Microwave-enhanced Friedl?nder synthesis for the rapid assembly of halogenated quinolines with antibacterial and biofilm eradication activities against drug resistant and tolerant bacteria

Article abstract of DOI:10.1039/c6md00381h

Herein, we disclose the development of a catalyst- and protecting-group-free microwave-enhanced Friedl?nder synthesis which permits the single-step, convergent assembly of diverse 8-hydroxyquinolines with greatly improved reaction yields over traditional oil bath heating (increased from 34% to 72%). This rapid synthesis permitted the discovery of novel biofilm-eradicating halogenated quinolines (MBECs = 1.0-23.5 μM) active against MRSA, MRSE, and VRE. These small molecules exhibit activity through mechanisms independent of membrane lysis, further demonstrating their potential as a clinically useful treatment option against persistent biofilm-associated infections.

Full text of DOI:10.1039/c6md00381h

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RESEARCH ARTICLE
Microwave-enhanced Friedländer synthesis for the
rapid assembly of halogenated quinolines with
antibacterial and biofilm eradication activities
against drug resistant and tolerant bacteria†‡
Cite this: DOI: 10.1039/c6md00381h
Aaron T. Garrison, Yasmeen Abouelhassan, Hongfen Yang, Hussain H. Yousaf,
*
Tho J. Nguyen and Robert W. Huigens III
Herein, we disclose the development of a catalyst- and protecting-group-free microwave-enhanced
Friedländer synthesis which permits the single-step, convergent assembly of diverse 8-hydroxyquinolines
with greatly improved reaction yields over traditional oil bath heating (increased from 34% to 72%). This
rapid synthesis permitted the discovery of novel biofilm-eradicating halogenated quinolines (MBECs = 1.0–
23.5 μM) active against MRSA, MRSE, and VRE. These small molecules exhibit activity through mechanisms
independent of membrane lysis, further demonstrating their potential as a clinically useful treatment option
against persistent biofilm-associated infections.
Received 8th July 2016,
Accepted 25th July 2016
DOI: 10.1039/c6md00381h
www.rsc.org/medchemcomm
The synthesis of quinoline-based scaffolds receives much at-
tention due to the ubiquity of these heterocycles among bio-
active molecules.1 In particular, 8-hydroxyquinolines have
seen widespread use in therapeutics, demonstrating an array
of medicinal applications (e.g., antibacterial, anti-cancer, anti-
neurodegenerative, anti-viral, etc.).2 However, many syntheses
of novel 8-hydroxyquinoline scaffolds utilize harsh acidic con-
ditions (Fig. 1a) and/or require O-methylated aniline starting
materials.3,4 These synthetic routes typically suffer from lim-
ited substrate scope, involve arduous workups and require
deprotection to obtain 8-hydroxyquinolines.
Of particular interest to our group are the biofilm-
eradicating properties of halogenated 8-hydroxyquinolines
(HQs). Biofilms (i.e. surface-adhered bacterial communities
encased within self-produced extracellular polymeric sub-
stances) are implicated in 80% of all bacterial infections
(Fig. 1c).5,6 Worse still, bacterial infections lead to the death
of more than 500 000 people annually in the U.S. alone.7,8
The prognosis of biofilm-related infections is exacerbated by
the presence of persister cells, a distinct subset of bacterial
populations which demonstrate non-dividing, dormant phe-
notypes.9 These bacterial populations are endowed with a
heightened degree of antibiotic tolerance which has proven
notoriously difficult to overcome using growth-dependent
conventional antibiotics.10
There are currently no clinically available agents that erad-
icate biofilm-associated infections; that is, agents which ef-
fectively kill biofilm-encased persister cells via growth-
independent mechanisms. Few small molecules are known to
eradicate bacterial biofilms. Lewis recently reported the eradi-
cation of Staphylococcus aureus persister cells and biofilms
using a mouse model with ADEP4 (an acyldepsipeptide ClpP
protease inhibitor) and Rifampicin co-treatment.11 Others
have employed antimicrobial peptides (AMPs) or mimics
thereof.12,13 Although these AMP-like agents show promise,
their mechanism of action involves the lysis of cellular mem-
branes, which may not lend itself to bacterial selectivity. Alas,
our current antimicrobial arsenal is completely incapable of
addressing persistent bacterial phenotypes and the advance-
ment of therapeutic agents able to combat them remains un-
der-pursued. Innovative approaches by which therapeutics
can be rapidly generated must be developed to counter these
pathogens and remedy our inability to treat biofilm-
associated infections.
Our group has recently identified a series of halogenated
quinolines capable of eradicating bacterial biofilms while
demonstrating minimal mammalian cytotoxicity and
haemolytic activities (HQ 1, Table 1).14–16 Our first efforts to-
ward the synthesis of HQs employed a Döbner–Miller reac-
tion (Fig. 1a). However, due to harsh conditions, the necessity
for demethylation, and the limitation of viable commercially
available α,β-unsaturated ketones or aldehydes, we surmised
Department of Medicinal Chemistry, Center for Natural Products Drug Discovery
and Development (CNPD3), University of Florida, Gainesville, Florida 32610, USA.
E-mail: rhuigens@cop.ufl.edu; Tel: +1 352 273 7718
† The authors declare no competing interests.
‡ Electronic supplementary information (ESI) available: Synthesis procedures,
characterization data (1H, 13C NMR, HRMS, MP), biological experimental proce-
dures, supporting biological images, NMR spectra for all new compounds. See
DOI: 10.1039/c6md00381h
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sitions (2a, 3a, 7a–10a) in low to moderate yield (Fig. 2). Ac-
cess to exclusively 3-substituted quinolines from aldehyde
starting materials, however, proved troublesome under both
basic and acidic conditions with traditional thermal heating
(i.e. oil bath). To our surprise, the utilization of microwave ir-
radiation afforded the previously unattainable 3-substituted
8-hydroxyquinolines (4a, 5a, and 6a) in 52% yield.
Further investigation into this microwave-assisted
Friedländer reaction of 12 diverse substrates revealed marked
improvements in yields for the majority of the quinoline li-
brary, from an overall average of 34% for traditional oil bath
heating compared to 72% under microwave irradiation
(Fig. 2b). Increased yields were observed in the condensation
of 20 with methyl ketones (8-hydroxyquinolines 1a, 2a, 3a),
1,3-dicarbonyls (7a, 8a, 9a), 3-pentanone (10a), and
malononitrile (12a). It is worth noting, however, that at-
tempts to improve the yield of 8-hydroxyquinoline 11a, which
requires a condensation with an acetophenone rather than a
benzaldehyde (see ESI‡), were unsuccessful. Following assem-
bly of the core scaffold, 8-hydroxyquinolines were brominated
at the 5- and 7-positions using 2.2 equivalents of
N-bromosuccinimide to afford the final halogenated quino-
line analogues in 22–94% yield (HQs 1–11, Fig. 2b, ESI‡). It
should be noted that difficulty with brominating 12a in addi-
tion to the poor solubility prompted the discontinuation of
its advancement to the corresponding HQ.
Our HQ library was screened for antibacterial activity
against a panel of human pathogens, including methicillin-
resistant Staphylococcus aureus (MRSA-2 clinical isolate),
methicillin-resistant Staphylococcus epidermidis (MRSE ATCC
35984), and vancomycin-resistant Enterococcus faecium (VRE
ATCC 700221). In preliminary MIC assays, several HQs
exhibited increased antibacterial activity compared to previ-
ously reported HQ 1 across our panel of pathogens (Table 1).
HQs 8 and 9 demonstrated improved activity against MRSA-2,
reporting MICs of 0.39 μM and 0.59 μM, respectively (see
ESI‡). Against MRSE 35984, HQs 8, 10, and 15 proved to be
more potent than HQ 1 with MICs of 0.10 μM, 0.15 μM, and
0.10 μM. Four HQ analogues (8–10, 15) reported improved
MIC activity compared to HQ 1 against VRE with 15 demon-
strating the highest potency against this strain (MIC = 0.30
μM). The MIC of these HQs permitted the identification of a
subset of the most potent compounds to be evaluated for bio-
film eradication activity.
Minimum bactericidal concentration (MBC), an evaluation
of activity against planktonic cells, and minimum biofilm
eradication concentration (MBEC) values were evaluated in
tandem by use of the Calgary biofilm device (CBD).18–20 This
apparatus allows the establishment of biofilms onto pegs
mounted to the lid of a 96-well plate. The pegs are sub-
merged in inoculated media and, following an incubation
and concomitant biofilm establishment, are rinsed and trans-
ferred to a second 96-well plate containing serially-diluted
test compound, allowing biofilm-eradicating agents to kill
bacterial biofilms. Following a second incubation, the pegs
are again transferred to a new 96-well plate containing only
Fig. 1 a) Common approaches for 8-hydroxyquinoline synthesis. b)
Microwave-enhanced Friedländer reaction from readily available
starting materials. c) Illustration of both free-floating planktonic cells
and surface-attached bacterial biofilms.
this approach was not amenable to library synthesis. Alterna-
tively, the Combes synthesis was explored, but attempts to
synthesize HQs were met with failure as significant quantities
of side products were observed and only trace amounts of the
desired 8-hydroxyquinoline were obtained. Still motivated to
further investigate HQs with greater structural diversity, we
turned to the Friedländer quinoline synthesis,17 which
proved to be a superior alternative. This synthesis proceeds
under mild conditions without the need for catalysts or hy-
droxyl protecting groups. Additionally, the Friedländer reac-
tion permits the use of readily available starting materials (al-
dehydes, β-keto esters, ketones and nitriles) to afford
quinolines with diverse substitution patterns at the 2-, 3-,
and 4-positions in a single step.
Thus, our synthesis began with the generation of 2-amino-
3-hydroxybenzaldehyde 20 on gram scale, which was first con-
densed with commercially available ketones to afford
8-hydroxyquinolines with substitution at the 2- or 2- and 3-po-
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Fig. 3 a) HQ 8 esters and amides. b) HQs with improved activities
compared to HQ 1 against MRSA-2, MRSE, and VRE. c) Calgary biofilm
device assay with HQs 1, 9, and front-running antibiotic vancomycin
against MRSA BAA 1707.
fresh media, wherein viable biofilms, after compound treat-
ment, are able to recover and disperse planktonic cells
contained within, resulting in turbid wells. The absence of
turbidity observed in this final 96-well plate represents eradi-
cated biofilms. Thus, the lowest concentration of test com-
pound that yields a non-turbid well is considered its MBEC.
The 96-well plate from which the pegged lid is last removed
allows the determination of MBC (see ESI‡).
HQ 9 proved to be one of the most potent biofilm eradica-
tors ever reported against both MRSA (MBEC = 3.9–23.5,
Fig. 3c, Table 1 and Table S1‡) and MRSE (MBEC = 1.0 μM).
The promising biofilm eradication activities of 8 against
MRSA-2 (MBEC = 31.3 μM) prompted the synthesis of a small
library of ester (HQs 14–16) and amide (HQs 17–19) ana-
logues of this HQ (Fig. 3a). However, these analogues, in ad-
dition to the carboxylic acid intermediate (HQ 13), exhibited
Fig.
2
a)
Microwave-enhanced
Friedländer
synthesis
of
8-hydroxyquinolines from a diverse array of starting materials. b) Yield
comparison of oil bath heating versus microwave irradiation for the
synthesis of 8-hydroxyquinolines.
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Table 1 Summary of biological investigations of HQ analogues (MIC, MBC, MBEC, and haemolysis)a
MRSA-2
MIC
MRSA-2
MBC/MBEC
MRSE 35984
MIC
MRSE 35984
MBC/MBEC
VRE 700221
MIC
VRE 700221
MBC/MBEC
% haemolysis
(200 μM)
Compound
1
2
3
4
5
6
7
8
9
10
11
13
14
15
16
17
0.78
25
23.5b/46.9b
—
0.20
0.39
4.69b
1.17b
0.59b
3.13
6.25
0.10d
3.13
0.15b
1.17b
25
7.8/3.0b
—
2.35b
>100
25
3.0b/2.0
—
≤1
1.9
≤1
≤1
2.4
4.6
2.3
2.2
2.5
≤1
≤1
2.5
1.5
7.9
≤1
7.0
13.0
≤1
>99
≤1
1.7
≤1
37.5b
3.13
6.25
50
—
—
—
15.6/46.9b
46.9b/188b
—
23.5b/31.3
4.69b
12.5
18.8b
25
7.8/5.9b
62.5/46.9b
31.3/23.5b
—
—
—
1.56
0.39
0.59b
6.25
6.25
50
1.56
6.25
12.5
37.5b
6.25
100
250/>1000
31.3/31.3
15.6c/23.5b
>1000/>1000
—
—
93.8a/23.5b
0.78
0.78
0.78
4.69b
>100
2.35b
0.30b
25
50
25
>100
2.35b
>100
—
3.0b/2.0
5.9b/1.0d
46.9b/23.5b
—
—
—
—
—
—
—
—
—
0.30b
0.10d
1.56
12.5
6.25
50
—
375b/>1000
—
46.9b/15.6c
3.9c/1.5b
—
—
—
—
—
—
—
—
—
18
19
>2000/>2000
—
31.3/125
3.0b/>2000
62.5b/>2000
15.6/>2000
QAC-10
Vancomycin
Daptomycin
Linezolid
3.13
0.59b
4.69b
3.13
2.35a
0.78
12.5
3.13
31.3/31.3
7.8/>2000
—
3.0b/3.0b
>200/200b
—
—
3.13
4.69c/1.56
a
All MIC, MBC, and MBEC values reported in μM. b Midpoint value of a two-fold range. c Midpoint value of a four-fold range. d Lowest concen-
tration tested. Each data point is the result of three independent experiments.
no improvement in biofilm eradication activity against any
pathogen with respect to 8. The most promising analogue
against VRE was HQ 15 (MBEC = 1.5 μM), exhibiting activity
which correlates with its potent MIC. Although we observed
improved MBEC activity for HQ 1 against MRSA-2 compared
to previous studies,15,16 the potency was similar to biofilm
eradication activities observed against other MRSA strains
during these investigations (Table S1‡).
The reported activities show promise, especially when in
comparison to known biofilm eradicator QAC-10 (a quater-
nary ammonium cation)13 and front-running MRSA treat-
ments vancomycin, linezolid, and daptomycin, which were
used as positive controls in our biofilm eradication assays
(Table 1). Despite demonstrating potent activity against
planktonic bacteria (MBC), the clinically-available antibiotics
exhibit no biofilm eradication activities (MBEC > 2000 μM)
against MRSA-2. Furthermore, active HQs report MBEC : MBC
ratios of ∼1, demonstrative of efficacious biofilm-eradicating
agents (Fig. 3c). Haemolysis assays were also conducted,
wherein we observed negligible haemolytic activity at 200
μM, with HQ 18 being the only analogue to report >10%
haemolysis.
reports of the modulation of antibacterial activities of HQs
following co-treatment with metalIJII) cations.15,16
Improved syntheses of 8-hydroxyquinolines are invaluable
to the discovery of therapeutic agents in multiple disease
areas important to human health. The incorporation of read-
ily available materials to assemble libraries in short, conver-
gent syntheses is paramount to drug discovery. This
microwave-mediated synthesis can incorporate a multitude of
available starting materials, including aldehydes, β-keto es-
ters,
ketones
and
nitriles
to
directly
generate
8-hydroxyquinolines with 2-, 3-, or 4-position substitutions
that may otherwise be unattainable via strongly basic or
acidic conditions and traditional oil bath heating. These ad-
vances permitted the rapid, facile synthesis of a series of
novel halogenated quinolines that potently eradicate drug-
resistant S. aureus, S. epidermidis, and E. faecium biofilms
without demonstrating significant haemolytic toxicity. These
data suggest that halogenated quinolines could prove to be a
promising class of compounds capable of treating biofilm-
associated infections.
It is understood that many 8-hydroxyquinolines with me-
dicinal utility demonstrate activity through mechanisms in-
volving metal chelation.2 Specifically, the phenolic oxygen
and adjacent nitrogen atoms have been shown to participate
in coordination to metalIJII) cations. Although further investi-
gation into the mode of action for our HQs is underway, we
believe these agents exhibit activity via a metalIJII)-dependent
mechanism. This hypothesis is corroborated by our previous
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