox-Q and HQ-
1; Scheme 1C), our strategy was to screen this library against
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film Device (CBD) assays[21] (Table 1). We began with an MIC
screen since these assays are operationally simple and HQs
that eradicate biofilms typically demonstrate potent antibacte-
rial activities. In addition, all HQ analogues were screened
against human red blood cells for haemolytic activity at
200 mm (Table 1) while select analogues were evaluated for
mammalian cytotoxicity against HeLa cells (see the Supporting
Information).
threefold more potent antibacterial activities (MIC=0.39 to
0.59 mm) compared to HQ-1 (MIC=1.17 mm). Eight new HQs
demonstrated equipotent antibacterial activities (MIC=0.78–
2.35 mm) while 15 new HQs reported partial or complete loss
in antibacterial activity against S. epidermidis (MIC=3.13 to
>100 mm). Select HQs were evaluated against methicillin-resist-
ant S. epidermidis (ATCC 35984), which demonstrated an in-
creased sensitivity towards HQ analogues as HQ-1 (MIC=
0.30 mm), A-HQ-1 (MIC=0.39 mm), A-HQ-3 (MIC=0.59 mm), RA-
HQ-5 (MIC=0.15 mm) and RA-HQ-9 (MIC=0.30 mm) demon-
strated the most potent antibacterial activities against MRSE
(Table 1; Supporting Information Table S1).
Comparing the 27 new HQs (Wave 1; Scheme 1C) to the
potent antibacterial activity of HQ-1 (MIC=0.59 mm; Support-
ing Information Table S1), we identified nine HQs that demon-
strated equipotent antibacterial activities (i.e., MIC values 0.39–
1.17 mm) against S. aureus 29213. Fifteen new HQs lost antibac-
terial activity (MIC values between 1.56 and 100 mm) and three
HQs were inactive (MIC>100 mm) against S. aureus 29213 in
our assays. Five of the seven A-HQs and three RA-HQs proved
to be the most potent antibacterial HQs against S. aureus
29213. When evaluated against MRSA-2 and MRSA BAA-44,
several HQs demonstrated potent antibacterial activities (MICꢀ
1.56 mm), including: A-HQ-1, A-HQ-3, RA-HQ-5 (Supporting In-
formation Table S1; Supporting Information Table S2 has addi-
tional MRSA clinical isolate MIC data).
Halogenated quinolines displayed a range of antibacterial
activities against VRE 700221. HQ-1 demonstrated good anti-
bacterial activity (MIC=2.35 mm) against VRE while eight new
HQs demonstrating increased antibacterial potencies (MIC=
0.39–0.78 mm) with A-HQ-4 and RA-HQ-9 proving to be the
most potent (MIC=0.39 mm; eightfold more potent than HQ-
1). From this series of wave 1 HQs, seven new HQs demon-
strated equipotent activity (MIC=1.17–3.13 mm) while 12 HQs
lost antibacterial activities (MIC=6.25–100 mm) against VRE
when compared to HQ-1.
Interestingly, against S. epidermidis 12228, four new HQs (A-
HQ-2, RA-HQ-7, RA-HQ-8, RA-HQ-9) demonstrated two- to
Similar antibacterial trends were observed between staphy-
lococcal pathogens (S. aureus and S. epidermidis) and E. faecium
Table 1. Summary of antibacterial, biofilm eradication and haemolysis activities for select halogenated quinoline (HQ) analogues, relevant conventional an-
tibiotics and controls. All concentrations are reported in mm.
Compound
MRSA-2
MIC
MRSA-2
MBC/MBEC
MRSA BAA-1707
MBC/MBEC
MRSE
MIC
MRSE
MBC/MBEC
VRE
MIC
VRE
MBC/MBEC
% Haemolysis
at 200 mm
HQ-1
A-HQ-1
A-HQ-3
A-HQ-4
A-HQ-5
A-HQ-7
0.78
0.78
0.78
1.56
0.78
9.38[a]
4.69[a]
2.35[a]
0.78
1.17[a]
3.13
1.56
18.8[a]
0.39
0.78
4.69[a]
1.56
1.56
3.13
>100
25
23.5[a]/188[a]
93.8[a]/1000
125/93.8[a]
62.5/46.9[a]
0.30[a]
0.39
31.3[b]/93.8[a]
31.3/>1000
9.38[a]/62.5
125/125
2.35[a]
0.78
0.78
0.39
100
2.35[a]
3.13
3.13
0.78
0.78
0.78
0.39
75[a]
0.78
0.78
4.69[a]
0.78
0.78
1.56
9.38[a]
25
2.0/1.5[a]
1.5[a]/2.0
2.0[b]/2.0[b]
7.8/7.8
ꢀ1
ꢀ1
ꢀ1
ꢀ1
12.3
ꢀ1
21.3
87.9
3.1
–
31.3/46.9[a]
0.59[a]
1.17[a]
0.59[a]
1.17[a]
2.35[a]
1.56
188[a]/188[a]
–
–
31.3/23.5[a]
–
–
375[a]/>1000
46.9[a]/>1000
31.3[a]/93.8
125/188[a]
62.5[b]/188[a]
125/125
62.5/750[a]
500/>1000
31.3[b]/93.8[a]
31.3/93.8[a]
250/750[a]
188[a]/750[a]
188[a]/500
750[a]/>1000
>1000/>1000
>1000/>1000
3.0/>2000
62.5[b]/>2000
15.6/>2000
31.3[b]/125
–
–
–
250/>1000
9.38[a]/62.5
23.5[a]/62.5
7.8/3.0[a]
9.38[a]/5.9[a]
5.9[a]/31.3
1.5[a]/23.5[a]
250/>1000
3.9/5.9[a]
31.3/46.9[a]
46.9[a]/188[a]
31.3[b]/93.8[a]
46.9[a]/125
188[a]/>1000
250/375[a]
125/375[a]
3.0[a]/>2000
–
9.38[a]/1.5[a]
15.6/3.9
15.6[b]/7.8
7.8[b]/1.5
1.5[a]/1.0[c]
3.9[b]/1.0[c]
2.0[b]/1.0[c]
125/9.38[a]
1.0[c]/1.0[c]
1.5[a]/1.5[a]
46.9[a]/1.5[a]
7.8[b]/23.5[a]
7.8[b]/1.5[a]
31.3[b]/3.0[a]
31.3[b]/7.8
31.3[b]/15.6
>200/150[a]
–
RA-HQ-1
RA-HQ-2
RA-HQ-5
RA-HQ-7
RA-HQ-8
RA-HQ-9
RA-HQ-11
RA-HQ-12
RA-HQ-13
RA-HQ-14
RA-HQ-15
RA-HQ-16
RA-HQ-17
RA-HQ-22
RA-HQ-23
vancomycin
daptomycin
linezolid
QAC-10
62.5[b]/93.8[a]
0.15[a]
0.30[a]
0.15[a]
0.30[a]
9.38[a]
0.39
0.30[a]
0.78
0.20
0.20
0.59[a]
4.69[a]
12.5
0.78
12.5
–
–
–
–
18.8
3.7
10.6
ꢀ1
ꢀ1
17.6
11.0
37.3
ꢀ1
7.4
15.6[b]/7.8
–
–
–
93.8[a]/375[a]
–
–
–
3.0
3.3
0.59[a]
4.69[a]
3.13
3.13
3.13
–
5.9[a]/>2000
>100
–
3.13
2.35[a]
–
–
–
–
ꢀ1
125/>2000
31.3/>2000
1.7
3.13
–
4.69[a]/1.56
3.0[a]/3.0[a]
–
>2000/>2000
–
ꢀ1
>99
3.5
–
ꢀ1
3.0
–
–
–
–
–
2.35[a]
6.25
31.3/31.3
31.3/93.8[a]
>2000/>2000
–
CCCP
NAC
pyrazinamide
EDTA
31.3/1000
>2000/>2000
>2000/>2000
2000/>2000
–
–
–
–
–
1000/>2000
–
[a] Midpoint value for a 2-fold range in independent experiments. [b] Midpoint value for a 4-fold range in independent experiments. [c] Lowest concentra-
tion tested. All MIC, MBC, MBEC values and haemolysis data were obtained from at least three independent experiments. MRSA=methicillin-resistant
Staphylococcus aureus, MRSE=methicillin-resistant Staphylococcus epidermidis, VRE=vancomycin-resistant Enterococcus faecium.
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for wave 1 HQs with several alkylated and reductive aminated
analogues demonstrating the most potent antibacterial activi-
ties. This observation guided our wave 2 synthesis efforts. In-
terestingly, a subpanel of 12 new HQs was found to be inactive
(MIC>100 mm; Supporting Information Table S3) against
a panel of Gram-negative pathogens, including: Klebsiella
pneumoniae, Pseudomonas aeruginosa, Escherichia coli and Aci-
netobacter baumannii. In addition, all HQs were screened
against RBCs at 200 mm and demonstrated a range of haemo-
lytic activities (ꢀ1 to 88% haemolysis; Table 1; Supporting In-
formation Table S1). Select HQs were evaluated against HeLa
cells (lactate dehydrogenase release assays) and demonstrated
good to excellent mammalian cytotoxicity (IC50 >50 to>
100 mm; Supporting Information Table S1).
HQ-8. In addition to new RA analogues that possess the 5,7-di-
bromo-8-hydroxyquinoline moiety, HQ-3 was used as a synthet-
ic template to yield diiodinated analogues RA-HQ-22 (55%
yield from HQ-3) and RA-HQ-23 (35% yield from HQ-3) for our
SAR investigations.
Antibacterial activities of wave 2 HQ analogues
Following the chemical synthesis of wave 2 RA-HQ analogues,
antibacterial assays revealed five new analogues (RA-HQ-12,
RA-HQ-13, RA-HQ-15, RA-HQ-16, RA-HQ-17) that possess
potent antibacterial activities (MICꢀ1.17 mm) against S. aureus,
S. epidermidis and E. faecium, including methicillin-resistant
staphylococcal isolates (Supporting Information Table S1). Im-
pressively, RA-HQ-12 and RA-HQ-16 demonstrate the highest
level of potency in antibacterial activities (MIC 0.20–1.56 mm)
against these pathogens, including drug-resistant isolates
while demonstrating no haemolysis against RBCs at 200 mm. In
general, the halogenated aniline moieties of the RA-HQ ana-
logues possessed good-to-highly potent antibacterial activities
while alkyl and methoxy aniline moieties generally gave de-
creased antibacterial activities. Diiodinated HQ analogues RA-
HQ-22 and RA-HQ-23 demonstrated a significant reduction of
antibacterial activities when compared to their corresponding
potent brominated analogues RA-HQ-5 and RA-HQ-8, respec-
tively.
Chemical synthesis of wave 2 HQ analogues
Following initial antibacterial studies with wave 1 HQs, we set
out to synthesize a second series (wave 2) of reductive aminat-
ed HQ analogues that contained diverse aniline moieties due
to the impressive biological activities of RA-HQ-5 (aniline
moiety) and RA-HQ-8 (4-bromoaniline moiety), which were
found to possess highly potent antibacterial activities against
S. aureus, S. epidermidis and E. faecium (MIC=0.59–0.78 mm)
while demonstrating <4% haemolytic activity against RBCs at
200 mm and low cytotoxicities against HeLa cells (Figure 3). In
addition to aniline moieties, diiodo-versions of RA-HQ-5 and
RA-HQ-8 were also synthesized (i.e., RA-HQ-22 and RA-HQ-
23).
Biofilm eradication investigations of wave 1 and 2 HQs
For the synthesis of wave 2 RA-HQs, we used our reductive
amination conditions that provided RA-HQs during wave 1 ana-
logue synthesis. Using these conditions, we were able to rapid-
ly generate ten new aniline-based RA analogues in 42 to 73%
yield (58% average yield; Scheme 2). These anilines were se-
lected to probe several structural features that would enable
a greater understanding of the structure–activity relationships
(SAR) of HQ analogues, including: 1) overall diversity of the
aniline moiety by using various substitutions of halogens, alkyl
and methoxy groups, 2) the role of the 4-bromine atom on the
aniline moiety by substituting this position with other halogen
atoms, alkyl and methoxy groups, and 3) alternative bromina-
tion patterns on the aniline moiety to compare to active RA-
Following antibacterial studies, 21 HQ analogues were evaluat-
ed for biofilm eradication activities using the Calgary Biofilm
Device (CBD), which has proven to be a useful tool for evaluat-
ing small molecules for antibiofilm activities.[21–23] CBD (biofilm
eradication) protocols involve three phases, including: Phase
1) establishment of bacterial biofilms in the absence of test
compound, Phase 2) challenging the biofilm with a test com-
pound (i.e., HQ), and Phase 3) recovery of viable biofilms in the
absence of test compounds (viable biofilms grow and disperse
planktonic bacteria into fresh media, resulting in turbidity).
Using the CBD, biofilms are established/treated/recovered on
pegs that are attached to a 96-well plate lid and submerged in
media. Eradicated biofilms following compound treatment are
Figure 3. Progression from wave 1 HQ analogue synthesis and biological evaluation to the design of wave 2 HQs focused primarily on diverse reductive ami-
nation analogues with aniline moieties.
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Scheme 2. Chemical synthesis of wave 2 RA-HQ analogues based on lead RA-HQ-5 and RA-HQ-8.
unable to recover, thus resulting in non-turbid microtiter wells
following the final incubation (i.e., recovery) period. The lowest
test concentration that results in eradicated biofilms (non-
turbid microtiter wells) corresponds to the MBEC value
(Figure 4).
biofilms are involved in endocarditis, catheter-associated urina-
ry tract infections and peridontitis.[28] Due to the significant
role these pathogens play in biofilm-related infections, we
evaluated our most promising HQs in biofilm eradication
assays against these bacteria.
In addition to determining biofilm-eradication activities, the
Calgary Biofilm Device can be used to quantify planktonic kill-
ing through the determination of minimum bactericidal con-
centrations (MBC) of compounds. This enables the assessment
of biofilm and planktonic eradication activities from the same
experiment through the evaluation of MBEC/MBC ratios. Bio-
film-eradicating agents should demonstrate MBEC/MBCꢁ1,
which is preferred to comparing MBEC and MIC values ob-
tained under different assay parameters.
HQ analogues displayed a range of biofilm eradication activi-
ties against MRSA-2 (a clinical isolate) biofilms (Table 1). HQ-
1 demonstrated good biofilm eradication activities against
MRSA-2 reporting an MBEC value of 188 mm using CBD assays.
A-HQ-3 (MBEC=93.8 mm), RA-HQ-8 (MBEC=125 mm), RA-HQ-
12 (MBEC=93.8 mm) and RA-HQ-13 (MBEC=93.8 mm) reported
improved biofilm eradication activities against MRSA-2 com-
pared to HQ-1. Using CBD assays, we found that lead biofilm-
eradicating HQs against MRSA-2 reported MBEC/MBC ratios be-
tween 1 and 3 demonstrating near equipotent biofilm and
planktonic killing efficiencies for these HQ analogues. Three
new HQs (A-HQ-4, RA-HQ-5, RA-HQ-7) demonstrated equipo-
tent biofilm eradication activities against MRSA-2 compared to
HQ-1 while 12 new HQs were found to be less active (MBEC=
500 to >1000 mm). Surprisingly, the 2-ethyl analogue A-HQ-
1 proved to have less potent biofilm eradication activity
against MRSA-2, despite the 2-methyl (HQ-1) and 2-propyl (A-
Staphylococcal pathogens, in particular S. aureus and S. epi-
dermidis, are the leading cause of biofilm-associated, hospital-
acquired bacterial infections and are often life-threatening.[24,25]
Staphylococcal biofilm infections are highly prevalent in in-
dwelling medical device-related infections (i.e., heart valve,
joint replacement).[25] S. epidermidis biofilms are a major clinical
problem as this pathogen is frequently encountered during
cerebral shunt[26] and catheter related treatments.[27] E. feacium
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a phenotype rarely encountered with HQ small molecules
against staphylococcal pathogens. In addition to A-HQ-1, we
observed a similar activity trend with A-HQ-7 which possess
a large 2-ethyl-4-bromophenyl moiety; however, this com-
pound is not as potent as the other more structurally simple 2-
alkyl HQ analogues.
In addition to new HQ analogues, we evaluated front-run-
ning MRSA treatments (i.e., vancomycin) in CBD assays against
MRSA-2 alongside new HQ analogues. From these experi-
ments, we found vancomycin, daptomycin and linezolid were
unable to eradicate MRSA-2 biofilms at the highest test con-
centration (MBEC >2000 mm) despite reporting moderate-to-
excellent planktonic killing (MBC=3.0–62.5 mm) in the same
experiment. These large planktonic versus biofilm killing activi-
ties (MBEC/MBC ratio for vancomycin is >667 against MRSA-2)
is illustrative of the high levels of tolerance that biofilms dis-
play towards conventional antibiotics. Against MRSA-2, lead
HQ analogues are >20-fold more potent as biofilm-eradicating
agents when compared to current anti-MRSA therapeutic
agents (Table 1). Other reported biofilm-eradicating agents and
persister cell killers (i.e., QAC-10,[9] CCCP,[12,15] NAC,[13] pyrazina-
mide;[1,29] Table 1) were also evaluated as positive controls
during our investigations. QAC-10, an antimicrobial peptide
mimic and membrane-lysing agent, proved to be the most
potent control in this panel; however, lead HQs identified
during these investigations were found to be more potent
than QAC-10 against MRSA, MRSE and VRE biofilms (Table 1).
We tested a small panel of active HQ analogues against
a second MRSA strain, ATCC BAA-1707, in CBD assays and
found this multi-drug resistant MRSA strain to be more sensi-
tive to HQ compounds than MRSA-2, which is a clinical isolate
from a patient at Shands Hospital (Gainesville, FL). Impressively,
RA-HQ-12 reported an MBEC value of 7.8 mm (Figure 4A),
which proved to be sixfold more potent than HQ-1 against
MRSA BAA-1707. Similar to MRSA-2, vancomycin, daptomycin
and linezolid all proved to be unable to eradicate MRSA BAA-
1707 biofilms despite demonstrating moderate to potent
planktonic killing activity in CBD assays (Table 1).
We found that MRSE biofilms demonstrated an increased
sensitivity to our HQs (compared to MRSA-2 results) as nine
new HQs showed improved biofilm eradication activities com-
pared to HQ-1 (MBEC=93.8 mm; Table 1) with RA-HQ-5
(MBEC=3.0 mm), RA-HQ-7 (MBEC=5.9 mm) and RA-HQ-12
(MBEC=5.9 mm; Figure 4) proving to be the most potent ana-
logues (MBEC/MBC ratios ꢁ1 for these analogues). In addition,
RA-HQ-9 (MBEC=23.5 mm), RA-HQ-8 (MBEC=31.3 mm) and
RA-HQ-13 (MBEC=46.9 mm) demonstrated excellent eradica-
tion activities against MRSE biofilms. Ten HQs demonstrated
equipotent or reduced biofilm eradication activities against
MRSE (MBEC=125 to >1000 mm) compared to HQ-1. Vanco-
mycin was unable to eradicate MRSE biofilms (MBEC>
2000 mm), despite effectively killing planktonic cells (MBC=
3.0 mm) in CBD assays (Figure 4A and B).
Figure 4. Calgary Biofilm Device (CBD) assays for RA-HQ-12. CBD is used to
determine planktonic and biofilm cell eradication in a single assay for evalu-
ating small molecules of interest.
HQ-3) analogues demonstrating potent activities. We were
puzzled by this result and repeated antibacterial and biofilm
eradication assay numerous times, further inspected multiple
samples of A-HQ-1 for stability and purity only to find this
compound to be both stable and pure. A-HQ-1 possesses
potent antibacterial activities against MRSA (and MRSE;
Table 1) without similar biofilm eradication activities, which is
Against VRE biofilms, 13 new HQ analogues were found to
be highly potent biofilm-eradicating agents (MBEC=1.0–
3.9 mm, Table 1) while demonstrating equipotent planktonic
and biofilm cell killing by comparing MBC/MBEC ratios. From
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our HQ panel, four analogues eradicated VRE biofilms at 1 mm
(i.e., RA-HQ-7, RA-HQ-8, RA-HQ-9, RA-HQ-12), which is the
lowest concentration tested during these investigations. Inter-
estingly, N-acetyl cystine (NAC) was unable to eradicate VRE bi-
ofilms at the highest test concentration (MBEC >2000 mm)
when tested alongside these HQ analogues (Figure 4) despite
a previous report that NAC eradicates E. faecium biofilms.[13]
During these studies, linezolid reported an MBEC value of
1.56 mm against VRE 700221 biofilms while QAC-10 (positive-
control) registered an MBEC value of 3.0 mm.
ties against S. aureus (including drug-resistant MRSA isolates),
S. epidermidis (including MRSE) and VRE while select analogues
demonstrated potent biofilm eradication activities without any
detectable haemolytic activity against RBCs with the exception
of A-HQ-5 which showed moderate levels of haemolysis at
high concentrations (12% RBC lysis at 200 mm).
Reductive amination HQ analogues demonstrated a broad
range of antibacterial activities, from highly potent to com-
pletely inactive against the Gram-positive pathogens, including
a panel of MRSA isolates. Unlike the 2-alkylated HQ series, RA-
HQ analogues registered drastically different haemolysis activi-
ties (ꢀ1 to 88% lysis of RBCs at 200 mm; Table 1 and Support-
ing Information Table S1). The aniline or 4-halogenated aniline
moieties on lead HQ scaffolds (i.e., RA-HQ-5, RA-HQ-8, RA-HQ-
12) proved to demonstrate potent biofilm eradication activi-
ties. This activity was sensitive to moving the halogen atoms
on the aniline ring (i.e., RA-HQ-15) and further modification
(i.e., RA-HQ-11). Biological profiles regarding the antibacterial,
biofilm eradication and haemolysis activities change dramati-
cally among related analogues as illustrated in Figure 5.
Biological activity profiles of HQ analogues
From these extensive biological investigations, critical insights
into the structure–activity relationships and biological activity
profiles integrating antibacterial, biofilm eradication and hae-
molysis activities of alkylated and reductive aminated ana-
logues at the 2-position of the HQ scaffold were gained. Alky-
lated HQ analogues proved to be promising as several ana-
logues in this subclass maintained potent antibacterial activi-
Figure 5. Activity profiles (antibacterial, biofilm eradication, haemolysis) of select HQ analogues from wave 1 and wave 2 against VRE. These activity profiles
will be useful in directing the development of future HQ biofilm-eradicating agents.
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Straight-chain aliphatic- along with select aniline-based reduc-
tive aminated analogues demonstrated moderate to high
levels of hemolytic activities against RBCs (>10% at 200 mm,
Figure 5). Overall, we are very encouraged by these findings
and feel that the ability to synthetically tune the 2-position of
the HQ scaffold will be critical in developing HQ biofilm-eradi-
cating agents as both small molecule probes and therapeutic
agents.
thank Ms. Gena Burch for initial biological investigations with
these HQ analogues.
Keywords: antibiotic tolerance
·
bacterial biofilms
·
halogenated quinolone · medicinal chemistry · structure–
activity relationships
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Experimental Section
Full experimental details concerning the chemical synthesis and
biological evaluation of these HQ compounds can be found in the
Supporting Information associated with this manuscript. All novel
compounds synthesized during these studies have H and 13C NMR
spectra, HRMS and melting point (for solids) are reported in the
Supporting Information. Images of select MIC and Calgary Biofilm
Device experiments are included in the Supporting Information
document.
1
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Acknowledgements
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We would like to thank the University of Florida for support of
these studies through start-up funds and a seed grant from
the Emerging Pathogens Institute (EPI at UF) awarded to
R.W.H. and S.J. V.M.N. is a graduate student fellow at UF. We
Received: February 26, 2016
Published online on June 1, 2016
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