Antimicrobial peptide-inspired NH125 analogues: Bacterial and fungal biofilm-eradicating agents and rapid killers of MRSA persisters

Article abstract of DOI:10.1039/c7ob01028a

During microbial infection, antimicrobial peptides are utilized by the immune response to rapidly eradicate microbial pathogens through the destruction of cellular membranes. Inspired by antimicrobial peptides, quaternary ammonium cationic (QAC) compounds have emerged as agents capable of destroying bacterial membranes leading to rapid bacterial death, including the eradication of persistent, surface-attached bacterial biofilms. NH125, an imidazolium cation with a sixteen membered fatty tail, was recently reported to eradicate persister cells and was our starting point for the development of novel antimicrobial agents. Here, we describe the design, chemical synthesis and biological investigations of a collection of 30 diverse NH125 analogues which provided critical insights into structural features that are important for antimicrobial activities in this class. From these studies, multiple NH125 analogues were identified to possess potent antibacterial and antifungal activities, eradicate both bacterial and fungal biofilms and rapidly eradicate MRSA persister cells in stationary phase. NH125 analogues also demonstrated more rapid persister cell killing activities against MRSA when tested alongside a panel of diverse membrane-active agents, including BAC-16 and daptomycin. NH125 analogues could have a significant impact on persister- and biofilm-related problems in numerous biomedical applications.

Full text of DOI:10.1039/c7ob01028a

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DOI: 10.1039/C7OB01028A
Antimicrobial Peptide-Inspired NH125 Analogues: Bacterial and Fungal Biofilm-
Eradicating Agents and Rapid Killers of MRSA Persisters
Akash Basak,a Yasmeen Abouelhassan,b Ran Zuo,b Hussain Yousaf,b Yousong Ding,b Robert W. Huigens IIIa,b*
a Department of Chemistry, University of Florida, Gainesville, FL 32610, USA.
b
Department of Medicinal Chemistry, Center for Natural Products, Drug Design and Development (CNPD3),
University of Florida, Gainesville, FL 32610, USA.
Abstract
During microbial infection, antimicrobial peptides are utilized by the immune response to rapidly eradicate
microbial pathogens through the destruction of cellular membranes. Inspired by antimicrobial peptides,
quaternary ammonium cationic (QAC) compounds have emerged as agents capable of destroying bacterial
membranes leading to rapid bacterial death, including the eradication of persistent, surface-attached bacterial
biofilms. NH125, an imidazolium cation with a sixteen membered fatty tail, was recently reported to eradicate
persister cells and was our starting point for the development of novel antimicrobial agents. Here, we describe
the design, chemical synthesis and biological investigations of a collection of 30 diverse NH125 analogues which
provided critical insights into structural features that are important for antimicrobial activities in this class. From
these studies, multiple NH125 analogues were identified to possess potent antibacterial and antifungal activities,
eradicate both bacterial and fungal biofilms and rapidly eradicate MRSA persister cells in stationary phase.
NH125 analogues also demonstrated more rapid persister cell killing activities against MRSA when tested
alongside a panel of diverse membrane-active agents, including BAC-16 and daptomycin. NH125 analogues
could have a significant impact on persister- and biofilm-related problems in numerous biomedical applications.

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DOI: 10.1039/C7OB01028A
Introduction
With the alarming concerns that pathogenic microorganisms (bacteria and fungi) have placed on mankind
through resistance1-5 and tolerance4,6-9, innovative strategies are needed to address such threats. Bacteria
employ several well-defined mechanisms that enable them to acquire resistance to antibiotics and antibacterial
agents following exposure, examples include: target modification, drug inactivation, changes in membrane
chemistry to inhibit drug penetration, overexpression of efflux pumps, and horizontal gene transfer of antibiotic
resistant genes.4,5 In contrast to bacterial resistance, tolerance does not result from antibiotic stress, rather this
phenotypic switch from metabolically active, rapidly-dividing bacteria to a dormant bacterial state results in non-
replicating “persister” cells.6-10 High levels of antibiotic tolerance are prevalent in bacterial biofilms, or surface-
attached bacterial communities encased in a slimy matrix of biological molecules that enables them to thrive in
hostile environments, leading to chronic and recurring bacterial infections in humans.11-17 In addition to bacteria,
pathogenic fungi demonstrate resistance18-20 and tolerance21,22 to antifungal agents due to acquired resistance
and fungal biofilm formation, respectively.
For many years, we considered bacteria to be individual free-floating, planktonic cells that rapidly replicated.
However, we have recently learned that bacteria use small organic signaling molecules to communicate with
one another in a process known as quorum sensing to coordinate group behaviors, including the simultaneous
attachment to a surface and formation of complex biofilm communities (Figure 1).23-26 Mature biofilms can
disperse planktonic cells back into their surroundings restarting the planktonic-biofilm cycle.
Figure 1. Free-floating planktonic bacteria use quorum sensing to attach to a surface and form a bacterial biofilm.
A mature biofilm can disperse planktonic cells back into it’s surrounding environment.
Each of the antibiotic/antibacterial classes that are used to treat bacterial infections was initially discovered as
growth inhibitors that target rapidly-dividing planktonic bacteria.16 As a consequence, non-replicative bacteria
within biofilms are tolerant to such agents, thus new small molecules operate through alternative mechanisms
to eradicate tolerant bacteria have uses in several sectors in our society, including: medicine/biomedical
applications (e.g., persistent biofilm infections13,17, hospital disinfectants27,28), industry (e.g., oil/gas pipeline
corrosion29,30, biofouling on ship hulls31), and agriculture (e.g., crop disease32,33).

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An array of antimicrobial peptides (AMP) are used by an extensive diversity of living organisms as a host defense
response to microbial infection.34-39 AMPs are successful against microorganisms due to their ability to target
and destroy bacterial membranes by causing pore formation, lysis and microbicidal death. Inspired by this
antimicrobial strategy, membrane-destroying small molecules have been identified that kill bacteria and fungi
through membrane destruction/lysis, including an extensive series of quaternary ammonium cations (QACs).40-
46
Wuest and Minbiole have reported several QAC small molecules that effectively eradicate bacterial
biofilms40,41,47, which caught our attention as impressive comparators (i.e., QAC-10)40 during our initial studies in
this area.15,48,49
Figure 2. NH125 as a starting point for the construction of new antimicrobial agents.
NH125, a benzylic imidazolium cation with a 16-membered aliphatic tail, was recently reported to eradicate
methicillin-resistant Staphylococcus aureus (MRSA) persister cells and biofilms through depolarization of the
outer membrane50, followed by a report of lipid bilayer destruction.51 NH125 is commercially available compound
with several interesting reports regarding its in vitro and in vivo anticancer52,53 and neuromodulatory54,55 activities.
With this activity profile, we set out to design improved NH125 analogues by probing various structural features
of this structurally simple scaffold. Our strategy was to maintain the optimized hydrophobic tail of NH125 and
utilize simple building blocks for library generation using standard synthetic transformations (e.g., C-N coupling,
alkylations), including: imidazoles, benzimidazoles, triazoles and aryl/alkyl halides. We recently reported the
identification of two N-arylated NH125 analogues (1 and 4, Figure 3), which proved to be rapid killers of MRSA
persister cells and potent biofilm-eradicating agents compared to other membrane-active antibacterial agents
(i.e., BAC-12).56 Here we report the full study of a new library of 30 NH125 analogues in addition to antimicrobial
investigations and structure-activity relationship (SAR) findings, which will drive future NH125 analogue
development for various biomedical applications.
Results and Discussion

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To follow up directly with our initial communication regarding N-arylated NH125 analogDuOeI:s1,0.w10e39/sCy7OnBt0h1e02s8iAzed a
focused library of seven N-arylated NH125 analogues assembled through a practical two-step sequence that
included a copper(I)-catalyzed C-N coupling between 2-methylimidazole and a panel of electronically diverse
aryl iodides (36-46% yield, Figure 3A), before a final alkylation reaction with 1-bromohexadecane (48-88% yield)
to give N-arylated NH125 analogues 1-7. This short synthetic sequence for N-alkylated NH125 analogues could
be carried out on multi-gram scale to yield pure analogues without the use of chromatography. The N-arylated
analogues were designed to probe the electronics of the aryl ring, containing a combination of electronic-
donating (e.g., 4-methoxy analogue 4, 4-phenoxy analogue 5) and electron-withdrawing (e.g., 4-nitro analogue
3, 4-chloro analogue 6) groups.
Figure 3. A-D) Synthetic routes used during these studies to generate a diverse NH125 library. E) Chemical
structures of NH125 library investigated during these studies. F) Membrane-active antibacterial comparators
used during these studies.

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We also synthesized a series of alkylated and benzylated NH125 analogues to probe theDnOeI:c1e0.s10s3i9t/yC7oOfBt0h10e28bAenzyl
group and to determine if we could modulate antimicrobial activities through benzyl group modification with
different substitution patterns and functional groups. All alkylated/benzylated NH125 analogues maintain the
charged imidazole head group and provided insights regarding structure-activity relationships of this class of
compounds during our biological investigations. Our synthetic approach was to perform an initial N-alkylation of
2-methylimidazole with 1-bromohexadecane (86% yield, Figure 3B), followed by a diversification alkylation (32-
82% yield) or benzylation (31-76% yield) reaction. A small series of analogues (8-11, 21) were alkylation products
that did not contain a benzyl group to determine if this moiety was critical/necessary for NH125’s antimicrobial
activities. Various benzylation reactions were carried out to probe the benzyl moiety on NH125 with various
functional groups and substitution patterns (12-20), including two dimeric benzyl analogues (28, 29). Truncated
NH125 analogues 22 and 23 were also synthesized to determine the requirement for both imidazole nitrogen
atoms to be substituted with carbon-containing groups.
Four 2-methylbenzimidazole analogues (24-27) were synthesized to probe the structural importance of the
imidazole heterocycle of NH125. Similar to the synthesis of benzylated NH125 analogues, benzimidazole
analogues were synthesized via an initial N-alkylation reaction between 2-methylbenzimidazole and 1-
bromohexadecane (57% yield, Figure 3C), followed by a diversifying alkylation/benzylation reaction (31-64%
yield). Finally, a triazole analogue 30 was synthesized via a copper(I)-catalyzed click reaction between
phenylacetylene and 1-azidohexadecane (52% yield, Figure 3D), before alkylation with methyl iodide yielded 30
(61% yield).
With 30 NH125 analogues in hand, we evaluated the entire series of compounds alongside a series of
membrane-active small molecules, including: NH125 (starting point for analogue synthesis/development), BAC-
12, BAC-16, QAC-10, 3157 (known QACs) and daptomycin (clinically used antibiotic). With a special interest in
hospital settings that are challenged by disinfecting MRSA isolates, we tested the entire membrane-active panel
in minimum inhibitory concentration (MIC) assays in 96-well plates against MRSA-2, a methicillin-resistant
Staphylococcus aureus clinical isolate from a patient treated at Shands Hospital (University of Florida,
Gainesville, FL). In addition to MRSA-2, we evaluated the entire panel of NH125 analogues and membrane-
active agents against methicillin-resistant S. epidermidis (MRSE 35984), vancomycin-resistant Enterococcus
faecium (VRE 700221), multidrug-resistant Acinetobacter baumannii (MDR ATCC 1794) and Pseudomonas
aeruginosa (PAO1). Select analogues of interest were then evaluated against additional S. aureus and MRSA
isolates (Supporting Table 1), Escherichia coli (clinical isolate UAEC-1, University of Arkansas for Medical
Sciences, Klebsiella pneumoniae (ATCC 13883) in addition to fungal pathogens Candida albicans (SC5314) and
C. neoformans (ATCC 66031). A brief summary of antimicrobial profiles is found in Table 1 with complete
antimicrobial activity profiles detailed in Supporting Table 2 (Supporting Information).
From our assessment of MIC results, alkylated NH125 analogues 8, 9 and 11 demonstrated 2- to 6-fold
improvements in antibacterial activities (MIC = 0.39 – 2.35 µM, Supporting Table 1) against multiple S. aureus
clinical isolates, including MRSA strains, and S. aureus 29213 (methicillin-sensitive) when directly compared to
parent NH125 (MIC = 2.35 – 6.25 µM; Table 1 and Supporting Table 1). In addition, these analogues elicited

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enhanced antibacterial activities against other bacterial pathogens, including: MRSE 35984 (8-11, MIC = 0.59 –
1.17 µM; NH125, MIC = 4.69 µM), VRE 700221 (8-11, MIC = 1.17 – 2.35 µM; NH125, MIC = 3.13 µM), MDR A.
baumannii (8-11, MIC = 6.25 – 12.5 µM; NH125, MIC = 37.5 µM). From this series of alkylated analogues, the
2-hydroxyethyl-NH125 analogue 11 demonstrated the most potent antibacterial profile from these studies, which
includes: MRSA-1707 (MIC = 0.39 µM), MRSA-2 (MIC = 1.17 µM), MRSE (MIC = 0.59 µM), VRE (MIC = 1.56
µM), MDR A. baumannii (MIC = 6.25 µM).
Table 1. Summary of antibacterial (MIC) and antifungal (MIC) studies with NH125, analogues and other
membrane-active antimicrobials and controls. All MIC values are recorded in micromolar (µM) concentrations.
C. albicans
Agent
MRSA-2
MRSA-1707
MRSE 35984
VRE 700221
A. bau. 1794
PAO1
SC5314
3.13
3.13
--
3.13
3.13
--
NH125
1
4.69a
3.13
3.13
6.25
4.69a
4.69a
2.35a
1.56
4.69a
3.13
3.13
4.69a
4.69a
1.17a
1.56
3.13
2.35a
--
3.13
3.13
1.56
2.35a
1.56
--
37.5a
12.5
18.8a
25
>100
>100
>100
>100
>100
>100
2
3
4
5
6
18.8a
37.5a
3.13
3.13
1.17a
1.56
12.5
>100
--
3.13
1.17a
2.35a
2.35a
1.17a
6.25
12.5
6.25
3.13
3.13
2.35a
2.35a
12.5
37.5
25
>100
6.25
3.13
3.13
3.13
6.25
>100
>100
3.13
25
4.69a
0.39
2.35a
--
0.39
--
--
--
18.8a
12.5
12.5
9.38a
6.25
>100
>100
>100
>100
>100
>100
25
>100
>100
>100
>100
>100
37.5a
>100
>100
>100
>100
>100
12.5
>100
75a
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9.38a
--
3.13
--
--
3.13
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
50
--
--
--
--
3.13
--
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
1.17a
1.17a
0.78
0.59a
2.35a
9.38a
6.25
3.13
3.13
2.35a
1.17a
12.5
12.5
>100
>100
3.13
4.69a
3.13
6.25
9.38a
>100
>100
1.56
9.38a
3.13
1.56
2.35a
2.35a
1.17a
2.35a
1.56
3.13
4.69a
4.69a
2.35a
1.56
1.56
1.56
9.38a
6.25
6.25
>100
2.35a
2.35a
1.56
1.56
3.13
>100
>100
3.13
50
--
--
--
4.69a
1.56
1.56
--
--
25
--
--
--
9.38a
--
--
>100
>100
3.13
12.5
6.25
1.56
4.69a
BAC-12
BAC-16
QAC-10
6.25
1.56
3.13
4.69a
--
25
3.13
6.25
6.25
>100
--
2.35a
--
Dapto.
3.13
--
3.13
--
125
--
>100
--
0.78
Amp. B
Note: a corresponds to a midpoint of a 2-fold range in values. Dapto. = Daptomycin. Amp. B = Amphotericin B.
A. bau. = A. baumannii. RBC = red blood cells. MIC = minimum inhibitory concentration (antibacterial activity).
All values were obtained from a minimum of three independent experiments.
NH125 analogues with various substitutions on the benzylated moiety did not generally lead to improvements in
antibacterial potency; however, 3-methoxybenzyl-NH125 analogue 17 and 3-fluorobenzyl-NH125 analogue 18
demonstrated up to 2-fold improvements in antibacterial potencies against MRSA, MRSE and VRE. Several

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benzylated NH125 analogues (12, 14, 15) were found to demonstrate equipotent antibacterial activities against
MRSA, MRSE and VRE, while benzylated NH125 analogues 13, 19 and 20 demonstrated a loss in antibacterial
activities. Truncated NH125 analogues 22 and 23 were synthesized and evaluated in antibacterial assays to
determine the structural importance of the benzyl group and the hexadecane chain to the activity of NH125
analogues. Benzylated analogue 22 (without hexadecane tail) demonstrated no antibacterial activities (MIC >
100 µM). Hexadecane analogue 23 (without benzyl group) displayed antibacterial activities within 2-fold of the
parent NH125 with the exception of losing activity against A. baumannii 1794 (MIC >100 µM) demonstrating the
absolute requirement of the aliphatic tail to activity, which was expected; however, we were surprised that 23
possessed near equipotent antibacterial activities as NH125, which contains a charged imidazolium as a polar
head group. Analogue 23 likely maintains most of the activity of NH125 as the imidazole heterocycle of this
compound should be protonated at pH 7 to provide a charged, polar head group and fatty aliphatic chain
analogous to other amphipathic antibacterial agents. Along this same line of thought, the dimeric NH125
analogues 28 and 29 were found to be completely inactive (MIC > 100 µM) against all bacterial pathogens during
these studies. This loss of antibacterial activity demonstrates the structural requirement of a charged head group
as the imidazolium moieties of 28 and 29 are embedded within the central region of these compounds, with long
aliphatic tails extending from both imidazole heterocycles. Alternatively, these dimeric agents may be inactive
due to inadequate charge associations between the positively-charged imidazolium moieties of 28 and 29 with
the negatively-charged phosphate groups of bacterial membranes (lipid bilayer), resulting in reduced penetration
of the hydrophobic tail that is required for membrane lysis and antibacterial activity.
To complement alkylated/benzylic NH125 analogues, we synthesized a small number of N-arylated analogues
1-7 to probe the structural requirement of the benzyl group of NH125. The N-arylated analogues demonstrated
equipotent antibacterial activities against MRSA with 2- to 3-fold in improvements in antibacterial potencies
against MRSE (1, 2 and 6), VRE (2, 4 and 6), MDR A. baumannii (1, 2, 4 and 6), UAEC-1 (1, 4 and 6) and K.
pneumoniae (1, 4 and 6) when compared to NH125. Although improvements in antibacterial activities for the N-
arylated analogues were slight, more significant enhancements in activities (compared to NH125) were observed
during our biofilm eradication and persister cell kinetic kill experiments discussed later.
We synthesized a small number of benzimidazoles (24-27) and a triazole (30) to determine the structural
importance of the imidazole nucleus. In general, these alternative heterocyclic agents demonstrated equipotent
antibacterial activity with benzimidazole analogues 25 and 26 displaying 2-fold more potent antibacterial activity
against VRE (MIC = 1.56 µM) while triazole analogue 30 demonstrates 3-fold more potent antibacterial activities
against MRSE (MIC = 1.56 µM) and MDR A. baumannii (MIC = 12.5 µM) compared to NH125. These analogues
demonstrates the overall flexibility of the imidazole group of NH125 for future design.
We evaluated our panel of NH125 analogues alongside several membrane-active antibacterial agents (Table 1)
and found our most active alkylated NH125 analogues, 8 and 11, to be more potent than the comparators (BAC-
12, BAC-16, QAC-10, 31 and daptomycin) against MRSA, MRSE and VRE strains. Interestingly, none of the
NH125 analogues and most comparators demonstrated no antibacterial activity against PAO1 (MIC >100 µM).
A small panel of our most potent NH125 analogues that were identified during our antibacterial studies (NH125,

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1, 3, 4, 8, 11, 28) were evaluated for antifungal activities against C. albicans and C. neoformans, two human
pathogenic fungi. All NH125 analogues that were investigated, with the exception of dimer 28, demonstrated
good antifungal activities against C. albicans SC5341 (MIC = 3.13 µM) and C. neoformans 66031 (MIC = 3.13 –
6.25 µM, Supporting Table 2).
Membrane-targeting agents, such as quaternary ammonium cations, often display general membrane-lysing
activities40-42,56; however, antimicrobial peptides (AMPs) can selectively target bacterial membranes for
destruction,34-39 which has inspired the development of small molecules with similar bacterial membrane
selectivity. When developing AMP or QAC-like compounds, it is important to determine the ability of new agents
to lyse red blood cells (RBCs) in haemolysis assays to assess general membrane destruction and lysis. We
evaluated several of our NH125 analogues in haemolysis assays and found these agents to display relatively
potent haemolysis activities that correspond to antimicrobial potency (e.g., 1 has an MIC = 1.56 µM against
MRSA-1707 and caused 50% haemolysis of RBCs (HC50) at 7.8 µM; Supporting Table 2), including NH125, and
BAC-16 (HC50 < 25 µM). Interestingly, NH125 displayed potent haemolytic activities during these investigations
(HC50 = 12.3 µM), yet has favorable in vitro and in vivo biological activities which were initially discussed.52-55
Likely, NH125 analogues could have immediate applications as disinfectants and biocides due to their potent
antimicrobial activities; however, in vivo applications for therapeutic potential (i.e., skin, systemic infections) will
require additional investigations.
Table 2. Summary of bacterial and fungal biofilm eradication (MBEC) investigations. All MBC, MFC and MBEC
values are reported in micromolar (µM) concentrations.
MRSA-2
MBC / MBEC
62.5b / 46.9a
11.7a / 15.6
15.6b / 31.3
11.7a / 15.6
11.7a / 23.5a
11.7a / 31.3
62.5 / 62.5
93.8a / 93.8a
375a / >1000
--
MRSA-1707
MBC / MBEC
23.5a / 46.9a
11.7a / 23.5a
25 / 25
7.8b / 11.7a
11.7a / 31.3
23.5a / 46.9a
62.5b / 93.8a
--
MRSE 35984
MBC / MBEC
15.6 / 5.9a
5.9a / 11.7a
--
5.9a / 5.9a
3.0a / 3.0a
3.9 / 3.9
23.5a / 23.5a
11.7a / 11.7a
--
VRE 700221
MBC / MBEC
5.9a / 11.7a
4.69a / 2.35a
--
3.0a / 5.9a
3.9 / 3.9
3.0a / 3.9
2.0 / 3.0a
5.9a / 11.7a
--
C. albicans
C. neoformans
Agent
MFC / MBEC
MFC / MBEC
NH125
6.25 / 62.5
6.25 / 62.5
6.25 / 15.6
6.25 / 7.8
1
3
4
6.25 / 15.6
6.25 / 7.8
6.25 / 7.8
3.13 / 3.13
8
6.25 / 15.6
6.25 / 7.8
11
16
6.25 / 7.8
3.13 / 3.13
--
--
18
--
--
21
23
--
--
--
--
--
100 / 150a
--
--
25
250 / 188a
31.3b / 31.3
31.3b / >1000
31.3 / 125
--
--
--
--
--
--
30
31
11.7a / 23.5a
62.5 / >1000
93.8a / 93.8a
15.6 / >1000
31.3b / 15.6
125 / >2000
--
--
--
--
--
--
--
5.9a / 7.8
31.3 / 31.3
11.7a / >2000
11.7a / 15.6
31.3 / >2000
--
46.9a / 46.9a
3.0a / 3.0a
31.3 / 23.5a
5.9a / 3.0a
375a / 93.8a
--
QAC-10
BAC-12
BAC-16
Dapto.
Amp. B
--
--
--
6.25 / 15.6
--
--
6.25 / 15.6
--
--
62.5a / >2000
--
1 / 12.5
1 / 6.25
b
Note: a corresponds to a midpoint of a 2-fold range in values. corresponds to a midpoint of a 4-fold range in
values (MBC values only). Dapto. = Daptomycin. Amp. B = Amphotericin B. Fungal strains used = C. albicans
(strain SC5314); C. neoformans (strain 66031). MBC = minimum bactericidal concentration. MFC = minimum
fungicidal concentration. MBEC = minimum biofilm eradication concentration. All values were obtained from a
minimum of three independent experiments.

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Figure 4. Calgary Biofilm Device (CBD) assays were used to demonstrate planktonic and biofilm eradication
activities of NH125 analogues and comparators against MRSA-1707 and VRE 700221.
Following antibacterial and antifungal growth inhibition studies, we advanced a sub-set of NH125 analogues to
biofilm eradication assays based on initial MIC assessments. Bacterial biofilm eradication assays were carried
out using Calgary Biofilm Device (CBD) assays56,58 while fungal biofilm eradication assays were carried out using
an MTT assay in 96-well plates.59 Biofilm eradication assays are divided into three distinct phases, which include:
1) initial biofilm establishment on a surface (i.e., submerged peg), 2) compound treatment (enabling killing of
biofilms) and 3) recovery in fresh media, allowing biofilm growth, dispersion and subsequent growth giving a
turbid or non-turbid microtiter well, corresponding to live or eradicated biofilms, respectively. Each phase of
these biofilm eradication assays involve an incubation period of 24 hours at 37 ºC. Between each of the biofilm
eradication assay phases, the respective surfaces (i.e., CBD pegs) were washed before advancing to the next
phase of the assay. Upon completion of the biofilm eradication assay, non-turbid microtiter wells result from
complete eradication of bacterial/fungal biofilms. The lowest compound test concentration that results in

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complete biofilm eradication is referred to as the minimum biofilm eradication concentratiDoOnI:(1M0.1B03E9/CC7)OvBa01l0u2e8A. The
Calgary Biofilm Device can also be used to determine minimum bactericidal concentrations to determine
planktonic toxicity from the same bacterial culture, while minimum fungicidal concentrations were carried out in
separate experiments from the fungal biofilm eradication assays (Table 2). We have found that bacterial biofilm-
eradicating agents typically demonstrate MBEC:MBC ratios of 1-3:1 for planktonic:biofilm killing, which is
dramatically different than conventional antibiotics (i.e., daptomycin) that typically demonstrate MBEC:MBC
ratios of >20:1, illustrative of planktonic-active agents that are essentially ineffective against tolerant
biofilms.15,49,56
1 2
A)
B)
D M S O
µ M
1 0
8
1
1 0 µ M
2 5 µ M
5 0 µ M
6
4
2
0
0
1
2
3
4
5
6
T im e (h )
C)
Figure 5. Kinetic kill curve experiments with stationary MRSA-1707 cultures enriched in persister cells,
demonstrating (A) the rapid and sustained killing activities of new NH125 analogues 1 and 11 compared to
NH125 (at 50 µM), (B) the dose-dependence persister killing for NH125 analogue 1 and (C) the comparative
killing kinetics of stationary MRSA-1707 cultures when 1 is tested alongside other membrane-active antibacterial
agents (at 50 µM, most data from Figure 5C was previously reported in a previous communication56).
NH125 was able to eradicate MRSA, MRSE and VRE biofilms at excellent levels of potency (MBEC = 5.9 – 62.5
µM); however, several new analogues demonstrated more impressive biofilm-killing activities. Against MRSA
biofilms, analogues 1, 3, 4, 8, 11 and 30 were found to display improved biofilm-eradicating activities (MBEC =
11.7 – 31.3 µM) compared to NH125 (MBEC = 46.9 µM), while analogues 16, 18, 21, 23 and 25 were found to
be less active (MBEC = 62.5 – >1,000 µM). Against MRSE biofilms, analogues 8 and 11 demonstrated enhanced
biofilm-eradicating activities (MBEC = 3.0 – 3.9 µM) compared to NH125 (MBEC = 5.9 µM). Analogues 1, 4, 8,
11 and 16 demonstrated more potent VRE biofilm eradication activities (MBEC = 3.0 – 5.9 µM) compared to
NH125 (MBEC = 11.7 µM). From these studies, NH125 analogues 4 and 8 demonstrated high bacterial biofilm
eradication potencies across four drug-resistant strains.

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DOI: 10.1039/C7OB01028A
We were delighted to see the improvements in biofilm eradication activities demonstrated by N-arylated NH125
analogues (1, 3, 4) and 11 compared to NH125. We then evaluated NH125 analogues 1, 3, 4 and 11 against
stationary cultures of MRSA-1707, which are known to be enriched with high concentrations of non-replicating
persister cells,60,61 and found each of these analogues to kill stationary cultures of MRSA-1707 more rapidly than
NH125 (Figure 5) at 50 µM. Stationary cell killing can be seen at the first time point (30 minutes), where NH125
analogues 1, 3, 4 and 11 demonstrate a 2-4 log reduction of viable MRSA-1707 stationary cells (99 to 99.99%
persister kill) and gradually continues to a 4-6 log reduction of viable stationary MRSA-1707 cells after 6 hours.
At 24 hours, analogue 11 gives >6-log reduction in viable MRSA-1707 stationary cells while no viable bacteria
were observed following treatment of 1 (>8-log reduction in viable MRSA-1707 stationary cells, Figure 5.A./C.).
This MRSA persister eradication activity is dose-dependent and persister-killing activities can be seen at 25 µM
and 50 µM, but not 10 µM for 1 (Figure 5.B.). When other membrane-active antibacterial agents were tested
alongside 1, only BAC-16 demonstrated rapid and effective MRSA-1707 persister cell killing response; however,
this effect was not as dramatic as seen with 1.
Against fungal biofilms, NH125 and 1, 3, 4, 8 and 11 demonstrated good to excellent biofilm eradication activities.
NH125 eradicated C. albicans SC5314 and C. neoformans 66031 biofilms with an MBEC value of 62.5 µM. All
NH125 analogues that were tested against C. albicans SC5314 biofilms demonstrated 4- to 8-fold increases in
biofilm eradication potencies compared to NH125 with 4 and 11 demonstrating the most potent fungal biofilm
eradication activities (MBEC = 7.8 µM). C. neoformans 66031 biofilms displayed enhanced sensitivity towards
analogues 4 and 11, which reported potent MBEC values of 3.13 - 7.8 µM, which was found to be the most
potent biofilm eradication activities against this fungal pathogen. NH125 analogues 4 and 11 proved more potent
fungal biofilm-eradicating agents than BAC-16 (MBEC = 15.6 µM) and amphotericin B (MBEC = 6.25 – 12.5 µM),
a clinically used antifungal agent.
This new series of NH125 analogues was critical in the establishment of detailed structure-activity relationship
analysis and antimicrobial activity profiles (Figure 6) for this class of antimicrobial agents capable of eradicating
persistent biofilms through the destruction of cellular membranes. Multiple membrane-active comparators (i.e.,
QAC antibacterials) were investigated and provided additional insights with respect to the potent antimicrobial
activities demonstrated by these new NH125 analogues. An overview of antibacterial, bacterial/fungal biofilm
eradication and MRSA persister cell killing kinetic profiles have been provided in Figure 6, which will be
instructive in the development of future NH125 analogues for various biofilm-related biomedical applications.

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Figure 6. Activity profiles for NH125 analogues and membrane-active comparators regarding (A) antibacterial
activities, (B) bacterial/fungal biofilm eradication activities and (C) MRSA persister cell killing kinetics.

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DOI: 10.1039/C7OB01028A
Conclusions
In conclusion, we have developed multiple chemical synthesis pathways to rapidly generate a diverse library of
30 NH125 analogues. Antimicrobial studies with NH125 analogues against a panel of pathogenic microbes that
have led to the identification of several antibacterial and antifungal agents with enhanced potency compared to
NH125 (e.g., 1, 8, 11 and 30). Analogues 1, 8, 11 and 30 also demonstrated excellent biofilm eradication
activities. In addition, 1, 2, 3 and 11 demonstrated more rapid MRSA persister cell killing kinetics compared to
NH125 and other membrane-active agents. NH125 analogues operate through a membrane-lysing mechanism
and have a multitude of promising biomedical applications as persistent microbial biofilms continue to threaten
human health.
Experimental
All synthetic procedures, characterization data for all new compounds (1H & 13C NMR, HRMS and melting points),
biological procedures (antibacterial and antifungal assays, biofilm eradication assays, MRSA stationary killing
kinetic experiments, haemolysis assays) are detailed in the ESI.
Acknowledgements
We would like to acknowledge the University of Florida for start-up funds to support this work. We would also
like to acknowledge Aaron Garrison for helpful discussions during the preparation of this manuscript. High-
resolution mass spectra (HRMS) were obtained from the Mass Spectrometry facility at the University of Florida
(Chemistry Department).
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