Identification of N-Arylated NH125 Analogues as Rapid Eradicating Agents against MRSA Persister Cells and Potent Biofilm Killers of Gram-Positive Pathogens

Article abstract of DOI:10.1002/cbic.201600622

Bacterial biofilms housing dormant persister cells are innately tolerant to antibiotics and disinfectants, yet several membrane-active agents are known to eradicate tolerant bacterial cells. NH125, a membrane-active persister killer and starting point for development, led to the identification of two N-arylated analogues (1 and 2) that displayed improved biofilm eradication potencies compared to the parent compound and rapid persister-cell-killing activities in stationary cultures of methicillin-resistant Staphylococcus aureus (MRSA). We found 1 and 2 to be superior to other membrane-active agents in biofilm eradication assays, with 1 demonstrating minimum biofilm eradication concentrations (MBEC) of 23.5, 11.7, and 2.35 μm against MRSA, methicillin-resistant Staphylococcus epidermidis (MRSE), and vancomycin-resistant Enterococcus faecium (VRE) biofilms, respectively. We tested our panel of membrane-active agents against MRSA stationary cultures and found 1 to rapidly eradicate MRSA stationary cells by 4 log units (99.99 %) in 30 min. The potent biofilm eradication and rapid persister-cell-killing activities exhibited by N-arylated NH125 analogues could have significant impact in addressing biofilm-associated problems.

Full text of DOI:10.1002/cbic.201600622

Accepted Article
Title: Identification of N-Arylated NH125 Analogues as Rapid
Eradicating Agents against MRSA Persister Cells and Potent
Biofilm Killers of Gram-Positive Pathogens
Authors: Yasmeen Abouelhassan, Akash Basak, Hussain Yousaf, and
Robert William Huigens III
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.201600622
Link to VoR: http://dx.doi.org/10.1002/cbic.201600622
A Journal of
www.chembiochem.org

10.1002/cbic.201600622
ChemBioChem
Identification of N-Arylated NH125 Analogues as Rapid Eradicating Agents against
MRSA Persister Cells and Potent Biofilm Killers of Gram-Positive Pathogens
Yasmeen Abouelhassan1, Akash Basak2, Hussain Yousaf1, Robert W. Huigens III1,2*
1. Department of Medicinal Chemistry, Center for Natural Products Drug Discovery and
Development (CNPD3), College of Pharmacy, University of Florida, 1345 Center Dr., Gainesville,
FL 32610.
2. Department of Chemistry, University of Florida, 1600 SW Archer Rd, Gainesville, FL 32610.
* Corresponding Author: Prof. Robert W. Huigens III, email: rhuigens@cop.ufl.edu
Keywords: N-arylated NH125 antibacterials, biofilms, biofilm eradication, persister cell killing,
drug-resistant bacteria
This article is protected by copyright. All rights reserved.

10.1002/cbic.201600622
ChemBioChem
Abstract
Bacterial biofilms housing dormant persister cells are innately tolerant to antibiotics and
disinfectants, yet several membrane-active agents are known to eradicate tolerant bacterial cells.
NH125, a membrane-active persister-killer and starting point for development, led to the
identification of N-arylated analogues 1 and 2 and displayed improved biofilm-killing potencies
and rapid persister-killing activities in stationary cultures of methicillin-resistant Staphylococcus
aureus (MRSA). We found 1 and 2 to be superior to other membrane-active agents in biofilm
eradication assays with 1 demonstrating minimum biofilm eradication concentrations (MBEC) of
23.5, 11.7 and 2.35 µM against MRSA, methicillin-resistant S. epidermidis (MRSE) and
vancomycin-resistant Enterococcus faecium (VRE) biofilms, respectively. We tested our panel of
membrane-active agents against MRSA stationary cultures and found 1 to rapidly eradicate
MRSA stationary cells by 4-logs (99.99%) in 30 minutes. The potent biofilm eradication and rapid
persister cell killing exhibited by N-arylated NH125 analogues could have significant impact
addressing biofilm-associated problems.
This article is protected by copyright. All rights reserved.

10.1002/cbic.201600622
ChemBioChem
Introduction
Bacterial biofilms are ubiquitous, surface-attached communities of slow- or non-replicating
bacteria (e.g., persister cells) housed within a protective extracellular matrix of biomolecules and
display high levels of tolerance to biocides, antibiotics and other chemical insults (i.e., host
immune response during infection).1-5 Although biofilm-associated infections have received much
attention over the past two decades, it should be no surprise that biofilms affect multiple sectors
in our society (i.e., medical1-7, industrial8,9 and agricultural10,11). Numerous applications exist for
small molecule biofilm-control strategies, a few of which include: disinfectants, therapeutic agents,
crop protection agents.12,13
Figure 1: The process of biofilm formation, maturation and dispersion.
Individual, free-swimming planktonic bacteria use organic signaling molecules to communicate
with each other in a process called quorum sensing to monitor their population density and
coordinate group behavior, including the simultaneous surface attachment, colonization and
development into a mature biofilm.14 Biofilms disperse some bacterial cells back into their
surrounding environment in a process called biofilm dispersion, which enables bacteria to
establish biofilms in new locations (Figure 1).2 Over the last several years, small molecules have
been identified that inhibit biofilm formation and disperse established biofilms through various
This article is protected by copyright. All rights reserved.

10.1002/cbic.201600622
ChemBioChem
mechanisms, including the modulation of quorum sensing.12,13 Alternatively, a much smaller
collection of compounds have been identified that eradicate biofilm cells, including non-dividing
persister cells.5,7,15,16
Antimicrobial peptides (AMPs) are produced by an extensive variety of organisms to defend
themselves against bacterial infection and operate through various membrane-destroying
mechanisms.17 A diverse array of AMP-inspired, membrane-active amphiphilic compounds have
been reported to demonstrate potent antibacterial and anti-biofilm activities while evading the
development of bacterial resistance (i.e., quaternary ammonium cations, QAC).16-23 Membrane-
targeting compounds often are challenging to develop for human therapeutic use due to
membrane selectivity issues19; however, such agents have diverse biomedical applications and
can be highly effective as disinfectants. For instance, a lack of sterilized hospital environments,
including surfaces and instruments, is a major problem that leads to life-threatening hospital-
acquired infections (HAI).24,25
Recently, NH125 (Figure 2A) was reported to eradicate methicillin-resistant Staphylococcus
aureus (MRSA) biofilms and persister cells in stationary cultures through the depolarization of the
outer membrane,26 then later lipid bilayer destruction.27 We found this to be interesting as NH125
also has reported anticancer28,29 and neuromodulatory30,31 activities in vitro and in vivo. The
charged nitrogen atom of the imidazole heterocycle along with the long aliphatic tail led us to
believe that NH125 elicits its antibacterial activities through the destruction and lysis of bacterial
membranes, similar to other quaternary ammonium cations.16,18,32 In addition, we felt new
synthetic analogues of NH125 could be rapidly synthesized and lead to the identification of more
effective persister- and biofilm-killing agents against drug-resistant and tolerant pathogenic
bacteria.
This article is protected by copyright. All rights reserved.

10.1002/cbic.201600622
ChemBioChem
Figure 2. A) Structures of membrane-targeting agents investigated. B) Synthesis of N-Arylated
NH125 analogues 1 and 2.
Results and Discussion
Here, we report the identification of N-arylated NH125 analogues 1 and 2 that outperform NH125
and other membrane-active agents (Figure 2A) in biofilm eradication assays against MRSA,
methicillin-resistant Staphylococcus epidermidis (MRSE) and vancomycin-resistant Enterococcus
faecium (VRE). In kill kinetic experiments against MRSA persister cells in stationary cultures,
which have enriched persister cell populations, 1 and 2 proved to be the most potent and rapid
killing agents in our panel of membrane-targeting antibacterial agents. We identified 1 and 2 from
a library of diverse NH125 analogues we prepared and evaluated against a panel of pathogenic
bacteria (a full paper detailing these findings will be reported in due course). The chemical
synthesis of 1 and 2 involves a copper-catalyzed coupling reaction between iodobenzene, or 4-
iodoanisole, and 2-methylimidazole 4 to yield N-arylated imidazoles 5 and 6, in 47% and 45%
yields, respectively (Figure 2B). Following the copper-catalyzed C-N coupling reactions, a final
This article is protected by copyright. All rights reserved.

10.1002/cbic.201600622
ChemBioChem
alkylation of the N-3-position with 1-bromohexadecane gives 1 in 85% yield (from 5) and 2 in 84%
yield (from 6). This 2-step route was carried out to generate 1.81 grams of 1 and 310 milligrams
of 2 on single runs to check the scalability of this route, which yields pure analogues without the
use of chromatography.
We were interested in evaluating a panel of chemically diverse membrane-targeting compounds
for antibacterial, biofilm eradication, MRSA persister killing and red blood cell lysis (hemolysis)
including: NH125, 1, 2, 333, QAC-1016, BAC-12, and daptomycin. This small but diverse
membrane-active panel was compiled for the following reasons: 1) BAC-12 is a commercial
disinfectant with antibacterial properties, 2) QAC-10 and 3 are membrane-active quaternary
ammonium cations (QAC)16,33, and 3) daptomycin is a clinically used lipopeptide antibiotic known
to depolarize bacterial membranes and eradicate Staphylococcal biofilms.34
We initially tested this panel of membrane-active agents against methicillin-resistant
Staphylococcus aureus (MRSA) isolates (MRSA-2, clinical isolate from Shands Hospital,
Gainesville, Florida; MRSA BAA-1707, a multidrug resistant strain purchased from ATCC),
methicillin-resistant Staphylococcus epidermidis (MRSE), vancomycin-resistant Enterococcus
faecium (VRE), multidrug-resistant Acinetobacter baumannii, Pseudomonas aeruginosa,
Klebsiella pneumoniae and Escherichia coli (UAEC-1, clinical isolate) for antibacterial activity in
minimum inhibitory concentration (MIC) assays (Table 1). We found 1 and 2 to have the most
potent antibacterial activities against two MRSA strains (MIC = 1.56-3.13 µM) in this panel edging
out NH125 (MIC = 2.35-4.69 µM), QAC-10 (MIC = 3.13-4.69 µM), daptomycin (MIC = 3.13-4.69
µM) and BAC-12 (MIC = 6.25 µM). We found compound 3 to have significantly reduced
antibacterial activities against these MRSA strains (MIC = 12.5-25 µM) in these assays. Similar
activity trends were observed against MRSE and VRE; however, daptomycin reported a
significantly higher MIC against VRE (MIC = 125 µM). Analogues 1 and 2 reported the most
potent antibacterial activities against K. pneumoniae and E. coli (MIC = 6.25 µM) among the panel
This article is protected by copyright. All rights reserved.

10.1002/cbic.201600622
ChemBioChem
with moderate activities against multidrug-resistant A. baumannii (MIC = 12.5 µM for 1; 18.8 µM
for 2). Only QAC-10 reported more potent antibacterial activities against A. baumannii in this
panel (MIC = 6.25 µM) and was found to be the only membrane-active compound to demonstrate
antibacterial activity against P. aeruginosa (MIC = 9.38 µM; all other compounds on this panel
reported MIC values >100 µM). As expected, daptomycin was inactive (MIC >100 µM) against
all gram-negative pathogens. Following antibacterial assays, we tested this panel in hemolysis
assays to determine hemolytic activities (HC50 values; the concentration at which 50% of red
blood cells are lysed) and found that 1, 2, QAC-10 and NH125 demonstrate potent hemolytic
activities, correlating with their antibacterial activities. Analogue 3, BAC-12 and daptomycin
reported HC50 values >100 µM (highest concentration tested).
Table 1. Summary of antibacterial activity and hemolysis results. All values are reported in µM.
MRSA
MRSE
35984
VRE
A. baumannii
K. pneumoniae
RBC
HC50
Compound
MRSA-2
PAO1
UAEC-1
BAA-1707
700221
1794
13883
NH125
4.69a
3.13
3.13
25
2.35a
1.56
3.13
12.5
4.69a
6.25
3.13
4.69a
1.17a
2.35a
9.38a
2.35a
3.13
3.13
3.13
1.56
50
37.5a
12.5
18.8a
>100
6.25
75a
>100
>100
>100
>100
9.38a
>100
>100
18.8a
6.25
6.25
100
25
18.8a
6.25
6.25
25
12.3
7.84
13.4
>100
7.92
>100
>100
1
2
3
QAC-10
BAC-12
Daptomycin
3.13
6.25
4.69a
2.35a
12.5
50
25
75a
3.13
125
>100
>100
>100
Notes: a Midpoint value for a 2-fold range in independent experiments. All MIC and HC50 values were obtained from
a minimum of three independent experiments. MRSA BAA-1707, MRSE 35984, VRE 700221, A. Baumannii 1794, K.
pneumoniae 13883 were purchased from ATCC. MRSA-2 and UAEC-1 are clinical isolates.
We then turned our attention to biofilm eradication assays using the Calgary Biofilm Device7,15,35
and tested our panel of membrane-active compounds against MRSA BAA-1707, MRSE ATCC
35984 and VRE ATCC 700221 (Table 2). Using a Calgary Biofilm Device, bacterial biofilms are
established on pegs suspended to the bottom of a 96-well plate lid. Following biofilm
establishment on lid pegs, lids are then transferred to new 96-well plates containing serial dilutions
of test compound and allowed to incubate. After compound treatment, 96-well plate lids with pegs
are once again transferred to 96-well plates containing fresh media. During this final phase of the
This article is protected by copyright. All rights reserved.

10.1002/cbic.201600622
ChemBioChem
assay, viable biofilms will grow and disperse planktonic cells into the media where they will grow
and give a turbid well result. Conversely, wells that have no growth are a result of pegs that had
eradicated bacterial biofilms. The lowest concentration at which no turbidity is observed is the
minimum biofilm eradication concentration (MBEC). Using the Calgary Biofilm Device, both
minimum biofilm eradication concentrations and minimum bactericidal concentrations (MBC) for
planktonic bacterial can be determined using one assay from a single bacterial culture (Figure
3A).
Effective biofilm-eradicating agents typically have MBEC:MBC ratios of 1-3 for
biofilm:planktonic killing.7,15
Table 2. Summary of biofilm eradication from CBD assays. All values are reported in µM.
MRSA BAA-
1707 MIC
2.35a
MRSA BAA-1707
MBC / MBEC
23.5a / 46.9a
11.7a / 23.5a
7.8b / 11.7a
MRSE 35984
MIC
MRSE 35984
MBC / MBEC
15.6 / 5.9a
5.9a / 11.7a
5.9a / 5.9a
5.9a / 7.8
VRE 700221
MIC
VRE 700221
MBC / MBEC
5.9a / 11.7a
4.69a / 2.35a
3.0a / 5.9a
46.9a / 46.9a
3.0a / 3.0a
Compound
4.69a
NH125
3.13
1
2
1.56
3.13
1.17a
3.13
1.56
2.35a
3
12.5
4.69a
6.25
62.5 / >1000
93.8a / 93.8a
15.6 / >1000
125 / >2000
9.38a
50
2.35a
25
QAC-10
BAC-12
Daptomycin
2.35a
31.3 / 31.3
11.7a / >2000
31.3 / >2000
3.13
31.3 / 23.5a
375a / 93.8a
3.13
3.13
125
Notes: a Midpoint value for a 2-fold range in independent experiments. All MIC, MBC, MBEC values were obtained
from a minimum of three independent experiments.
We found analogues 1 and 2 to be the most potent analogues against MRSA BAA-1707 biofilms
and reported minimum biofilm eradication concentration (MBEC) values of 23.5 µM and 11.7 µM,
respectively (Table 2). The biofilm-killing activities of 1 and 2 were 2- to 4-fold more potent than
NH125 (MBEC = 46.9 µM) and 4- to 8-fold more potent than QAC-10 (MBEC = 93.8 µM) against
MRSA BAA-1707 (Figure 3A). Interestingly, 3, BAC-12 and daptomycin all reported MBECs
>1,000 µM, despite reporting MBC values of 15.6-125 µM against planktonic cells in the same
assays. When viable biofilm cell counts were assessed using the Calgary Biofilm Device, the
entire panel reduced MRSA BAA-1707 ~2-logs at sub-MBEC values (Figure 3B). NH-125 and 1
showed a clear dose-response in these assays while 1 reduced viable MRSA BAA-1707 biofilm
cells by >4-logs at 15.6 µM (MBEC = 23.5 µM), which was a more dramatic biofilm killing effect
This article is protected by copyright. All rights reserved.

10.1002/cbic.201600622
ChemBioChem
than NH-125 at 62.5 (MBEC = 46.9 µM; 3 to 4-log reduction of viable biofilm cells). Live/Dead
staining of MRSA BAA-1707 biofilms treated with NH-125 and 2 show that these analogues have
an effective clearance of established biofilms at 31.3 µM (Figure 4).
This article is protected by copyright. All rights reserved.

10.1002/cbic.201600622
ChemBioChem
MRSA Planktonic
A.)
MBC (µM)
--
A) DMSO
31.3
7.8
B) NH125 (a)
C) 1 (a)
125
D) QAC-10 (b)
E) 2 (a)
7.8
31.3
15.6
125
F) 3 (a)
G) BAC-12 (a)
H) Daptomycin (b)
MRSA Biofilms
MBEC (µM)
--
A) DMSO
B) NH125 (a)
C) 1 (a)
31.3
15.6
D) QAC-10 (b)
E) 2 (a)
62.5
15.6
F) 3 (a)
>1000
>1000
>2000
G) BAC-12 (a)
H) Daptomycin (b)
DMSO stock (with test range): (a) 50 mM (1 - 1000 µM), (b) 100 mM (2 - 2000 µM).
Colony Count Results (MRSA Biofilms on CBD Pegs)
B.)
D M S O
1 0 0
N H -1 2 5
1
1 0
1
Q A C -10
3
0 .1
B A C -12
0 .0 1
0 .0 0 1
0 .0 0 0 1
8
0
5
3
0
6
0
5
5
0
0
3
0
0
5
2
3
.
O
.
7
.
.
.
.
.
0
0
0
2
0
5
0
S
2
1
5
2
1
1
3
0
0
0
1
0
2
0
1
6
3
1
6
3
M
1
1
2
1
D
C o n c e n tra tio n (µ M )
Figure 3: A.) Image of Calgary Biofilm Device (CBD) assay against MRSA BAA-1707. This assay
can be used to determine the planktonic and biofilm killing effects of antibacterial agents from a
single bacterial culture. B) Viable MRSA BAA-1707 biofilm cells from colony counts of CBD pegs.
This article is protected by copyright. All rights reserved.

10.1002/cbic.201600622
ChemBioChem
A similar trend in biofilm eradication was observed against MRSE ATCC 35984 although N-
arylated NH125 analogues 1 and 2 did not demonstrate improvements in potencies against MRSE
biofilms compared to NH125 (MBECs 5.9-11.7 µM). Interestingly, 3 and QAC-10 demonstrated
potent biofilm eradication activities against MRSE biofilms with MBEC values of 7.8 µM and 31.3
µM, respectively. Against VRE biofilms, 1 and QAC-10 proved to be the most potent reporting
MBEC values of 2.35 µM (>4-fold more potent than NH-125) and 3.0 µM, respectively.
NH125 (31.3 µM)
DMSO (control)
2 (31.3 µM)
Figure 4. Live/Dead stain of MRSA BAA-1707 biofilms 24 hours after treatment with vehicle
(DMSO), NH125 and 2. Fluorescence images show potent clearance of MRSA biofilms for
NH125 and 2.
We then evaluated the panel of membrane-active compounds head-to-head at 50 µM against
MRSA BAA-1707 stationary cultures in kinetic killing experiments. Stationary cultures of S.
aureus have elevated populations of metabolically dormant persister cells36,37 and using this
experiment, we could get multiple early time points to see if 1 and 2 were rapid killers of MRSA
persister cells. The entire panel reduced the number of viable stationary cultures after 24 hours;
however, 1 and 2 were the only compounds to elicit a rapid killing effect of 4-logs (99.99% killing
of stationary cells) in 30 minutes and >5-logs after 90 minutes. The remaining compounds on the
This article is protected by copyright. All rights reserved.

10.1002/cbic.201600622
ChemBioChem
panel, showed very little activity at 30 minutes and only NH125 showed ~1-log reduction of
stationary MRSA cells after 90 minutes. The rapid killing of stationary MRSA cultures with 1 and
2 was sustained for 6 hours and no viable MRSA cells could be found at 24 hours upon treatment
with 1 whereas there was a slight recovery of MRSA cultures after 24 hours with 2.
Figure 5. Experimental results for the killing kinetics of MRSA BAA-1707 stationary cultures,
which are highly populated with persister cells. All compounds were tested at 50 µM from three
independent experiments.
In conclusion, we have identified two new N-arylated NH125 analogues 1 and 2, designed from
NH125, that demonstrate broad-spectrum antibacterial activities against multiple pathogenic
bacteria, including drug-resistant strains. We found 1 and 2 to demonstrate potent hemolysis
activity, similar to quaternary ammonium cationic agents (i.e., QAC-10), and eradicate MRSA,
MRSE and VRE biofilms with the most potent activity in a panel of diverse membrane-active
agents. Interestingly, 1 and 2 were found to be rapid killers of MRSA persister cells in stationary
cultures. N-Arylated NH125 analogues are promising agents for the development of new
disinfectants and antiseptics to effectively eradicate bacteria, biofilms and persister cells.
Materials and Methods
This article is protected by copyright. All rights reserved.

10.1002/cbic.201600622
ChemBioChem
General. Reported biological data result from a minimum of three independent experiments.
Bacterial strains used during these investigations include: methicillin-resistant Staphylococcus
aureus (Clinical Isolate from Shands Hospital in Gainesville, FL: MRSA-2; ATCC strain: BAA-
1707) methicillin-resistant Staphylococcus epidermidis (MRSE strain ATCC 35984), vancomycin-
resistant Enterococcus faecium (VRE strain ATCC 700221), Acinetobacter baumannii (ATCC
1794), Pseudomonas aeruginosa (PAO1), Klebsiella pneumonia (ATCC 13883) and Escherichia
coli clinical isolate (UAEC-1). All compounds were stored as DMSO stocks at room temperature
in the absence of light for several months at a time without observing any loss in biological activity.
Minimum Inhibitory Concentration (MIC) Microdilution Assays. The minimum inhibitory
concentration (MIC) for each tested compound was determined by the broth microdilution method
as recommended by the Clinical and Laboratory Standards Institute (CLSI). In a 96-well plate,
eleven two-fold serial dilutions of each compound were made in a final volume of 100 μL Luria
Broth. Each well was inoculated with ~105 bacterial cells at the initial time of incubation, prepared
from a fresh log phase culture (OD600 of 0.5 to 1.0 depending on bacterial strain). The MIC was
defined as the lowest concentration of compound that prevented bacterial growth after incubating
16 to 18 hours at 37 °C (MIC values were supported by spectrophotometric readings at OD600).
DMSO served as our vehicle and negative control in each microdilution MIC assay. DMSO was
serially diluted with a top concentration of 1% v/v.
Biofilm Eradication Assays (MBEC Determination; Calgary Biofilm Device Assays). Biofilm
eradication experiments were performed using the Calgary Biofilm Device to determine
MBC/MBEC values for various compounds of interest (Innovotech, product code: 19111). The
Calgary device (96-well plate with lid containing pegs to establish biofilms on) was inoculated with
125 µL of a mid-log phase culture diluted 1,000-fold in tryptic soy broth with 0.5% glucose (TSBG)
to establish bacterial biofilms after incubation at 37 °C for 24 hours. The lid of the Calgary device
was then removed, washed and transferred to another 96-well plate containing 2-fold serial
This article is protected by copyright. All rights reserved.

10.1002/cbic.201600622
ChemBioChem
dilutions of the test compounds (the “challenge plate”). The total volume of media with compound
in each well in the challenge plate is 150 µL. The Calgary device was then incubated at 37 °C for
24 hours. The lid was then removed from the challenge plate and MBC/MBEC values were
determined using different final assays. To determine MBC values, 20 µL of the challenge plate
was transferred into a fresh 96-well plate containing 180 µL TSBG and incubated overnight at 37
°C. The MBC values were determined as the concentration giving a lack of visible bacterial growth
(i.e., turbidity). For determination of MBEC values, the Calgary device lid (with attached
pegs/treated biofilms) was transferred to a new 96-well plate containing 150 µL of fresh TSBG
media in each well and incubated for 24 hours at 37 °C to allow viable biofilms to grow and
disperse resulting in turbidity after the incubation period. MBEC values were determined as the
lowest test concentration that resulted in eradicated biofilm (i.e., wells that had no turbidity after
final incubation period). In select experiments, pegs from the Calgary device were removed from
lead biofilm eradicators after final incubation, sonicated for 30 minutes in PBS and plated out to
determine biofilm cell killing of lead biofilm-eradicating agents (i.e., colony forming unit per
milliliter, CFU/mL).
MRSA Persister Cell Kill Kinetics. An overnight culture of MRSA BAA-1707 was diluted in fresh
TSBG (1:13 to 1:20 fold) and allowed to grow with shaking. Once the culture reached stationary
phase (4-6 hours), test compounds were added at a final test concentration of 50 µM. The
cultures were incubated with shaking at 250 rpm and aliquots were removed and plated out at
predetermined time points. Colony forming units (CFU) per milliliter data was recorded and
plotted using Graphpad Prism 6.0.
Live/Dead Staining of MRSA Biofilms. A mid-log culture of MRSA BAA-1707 was diluted
1:1000-fold and 500 µL was transferred to each compartment of a four-compartment CELLview
dish (Greiner Bio-One 627871). The dish was then incubated for 24 hours at 37 °C to establish
MRSA-1707 biofilms. After this time, the cultures were removed and the plate was washed with
This article is protected by copyright. All rights reserved.

10.1002/cbic.201600622
ChemBioChem
0.9% saline. The dish was then treated with test compounds in fresh media at various
concentrations. DMSO was used as a negative control in this assay. The dish was incubated
with the test compound for 24 hours at 37 °C. After this time, the cultures were removed and the
dish was washed with 0.9% saline for 2 minutes. Saline was then removed and 500 µL of the
stain (Live/Dead BacLight Viability Kit, Invitrogen) were added for 15 minutes and left in the dark.
After this time, the stain was removed and the dish was washed twice with 0.9% saline. Then the
dish was fixed with 500 µL 4% paraformaldehyde in PBS for 30 minutes. Images of remaining
MRSA biofilms were then taken with a fluorescence microscope.
Hemolysis of Red Blood Cells. Freshly drawn human red blood cells (hRBC) with
ethylenediaminetetraacetic acid (EDTA) as an anticoagulant were washed with Tris-buffered
saline (0.01M Tris-base, 0.155 M sodium chloride, pH 7.2) and centrifuged for 5 minutes at 3,500
rpm. The washing was repeated three times with the buffer. In a 96-well plate, test compounds
were added to the buffer from DMSO stocks. Then 2% hRBCs (50 µL) in buffer were added to
test compounds to give a final concentration of 200 µM. The plate was then incubated for 1 hour
at 37 °C. After incubation, the plate was centrifuged for 5 minutes at 3,500 rpm. Then 80 µL of
the supernatant was transferred to another 96-well plate and the optical density (OD) was read at
405 nm. DMSO served as our negative control (0% hemolysis) while Triton X served as our
positive control (100% hemolysis). The percent of hemolysis was calculated as (OD405 of the
compound- OD405 DMSO) / (OD405 Triton X- OD405 buffer). All reported data correspond to three
independent experiments. Ten-point dose-response curves were generated in 96-well plates to
determine 50% hemolysis (HC50 values) of red blood cells.
General C-N Coupling Procedure for the Synthesis of 5 and 6. To a stirring solution of 2-
methyl-1H-imidazole 4 (800 mg, 9.74 mmol) in 6 mL anhydrous dimethyl sulfoxide under argon
was added iodobenzene (1.09 mL, 9.74 mmol), then copper(I) iodide (185 mg, 0.97 mmol) and
finally anhydrous potassium carbonate (2.69 g, 19.49 mmol). The reaction mixture was allowed
This article is protected by copyright. All rights reserved.

10.1002/cbic.201600622
ChemBioChem
to stir at 130 °C for 48 hours in a sealed tube. After the completion of the reaction, the contents
of the reaction mixture were transferred to a separatory funnel containing ethyl acetate (200 mL).
The organic layer was washed with water (4x40 mL), then brine (2x30 mL) before the organic
layers were collected, dried with anhydrous sodium sulfate, filtered, and concentrated in vacuo to
give the crude product which was then purified via flash column chromatography using
hexanes:ethyl acetate (1:1 to 1:4) to elute pure 2-methyl-1-phenyl-1H-imidazole 5 as a clear oil
(730 mg, 47%).
General Alkylation Procedure for the Synthesis of 1 and 2. To a stirring solution of 2-methyl-
1-phenyl-1H-imidazole 5 (700 mg, 4.46 mmol) in 5 mL anhydrous acetonitrile in a glass tube at
room temperature was added 1-bromohexadecane (1.42 mL, 4.87 mmol) and the mixture was
sealed and heated at 110 C for 24 hours. The reaction was then allowed to cool to room
temperature before being concentrated via rotovap. The crude product was stirred in anhydrous
ether under argon for 5 hours and the resulting white precipitate was filtered and washed with
anhydrous ether and dried under vacuum to obtain 1 as a pure white solid (1.81 g, 85%).
Associated Content
The Supporting Information is available free of charge on the ACS Publications website at DOI:
(insert DOI here). Biological images of biofilm eradication assays, synthetic procedures for new
compounds synthesized during these experiments are characterized by 1H and 13C NMR, HRMS
and MP (solids) can be found in the supporting information document associated with this
publication.
Author Information
Corresponding Author
(R.W.H) E-mail: rhuigens@cop.ufl.edu
This article is protected by copyright. All rights reserved.

10.1002/cbic.201600622
ChemBioChem
Notes
The authors declare no competing financial interests.
Acknowledgements
We would like to thank the Departments of Medicinal Chemistry and Chemistry at the University
of Florida for the support of this work. All high resolution mass spectrometry (HRMS) were
obtained from the Chemistry Department at the University of Florida.
References
1. R. M. Donlan, J. W. Costerton, Clin. Microbiol. Rev. 2002, 15, 167-193.
2. L. Hall-Stoodley, J. W. Costerton, P. Stoodley, Nat. Rev. Microbiol. 2004, 2, 95-108.
3. K. Lewis, Nat. Rev. Microbiol. 2007, 5, 48-56.
4. R. Wolcott, S. Dowd, Plast. Reconstr. Surg. 2011, 127, Suppl 1: 28S-35S.
5. T. K. Wood, Biotechnol. Bioengineer. 2016, 113, 476-483.
6. J. D. Bryers, Biotechnol. Bioeng. 2008, 100, 1-18.
7. A. T. Garrison, Y. Abouelhassan, V. M. Norwood IV, D. Kallifidas, F. Bai, M. T. Nguyen, M.
Rolfe, G. M. Burch, S. Jin, H. Luesch, R. W. Huigens III, J. Med. Chem. 2016, 59, 3808-3825.
8. C. N. Street, A. Gibbs, Corros. Sci. 2010, 52, 1447-1452.
9. C. U. Schwermer, G. Lavik, R. M. M. Abed, B. Dunsmore, T. G. Ferdelman, P. Stoodley, A.
Gieseke, D. de Beer, Appl. Environ. Microbiol. 2008, 74, 2841-2851.
10. T. Danhorn, C. Fuqua, Annu. Rev. Microbiol. 2007, 61, 401-422.
11. L. S. Muranaka, M. A. Takita, J. C. Olivato, L. T. Kishi, A. A. de Souza, J. Bacteriol. 2012,
194, 4561-4569.
12. M. H. Fletcher, M. C. Jennings, W. M. Wuest, Tetrahedron 2014, 70, 6373-6383.
13. R. J. Worthington, J. J. Richards, C. Melander, Org. Biomol. Chem. 2012, 10, 7457-7474.
14. M. B. Miller, B. L. Bassler, Annu. Rev. Microbiol. 2001, 55, 165-199.
This article is protected by copyright. All rights reserved.

10.1002/cbic.201600622
ChemBioChem
15. A. Basak, Y. Abouelhassan, V. M. Norwood IV, F. Bai, M. Nguyen, S. Jin, R. W. Huigens
III, Chem. Eur. J. 2016, 22, 9181-9189.
16. M. C. Jennings, L. C. Ator, T. J. Paniak, K. P. C. Minbiole, W. M. Wuest, ChemBioChem 2014,
15, 2211-2215.
17. K. A. Brogden, Nat. Rev. Microbiol. 2015, 3, 238-250.
18. M. C. Jennings, B. A. Buttaro, K. P. C. Minbiole, W. M. Wuest, ACS Infect. Dis. 2015, 1, 304-
309.
19. Y. J. Gordon, E. G. Romanowski, Curr. Eye Res. 2005, 30, 505-515.
20. T. Böttcher, I. Kolodkin-Gal, R. Kolter, R. Losick, J. Clardy, J. Am. Chem. Soc. 2013, 135,
2927-2930.
21. R. Joseph, A. Naugolny, M. Feldman, I. M. Herzog, M. Fridman, Y. Cohen, J. Am. Chem. Soc.
2016, 138, 754-757.
22. I. M. Herzog, K. D. Green, Y. Berkov-Zrihen, M. Feldman, R. R. Vidavski, A. Eldar-Boock, R.
Satchi-Fainaro, A. Eldar, S. Garneau-Tsodikova, M. Fridman, Angew. Chem. Int. Ed. 2012,
51, 5652-5656.
23. G. Guchhait, A. Altieri, B. Gorityala, X. Yang, B. Findlay, G. G. Zhanel, N. Mookherjee, F.
Schweizer, Angew. Chem. Int. Ed. 2015, 54, 6278-6282.
24. G. McDonnell, A. D. Russell, Clin. Microbiol. Rev. 1999, 12, 147-179.
25. A. C. Abreu, R. R. Tavares, A. Borges, F. Mergulhão, M. Simões, J. Antimicrob. Chemother.
2013, 68, 2718-2732.
26. W. Kim, A. L. Conery, R. Rajamuthiah, B. B. Fuchs, F. M. Ausubel, E. Mylonakis, PLoS One
2015, 10, 15 p.
27. W. Kim, N. Fricke, A. L. Conery, B. B. Fuchs, R. Rajamuthiah, E. Jayamani, P. M. Vlahovska,
F. M. Ausubel, E. Mylonakis, Future Med. Chem. 2016, 8, 257-269.
28. S. Arora, J. M. Yang, R. Utsumi, T. Okamoto, T. Kitayama, W. N. Hait, Mol. Pharmacol. 2004,
66, 460-467.
This article is protected by copyright. All rights reserved.

10.1002/cbic.201600622
ChemBioChem
29. L. Liu, P. Huang, Z. Wang, N. Chen, C. Tang, Z. Lin, P. Peng, BMC Cancer 2016,16, 813-
820.
30. A. E. Autry, M. Adachi, E. Nosyreva, E. S. Na, M. F. Los, P. Cheng, E. T. Kavalali, L. M.
Monteggia, Nature 2011, 475, 91-95.
31. C. L. Bender, Q. Yang, L. Sun, S. J. Liu, Neuropharmacol. 2016, 101, 531-537.
32. L. A. T. Asri, M. Crismaru, S. Roest, Y. Chen, O. Ivashenko, P. Rudolf, J. C. Tiller, H. C. van
der Mei, T. J. A. Loontjens, H. J. Busscher, Adv. Funct. Mater. 2014, 24, 346-355.
33. J. Hoque, M. M. Konai, S. Samaddar, S. Gonuguntala, G. B. Manjunath, C. Ghosh, J. Haldar,
Chem. Commun. 2015, 51, 13670-13673.
34. S. Roveta, A. Marchese, G. C. Schito, Int. J. Antimicrob. Agents 2008, 31, 321-328.
35. J. J. Harrison, C. A. Stremick, R. J. Turner, N. D. Allan, M. E. Olson, H. Ceri, Nat. Protoc.
2010, 5, 1236-1254.
36. I. Keren, N. Kaldalu, A. Spoering, Y. Wang, K. Lewis, FEMS Microbiol. 2004, 230, 13-18.
37. S. Lechner, K. Lewis, R. Bertram, J. Mol. Microbiol. Biotechnol. 2012, 22, 235-244.
This article is protected by copyright. All rights reserved.