Benzimidazolium salts prevent and disrupt methicillin-resistant: Staphylococcus aureus biofilms

Article abstract of DOI:10.1039/d0ra00738b

Emergence of resistant bacteria encourages us to develop new antibiotics and strategies to compensate for the different mechanisms of resistance they acquire. One of the defense mechanisms of resistant bacteria is the formation of biofilms. Herein we show

Full text of DOI:10.1039/d0ra00738b

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Benzimidazolium salts prevent and disrupt
methicillin-resistant Staphylococcus aureus
biofilms†
Cite this: RSC Adv., 2020, 10, 9420
*
Jeremie Tessier and Andreea R. Schmitzer
´ ´
Emergence of resistant bacteria encourages us to develop new antibiotics and strategies to compensate for
the different mechanisms of resistance they acquire. One of the defense mechanisms of resistant bacteria is
the formation of biofilms. Herein we show that benzimidazolium salts with various flexible or rigid side
chains act as strong antibiotic and antibiofilm agents. We show that their antibiofilm activity is due to
their capacity to destroy the biofilm matrix and the bacterial cellular membranes. These compounds are
able to avoid the formation of biofilms and disperse mature biofilms showing a universal use in the
treatment of biofilm-associated infections.
Received 23rd January 2020
Accepted 24th February 2020
DOI: 10.1039/d0ra00738b
rsc.li/rsc-advances
Subsequent steps involve biolm maturation and growth, which
results in the formation of a bacterial community embedded in
Introduction
the EPS. The detachment of the microorganisms from the biolm
occurs in the last step where planktonic bacteria are released from
the biolm matrix in order to colonize new sites and enlarge the
overall structure of the biolm. Once a biolm has reach maturity,
the bacteria trapped inside the protective layer are less accessible
for antibiotics. As the negatively charged extracellular DNA is
a major component of these biolms' matrix,10,11 many new
cationic amphiphile agents have been designed to interact with
the DNA and achieve a biolm penetration/destruction. Recent
synthesis of cationic polymers,12–14 cationic peptides,15 cationic
pillararenes,16 cationic porphyrins,17 cationic dendrimers,18
biomimetic biguanidinylated polyamines,19 and amphiphilic
small molecules20 showed interesting results toward the inhibition
of biolm formation and its destruction. Commercially available
quaternary ammonium salts and benzalkonium chlorides (BAC)
in particular, disrupt the interactions of the extracellular DNA and
other components of the bacterial EPS, induce bacterial death,
while showing very low toxicity on eukaryotic cells.21 We previously
showed that BAC has no activity on MRSA biolms, even at
concentrations higher than its planktonic MIC (330
mg mLꢀ1).22 We previously designed and studied amphiphilic
imidazolium and benzimidazolium salts with various exible or
rigid side chains that showed better antibiolm activities than
BAC.21,22 The unique property of benzimidazolium salts is that they
can be converted into many structural analogs with completely
new properties, for example by only changing the hydrophilic/
hydrophobic distribution in the structure.21 The electrostatic
interactions with the cellular membranes and their capacity to
produce reactive oxygen species are general features that make
imidazolium- and benzimidazolium compounds to alter the
integrity of microorganisms. Herein, we modied the previously
reported bis-phenylethynylbenzyl (bis-PEB) benzimidazolium
For several years now, researchers have been studying the increase
of bacterial resistance,1,2 which plays an increasingly important
role in chronic diseases, namely in lung infection and inam-
mation.3 Deaths from related infections caused by Gram positive
resistant bacteria such as methicillin-resistant Staphylococcus
aureus (MRSA) are now a major concern in hospital environ-
ments.4,5 In addition to developing resistance in their planktonic
form, this specic strain of bacteria is known to form biolms.6
These biolms are microbial communities attached to a surface
and protected from the external environment by a matrix named
extracellular polymeric substance (EPS).7 This matrix is composed
of polysaccharides, DNA and proteins, surrounded by channels
responsible for air, water and nutrient transport into the entire
biolm. This mixture of biopolymers and nutriments provides
a viable environment for any microorganism to survive and
proliferate. While in the biolms, bacteria are less sensitive to
antibiotics and antimicrobials due to the limited permeation of
these compounds into the biolm and impossibility to reach their
site of action. Development of compounds able to destroy the
biolm matrix and possessing bacterial membrane permeation
properties and antibacterial properties at the same time seems
a good strategy to eradicate these MRSA biolms. The formation of
a biolm is a multistep process which involves four different
stages.8 First, the planktonic bacteria attach to an abiotic or biotic
surface. In a second stage, the attachment of the planktonic
bacteria to a surface becomes irreversible because of the synthesis
of surface proteins, and then bacterial proliferation can occur.9
Department of Chemistry, University of Montreal, PO Box 6128, Succursale
Centre-Ville Montreal, QC, H3C 3J7, Canada. E-mail: ar.schmitzer@umontreal.ca
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra00738b
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motif (I) that showed very good planktonic antimicrobial activity, this regard we synthesized a semi-rigid analogue (21) bearing
but modest antibiolm activity,21,22 due to their property to self- one octyl chain and one PEB.
assemble in channel-like aggregates. The p-stackable bis-PEB
This new compound also showed general antibacterial
moiety allows the formation of empty channels inside the bacterial activity, and low toxicity similar to 6, but also a lower selectivity
membranes which leads to electrolytic imbalance and causes compared to 11. With these results in hand, we studied the
bacterial death.21,22 Once formed inside the biolm, these chan- ability of the most active compounds 11, 14, and 21 to prevent
nels have low probability to affect the overall electrolytic potential the formation of MRSA biolms using a commercially available
of the isolated biolm system, which leads to a low or no anti- uorescent assay (Baclight Live/Dead).26 Simultaneously, we
biolm activity. Benzimidazolium-based mobile synthetic trans- studied the effect of these analogues on the morphology of the
porters which have very low mobility inside the well packed and biolms using scanning electron microscopy (SEM). Using
organized biolm matrix also showed only modest antibiolm these two techniques, we were able not only to measure the
activity.23
bacterial Live/Dead ratio in the biolm, but we were also able to
observe the effect of those compounds on the spatial organi-
zation of the bacterial communities inside the biolm (Fig. 2).27
Confocal and SEM images of an untreated bacterial biolms
showed the formation of well-dened and homogenous bio-
lms (Fig. 2(1a and 2a)). Once treated with compound 6 at its
MIC, a stripe organization is observed (Fig. 2(1b and 2b)). This
change of morphology is the consequence of the bacterial stress
response inside the biolm. This change of morphology is the
consequence of the bacterial stress response inside the biolm.
When treated with 6, the bacteria regroup to form a tighter and
stronger biolm, decreasing the ability of the antibiotic to
penetrate. As we previously showed, a 20-fold MIC concentra-
tion is needed for this type of synthetic transporter to
completely inhibit the formation of the biolm.23 However,
once treated with compounds 11 and 14 at their MIC, we
observed the formation of overall thinner biolms (Fig. 2(1c and
d, 2c and d)). These fast acting anti-biolm agents may have an
increased capacity to penetrate, weaken and disrupt bacterial
biolms and prevent bacterial stress response. Bacterial biolm
disruption was even more pronounced when the biolm was
treated with the semi-rigid compound 21. As shown by both
confocal and SEM images (Fig. 2(1e and 2e)), this antibiolm
agent can disrupt and weaken the bacterial biolm at its MIC
concentration. Not only does this compound have an effect on
the thickness and the density of the biolm, but complete death
of the bacterial community can be observed inside the biolm.
These results suggest that the octyl chain can strongly increase
the antibiolm activity and the overall efficiency of those anti-
bacterial agents, acting both on the biolm matrix and the
bacterial membranes.
Results and discussion
We designed and synthesized new amphiphilic analogues in
order to decrease their self-assembling properties and increase
their biolm penetration (Fig. 1) while keeping an overall
molecular rigidity. The general synthetic routes for these
compounds are summarized in Scheme 1. With this synthetic
method, we were able to synthesize a library of compounds
bearing different aromatic and aliphatic groups side chains. We
substituted one of the highly p-stackable PEB side chain with
various benzyl derivative (II) in order to maintain their global
hydrophobicity while reducing their channel formation
potential.
We rst investigated the ability of the rigid benzimidazolium
salts (II) to kill planktonic bacteria (Table 1). Most of the newly
synthesized compounds showed good antimicrobial activity
compared to the previously reported generation (I) or
commercially available BAC on all the studied bacterial stains.
Compounds 11 and 14 possess not only activity on planktonic
Gram-positive bacteria (MRSA and VRE) similar to compound 6,
but also a 4-fold increased activity on Gram-negative bacteria.
Gram-negative bacteria possess an outer membrane acting as
an extra protective layer that usually reduces the antibacterial
power of synthetic compounds.25
Our new analogues may act on the outer cellular membrane
inducing its permeabilization. In addition to their interesting
antibacterial activity, these new analogues show low toxicity on
red blood cells (Table 1, HC50 values). Indeed, we observed
different specicities of these compounds on MRSA, versus red
blood cells. We recently showed that the addition of an octyl
chain to the benzimidazole fragment of miconazole increased
the capacity of these drugs to penetrate phospholipid
membranes and increased their antimicrobial activities.24 In
As compound 21 showed the most promising activity on
MRSA biolms, we designed a new series of analogues bearing
octyl chains (Scheme 1, generation III). The decrease of the
lipophilic character of an antibiotic oen comes with a decrease
of its toxicity on red blood cells,28 but these new compounds
showed lower specicity for MRSA bacteria versus red blood
cells (Table 2).
We selected the most actives compounds 34, 39, and 42 and
studied their ability to prevent the formation of MRSA biolms.
Once treated with these compounds at their planktonic MICs,
we observed a fast and strong inhibition of MRSA biolm
compared to the negative control (Fig. 3). Those amphiphilic
salts have the capacity not only to kill bacteria, but also to
penetrate and disrupt the biolm and kill the present bacteria.
These compounds are also able to interfere with the biolm
Fig. 1 Structure of benzimidazolium salts.
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Scheme 1 Synthesis of second and third generation analogues.
formation and disperse mature biolms having the potential purication. Fresh human red blood cells (blood type O)
for universal use in the treatment of biolm-associated purchased from Innovative Research in Alserver's solution.
infections.
(4-Phenylethynyl)benzyl alcohol (2)
Conclusions
To a carefully degassed solution of 4-iodobenzyl alcohol (4.0 g,
17.1 mmol), PPh3 (90 mg, 0.343 mmol), and PdCl2(PPh3)2
In regard to the rising MRSA related infections around the
(120 mg, 0.171 mmol) in 50 mL of dry THF was added CuI
world and knowing that the primary defence mechanism of
(65 mg, 0.343 mmol) and Et3N (19.1 mL, 137 mmol). The
these bacteria is the formation of biolms, we believe that the
mixture was degassed for another 5 min before phenylacetylene
inhibition and destruction of those biolms at low antibiotic
(1.98 mL, 18 mmol) was added dropwise. The reaction was
concentrations is the key to overcome the challenges brought by
stirred overnight at 50 ꢂC under nitrogen atmosphere. Then,
the appearance of resistant bacteria. For example, compound 11
50 mL of water was added and the aqueous layer was extracted
showed a 100-fold selectivity to MRSA compared to RBC and
three times with EtOAc. The combined organic fractions were
a great ability to disrupt mature biolms (Fig. 4). Herein we
washed with NH4Cl (sat.) where a blue aqueous phase was
show that benzimidazolium salts have antibiotic, antibiolm
observed, brine, and dried over Na2SO4. The solvent was
activities and good selectivity. Our readily available intermedi-
removed under reduced pressure and the resulting brownish
ates and relatively simple synthesis of new analogues make
solid was triturated with hexane until no more apolar (solvent
them good candidates for diversication and screening for
front) product was observed by TLC (40% EtOAc/hexane).
stronger new activities on bacterial resistant strains.
Filtration of the resulting solid afforded (4-phenylethynyl)
benzyl alcohol (2) as a white solid in 90% isolated yield (3.21 g,
15.4 mmol). 1H NMR (300 MHz, CDCl3) d 7.54–7.50 (m, 4H),
7.37–7.24 (m, 5H), 4.66 (s, 2H), 2.00 (b, 1H). 13C NMR (75 MHz,
CDCl3) d 140.9, 131.7, 131.5, 128.3, 128.2, 126.7, 123.1, 122.3,
89.3, 89.1, 64.8. HRMS (ESI) calcd for C15H13O+[M + H]+:
209.0961, found 209.0969.
Experimental
1H- and 13C-NMR spectra were recorded on 400 and 75 MHz
spectrometers, respectively, in the indicated solvent. Chemical
shis are reported in ppm with internal reference to TMS, and J
values are given in hertz. The purity of nal compounds used in
biological assays was determined by ESI/LC-MS on Quantum TSQ
Ultra instruments (C18 (2.1 ꢁ 100 mm) column, water +0.1%
(4-Phenylethynyl)benzyl bromide (3)
formic acid). Benzimidazole (4) and 1-methylbenzimidazole (22) (4-Phenylethynyl)benzyl alcohol (2) (1.0 g, 4.8 mmol) was dis-
were purchase from Sigma-Aldrich and use without further solved in 20 mL of dry CH2Cl2. The mixture was kept at 0 ꢂC and
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Table 1 Minimal inhibitory concentration (MIC) of flexible benzimi- Table 2 Minimal inhibitory concentration (MIC) of flexible benzimi-
dazolium salts (II)a
dazolium salts (III)a
MIC (mg mLꢀ1
)
MIC (mg mLꢀ1
)
HC50 (mg mLꢀ1
)
E.
MRSA VRE coli HC50 (mg mLꢀ1
Antimicrobials
MRSA
VRE
E. coli
Entry Antimicrobials (–R)
)
BAC
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
100
1.25
5
4
5
2.5
1.25
2.5
2.5
1.25
1.25
2.5
5
—
1.25
20
10
10
5
5
5
5
2.5
1.25
2.5
10
10
5
100
100
50
20
25
20
10
25
20
25
100
20
50
50
10
50
10
95
70
33
34
35
36
37
38
39
40
41
42
43
Methyl
Octyl
Benzyl
400
5
50
10
25
6
6
10
6
400 400 >200
2.5 50 50
100 200 >200
200
200
200
200
120
145
95
4-Methylbenzyl
4-Fluorobenzyl
4-Chlorobenzyl
4-Bromobenzyl
4-Iodobenzyl
4-(Triuoromethyl)benzyl
4-tert-Butylbenzyl
4-Phenylbenzyl
25
50
15
15
25
25
2.5
2.5
200 150
200 >200
100 100
100
75
100 150
100 >200
60
70
2.5
3
50
50
25
25
120
150
200
140
200
50
a
MRSA
¼
methicillin-resistant S. aureus (ATCC 43300), VRE
¼
5
5
5
2
vancomycin-resistant E. faecium (BAA-2316), E. coli (ATCC 25922),
HC50: concentration of the antimicrobials at which 50% of human red
blood cells get lysed.
10
2.5
a
MRSA
¼
methicillin-resistant S. aureus (ATCC 43300), VRE
¼
vancomycin-resistant E. faecium (BAA-2316), E. coli (ATCC 25922),
HC50: concentration of the antimicrobials at which 50% of human red
blood cells get lysed.
with CH2Cl2. Then, silica gel was added before the solvent was
removed under reduced pressure. The product was puried by
ltration on silica gel (hexane 100%) to afford (4-phenylethynyl)
benzyl bromide (3) as a white solid in 100% isolated yield (1.3 g,
4.8 mmol). 1H NMR (300 MHz, CDCl3) d 7.54–7.48 (m, 4H), 7.37–
7.24 (m, 5H), 4.48 (s, 2H). 13C NMR (75 MHz, CDCl3) d 137.6,
131.9, 131.5, 129.0, 128.3, 128.2, 123.3, 122.9, 90.2, 88.8, 32.9.
phosphorus tribromide (0.68 mL, 7.20 mmol) was added
dropwise. Then the mixture was stirred 2 hours at 0 ꢂC then
water was added and the aqueous phase was extracted 3 times
Fig. 2 (Top) MRSA biofilms labeled with Live/Dead stains after a 12 h incubation at the MIC concentration of the antimicrobials. (1a) Negative
control (DMSO); (1b) compound 6; (1c) compound 11; (1d) compound 14; (1e) compound 21; (bottom) SEM images of MRSA biofilms after a 12 h
incubation with MIC concentration of the antimicrobials. (2a) Negative control (DMSO); (2b) compound 6; (2c) compound 11; (2d) compound 14;
(2e) compound 21.
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Fig. 3 (Top) MRSA biofilms labeled with Live/Dead stains after a 12 h incubation with MIC concentration of the antimicrobials. (1a) Negative
control (DMSO); (1b) compound 34; (1c) compound 39; (1d) compound 42; (bottom) SEM images of MRSA biofilms after a 12 h incubation with
MIC concentration of the antimicrobials. (2a) Negative control (DMSO); (2b) compound 34; (2c) compound 39; (2d) compound 42.
HRMS (ESI) calcd for C15H12Br+[M + H]+: 271.0117, found more starting benzimidazole was observed by TLC (100%
271.0109.
EtOAc) (2–3 hours). The reaction was then quenched with water
(200 mL) and the resulting precipitate was ltered and washed
with water, EtOAc, and hexane to afford (4-phenylethynyl)benzyl
benzimidazole (5) as a white solid in 93% yield (2.0 g, 6.5
mmol). 1H NMR (500 MHz, DMSO-d6) d 7.66–7.51 (m, 7H), 7.44–
7.38 (m, 3H), 7.33 (d, J ¼ 8.0 Hz, 2H), 7.21 (d, J ¼ 6.2 Hz, 2H),
5.56 (s, 2H). 13C NMR (126 MHz, DMSO-d6) d 137.6, 131.7, 131.5,
131.4, 131.3, 128.8, 128.8, 128.8, 127.7, 122.5, 122.1, 121.6, 89.5,
88.9, 47.5. HRMS (ESI) calcd for C22H16N2[M + H]+: 309.1386,
found 309.1380.
(4-Phenylethynyl)benzyl benzimidazole (5)
To a solution of benzimidazole (869 mg, 7.36 mmol) in THF
(30.0 mL) at 0 ꢂC was added sodium hydride (336 mg, 8.4 mmol)
portion wise. Aer stirring for 15 minutes, (4-phenylethynyl)
benzyl bromide (3) (1.9 g, 7.00 mmol) was slowly added. The
reaction was allowed to warm up to R.T. and stirred until no
General procedure for generation I and II of benzimidazolium
salt (6–21)
To a solution of (4-phenylethynyl)benzyl benzimidazole (5)
(135 mg, 0.438 mmol) in MeCN (2 mL) was added the corre-
sponding alkyl or benzyl bromide (1.31 mmol, 3 eq.). The
reaction was stirred until LC-MS analysis showed completion of
the reaction (24–48 h). MeCN was evaporated and the resulting
solid was triturated with EtOAc to remove the excess alkyl or
benzyl bromide. The resulting white solid was ltered, washed
with hexane, and dried to afford the corresponding benzimi-
dazolium salt (6–21) as a white powder.
1,3-Bis(4-(phenylethynyl)benzyl)-1H-benzo[d]imidazol-3-ium
bromide (6)
Yield 95%. Purity 98.99%. 1H NMR (300 MHz, CDCl3): d ppm ¼
9.97 (s, 1H), 7.90–7.96 (m, 1H), 7.52 (m, 14H), 7.38–7.45 (m, 7H),
5.81 (s, 4H). 13C NMR (126 MHz, CDCl3) d 143.5, 137.3, 131.7,
131.5, 131.4, 131.3, 128.8, 127.6, 122.5, 122.1, 121.1, 89.5, 88.9,
47.5. HRMS (ESI): m/z calcd for C37H27N2[M]+: 499.2169, found
499.2175.
Fig.
4 Antibiofilm activity of studied compounds. Compared to
commercially available BAC, all the benzimidazolium salts possess the
ability to prevent the formation and disrupt mature biofilms. The
dotted line represents the 25-fold selectivity towards MRSA bacteria
compared to RBC.
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3-Benzyl-1-(4-(phenylethynyl)benzyl)-1H-benzo[d]imidazol-3-
3-(4-Iodobenzyl)-1-(4-(phenylethynyl)benzyl)-1H-benzo[d]
ium bromide (7)
imidazol-3-ium bromide (12)
1
1
Yield 65%. Purity 100%. H NMR (400 MHz, DMSO-d6) d 10.11 Yield 99%. Purity 100%. H NMR (400 MHz, DMSO-d6) d 10.04
(s, 1H), 7.98 (m, J ¼ 8.8, 6.4, 3.0 Hz, 3H), 7.75–7.51 (m, 12H), (s, 1H), 7.95 (s, 2H), 7.81 (m, J ¼ 6.9 Hz, 2H), 7.70–7.51 (m, 8H),
7.50–7.34 (m, 8H), 5.84 (d, J ¼ 15.6 Hz, 4H). 13C NMR (126 MHz, 7.49–7.24 (m, 5H), 5.80 (d, J ¼ 28.5 Hz, 4H). 13C NMR (126 MHz,
DMSO-d6) d 143.3, 134.9, 134.4, 132.4, 131.9, 131.6, 129.5, 129.3, DMSO) d 142.9, 137.8, 134.4, 133.5, 131.9, 131.4, 131.1, 131.1,
129.2, 129.2, 128.8, 127.3, 123.1, 122.4, 114.5, 114.5, 90.6, 89.2, 130.7, 129.0, 128.8, 128.7, 126.9, 122.6, 122.0, 114.0, 95.5, 90.1,
50.5, 50.2. HRMS (ESI): m/z calcd for C29H23N2[M]+: 399.1856, 88.7, 49.7, 49.5. HRMS (ESI): m/z calcd for C29H22IN2[M]+:
found 399.1856.
525.0822, found 525.0804.
1-(4-(Phenylethynyl)benzyl)-3-(4-(triuoromethyl)benzyl)-1H-
benzo[d]imidazol-3-ium bromide (13)
3-(4-Methylbenzyl)-1-(4-(phenylethynyl)benzyl)-1H-benzo[d]
imidazol-3-ium bromide (8)
Yield 65%. Purity 99.57%. 1H NMR (500 MHz, DMSO-d6) d 10.11
(s, 1H), 8.09–7.90 (m, 2H), 7.83 (d, J ¼ 8.3 Hz, 2H), 7.76 (d, J ¼
8.2 Hz, 2H), 7.71–7.55 (m, 6H), 7.55 (m, 2H), 7.48–7.37 (m, 3H),
5.91 (d, J ¼ 39.2 Hz, 4H). 13C NMR (126 MHz, DMSO) d 143.4,
138.7, 134.5, 132.1, 131.6, 131.3, 129.3, 129.2, 129.0, 128.9,
127.1, 127.1, 126.1, 126.0, 122.8, 122.1, 114.2, 114.1, 90.2, 88.8,
49.9, 49.6. 19F NMR (471 MHz, DMSO) d ꢀ63.71. HRMS (ESI): m/
z calcd for C30H22F3N2[M]+: 467.1729, found 467.1727.
Yield 65%. Purity 99.48%. 1H NMR (400 MHz, DMSO-d6) d 10.03
(s, 1H), 7.96 (m, J ¼ 12.2, 6.8, 3.1 Hz, 2H), 7.69–7.60 (m, 4H),
7.60–7.52 (m, 4H), 7.48–7.40 (m, 5H), 7.24 (d, J ¼ 7.9 Hz, 2H),
5.83 (s, 2H), 5.74 (s, 2H), 2.30 (s, 3H).$13C NMR (126 MHz,
DMSO-d6) d 142.7, 138.3, 134.5, 131.9, 131.4, 131.1, 130.8, 129.6,
129.0, 128.8, 128.7, 128.4, 126.9, 122.7, 122.0, 114.1, 114.0, 90.1,
88.7, 49.9, 49.7, 20.7. HRMS (ESI): m/z calcd for C30H25N2[M]+:
413.2001, found 413.2012.
3-(4-(tert-Butyl)benzyl)-1-(4-(phenylethynyl)benzyl)-1H-benzo
[d]imidazol-3-ium bromide (14)
3-(4-Fluorobenzyl)-1-(4-(phenylethynyl)benzyl)-1H-benzo[d]
imidazol-3-ium bromide (9)
Yield 62%. Purity 99.52%. 1H NMR (500 MHz, DMSO-d6) d 10.09
(s, 1H), 8.06–8.02 (m, 1H), 7.98–7.93 (m, 1H), 7.68–7.52 (m, 8H),
7.50–7.41 (m, 7H), 5.80 (d, J ¼ 40.9 Hz, 4H), 2.08 (s, 1H), 1.26 (s,
9H). 13C NMR (126 MHz, DMSO) d 151.3, 142.7, 134.5, 131.9,
131.4, 131.1, 131.1, 131.0, 129.0, 128.8, 128.2, 126.8, 125.8,
122.6, 122.0, 114.1, 114.0, 90.1, 88.7, 49.7, 34.4, 31.0. HRMS
(ESI): m/z calcd for C33H31N2[M]+: 455.2482, found 455.2483.
Yield 79%. Purity 98.65%. 1H NMR (400 MHz, DMSO-d6) d 10.07
(s, 1H), 8.08–7.90 (m, 2H), 7.68–7.49 (m, 10H), 7.46–7.39 (m,
3H), 7.29 (t, J ¼ 8.9 Hz, 2H), 5.82 (d, J ¼ 17.6 Hz, 4H). 13C NMR
(126 MHz, DMSO-d6) d 163.7, 161.7, 143.3, 134.9, 132.4, 131.9,
131.6, 131.5, 131.4, 131.3, 130.6, 129.5, 129.3, 129.2, 127.3,
123.1, 122.4, 116.5, 116.3, 114.5, 114.5, 90.6, 89.2, 65.4, 62.5,
50.2, 49.8. 19F NMR (471 MHz, DMSO-d6) d ꢀ114.73. HRMS
(ESI): m/z calcd for C29H22FN2[M]+: 417.1762, found 417.1760.
3-([1,10-Biphenyl]-4-ylmethyl)-1-(4-(phenylethynyl)benzyl)-1H-
benzo[d]imidazol-3-ium bromide (15)
3-(4-Chlorobenzyl)-1-(4-(phenylethynyl)benzyl)-1H-benzo[d]
imidazol-3-ium bromide (10)
1
Yield 69%. Purity 100%. H NMR (500 MHz, DMSO-d6) d 10.09
(s, 1H), 8.09–8.03 (m, 1H), 8.00–7.95 (m, 1H), 7.79–7.71 (m, 2H),
7.71–7.52 (m, 12H), 7.51–7.32 (m, 6H), 5.85 (s, 4H). 13C NMR
(126 MHz, DMSO) d 142.9, 140.6, 139.3, 134.4, 133.0, 131.9,
131.4, 131.1, 129.0, 129.0, 128.8, 128.7, 127.8, 127.2, 126.9,
126.7, 122.7, 122.0, 114.1, 114.0, 90.1, 88.7, 49.7. HRMS (ESI): m/
z calcd for C35H27N2[M]+: 475.2169, found 455.2159.
Yield 75%. Purity 96.89%. 1H NMR (400 MHz, DMSO) d 10.07 (s,
1H), 8.04–7.90 (m, 2H), 7.76–7.49 (m, 12H), 7.47–7.35 (m, 3H),
5.83 (d, J ¼ 12.9 Hz, 4H). 13C NMR (126 MHz, DMSO-d6) d 143.0,
134.4, 133.5, 132.8, 131.9, 131.4, 131.1, 131.0, 130.4, 129.0,
128.8, 128.7, 126.9, 122.6, 122.0, 114.0, 90.1, 88.7, 49.7, 49.3.
HRMS (ESI): m/z calcd for C29H22ClN2[M]+: 433.1466, found
433.1456.
3-(3,5-Bis(triuoromethyl)benzyl)-1-(4-(phenylethynyl)benzyl)-
1H-benzo[d]imidazol-3-ium bromide (16)
Yield 68%. Purity 97.62%. 1H NMR (500 MHz, DMSO-d6) d 10.03
(s, 1H), 8.35 (s, 2H), 8.19 (s, 1H), 8.07–8.01 (m, 1H), 7.99–7.91
3-(4-Bromobenzyl)-1-(4-(phenylethynyl)benzyl)-1H-benzo[d]
imidazol-3-ium bromide (11)
Yield 80%. Purity 99.53%. 1H NMR (400 MHz, DMSO-d6) d 10.09 (m, 1H), 7.74–7.51 (m, 8H), 7.48–7.39 (m, 3H), 5.97 (s, 2H), 5.84
(s, 1H), 7.97 (s, 2H), 7.72–7.48 (m, 12H), 7.43 (s, 3H), 5.83 (d, J ¼ (s, 2H). 13C NMR (126 MHz, DMSO) d 143.5, 137.0, 134.4, 131.8,
19.5 Hz, 4H). 13C NMR (126 MHz, DMSO) d 143.0, 134.4, 133.2, 131.4, 131.1, 131.1, 130.8, 130.6, 129.8, 129.0, 128.8, 128.7,
131.9, 131.4, 131.1, 131.0, 130.7, 129.0, 128.8, 128.7, 126.9, 127.0, 126.9, 122.6, 122.0, 114.1, 113.8, 90.1, 88.7, 49.8, 48.9. 19
F
122.6, 122.1, 122.0, 114.0, 90.1, 88.7, 49.7, 49.4. HRMS (ESI): m/z NMR (471 MHz, DMSO) d ꢀ62.79. HRMS (ESI): m/z calcd for
calcd for C29H22CBrN2[M]+: 477.0961, found 477.0959.
C
31H21F6N2[M]+: 535.1603, found 535.1609.
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3-(Benzo[d][1,3]dioxol-5-ylmethyl)-1-(4-(phenylethynyl)
benzyl)-1H-benzo[d]imidazol-3-ium bromide (17)
General procedure for benzimidazole alkylation (23–32)
To a solution of benzimidazole (4) (1.00 g, 8.46 mmol) in THF
Yield 87%. Purity 98.85%. 1H NMR (500 MHz, DMSO-d6) d 10.07 (25.0 mL) at 0 ꢂC was added sodium hydride (406 mg, 10.2
(s, 1H), 8.05–7.99 (m, 1H), 7.99–7.88 (m, 1H), 7.70–7.49 (m, 8H), mmol) portion wise. Aer stirring for 15 minutes, a solution of
7.47–7.36 (m, 3H), 7.20 (d, J ¼ 1.7 Hz, 1H), 7.13 (dd, J ¼ 8.0, the corresponding benzyl or alkyl bromide (8.89 mmol, 1.05 eq.)
1.8 Hz, 1H), 6.98 (d, J ¼ 8.0 Hz, 1H), 6.04 (s, 2H), 5.84 (s, 2H), in THF (5 mL) was slowly added. The reaction was allowed to
5.69 (s, 2H). 13C NMR (126 MHz, DMSO) d 147.7, 147.7, 142.7, warm up to R.T. and stirred until no more starting benzimid-
134.5, 131.9, 131.4, 131.1, 131.0, 129.0, 128.8, 128.7, 127.2, azole was observed by TLC (100% EtOAc) (2–3 hours). The
126.8, 126.8, 122.7, 122.6, 122.0, 114.1, 113.9, 109.0, 108.6, reaction was then quenched with water and the aqueous layer
101.4, 90.1, 88.7, 50.0, 49.7. HRMS (ESI): m/z calcd for was extracted three times with EtOAc, dried over Na2SO4 and
C
30H23N2O2[M]+: 443.1754, found 443.1756.
puried using silica gel chromatography (100% EtOAc) to afford
the corresponding alkylated benzimidazole (23–32).
3-(3,5-Dimethoxybenzyl)-1-(4-(phenylethynyl)benzyl)-1H-
benzo[d]imidazol-3-ium bromide (18)
1-Octyl-1H-benzo[d]imidazole (23)
1
Yield 87%. Purity 99.09%. 1H NMR (400 MHz, DMSO-d6) d 10.15
(s, 1H), 8.15–7.86 (m, 2H), 7.76–7.51 (m, 8H), 7.49–7.41 (m, 3H),
6.75 (s, 2H), 6.51 (s, 1H), 5.87 (s, 2H), 5.72 (s, 2H), 3.74 (s, 6H).
13C NMR (126 MHz, DMSO) d 160.9, 142.9, 135.9, 134.5, 131.9,
131.4, 131.1, 131.0, 129.0, 128.8, 128.7, 126.8, 122.6, 122.0,
114.1, 114.0, 106.7, 100.0, 90.1, 88.7, 55.4, 50.1, 49.7. HRMS
(ESI): m/z calcd for C31H27N2O2[M]+: 459.2067, found 459.2078.
Yield 96%, H NMR (300 MHz, CDCl3) d 7.81 (s, 1H), 7.76–7.71
(m, 1H), 7.35–7.31 (m, 1H), 7.25–7.20 (m, 2H), 4.17 (d, 6.3 Hz,
2H), 1.86 (m, 2H), 1.19–1.13 (m, 10H), 0.80 (t, J ¼ 1.8 Hz, 3H) 13
C
NMR (75 MHz, CDCl3) d 143.1, 142.6, 133.8, 122.7, 121.2, 120.2,
109.8, 45.3, 31.6, 29.2, 29.3, 26.7, 22.5, 14.0. HRMS (ESI): m/z
calcd for C15H22N2[M + H]+: 231.1863, found 231.1867.
1-Benzyl-1H-benzo[d]imidazole (24)
1
Yield 91%, H NMR (500 MHz, DMSO-d6) d 8.41 (s, 1H), 7.68–
1-(4-(Phenylethynyl)benzyl)-3-(4-(triuoromethoxy)benzyl)-
1H-benzo[d]imidazol-3-ium bromide (19)
7.64 (m, 1H), 7.53–7.49 (m, 1H), 7.35–7.25 (m, 5H), 7.23–7.16
(m, 2H), 5.50 (s, 2H). 13C NMR (126 MHz, DMSO) d 144.25,
143.58, 136.98, 133.67, 128.71, 127.75, 127.40, 122.41, 121.60,
119.51, 110.73, 47.64. HRMS (ESI): m/z calcd for C14H13N2[M +
H]+: 209.1073, found 209.1079.
Yield 75%. Purity 99.63%. 1H NMR (400 MHz, DMSO-d6) d 10.09
(s, 1H), 8.06–7.89 (m, 2H), 7.72–7.51 (m, 10H), 7.48–7.38 (m,
5H), 5.85 (d, J ¼ 2.7 Hz, 4H). 13C NMR (126 MHz, CDCl3) d 148.3,
142.9, 134.2, 133.2, 131.7, 131.2, 130.9, 130.9, 130.4, 128.8,
128.6, 128.6, 126.7, 122.5, 121.8, 121.4, 113.9, 113.8, 89.9, 88.5,
49.6, 49.0. 19F NMR (471 MHz, CDCl3) d ꢀ52.03. HRMS (ESI): m/
z calcd for C30H22F3N2O[M]+: 483.1679, found 483.1688.
1-(4-Methylbenzyl)-1H-benzo[d]imidazole (25)
1
Yield 40%, H NMR (500 MHz, DMSO-d6) d 8.39 (s, 1H), 7.70–
7.60 (m, 1H), 7.55–7.40 (m, 1H), 7.27–7.04 (m, 6H), 5.44 (s, 2H),
2.24 (s, 3H). 13C NMR (126 MHz, DMSO) d 144.2, 143.6, 137.0,
133.9, 133.6, 129.2, 127.4, 122.3, 121.5, 119.5, 110.7, 47.4, 20.6.
HRMS (ESI): m/z calcd for C15H15N2[M + H]+: 223.1230, found
223.1232.
3-(4-(Methoxycarbonyl)benzyl)-1-(4-(phenylethynyl)benzyl)-
1H-benzo[d]imidazol-3-ium bromide (20)
1
Yield 84%. Purity 100%. H NMR (400 MHz, DMSO-d6) d 10.19
(s, 1H), 8.14–7.85 (m, 4H), 7.78–7.49 (m, 10H), 7.49–7.25 (m,
3H), 5.92 (d, J ¼ 23.2 Hz, 4H), 3.85 (s, 3H). 13C NMR (126 MHz,
CDCl3) d 165.6, 143.0, 139.0, 134.2, 131.7, 131.2, 130.9, 129.6,
129.5, 128.8, 128.6, 128.4, 126.7, 122.5, 121.8, 113.9, 113.8, 89.9,
88.5, 52.1, 49.6, 49.5. HRMS (ESI): m/z calcd for C31H25N2O2[M]+:
457.1911, found 457.1912.
1-(4-Fluorobenzyl)-1H-benzo[d]imidazole (26)
1
Yield 97%, H NMR (500 MHz, DMSO-d6) d 8.44 (s, 1H), 7.71–
7.66 (m, 1H), 7.57–7.51 (m, 1H), 7.43–7.36 (m, 2H), 7.27–7.13
(m, 4H), 5.50 (s, 2H). 13C NMR (126 MHz, DMSO) d 162.59,
160.65, 144.15, 143.62, 133.55, 133.20, 133.17, 129.62, 129.56,
122.42, 121.61, 119.53, 115.58, 115.40, 110.66, 46.87. 19F NMR
(471 MHz, DMSO) d ꢀ114.63. HRMS (ESI): m/z calcd for
3-Octyl-1-(4-(phenylethynyl)benzyl)-1H-benzo[d]imidazol-3-
ium bromide (21)
Yield 66%. Purity 99.06%. 1H NMR (500 MHz, DMSO-d6) d 9.97
(s, 1H), 8.27–8.07 (m, 1H), 7.94 (dd, J ¼ 7.3, 1.2 Hz, 1H), 7.76–
7.50 (m, 8H), 7.43 (d, J ¼ 2.2 Hz, 3H), 5.82 (s, 2H), 4.51 (t, J ¼
C
14H12FN2[M + H]+: 227.0979, found 227.0973.
1-(4-Chlorobenzyl)-1H-benzo[d]imidazole (27)
1
7.3 Hz, 2H), 2.10–1.73 (m, 2H), 1.41–1.09 (m, 10H), 0.84 (t, J ¼ Yield 92%, H NMR (500 MHz, DMSO-d6) d 8.42 (s, 1H), 7.73–
7.0 Hz, 3H). 13C NMR (126 MHz, DMSO) d 142.4, 134.5, 131.8, 7.62 (m, 1H), 7.55–7.47 (m, 1H), 7.43–7.36 (m, 2H), 7.36–7.26
131.3, 131.2, 130.7, 128.9, 128.7, 128.5, 126.7, 126.6, 122.5, (m, 2H), 7.25–7.15 (m, 2H), 5.50 (s, 2H). 13C NMR (126 MHz,
121.8, 113.8, 113.7, 90.0, 88.5, 49.5, 46.8, 31.0, 28.4, 28.3, 25.7, DMSO) d 144.23, 143.59, 135.98, 133.55, 132.41, 129.30, 128.70,
22.0, 13.8. HRMS (ESI): m/z calcd for C30H33N2[M]+: 421.2638, 122.50, 121.69, 119.56, 110.66, 46.90. HRMS (ESI): m/z calcd for
found 421.2645.
C
14H12ClN2[M + H]+: 243.0684, found 243.0680.
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1-(4-Bromobenzyl)-1H-benzo[d]imidazole (28)
1-Methyl-3-octyl-1H-benzo[d]imidazol-3-ium bromide (33)
1
Yield 62%, H NMR (500 MHz, DMSO-d6) d 8.42 (s, 1H), 7.71– Yield 94%. Purity 99.36%. 1H NMR (500 MHz, CDCl3) d 11.20 (s,
7.63 (m, 1H), 7.57–7.46 (m, 3H), 7.29–7.24 (m, 2H), 7.24–7.15 1H), 7.77–7.73 (m, 1H), 7.71–7.66 (m, 1H), 7.64–7.59 (m, 2H),
(m, 2H), 5.49 (s, 2H). 13C NMR (126 MHz, DMSO) d 144.2, 143.6, 4.55–4.48 (m, 2H), 4.26 (s, 3H), 1.98 (q, J ¼ 7.6 Hz, 2H), 1.38–1.31
136.4, 133.6, 131.6, 129.6, 122.5, 121.7, 120.9, 119.6, 110.7, 46.9. (m, 2H), 1.31–1.22 (m, 2H), 1.22–1.11 (m, 6H), 0.76 (t, J ¼ 7.0 Hz,
HRMS (ESI): m/z calcd for C14H12BrN2[M + H]+: 287.0178, found 3H). 13C NMR (126 MHz, CDCl3) d 142.72, 131.97, 131.02,
287.0173.
127.28, 127.25, 113.10, 112.90, 47.68, 33.86, 31.58, 29.46, 28.92,
28.91, 26.49, 22.47, 13.97. HRMS (ESI): m/z calcd for
C
16H25N2[M]+: 245.2012, found 245.2012.
1-(4-Iodobenzyl)-1H-benzo[d]imidazole (29)
1
Yield 71%, H NMR (400 MHz, DMSO-d6) d 8.40 (s, 1H), 7.77–
1,3-Dioctyl-1H-benzo[d]imidazol-3-ium bromide (34)
7.56 (m, 3H), 7.48 (d, J ¼ 5.7 Hz, 1H), 7.29–6.92 (m, 4H), 5.47 (s,
2H). 13C NMR (126 MHz, DMSO) d 143.7, 143.1, 135.9, 133.0,
131.1, 129.1, 122.0, 121.2, 120.4, 119.1, 110.2, 46.5. HRMS (ESI):
m/z calcd for C14H12IN2[M + H]+: 335.0040, found 335.0041.
Yield 81%. Purity 95.46%. 1H NMR (400 MHz, DMSO-d6) d 9.88
(s, 1H), 8.11 (dd, J ¼ 6.2, 3.1 Hz, 2H), 7.69 (dd, J ¼ 6.3, 3.1 Hz,
2H), 4.49 (t, J ¼ 7.1 Hz, 4H), 1.98–1.78 (m, 4H), 1.47–1.07 (m,
20H), 0.83 (t, J ¼ 6.7 Hz, 6H). 13C NMR (126 MHz, DMSO)
d 142.07, 131.10, 126.55, 113.74, 46.67, 39.52, 31.14, 28.50,
28.45, 28.39, 25.73, 22.04, 13.93. HRMS (ESI): m/z calcd for
1-(4-(Triuoromethyl)benzyl)-1H-benzo[d]imidazole (30)
C
23H39N2[M]+: 343.3113, found 343.3115.
1
Yield 96%, H NMR (500 MHz, DMSO-d6) d 8.45 (s, 1H), 7.79–
7.60 (m, 3H), 7.56–7.43 (m, 3H), 7.28–7.09 (m, 2H), 5.63 (s, 2H).
13C NMR (126 MHz, DMSO) d 144.3, 143.6, 141.8, 133.6, 128.0,
125.6, 125.6, 125.6, 122.6, 121.7, 119.6, 110.6, 47.0. 19F NMR
(471 MHz, DMSO) d ꢀ64.06. HRMS (ESI): m/z calcd for
1-Benzyl-3-octyl-1H-benzo[d]imidazol-3-ium bromide (35)
Yield 67%. Purity 95.83%. 1H NMR (500 MHz, DMSO-d6) d 10.03
(s, 1H), 8.13 (dd, J ¼ 7.2, 1.3 Hz, 1H), 7.98 (dd, J ¼ 7.1, 1.3 Hz,
1H), 7.77–7.61 (m, 2H), 7.58–7.48 (m, 2H), 7.47–7.34 (m, 3H),
5.80 (s, 2H), 4.53 (t, J ¼ 7.3 Hz, 2H), 1.98–1.90 (m, 2H), 1.37–1.17
(m, 10H), 0.91–0.82 (m, 3H). 13C NMR (126 MHz, DMSO) d 142.4,
134.1, 131.3, 130.8, 129.0, 128.7, 128.2, 126.7, 126.6, 113.9,
113.9, 49.8, 46.8, 31.1, 28.5, 28.4, 28.4, 25.8, 22.0, 13.9. HRMS
(ESI): m/z calcd for C22H29N2[M]+: 321.2325, found 321.2326.
C
15H12F3N2[M + H]+: 277.0947, found 277.0942.
1-(4-(tert-Butyl)benzyl)-1H-benzo[d]imidazole (31)
Yield 84%, 1H NMR (500 MHz, DMSO-d6) d 8.41 (s, 1H), 7.66 (d, J
¼ 7.3 Hz, 1H), 7.55 (d, J ¼ 7.3 Hz, 1H), 7.38–7.32 (m, 2H), 7.26–
7.16 (m, 4H), 5.45 (s, 2H), 1.22 (s, 9H). 13C NMR (126 MHz,
DMSO) d 150.1, 134.0, 127.1, 125.4, 122.3, 121.5, 119.5, 110.7,
47.2, 34.2, 31.0. HRMS (ESI): m/z calcd for C18H21N2[M + H]+:
265.1699, found 265.1697.
1-(4-Methylbenzyl)-3-octyl-1H-benzo[d]imidazol-3-ium
bromide (36)
Yield 99%. Purity 99.69%. 1H NMR (400 MHz, DMSO-d6) d 10.13
(s, 1H), 8.12 (d, J ¼ 6.9 Hz, 1H), 8.04–7.94 (m, 1H), 7.65 (m, 2H),
7.44 (d, J ¼ 7.9 Hz, 2H), 7.20 (d, J ¼ 7.8 Hz, 2H), 5.76 (s, 2H), 4.53
(t, J ¼ 7.2 Hz, 2H), 2.27 (s, 3H), 1.92 (m, 2H), 1.38–1.13 (m, 10H),
0.83 (t, J ¼ 6.7 Hz, 3H). 13C NMR (126 MHz, DMSO) d 142.2,
138.1, 131.3, 131.1, 130.7, 129.4, 128.3, 126.6, 126.6, 113.9,
113.9, 49.6, 46.8, 31.1, 28.5, 28.4, 28.4, 25.7, 22.0, 20.7, 13.9.
HRMS (ESI): m/z calcd for C23H31N2[M]+: 335.2482, found
335.2473.
1-([1,10-Biphenyl]-4-ylmethyl)-1H-benzo[d]imidazole (32)
1
Yield 44%, H NMR (500 MHz, DMSO-d6) d 8.45 (s, 1H), 7.70–
7.54 (m, 6H), 7.47–7.32 (m, 5H), 7.26–7.11 (m, 2H), 5.55 (s, 2H).
13C NMR (126 MHz, DMSO) d 144.4, 143.8, 139.8, 136.3, 133.8,
129.1, 128.1, 127.7, 127.2, 126.8, 122.6, 121.8, 119.7, 110.9, 47.4.
HRMS (ESI): m/z calcd for C20H17N2[M + H]+: 285.1386, found
285.1386.
General procedure for generation III of benzimidazolium salt
formation (33–43)
1-(4-Fluorobenzyl)-3-octyl-1H-benzo[d]imidazol-3-ium
bromide (37)
To a solution of corresponding alkylated benzimidazole (22–32)
1
(0.438 mmol) in MeCN (2 mL) was added octyl bromide Yield 81%. Purity 100%. H NMR (500 MHz, DMSO-d6) d 10.00
(1.31 mmol, 3 eq.). The reaction was stirred until LC-MS analysis (s, 1H), 8.12 (dd, J ¼ 7.0, 1.7 Hz, 1H), 8.06–7.94 (m, 1H), 7.74–
showed completion of the reaction (24–48 h). Another 3 equiv- 7.57 (m, 4H), 7.35–7.22 (m, 2H), 5.77 (s, 2H), 4.51 (t, J ¼ 7.3 Hz,
alents of octyl bromide were added if the reaction was not 2H), 1.93 (p, J ¼ 7.6 Hz, 2H), 1.43–1.18 (m, 10H), 0.84 (t, J ¼
completed aer 48 h and stirred for an additional 24 h. MeCN 6.7 Hz, 3H). 13C NMR (126 MHz, DMSO) d 206.5, 163.1, 161.2,
was then evaporated and the resulting solid was triturated with 142.4, 131.3, 130.8, 130.7, 130.3, 130.3, 126.7, 126.7, 115.9,
EtOAc to remove the excess reagents. The resulting white solid 115.8, 113.9, 113.8, 49.1, 46.8, 31.1, 30.7, 28.5, 28.4, 25.8, 22.0,
was ltered, washed with hexane, and dried to afford the cor- 13.9. 19F NMR (471 MHz, DMSO) d ꢀ114.68. HRMS (ESI): m/z
responding benzimidazolium salt (33–43) as a white powder.
calcd for C22H28FN2[M]+: 339.2231, found 339.2232.
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1-(4-Chlorobenzyl)-3-octyl-1H-benzo[d]imidazol-3-ium
1-([1,10-Biphenyl]-4-ylmethyl)-3-octyl-1H-benzo[d]imidazol-3-
bromide (38)
ium bromide (43)
Yield 88% Purity 100%. 1H NMR (500 MHz, DMSO-d6) d 9.99 (s, Yield 97%. Purity 98.66%. 1H NMR (500 MHz, DMSO-d6) d 10.02
1H), 8.21–8.05 (m, 1H), 8.05–7.93 (m, 1H), 7.75–7.64 (m, 2H), (s, 1H), 8.12 (dd, J ¼ 6.5, 2.4 Hz, 1H), 8.03 (dd, J ¼ 6.5, 2.4 Hz,
7.57 (d, J ¼ 8.5 Hz, 2H), 7.53–7.39 (m, 2H), 5.79 (s, 2H), 4.51 (t, J 1H), 7.74–7.60 (m, 8H), 7.46 (t, J ¼ 7.6 Hz, 2H), 7.41–7.32 (m,
¼ 7.3 Hz, 2H), 1.96–1.89 (m, 2H), 1.37–1.20 (m, 10H), 0.84 (t, J ¼ 1H), 5.82 (s, 2H), 4.52 (t, J ¼ 7.3 Hz, 2H), 2.02–1.83 (m, 2H), 1.47–
7.0 Hz, 3H). 13C NMR (126 MHz, DMSO) d 142.9, 133.9, 133.5, 1.18 (m, 10H), 0.91–0.66 (m, 3H). 13C NMR (126 MHz, DMSO)
131.8, 131.3, 130.8, 129.4, 127.2, 127.1, 114.4, 114.3, 49.6, 47.3, d 142.9, 141.0, 139.8, 133.7, 131.8, 131.4, 129.5, 129.4, 128.2,
31.6, 29.0, 28.9, 26.2, 22.5, 14.4. HRMS (ESI): m/z calcd for 127.7, 127.2, 127.2, 114.4, 50.0, 47.3, 31.6, 29.0, 28.9, 26.3, 22.5,
C
22H28ClN2[M]+: 355.1936, found 355.1941.
14.4. HRMS (ESI): m/z calcd for C28H33N2[M]+: 397.2638, found
397.2638.
1-(4-Bromobenzyl)-3-octyl-1H-benzo[d]imidazol-3-ium
bromide (39)
Bacterial strains, culture conditions, and viability assays
Yield 93%. Purity 100%. 1H NMR (500 MHz, DMSO-d6) d 9.88 (s,
1H), 8.12 (dd, J ¼ 7.1, 1.3 Hz, 1H), 7.95 (dd, J ¼ 7.0, 1.3 Hz, 1H),
7.75–7.54 (m, 4H), 7.48 (d, J ¼ 8.5 Hz, 2H), 5.74 (s, 2H), 4.50 (t, J
¼ 7.3 Hz, 2H), 1.92 (d, J ¼ 7.1 Hz, 2H), 1.40–1.15 (m, 10H), 0.85
(t, J ¼ 6.9 Hz, 3H). 13C NMR (126 MHz, DMSO) d 142.9, 133.9,
132.3, 131.8, 131.2, 131.1, 127.2, 127.1, 122.5, 114.4, 114.3, 49.6,
47.3, 31.6, 29.0, 28.9, 26.2, 22.5, 14.4. HRMS (ESI): m/z calcd for
MICs were determined in 96-well microtiter plates. Assays were
conducted in Luria Broth (LB) medium at 37 ꢂC in triplicate.
Bacterial cell density (OD at 600 nm) was measured using
a Fischer Scientic cell density meter model 40. UV-vis and
uorescence spectroscopy experiments were performed on
a Tecan Innite M200 microplate reader.
C
22H28BrN2[M]+: 399.1430, found 399.1424.
Hemolytic assay
Red blood cells in Alserver's solution were centrifuged 10 min at
300 ꢁ g, washed 3 times with PBS buffer and resuspended in
PBS at 2% v/v. In a 96 wells plate was added 195 mL of red blood
cells solution and 5 mL of compound in DMSO, and the plate
was incubated with light agitation for 1 h at 37 ꢂC. The plate was
then centrifuged for 10 min at 300 ꢁ g and 50 mL of the
supernatant of each well was transferred to another plate.
Absorbance was measured at l ¼ 405 nm.
1-(4-Iodobenzyl)-3-octyl-1H-benzo[d]imidazol-3-ium bromide
(40)
Yield 46%. Purity 97.73%. 1H NMR (500 MHz, DMSO-d6) d 9.89
(s, 1H), 8.11 (dd, J ¼ 7.3, 1.3 Hz, 1H), 7.94 (dd, J ¼ 7.2, 1.2 Hz,
1H), 7.86–7.73 (m, 2H), 7.73–7.55 (m, 2H), 7.32 (d, J ¼ 8.4 Hz,
2H), 5.72 (s, 2H), 4.50 (t, J ¼ 7.3 Hz, 2H), 1.98–1.84 (m, 2H), 1.40–
1.14 (m, 10H), 0.84 (d, J ¼ 6.9 Hz, 3H). 13C NMR (126 MHz,
DMSO) d 142.5, 133.4, 131.8, 131.3, 130.8, 130.6, 126.7, 126.6,
122.0, 113.9, 113.8, 49.1, 46.8, 31.1, 28.5, 28.4, 25.8, 22.0, 13.9.
HRMS (ESI): m/z calcd for C22H28IN2[M]+: 447.1292, found
447.1281.
Biolm inhibition assay
S. aureus cells were incubated in LB medium at 37 ꢂC for 5 h and
rediluted in LB medium to the desired nal concentration
(OD600 nm ¼ 0.1–0.15). S. aureus biolms were labeled with Live/
Dead stain aer 12 h incubation with antibiotics in growth
media (LB broth). Negative control: DMSO (nal concentration
not exceeding 10% volume). Positive control: 70% ethanol.
3-Octyl-1-(4-(triuoromethyl)benzyl)-1H-benzo[d]imidazol-3-
ium bromide (41)
1
Yield 71%. Purity 100%. H NMR (400 MHz, DMSO-d6) d 10.05
(s, 1H), 8.14 (d, J ¼ 7.5 Hz, 1H), 7.97 (d, J ¼ 7.5 Hz, 1H), 7.83–
7.58 (m, 6H), 5.92 (s, 2H), 4.52 (t, J ¼ 7.2 Hz, 2H), 2.10–1.81 (m,
2H), 1.27 (d, J ¼ 35.6 Hz, 10H), 0.84 (t, J ¼ 6.6 Hz, 3H). 13C NMR
(126 MHz, DMSO) d 143.1, 139.2, 131.8, 131.3, 129.5, 127.3,
127.2, 126.3, 126.3, 114.4, 114.2, 49.7, 47.4, 31.6, 29.0, 28.9, 26.2,
22.5, 14.4. 19F NMR (471 MHz, DMSO) d ꢀ63.87. HRMS (ESI): m/
z calcd for C23H28F3N2[M]+: 389.2199, found 389.2206.
Biolm staining and confocal laser scanning microscopy
(LSM) analysis
Biolms stained with FilmTracer™ LIVE/DEAD® Biolm
viability kit (Molecular Probes, Life Technologies Ltd.). Briey,
a working solution of uorescent stains was prepared by adding
1 mL of SYTO® 9 stain and 1 mL of PI stain to 1 mL of lter-
sterilized water. Two hundred mL of staining solution were
deposited on each well of a chambered coverglass (8-well), aer
1-(4-(tert-Butyl)benzyl)-3-octyl-1H-benzo[d]imidazol-3-ium
bromide (42)
Yield 83%. Purity 98.83%. 1H NMR (400 MHz, DMSO-d6) d 10.00 15 min at room temperature in the dark, samples were washed
(s, 1H), 8.18–8.08 (m, 1H), 8.08–7.96 (m, 1H), 7.74–7.62 (m, 2H), with sterile saline (0.9% NaCl) from base of the support mate-
7.54–7.37 (m, 4H), 5.73 (s, 2H), 4.51 (t, J ¼ 7.2 Hz, 2H), 1.96–1.76 rial. Then, biolms were examined with a confocal laser
(m, 2H), 1.24 (s, 19H), 0.96–0.67 (m, 3H). 13C NMR (126 MHz, microscope (Leica model TCS SP5; Leica Microsystems CMS
DMSO) d 151.2, 142.3, 131.3, 131.2, 130.9, 128.0, 126.7, 126.6, GmbH, Mannheim, Germany) using a 20ꢁ dry objective (HC PL
125.7, 113.9, 49.5, 46.8, 34.4, 31.1, 31.0, 28.5, 28.4, 25.8, 22.0, FLUOTAR 20.0ꢁ 0.50 DRY). A 488 nm laser line was used to
13.9. HRMS (ESI): m/z calcd for C26H37N2[M]+: 377.2951, found excite SYTO® 9, while the uorescent emission was detected
377.2954.
from 500 to 540 nm. PI was sequentially excited with 561 nm
9428 | RSC Adv., 2020, 10, 9420–9430
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RSC Advances
laser line and its uorescent emission was detected from 600 to
antibiotic-resistance
and
the
biolm
phenotype,
695 nm.
MedChemComm, 2019, 10, 1231–1241.
7 H. C. Flemming, T. R. Neu and D. J. Wozniak, The EPS
matrix: the “house of biolm cells”, J. Bacteriol., 2007, 189,
7945–7947.
8 Y. Z. Nira Rabin, C. Opoku-Temeng, Y. Du, E. Bonsu and
H. O. Sintim, Biolm formation mechanisms and targets
for developing antibiolm agents, Future Med. Chem., 2015,
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9 K. H. Dakheel, R. Abdul Rahim, V. K. Neela, J. R. Al-Obaidi,
T. G. Hun and K. Yusoff, Methicillin-resistant
Staphylococcus aureus biolms and their inuence on
cacterial adhesion and cohesion, BioMed Res. Int., 2016, 1,
1–14.
10 N. K. Archer, M. J. Mazaitis, J. W. Costerton, J. G. Leid,
M. E. Powers and M. E. Shirtliff, Virulence, 2011, 2, 445.
11 S. Sugimoto, F. Sato, R. Miyakawa, A. Chiba, S. Onodera,
S. Hori and Y. Mizunoe, Broad impact of extracellular DNA
on biolm formation by clinically isolated Methicillin-
resistant and -sensitive strains of Staphylococcus aureus,
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Scanning electron microscopy
Bacterial samples were applied to titanium surfaces for 12 hours
in order to form biolms, following by a xation for 1 h at 4 ꢂC
in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M
phosphate buffer (PB), pH 7.3, and subsequently rinsed three
times with PB. Following xation, samples were incubated for 1
hour in 1% osmium tetroxide at RT and then dehydrated
through an ethanol series (30%, 50%, 70%, 90%, 95% and two
times 100%) followed by drying using a Critical Point Drier
CPD300 (Leica Biosystems, Concord, ON, Canada). A JEOL JSM-
7400F (JEOL Ltd, Tokyo, Japan) eld-emission scanning elec-
tron microscope (FE-SEM) operated at 1.5 kV was used to image
the samples.
Conflicts of interest
There are no conicts to declare.
12 D. Borisova, E. Haladjova, M. Kyulavska, P. Petrov, S. Pispas,
S. Stoitsova and T. Paunova-Krasteva, Application of cationic
polymer micelles for the dispersal of bacterial biolms, Eng.
Life Sci., 2018, 18, 943–948.
13 Z. Jing, K. Xiu and Y. Sun, Amide-based cationic polymeric N-
halamines: synthesis, characterization and antimicrobial
and biolm-binding properties, Ind. Eng. Chem. Res., 2019,
58, 6218–6225.
14 P. Zhang, S. Li, H. Chen, X. Wang, L. Liu, F. Lv and S. Wang,
Biolm inhibition and elimination regulated by cationic
conjugated polymers, ACS Appl. Mater. Interfaces, 2017, 9,
16933–16938.
Acknowledgements
We gratefully acknowledge the Natural Sciences and Engi-
neering Research Council of Canada (NSERC) and the Uni-
´
´
versite de Montreal for nancial support. We thank J. N.
´
Pelletier and K. J. Wilkinson from the Departement de Chimie-
´
´
Universite de Montreal for access to their laboratories and
instruments. We thank A. Fouillen (PhD) from the electron
microscopy platform of the University of Montreal for the SEM
preparation and analysis of the samples.
15 B. C. Koppen, P. P. G. Mulder, L. de Boer, M. Riool,
J. W. Drijout and S. A. J. Zaat, Synergistic microbicidal
effect of cationic antimicrobial peptides and teicoplanin
against planktonic and biolm-encased Staphylococcus
aureus, Int. J. Antimicrob. Agents, 2019, 53, 143–151.
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