Amphipathic sulfonamidobenzamides mimicking small antimicrobial marine natural products; investigation of antibacterial and anti-biofilm activity against antibiotic resistant clinical isolates

Article abstract of DOI:10.1016/j.bmc.2018.08.032

There is an urgent need for novel antimicrobial agents to address the threat of bacterial resistance to modern society. We have used a structural motif found in antimicrobial marine hit compounds as a basis for synthesizing a library of antimicrobial sulf

Full text of DOI:10.1016/j.bmc.2018.08.032

Accepted Manuscript
Amphipathic sulfonamidobenzamides mimicking small antimicrobial marine
natural products; investigation of antibacterial and anti-biofilm activity against
antibiotic resistant clinical isolates
Elizaveta M. Igumnova, Ekaterina Mishchenko, Tor Haug, Hans-Matti Blencke,
Johanna U. Ericson Sollid, Elizabeth G. Aarag Fredheim, Silje Lauksund, Klara
Stensvåg, Morten B. Strøm
PII:
S0968-0896(18)30897-6
https://doi.org/10.1016/j.bmc.2018.08.032
BMC 14519
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 May 2018
22 August 2018
26 August 2018
Please cite this article as: Igumnova, E.M., Mishchenko, E., Haug, T., Blencke, H-M., Sollid, U.E., Fredheim, G.A.,
Lauksund, S., Stensvåg, K., Strøm, M.B., Amphipathic sulfonamidobenzamides mimicking small antimicrobial
marine natural products; investigation of antibacterial and anti-biofilm activity against antibiotic resistant clinical
isolates, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.08.032
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1
Manuscript for submission
Amphipathic sulfonamidobenzamides mimicking small antimicrobial marine natural
products; investigation of antibacterial and anti-biofilm activity against antibiotic
resistant clinical isolates
Elizaveta M. Igumnova a,†, Ekaterina Mishchenko b,†, Tor Haug b, Hans-Matti Blencke b, Johanna U.
Ericson Sollid c, Elizabeth G. Aarag Fredheim a, Silje Lauksund a, Klara Stensvåg b, Morten B. Strøm a,*
a Department of Pharmacy, Faculty of Health Sciences, UiT – The Arctic University of Norway, NO-9037 Tromsø, Norway
b The Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, UiT – The Arctic University of Norway, NO-9037 Tromsø,
Norway
c Department of Medical Biology, Faculty of Health Sciences, UiT – The Arctic University of Norway, NO-9037 Tromsø, Norway
† These authors contributed equally to this work.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
There is an urgent need for novel antimicrobial agents to address the threat of bacterial resistance
to modern society. We have used a structural motif found in antimicrobial marine hit compounds
as a basis for synthesizing a library of antimicrobial sulfonamidobenzamide lead compounds.
Potent in vitro antimicrobial activity against clinically relevant bacterial strains was demonstrated
for two compounds, G6 and J18, with minimal inhibitory concentrations (MIC) of 4–16 g/ml
against clinical methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant
Enterococcus faecium (VRE). The two compounds G6 and J18, together with several other
compounds of this library, also caused ≥90% eradication of pre-established biofilm of methicillin-
resistant S. epidermidis (MRSE) at 40 g/ml. Using a luciferase assay, the mechanism of action
of G6 was shown to resemble the biocide chlorhexidine by targeting the bacterial cell membrane.
Available online
Keywords:
Antibacterial
Antimicrobial
ESBL – CARBA
Marine natural product mimics
Multi-resistant bacteria
Peptidomimetics
Sulfonamidobenzamides
formed the motivating hit molecules for the design, such as
ianthelline4 and the synoxazolidinones.5 This strategy has also
been successfully explored in synthesis of amphipathic 1,2,3-
1.Introduction
Development of novel antimicrobial drugs is a high-risk
business due to the general massive costs of any drug R&D
program, potential limited sales volume, and restricted use of any
innovative antimicrobial to avoid development of resistance.1
Most antimicrobial drugs on the market interfere with highly
specific targets in bacteria, and few novel unique targets have
been identified. Design of antimicrobial agents killing bacteria
through interactions with non-specific targets can be a valuable
strategy to encounter the challenges of antimicrobial resistance.
Cationic antimicrobial peptides (AMPs) have gained much
attention in recent decades since they attack the cell membrane of
bacteria.2 The pharmacokinetics of AMPs may however disfavor
them as drugs because of poor bioavailability and low proteolytic
stability.
triazole MNPM antimicrobials.6,7
Synoxazolidinone A
E23
We have previously prepared a library of small cationic
amphipathic aminobenzamides based on a common structural
motif found in several isolated marine antimicrobials and that
may also be considered as peptidomimetics of small AMPs
(Figure 1).3 The pharmacophore or active motif of this class of
antimicrobial marine natural product mimics (MNPMs) was
explored by investigating a central benzamide group linked to a
lipophilic 3,5-di-tert-butyl-benzyl group and various cationic
groups through amide bonds. Improved antimicrobial activity
was achieved compared to the marine natural products that
Ianthelline
Figure 1. Isolated cationic amphipathic marine natural products
synoxazolidinone A5 and ianthelline,4 and the synthetic
aminobenzamide marine natural product mimic E23.3

2
In the present study we report a library of related molecules
that contain a sulphonamide linker between the lipophilic and
central spacer group, and we have further explored the
reference strains of Gram-positive Staphylococcus aureus,
methicillin resistant Staphylococcus epidermidis (MRSE),
Bacillus subtilis and Corynebacterium glutamicum, and Gram-
negative Escherichia coli and Pseudomonas aeruginosa. These
studies were followed up by screening promising compounds
from the G- to L-series against 275 clinical isolates deposited at
the Norwegian Organization for Surveillance of Resistant
Microorganisms (NORM) (2012-2014), and a panel of 30 multi-
resistant clinical isolates from the strain collection at The
Norwegian National Advisory Unit on Detection of Antimicrobial
Resistance.8-10 Promising compounds were also screened for their
ability to eradicate preformed polysaccharide biofilm of MRSE
and the mode-of-action was determined for two representative
compounds.
importance of the central spacer group by varying the
substitution pattern, and also including aliphatic spacer groups
(Figure 2). The sulphonamide group is bioisosteric to an amide
group but has also other characteristics, such as different
conjugation properties and contains an acidic proton with
possibility of ionization. An expanded panel of lipophilic groups
were included together with the promising 3,5-di-tert-butyl-
benzyl group. Primary amine and guanidine groups were used as
cationic groups and varied in distance by two or three methylene
groups in accordance to our previous results.3 A structure-activity
relationship (SAR) study was done by screening against bacterial
Amine derivatives:
Guanidine derivatives:
G1
G2
G3
G4
G5
G6
H7
I11
H8
I12
H9
I13
H10
114
J15
J16
J17
J18
Variations in spacer group – guanidine derivatives:
K19
K20
K21
K22
Variations in lipophilic group – guanidine derivatives of G2:
L23
L24
L25
L26
Figure 2. Library of sulfonamidobenzamides synthesised and investigated for antimicrobial activity. All compounds were isolated and tested as
HCl salts.

3
(non-methylated guanidine) with the more potent G5 (methylated
guanidine) against P. aeruginosa. The chemical implications of
methylation of the sulphonamide group are first of all abolishing
its ability to ionize. The sulphonamide proton of G1 is weakly
acidic (calculated pKa 7.71, ChemBioDraw version 13.0.2) and
G1 is likely ~30% ionized at physiological conditions (pH 7.4).
Methylation increases overall lipophilicity and may also have an
impact on overall spatial structure, i.e., by restricting rotation and
forcing the two adjacent aromatic rings of the lipophilic and
spacer groups out of plane.
Results and discussion
2.1 Synthesis
An outline for synthesis of the library of
sulfonamidobenzamides is shown in Scheme 1. For all
compounds except K22 the first step was formation of a
sulphonamide bond linking the lipophilic group to the spacer
group by reaction of the corresponding sulfonyl chlorides with
the appropriate amines. The reactions were conducted without
solvent at 150 C and with 2 equivalents of the appropriate
amines. In case of secondary amines; 1 equivalent of NaHCO3
was added. The amide bond between the spacer group and
cationic group was formed by amidation of the ester group with
the appropriate aliphatic di-amines. Guanidine derivatives were
prepared by reaction with 1-amidino-1H-1,2,4-triazole
hydrochloride. In order to ensure optimal solubility in aqueous
test media, the final compounds were isolated as HCl salts and
purified by recrystallisation from MeOH and Et2O.
O
O
O
H2N
CH3
t-Bu
S
1
Cl
O
+
t-Bu
Lipophilic group
Spacer group
O
O
O
S
NH
t-Bu
CH3
O
2
3
2.2 Antimicrobial activity against bacterial reference strains and
toxicity against human cells
t-Bu
O
O
The library of compounds constituted six different structural
series (G – L) as governed by the structures of the central spacer
groups (Figure 2). Minimum inhibitory concentrations (MICs)
were initially determined against a panel of Gram-positive and
Gram-negative bacterial reference strains (Table 1). Hemolytic
activity (EC50) against human red blood cells (RBCs) and
cytotoxicity (EC50) against normal human lung fibroblasts
O
S
NH
t-Bu
NH2
N
H
n
t-Bu
Amine derivatives
(See Fig. 2 for complete structures)
O
O
O
(MRC-5) cells were used as a measurement of toxicity.11
selectivity index SI was calculated by comparing toxicity against
RBC and MIC against MRSE (EC50 RBC / MICMRSE).
A
H
S
NH
t-Bu
N
NH
N
H
n
NH2
As observed for the previously reported aminobenzamides,3
the results showed in general higher antimicrobial activity against
Gram-positive strains than against Gram-negative strains.
Against Gram-positive strains the most potent compounds
displayed MIC values of 1–4 µg/ml. Lower activity was obtained
against Gram-negative strains with MIC values of 8–16 µg/ml for
the most potent derivatives (Table 1).
t-Bu
Guanidine derivatives
(See Fig. 2 for complete structures)
O
CH3
O
Variations in the
lipophilic group:
Variations in the
spacer group:
HN
R1
A positive effect of guanylation on antimicrobial activity
against Gram-positive strains was revealed by the G-series. The
G-series contained a central 4-aminobenzamide spacer group and
the guanidine derivatives G2, G5 and G6 clearly displayed a
higher antimicrobial activity (MIC: 2–8 µg/ml) against Gram-
positive strains than the corresponding amine derivatives G1, G3
and G4 (MIC: 8–32 µg/ml). The effect of guanylation was less
obvious against Gram-negative strains and relatively small
variations in antimicrobial potencies were observed (MIC: 16–64
µg/ml).
Another important implication of guanylation that was
obvious throughout the G- to L-series was lower RBC toxicity
and MRC-5 cell cytotoxicity of the guanylated compounds
compared to the amine derivatives (Table 1). An exception was
G3 (amine), which was less hemolytic than its guanidine
counterpart G5. For all other amine and guandidine analogues of
the G- to L-series the guanidine derivatives were less toxic than
the amines.
O
O
O
O
O
O
CH3
CH3
O
H3C
S
S
O
H2N
H2N
CH3
R1
HN
NH2
O
O
O
CH3
F3C
CH3
O
O
CF3
O
O
O
NH2
O
CH3
CH3
S
O
H2N
CH3
O
O
O
O
H
CH3
CH3
t-Bu
H3C
S
N
O
O
HN
O
t-Bu
A structural variation within the G-series was methylation of
the sulphonamide group that resulted in improved antimicrobial
activity against Gram-negative strains but had less effect on
improving activity against Gram-positive strains. All the
compounds G3, G4, G5, and G6 had a methylated sulphonamide
group, whereas G1 and G2 were non-methylated. The positive
effect of methylation of the sulphonamide group was evident
when comparing G1 (non-methylated amine derivative) with G3
(methylated amine derivative) where the activity against E. coli
was improved by methylation. Similarly, when comparing G2
Scheme 1. Reactants and conditions are shown in the scheme for
synthesis of sulfonamidobenzamides investigated for antimicrobial
and antibiofilm activity, and together with variations in spacer and
lipophilic groups (R1 = H or CH3). Step 1. Sulfonyl chloride was
reacted with 2 equivalents of the appropriate amines at 150 C
(thermal fusion) for 1 h. 1 equivalent of NaHCO3 was added when
using secondary amines. Step 2. Excess of H2N-CH2(CH2)nNH2 (n =
1, 2, or 3) stirred at 100 C for 18 h. Step 3. 1-Amidino-1H-1,2,4-
triazole hydrochloride in DMF at room temperature for 18 h.

4
toxicity against RBC and MRC-5 cells (EC50: ≥92 and SI: 12).
As observed for the G-series, the guanylated compounds I13 and
I14 showed both higher overall antimicrobial activity and lower
RBC and MRC-5 cell toxicity compared to the amine analogues
I11 and I12.
The non-methylated derivative G1 was less toxic against RBC
and MRC-5 cells than its methylated analogue G3, and similarly
was G2 (non-methylated) less toxic than G5 (methylated
analogue). The hemolytic activity within the G-series varied with
EC50 values of 28–64 µg/ml and against MRC-5 with EC50 values
of 5–>100 µg/ml. This resulted in the guanylated G2 having the
highest SI: 16 within the G-series. The two guanylated and
methylated derivatives G5 and G6 displayed also high SI of 11–
14 that was close to the SI: 16 obtained for G2.
Varying the length of the methylene chain between the spacer
group and cationic group was ineffective with respect to
antimicrobial activity. Almost identical MIC values were
obtained for G3 and G4, which differed in cationic side-chain
length by a methylene group. This was also observed for G5 and
G6. A short ethylene chain was therefore selected for the
following H- to L-series except for K22.
Exploring 1,2-substitution of the aromatic spacer in the J-
series did not result in further improvement in antimicrobial
potency for J15 and J17 compared to the previous 1,3-
substitution of the I-series. Compounds J15 and J17 had a 2-
aminobenzamide spacer group, in which the guanylated 2-
aminobenzamide J17 (MIC: 4–8 µg/ml) was clearly more potent
than the corresponding amine derivative J15 (MIC: 16–32
µg/ml) against Gram-positive strains. Furthermore, J17 displayed
lower hemolytic activity and MRC-5 cell cytotoxicity than J15,
and the advantage of guanylation was thereby once more
demonstrated by J17 (guanidine) compared to J15 (amine).
Increasing lipophilicity and size by having a 3-sulfonamido-2-
naphthamide spacer group in J16 and J18 improved
Increasing flexibility by introducing a methylene group in the
H-series was of little benefit for improving antimicrobial potency
compared to the G-series. The flexible H-series displayed
however very low hemolytic activity with EC50 values of 73–113
µg/ml. The H-series differed structurally from the G-series by
having an additional methylene group positioned either between
the sulphonamide functionality and the spacer group (H7 and
H9), or between the spacer group and the carboxamide
functionality (H8 and H10). The MIC values varied from 4–32
µg/ml against the Gram-positive strains, and from 32–64 µg/ml
against the Gram-negative strains. The guanidine derivatives H9
and H10 displayed highest antimicrobial activity with MIC
values of 4–16 µg/ml against Gram-positive strains, and low
toxicity against RBCs resulting in SI: 6–7. The guanylated
derivatives H9 and H10 (EC50: 55–>100 µg/ml) were also clearly
less cytotoxic to MRC-5 cells than their amine analogues H7 and
H8 (EC50: 20 µg/ml). Introduction of a spatial methylene group
in the H-series increases the flexibility of the compounds and
may also disrupt -bond interactions between the functional
sulphonamide and carboxamide groups connected to the aromatic
spacer group. Clearly, this was not beneficial for antimicrobial
activity, but advantageous for reducing toxicity against RBC and
MRC-5 cells.
Apparently 1,3-substitution of the aromatic spacer group in
the I-series was more favorable than 1,4-substitution of the G-
series, especially when evaluating toxicity (Figure 2). No
increased hemolytic activity was observed following methylation
of the sulphonamide group in I13 and I14 compared to what was
observed for the previous G-series. Compounds of the I-series
had a 3-aminobenzamide spacer group and displayed MIC: 2–16
µg/ml against Gram-positive strains and MIC: 16–64 µg/ml
against Gram-negative strains. The most potent compound of the
I-series was the methylated and guanylated derivative I14 with
MIC: 2–4 µg/ml against the Gram-positive strains. I14 thereby
displayed similar high antimicrobial activity against the Gram-
positive strains as the most potent compound G6 of the first
series, but much lower toxicity against RBC and MRC-5 cells
than G6. Structurally it’s important to emphasize that I14 had a
methylated sulphonamide group, which in the G-series gave
increased RBC and MRC-5 cell toxicity. Similar increased
toxicity following methylation was thus not observed for I14 and
it thereby had the second highest SI: 24 of all studied
antimicrobial activity against the Gram-negative strains, and
especially against E. coli (MIC: 8 µg/ml). High antimicrobial
activity against Gram-positive strains was also maintained (MIC:
2–4 µg/ml). RBC toxicity was however high for J16 and J18
with EC50: 21–25 µg/ml that resulted in low SI: 5–6. The 3-
sulfonamido-2-naphthamide derivatives J16 and J18 were more
potent than J17 against the Gram-positive strains and E. coli. The
positive effect of guanylation was however lost by the increased
lipophilicity provided by the 3-sulfonamido-2-naphthamide
spacer group. Further optimizations are needed for compounds
with the naphthalenic spacer group to reduce RBC toxicity.
Toxicity against MRC-5 cells was less affected by the
naphthalenic spacer group in J16 and J18. This resulted in an
important observation; for the whole G to L-series toxicity
against MRC-5 cells of the amine derivatives was within a
narrow range of EC50: 5–25 µg/ml, whereas toxicity against
MRC-5 cells for the guanidine derivatives was EC50: 42–>100
µg/ml. In comparison, hemolytic activity of both the amine and
guanidine derivatives varied more, displaying EC50values of 21–
92 µg/ml for the amine derivatives, and 25–>100 µg/ml for the
guanidine derivatives. I.e. the differences in EC50 values between
amine and guanidine derivatives were larger against the
nucleated MRC-5 cells compared to the non-nucleated RBCs.
Since compounds with an amine group in general diffuse more
easily across cell membranes than compounds with a guanidine
group, this observation may imply that the amine derivatives may
reach additional intracellular targets in nucleated cells such as
MRC-5 cells explaining the higher MRC-5 cell cytotoxicity of
the amine derivatives. The differences in diffusion rate is
attributed to the general lower average pKa value and lower
ionization state of amines, favoring a higher proportion un-
ionized amine, compared to guanidines being fully ionized at
physiological conditions.
Alteration of the central spacer group showed a positive effect
on reducing RBC toxicity. The K-series contained a variety of
spacer groups and all compounds were guanylated. Compound
K19 had an additional methyl substituent on the aromatic spacer
group. Compared to G5 having a methylated sulphonamide
group, K19 was comparable potent against the Gram-positive
strains (MIC: 4–8 µg/ml) and E. coli (MIC: 32 µg/ml).
Compound K19 showed also lower hemolytic activity and MRC-
5 cell cytotoxicity than G5. Strangely K19 was inactive against
P. aeruginosa as opposed to G5. As for methylation of the
sulphonamide group in G5, the methyl group in K19 may
similarly restrict rotation by introducing steric hindrance and
increase overall lipophilicity.
compounds. A repulsive steric effect or less optimal conjugation
between the methylated sulphonamide and amide functionalities
in the case of 1,3-substitution could be a plausible reason for this
beneficial shift in activity of I14. The second most potent
derivative against the Gram-positive strains within the I-series
was the guanylated I13 (MIC: 4–8 µg/ml) that also displayed low

5
Introduction of aliphatic cyclic spacer groups resulted in low
antimicrobial activity. Compound K20 contained an aliphatic
cyclic piperidine-4-carboxamide spacer group and K21 an L-
proline spacer group (Figure 2). Both K20 and K21 showed low
antimicrobial activity except against C. glutamicum. The low
antimicrobial activity correlated also with no detectable RBC
toxicity or MRC-5 cell cytotoxicity for K20 and K21. The low
antimicrobial activity following introduction of aliphatic cyclic
spacer groups demonstrated the superiority of the previous
aromatic spacer groups investigated.
although RBC toxicity was reduced in the H-series. Aromatic
spacer groups were more efficient for high antimicrobial activity,
whereas cyclic aliphatic spacers such as piperidine-4-
carboxamide (K20) or L-proline (K21) were less efficient.
Clearly, a large size of the lipophilic group mattered as follows
from comparison of G2 and the L-series. Of special interest was
K22, which had no cyclic spacer at all, but to our surprise
showed good antimicrobial activity and low RBC toxicity.
Compared to the previous reported aminobenzamide E23,3 both
the novel promising compounds I14 and K19 showed
To our surprise the highly flexible derivative K22 having no
spacer group displayed high antimicrobial activity against Gram-
positive strains (MIC: 4–16 µg/ml) and low hemolytic activity
(EC50: 110 µg/ml and SI: 14). Although cytotoxicity against
MRC-5 cells was observed (EC50: 42 µg/ml), the highly flexible
K22 is an attractive model compound for synthesis of mimics of,
e.g., the marine antimicrobial ianthelline4, which has a flexible 2-
(hydroxyimino)acetamide spacer group linking its lipophilic and
cationic groups (Figure 1).
antimicrobial activity in the same range against MRSE (Table 1).
Importantly, both I14 and K19 were less hemolytic than E23 and
had a higher SI: 24 for I14 and SI: 26 for K19 than E23 (SI: 19).
We therefore consider the present sulfonamidobenzamides as a
promising class of antimicrobials for further optimization studies.
The L-series showed that reducing the lipophilicity and
bulkiness of the lipophilic group decreased antimicrobial activity
compared to the structurally related G2 as reference. The L-
series contained a 4-aminobenzamide spacer-group connected to
four different lipophilic groups (Figure 2). The L-series showed
highly variable MIC: 32–≥128 µg/ml for the least lipophilic
members L23, L24 and L25, and with no antimicrobial activity
against Gram-negative strains. No cytotoxicity against MRC-5
cells was observed for any of the gyanylated derivatives in the L-
series. Compound L26 was most structural similar to G2 and had
an additional 4-methoxy-group that increased overall
lipophilicity and bulkiness. The two compounds L26 and G2
showed similar high antimicrobial activity against Gram-positive
strains (MIC: 4–8 µg/ml), but both encountered solubility
challenges, as observed when evaluating RBC toxicity
(precipitations above 64 µg/ml in the hemolytic assay).
In summary, the SAR study against bacterial reference strains
showed that the guanylated analogues were both more potent and
less toxic against RBC and MRC-5 cells than the amine
analogues as first shown by the G-series (Figure 3). Methylation
of the sulphonamide group had different effects on antimicrobial
activity and toxicity depending on the substitution pattern of the
spacer group, as shown when comparing the G- and I-series.
Increased flexibility did not improve antimicrobial activity,
Figure 3. Summary of SAR resulting in high antimicrobial activity
of synthesised sulfonamidobenzamides (exemplified by G6). *Lower
toxicity against RBC and MRC-5 cells was observed for guanidine
compared to amine derivatives. 2*An exception to the low
antimicrobial potency of compounds with an aliphatic spacer group
was K22, which had no spacer group and still displayed high
antimicrobial activity against Gram-positive bacteria and low
hemolytic activity.

6
Table 1. Antimicrobial activity (MIC in µg/ml) against bacterial reference strains and hemolytic activity against human RBCs (EC50 in µg/ml)
of synthesized amphipathic sulfonamidobenzamides.
Cationic
Series Comp group
Antimicrobial activity a - µg/ml
RBC
MRC-5
EC50
25
>100
5
9
36
36
20
20
>100
55
16
10
>100
91
10
22
42
SI c
RBC/MRSE
2
16
3
2
11
14
6
Mw b MRSE
468.1 32
S. aureus B. subtilis C. glutamicum E. coli P. aeruginosa EC50
G1
Amine
32
4
16
8
8
8
4
4
8
16
8
8
8
8
8
4
32
4
8
4
8
8
4
16
8
2
2
8
16
4
8
8
8
4
2
16
2
4
2
4
64
32
16
16
32
16
32
64
32
64
32
16
32
32
64
8
64
64
32
32
16
16
32
64
32
64
64
32
32
32
128
32
64
63
G2
Guanidine 510.1
4
64d
53
G3
G4
G5
G6
H7
H8
H9
482.1 16
496.1 16
16
16
4
G
Amine
28
45
55
92
524.1
538.2
4
4
Guanidine
Amine
4
482.1 16
482.1 16
32
32
16
16
16
16
8
4
32
4
73
5
H
I
524.1
524.1
468.1
8
8
8
113
101
58
26
92
95
56
21
77
14
13
7
Guanidine
Amine
H10
I11
I12
I13
I14
J15
J16
J17
J18
K19
K20
K21
K22
L23
L24
L25
L26
482.1 16
2
510.1
524.1
8
4
12
24
2
5
10
6
26
8
>4
14
>2
-
>4
16
19
Guanidine
Amine
468.1 32
518.1
510.1
560.2
524.1
4
8
4
4
J
4
4
8
32
8
Guanidine
32
25
55
56
32
64
>128
64
>128
>128
>128
32
8
>128
128
128
64
>128
>128
>128
64
104
124
>128
110
>128
64d
>128
64d
37
502.1 16
488.1 32
16
64
8
128
>128
64
4
16
32
16
64
>128
64
4
4
8
4
>100
>100
42
>100
>100
>100
>100
22
K
L
Guanidine
Guanidine
419.0
8
425.9 64
533.9 >128
447.9 32
64
>128
32
4
540.1
4
2
E23 e Guanidine 474.1
1
1
0.5
16
a Staphylococcus aureus ATCC 9144; methicillin resistant Staphylococcus epidermidis RP62A (MRSE),50 CCUG 31568 (ATCC 35984); Bacillus subtilis 168,18
laboratory collection (ATCC 23857); Corynebacterium glutamicum ATCC 13032; Escherichia coli ATCC 25922 and Pseudomonas aeruginosa PA01, DSM
19880 (ATCC 15692). b Mw including 1 eq. of HCl. c Selectivity index (SI) calculated as the EC50 value against RBC divided by the MIC value against MRSE. d
Precipitation observed in test buffer used for RBC determination at higher concentrations. e Antimicrobial activity and RBC toxicity previously reported for the
synthetic cationic amphipathic aminobenzamide E23 (Figure 1).3
Table 2. Antimicrobial activity (MIC in g/ml) against 25 randomly
2.3 Antimicrobial profile against clinical isolates
chosen clinical isolates. MIC90 is the concentration of the compounds
that inhibited ≥90% of all clinical isolates when screened against 50
additional isolates within each group of bacteria (in total 250
isolates).
We continued addressing clinical relevance by screening 15 of
the most potent sulfonamidobenzamides from all six series
against 25 clinical isolates (Table 2). Gram-positive bacteria
were represented by S. aureus and Enterococcus spp., and Gram-
negative bacteria were E. coli, P. aeruginosa, and Klebsiella
pneumoniae. Five isolates of each group of bacteria were used.
The antimicrobial activity against the clinical isolates was in
general similar or a little lower (by one titer step) compared to
the initial screening against the reference strains (Table 2 – top
section). The overall most potent and broad-spectrum compounds
against the 25 clinical isolates were G6 and J18. The activity
against the Gram-positive clinical isolates was MIC: 4–32 g/ml,
and MIC: 8–128 µg/ml against the Gram-negative clinical
isolates. Relatively high antimicrobial activity against clinical
isolates of Enterococcus spp. was also maintained for compounds
G6, I14, J16, and J18 (MIC: 8–16 µg/ml). The effect of the same
compounds against clinical isolates of K. pneumonia were
however variable (MIC: 16–128 g/ml).
MIC - µg/ml
S.
aureus
E.
coli
P.
K.
Enterococcus
spp.
Comp
aeruginosa pneumoniae
No. of
isolates:
G2
G5
G6
5
5
5
5
5
8
4
4
32
32
8
>128
32
16
>128
16
8
>128
64
32
H9
H10
I13
I14
J15
J16
J17
J18
16
16
8
8
32
8
64
32
32
16
32
8
128
64
64
32
128
32
64
64
64
32
128
32
32
128
128
128
64
128
128
128
16
8
4
32
8
64
8
64
K19
K22
L25
L26
8
32
32
>128
64
64
64
64
>128
32
>128
128
>128
>128
16
128
8
128
>128
>128
The two most broad-spectrum compounds G6 and J18 were
furthermore tested against an expanded panel of additional 50
clinical isolates of each bacterial group (250 isolates; lower
section Table 2). MIC90 values, i.e., the minimum concentrations
that inhibited 90% of these isolates (45 out of 50 isolates), were
in line with the preceding screening results (Table 2 – top
section). Thus, MIC90 against the Gram-positive isolates did not
exceed 8 g/ml for J18 and 16 g/ml for G6. Against the Gram-
negative isolates, only J18 had MIC90 higher than 32 g/ml
against P. aeruginosa. Thus, the antimicrobial activity of G6 and
J18 against the reference strains was confirmed when tested
against the 275 clinical isolates.
MIC90 - µg/ml
No. of
isolates:
G6
50
50
50
50
50
4
4
16
8
16
8
16
>32
32
32
J18

7
2.4 Antimicrobial activity against 30 multi-resistant isolates
The clinical relevance of G6 and J18 was finally tested by
determination of their antimicrobial activity against a panel of 30
multi-resistant clinical isolates from the strain collection at The
Norwegian National Advisory Unit on Detection of Antimicrobial
Resistance.8-10 (Table 3). Included in the screening were
methicillin resistant S. aureus (MRSA), vancomycin resistant
enterococci (VRE), and Gram-negative bacteria producing
extended spectrum -lactamases–carbapenemases (ESBL–
CARBA). As shown in Table 3 compounds G6 and J18 were
potent against MRSA and VRE (MIC: 4–16 µg/ml) but lost their
activity against the majority of the Gram-negative isolates. The
P. aeruginosa isolates were still comparatively susceptible to G6
with MIC: 32 g/ml and J18 had similar MIC: 32 g/ml against
the included Acinetobacter baumannii isolates.
polysaccharide biofilm of MRSE (Table 4). The threshold was
set as ≥90% biofilm eradication, which considers the non-
specific binding of growth medium components. The compounds
were tested with main emphasis to determine whether this class
of amphipathic sulfonamidobenzamides could eradicate MRSE
biofilm on a general basis.
Overall, the results showed that this class of small
amphipathic molecules was able to enter into the complex
biofilm matrix, presumably kill the bacteria, and effectuate the
disintegration of the biofilm. The results revealed that an overall
5–10 times higher concentration than the MIC value of individual
derivatives against planktonic MRSE was required for achieving
≥90% biofilm eradication, which corresponded to concentrations
of 40–80 µg/ml of the compounds tested (Table 4). However, due
to variation between individual biofilm eradication experiments,
it was challenging to differentiate between individual compounds
with respect to SAR on biofilm eradication. The challenges with
reproducibility of the results and complexity of eradicating
biofilms produced by MRSE may be explained by the complex
biofilm matrix and phenotypic variation reported for the MRSE
strain.27,28 However, when evaluating the combined effects of
high antimicrobial activity against planktonic MRSE (i.e. MIC
≤4 µg/ml), a maximum concentration of 40 µg/ml for ≥90%
biofilm eradication, and low RBC toxicity (EC50: >90 µg/ml), the
most promising compounds for biofilm treatment were I14 and
K19. If a higher MIC (8 µg/ml) against planktonic MRSE is
tolerated, compounds H10, I13 and K22 can be added to this list
of selected compounds for further optimizations.
Table 3. Antimicrobial activity (MIC in µg/ml) of G6 and J18
against 30 multi-resistant isolates.
Multi-resistant isolate
G6
J18
4
ESBL-CARBAa
S. aureus N315
8
S. aureus NCTC 10442
S. aureus strain 85/2082
S. aureus strain WIS
S. aureus IHT 99040
E. faecium 50673722
E. faecium 50901530
E. faecium K36-18
E. faecium 50758899
E. faecium TUH50-22
E. coli 50579417
8
8
8
8
16
16
16
8
8
8
8
8
8
8
8
8
16
4
≥32 ≥32 OXAb-48
E. coli 50639799
E. coli 50676002
E. coli 50739822
E. coli 50857972
32
≥32 VIMc-29
Table 4: Antimicrobial activity (MIC, µg/ml) compared to anti-
biofilm activity (concentration required for ≥90% eradication of
biofilm, µg/ml) of selected compounds against MRSE.
≥32 >32 NDMd-1
≥32 >32 NDM-1
32
>32 IMPe-26
MIC a
S. epiderimidis
4
4
4
8
8
8
≥90% Biofilm eradication b
RBC a
EC50
64
45
55
113
101
92
P. aeruginosa K34-7
P. aeruginosa K34-73
P. aeruginosa K44-24
P. aeruginosa 50692172
P. aeruginosa 50692520
K. pneumoniae K47-25g
K. pneumoniae K66-45
K. pneumoniae 50531633g
K. pneumoniae 50625602
K. pneumoniae 50667959
A. baumannii K12-21
A. baumannii K44-35
A. baumannii K47-42
A. baumannii K55-13
A. baumannii K63-58g
>32 >64 VIM-2
Comp
G2
G5
G6
H9
µg/ml
40
40
40
80
x MIC
10
10
10
10
5
32
32
32
32
≥32 VIM-4
>64 IMP-14
>64 NDM-1
≥64 VIM
≥32 >32 KPCf-2
32 32 NDM-1
H10
I13
40
40
5
≥32 >32 NDM-1+OXA-181
≥32 ≥32 OXA-245
≥32 ≥32 VIM-1
I14
4
4
4
16
8
40
40
40
80
40
160
40
10
10
10
5
5
5
95
25
104
124
110
>128
64
J18
K19
K20
K22
L25
L26
≥32 32
≥32 32
OXA-58
OXA-23
OXA-23
OXA-24
OXA-23
32
32
32
4
≥32 32
≥32 32
10
a MIC planktonic MRSE and RBC values are from Table 1.
b Concentrations tested: 2x, 5x and 10x MIC.
a ESBL-CARBA: Extended spectrum β-lactamase-carbapenemase-producing
isolates. b OXA, oxacillinase; c VIM, Verona integron-encoded metallo-β-
lactamase; d NDM, New Delhi metallo-β-lactamase; e IMP, imipenem-type
carbapenemase; f KPC, K. pneumoniae carbapenemase; g Clinical isolates
resistant to the antibiotic colistin.
2.6 Membrane integrity investigations
We tested also two representative compounds G6 and I14 for
their ability to alter bacterial cell membrane integrity. A
luciferase-expressing Bacillus subtilis strain was used that emits
luminescence upon the entrance of the substrate D-luciferin into
bacterial cells if membrane integrity is compromised.29 Both
compounds G6 and I14 displayed a dose-response effect against
B. subtilis (Figure 4). A clear membranolytic effect was observed
for G6 at a concentration close to the MIC value against B.
subtilis (MIC: 4 µg/ml, Table 1), whereas for I14 a concentration
of 12.5 µg/ml (i.e. 3x MIC: 4 µg/ml) was needed to induce
luminescence (Figure 4). The different kinetics of luminescence
emission shown for the two compounds could indicate
2.5 Anti-biofilm activity
Serious human infections are frequently associated with
formation of bacterial biofilms hampering antibiotic
treatment.19,20 Medical device-associated infections are often
caused by coagulase-negative staphylococci and particularly
MRSE presumably due to its prevalence for biofilm formation.21-
23 Mechanical properties, physiological heterogeneity and
flexibility24,25 make biofilm cultures considerably more tolerant
to biocides and antibiotics than their planktonic counterparts. For
example, the rate of killing MRSE in biofilm by the antibiotic
rifampin was shown to be seven times slower and 157 times
slower by vancomycin, compared to rate of killing MRSE in the
planktonic state.26
differences in the way they interacted with the cell membrane
(Figure 5). The membrane-disruptive activity of the compounds
appeared to be comparable to that of chlorhexidine, a biocide
known for its membranolytic activity.30 The results were similar
In the present study, the most potent guanidine derivatives
were also screened for effects on eradicating preformed

8
to the effects observed for the amidobenzamide derivatives3 and
small marine natural antimicrobials reported previously.4
by an arrow. The Fold LUM induction compared to the maximum
LUM of the untreated control after chlorhexidine addition is shown.
Data from a single experiment, representative of two independent
experiments, are displayed.
3. Conclusions
To conclude, a promising class of amphipathic
sulfonamidobenzamides have been designed and synthesized
with potential for further drug development of antimicrobial and
anti-biofilm efficient drugs. Since bacterial biofilms are known to
be associated with wound infections,31-33 topical wound healing is
in that respect an area of interest, especially for treatment of
chronic wound infections, which are of particular importance in
aged and immobilized patients.31,34 The broad-spectrum activity
of the described class of compounds indicated by the potencies of
selected derivatives G6 and J18 against clinical isolates,
including multi-drug resistant isolates, makes them promising
candidates for treatment of polymicrobial wound infections.31,35
Although MRSE is not the leading causative agent in such
infections,23 it nevertheless served well as a pilot biofilm model.
Apart from G6 and J18, the guanidine derivatives, G2 and I14,
displayed both potent activity against MRSE biofilms and
favorable selectivity for staphylococci. This combination of anti-
staphylococcal properties makes G2 and I14 promising
candidates for further investigation with clinically relevant
biofilm-forming staphylococci, commonly associated with
wounds and implant associated infections.
Figure 4. Luminescence (LUM) induction in B. subtilis
168/pCSS962 treated with G6 and I14 at concentrations 25–1.6
µg/ml. Data for the first 30 sec after injection of the bacterial
suspension (containing D-luciferin) is presented. Chlorhexidine was
used as a membrane-disruptive control. The Fold LUM induction
was the ratio between the maximum luminescence in compound-
treated samples compared to the chlorhexidine-treated control (31
µg/ml of chlorhexidine was added in case of I14 and 20 µg/ml for
G6). The mean of two experiments ± SD is displayed.
4.Experimental section
4.1 Chemicals and equipment
All chemicals used for synthesis were purchased from Sigma-
Aldrich Inc., USA. The course of the reactions was monitored using
a Waters Alliance 2695 Separation Module HPLC system (Waters
Inc., USA) accompanied by Micromass Qattro LC (Micromass, UK)
MS system. 1H and 13C NMR spectra were recorded on a 400 MHz
NMR spectrometer (Varian, USA). Chemical shifts are expressed in
ppm relative to methanol-d4 (δH: 3.310 ppm and δC: 49.000 ppm).
High-resolution MS spectra were acquired on a Waters LC-MS
system (Milford, MA, USA) composed of an Acquity UPLC coupled
to an LCT-Premier TOF MS with electrospray ionization (ESI). The
MS data were obtained in positive ESI mode. Melting points were
determined in open capillary tubes with a Büchi B-540 melting point
apparatus.
4.2 Synthesis of starting materials
3,5-Di-tert-butylbenzene-1-sulfonyl chloride was synthesised
according to Hall.36 Briefly, 1,3-di-tert-butylbenzene (1 g, 5 mmol)
was dissolved in anhydrous amylenes stabilised chloroform (20 ml).
Chlorosulfonic acid (5.83 g, 50 mmol) was dissolved in anhydrous
amylenes stabilised chloroform (40 ml) and added drop-wise to the
reaction mixture with cooling and vigorous stirring for 3 h. The
reaction mixture was stirred additionally at room temperature for 1 h
before it was poured into crushed ice (300 g). The organic phase was
dried with CaCl2. Chloroform was removed in vacuo and 3,5-di-tert-
butylbenzene-1-sulfonyl chloride was obtained in 86% yield and
used without further purification. Spectroscopic data were in
accordance to Ris et al.37
3,5-Di-tert-butyl-4-methoxybenzene-1-sulfonyl chloride: 1,3-Di-
tert-butyl-2-methoxybenzene was synthesised according to Bai et
al.38 Briefly, 2,6-di-tert-butylphenol (5.0 g, 24 mmol), NaH (1.15 g,
29 mmol), and iodomethane (4.11 g, 29 mmol) were added to DMF
(25 ml) at 0 °C for 2 h. The reaction mixture was poured into cold
Figure 5. The figure shows kinetics of luminescence (LUM)
induction in B. subtilis 168/pCSS962 in presence of D-luciferin after
the addition of different concentrations (µg/ml) of the compounds;
A) G6, B) I14, and C) chlorhexidine. Data for chlorhexidine are
from reference.3 LUM was monitored for up to 180 sec, but only the
first 60 sec are presented. Water was added to an untreated control.
Complete membrane disruption was achieved by addition of
chlorhexidine (31 µg/ml) to the samples at the time point indicated
water, extracted with diethyl ether, and dried with Na SO . Diethyl
2
4
ether was evaporated under reduced pressure. Spectroscopic data
were in accordance to Bai et al.38 1,3-Di-tert-butyl-2-
methoxybenzene (1 g, 4.5 mmol) was dissolved in anhydrous
amylenes stabilised chloroform (20 ml). Chlorosulfonic acid (5.83 g,
50 mmol) was dissolved in anhydrous amylenes stabilised
chloroform (40 ml) and added drop-wise to the reaction mixture with

9
cooling and vigorous stirring for 3 h. The reaction mixture was
stirred additionally at room temperature for 1 h before it was poured
into crushed ice (300 g). The organic phase was dried with CaCl2.
Chloroform was removed in vacuo and 3,5-di-tert-butyl-4-
methoxybenzene-1-sulfonyl chloride was obtained in 80% yield. It
was used in subsequent reactions without further purification. 1H
NMR (CDCl3): 7.90 (s, 2H), 3.77 (s, 3H), 1.46 (s, 18H).
Methyl 2-(4-aminophenyl)acetate 4-methylbenzenesulfonate (or
4-(2-methoxy-2-oxoethyl)benzenaminium 4-methylbenzenesulfonate):
4-Aminophenylacetic acid (5 g, 33 mmol) was heated under reflux in
an excess of methanol (100 ml) in the presence of para-toluene
sulfonic acid (7.6 g, 40 mmol) for 18 h. Methanol was evaporated
under reduced pressure. Methyl 2-(4-aminophenyl)acetate 4-
methylbenzenesulfonate was obtained in approx. quantitative yield
and was used in further reactions without purification.
Methyl 4-(methylamino)benzoate: 4-Methylaminobenzoic acid (5
g, 33 mmol) was refluxed in an excess of methanol (100 ml) in the
presence of para-toluene sulfonic acid (7.6 g, 40 mmol) for 18 h39.
Methanol was evaporated in vacuo and a saturated solution of
NaHCO3 was added to the residue. The product was isolated by
extraction with ethyl acetate. The organic solvent was evaporated
under reduced pressure and methyl 4-methylaminobenzoate was
obtained in approx. quantitative yield. Spectroscopic data were in
accordance to Jones et al.39
Methyl 3-(methylamino)benzoate and methyl 4-amino-3-
methylbenzoate were synthesised by the same method and with
approx. quantitative yield. Spectroscopic data for methyl 3-
(methylamino)benzoate was in accordance to Gao et al.40 and methyl
4-amino-3-methylbenzoate was in accordance to Yang et al.41
Methyl isonipecotate was of technical grade (Sigma-Aldrich;
purity approx. 80%) and was purified by vacuum distillation before
use.
Methyl 3-amino-2-naphthoate: 3-Amino-2-naphthoic acid was of
technical grade (Sigma-Aldrich; purity approx. 80%) and was
purified before use. Crude 3-amino-2-naphthoic acid (5 g, 27 mM)
was dissolved in 500 ml of saturated solution of NaHCO3. The
solution was filtered and concentrated HCl was added drop wise to
the solution until pH 7, in which 3-amino-2-naphthoic acid started to
precipitate. The yellowish precipitate was isolated by filtration. Yield
of purified 3-amino-2-naphtoic acid was 70% (3.5 g). Methyl 3-
amino-2-naphthoate was synthesized according to a modified method
described elsewhere.42 Briefly, concentrated H2SO4 (12 ml) was
added to methanol (70 ml), followed by 3-amino-2-naphthoic acid
(3.5 g, 19 mmol). The solution was refluxed for 18 h, the mixture
was cooled and methanol evaporated in vacuo. The residue was
poured onto ice and neutralized with saturated NaHCO3. The
resulting yellowish precipitate was filtered, washed with water, and
dried. An amount of 2.7 g of methyl 3-amino-2-naphthoate was
obtained (70% yield). Spectroscopic data were in accordance to
Theeraladanon et al.43
biological screenings, N-(2-aminoethyl)-4-((3,5-di-tert-
butylphenyl)sulfonamido)benzamide (200 mg, 0.5 mmol) was
further dissolved in MeOH (1 ml), an excess of 4 M solution of HCl
in dioxane (0.5 ml) was added, and the mixture stirred for 30 min at
room temperature to achieve the HCl salt. Solvents were removed in
vacuo. Obtained N-(2-aminoethyl)-4-((3,5-di-tert-
butylphenyl)sulfonamido)benzamide hydrochloride was purified in
quantitative yield by crystallisation from MeOH/Et2O. Briefly, N-(2-
aminoethyl)-4-((3,5-di-tert-butylphenyl)sulfonamido)benzamide
hydrochloride (100 mg, 0.2 mmol) was dissolved in 1 ml of MeOH
under reflux. Et2O was added drop wise until precipitation was
complete. 1H NMR (CD3OD): 7.75 (d, 2H, J = 8.8 Hz), 7.63 (s, 3H),
7.20 (d, 2H, J = 8.8), 3.61 (t, 2H, J = 6 Hz), 3.12 (t, 2H, J = 6 Hz),
1.27 (s, 18H). 13C NMR (CD3OD): 168.9, 152.0, 141.6, 138.9, 128.8,
128.3, 126.7, 120.9, 119.1, 39.7, 37.3, 34.6, 30.1. HRMS-ESI:
C23H34N3O3S [M+H]+ calcd: 432.2321, found: 432.2324; mp 264-
267 C (decomp.).
Compounds H7, H8, I11, J15, and J16 were synthesized in a
similar manner (Scheme 1).
N-(2-Aminoethyl)-4-(((3,5-di-tert-
butylphenyl)sulfonamido)methyl)benzamide hydrochloride (H7) 1H
NMR (CD3OD): 7.79 (d, 2H, J = 8 Hz), 7.71 (s, 2H), 7.70 (s, 1H),
7.33 (d, 2H, J = 8 Hz), 4.15 (s, 2H), 3.67 (t, 2H, J = 6 Hz), 3.17 (t,
2H, J = 6 Hz), 1.36 (s, 18H). 13C NMR (CD3OD): 169.2, 152.1,
141.9, 140.1, 132.3, 127.4, 127.2, 126.4, 120.7, 45.9, 39.7, 37.4,
34.7, 30.2. HRMS-ESI: C24H36N3O3S [M+H]+ calcd: 446.2477,
found: 446.2460; mp 133-135 C (decomp.).
N-(2-Aminoethyl)-2-(4-((3,5-di-tert-
butylphenyl)sulfonamido)phenyl)acetamide hydrochloride (H8) 1H
NMR (CD3OD): 7.63 (t, 1H, J1 = 1.6 Hz), 7.56 (s, 2H, J1 = 1.6 Hz),
7.20 (d, 2H, J = 8 Hz), 7.06 (d, 2H, J = 8 Hz), 3.48 (s, 2H), 3.43 (t,
2H, J1 = 6 Hz), 3.05 (t, 2H, J1 = 6 Hz), 1.28 (s, 18H). 13C NMR
(CD3OD): 173.8, 151.8, 138.8, 136.7, 131.7, 129.5, 126.4, 121.6,
120.9, 41.5, 39.5, 37.0, 34.6, 30.1. HRMS-ESI: C24H36N3O3S
[M+H]+ calcd: 446.2477, found: 446.2462; mp > 80-83 C
(decomp.).
N-(2-Aminoethyl)-3-((3,5-di-tert-
butylphenyl)sulfonamido)benzamide hydrochloride (I11) 1H NMR
(CD3OD): 7.71 (t, 1H, J = 2 Hz), 7.62 (t, 1H, J = 1.6 Hz), 7.56 (d,
2H, J = 1.6 Hz), 7.56 (dd, 1H, J1 = 8 Hz, J2 = 2 Hz), 7.32 (t, 1H, J =
8 Hz), 7.19 (d, 1H, J1 = 8 Hz, J2 = 2 Hz), 3.63 (t, 2H, J = 6 Hz), 3.14
(t, 2H, J = 6 Hz), 1.26 (s, 18H). 13C NMR (CD3OD): 168.9, 151.9,
138.7, 138.5, 134.6, 128.9, 126.6, 124.4, 122.9, 120.9, 120.5, 39.7,
37.7, 34.6, 30.1. HRMS-ESI: C23H34N3O3S [M+H]+ calcd: 432.2321,
found: 432.2300; mp > 79-82 C (decomp.).
N-(2-Aminoethyl)-2-((3,5-di-tert-
butylphenyl)sulfonamido)benzamide hydrochloride (J15) 1H NMR
(CD3OD): 7.73 (d, 1H, J1 = 8 Hz), 7.64 (s, 1H), 7.56 (s, 2H), 7.49 (d,
1H, J1 = 8 Hz), 7.44 (t, 1H, J1 = 8 Hz), 7.18 (t, 1H, J1 = 8 Hz), 3.64
(t, 2H, J1 = 6 Hz), 3.18 (t, 2H, J1 = 6 Hz), 1.27 (s, 18H). 13C NMR
(CD3OD): 170.1, 152.0, 138.4, 138.1, 132.2, 128.2, 126.8, 124.1,
122.5, 121.7, 121.0, 39.6, 37.1, 34.6, 30.1. HRMS-ESI:
C23H34N3O3S [M+H]+ calcd: 432.2321, found: 432.2310; mp > 112-
114 C (decomp.).
4.3 Synthesis of MNPM test compounds
N-(2-Aminoethyl)-4-((3,5-di-tert-
butylphenyl)sulfonamido)benzamide hydrochloride (G1): Methyl 4-
aminobenzoate (604 mg, 4 mmol) and 3,5-di-tert-butylbenzene-1-
sulfonyl chloride (549 mg, 1.9 mmol) were heated together at 150 C
with stirring until the melt solidified. The reaction mixture was
allowed to cool down to room temperature, and 2 M solution of HCl
was added. The solid residue was filtered out and crystallised from
MeOH/H2O. Methyl 4-((3,5-di-tert-
butylphenyl)sulfonamido)benzoate was obtained in 85% yield.
Methyl 4-((3,5-di-tert-butylphenyl)sulfonamido)benzoate (300 mg,
0.7 mmol) was dissolved in an excess of ethylenediamine (3 ml, 45
mmol) and the reaction mixture was stirred at 100 C for 18 h. The
excess of ethylenediamine was removed in vacuo and the residue
was dissolved in acetonitrile and washed with hexane. The solvent
was removed in vacuo and N-(2-aminoethyl)-4-((3,5-di-tert-
butylphenyl)sulfonamido)benzamide was obtained in 92% yield and
used directly in the next step without further purification for
synthesis of its guanylated analogue G2 (see below for G2). Prior to
N-(2-Aminoethyl)-3-((3,5-di-tert-butylphenyl)sulfonamido)-2-
naphthamide hydrochloride (J16) 1H NMR (CD3OD): 8.27 (s, 1H),
7.86 (d, 1H, J = 8 Hz), 7.71 (d, 1H, J = 8 Hz), 7.70 (s, 1H), 7.56 (s,
1H), 7.55 (t, 1H, J = 8 Hz), 7.53 (s, 2H), 7.48 (t, 1H, J = 8 Hz), 3.68
(t, 2H, J = 6 Hz), 3.23 (t, 2H, J = 6 Hz), 1.15 (s, 18H). 13C NMR
(CD3OD): 170.2, 151.9, 137.5, 134.6, 133.7, 129.8, 129.6, 128.5,
128.2, 126.7, 126.7, 126.1, 123.6, 121.2, 120.0, 39.8, 37.2, 34.5,
30.0. HRMS-ESI: C27H36N3O3S [M+H]+ calcd: 482.2477, found:
482.2466; mp 165-167 C (decomp.).
N-(2-Aminoethyl)-4-((3,5-di-tert-butyl-N-
methylphenyl)sulfonamido)benzamide hydrochloride (G3): 4-
Methylaminobenzoate (330 mg, 2 mmol), 3,5-di-tert-butylbenzene-
1-sulfonyl chloride (549 mg, 1.9 mmol) and NaHCO3 (170 mg, 2
mmol) were heated together at 150 C with stirring until the melt
solidified. The reaction mixture was allowed to cool down to room

10
temperature, and 2 M solution of HCl was added. The solid residue
was filtered out and recrystallised from MeOH/H2O. Methyl 4-(3,5-
di-tert-butyl-N-methylphenylsulfonamido)benzoate was obtained in
87% yield. The rest of the synthesis was performed according to the
method described for G1.
8.8 Hz), 3.48 (t, 2H, J = 6.4 Hz), 3.28 (t, 2H, J = 6.4 Hz), 3.16 (s,
3H), 1.90 (p, 2H, J = 6.4 Hz), 1.28 (s, 18H). 13C NMR (CD3OD):
167.9, 157.3, 152.0, 144.8, 135.0, 132.2, 127.5, 127.0, 125.5, 121.5,
38.6, 36.6, 36.6, 34.6, 30.1, 28.7. HRMS-ESI: C26H40N5O3S [M+H]+
calcd: 502.2852, found: 502.2858; mp 119-121 C (decomp.).
4-(((3,5-Di-tert-butylphenyl)sulfonamido)methyl)-N-(2-
N-(2-Aminoethyl)-4-((3,5-di-tert-butyl-N-
methylphenyl)sulfonamido)benzamide hydrochloride (G3) 1H NMR
(CD3OD): 7.84 (d, 2H, J = 8.8 Hz), 7.72 (t, 1H, J = 1.6 Hz), 7.29
(d, 2H, J = 1.6 Hz), 7.24 (2, 2H, J = 8.8 Hz), 3.65 (t, 2H, J = 6
Hz), 3.16 (t, 2H, J = 6 Hz), 3.14 (s, 3H), 1.26 (s, 18H). 13C NMR
(CD3OD): 168.6, 152.0, 145.1, 135.1, 131.7, 127.7, 127.1, 125.5,
121.5, 39.6, 37.4, 36.7, 34.6, 30.1. HRMS-ESI: C24H36N3O3S
[M+H]+ calcd: 446.2477, found: 446.2480; mp 137-139 C
(decomp.).
guanidinoethyl)benzamide hydrochloride (H9) 1H NMR (CD3OD):
7.75 (d, 2H, J = 8 Hz), 7.70 (s, 3H), 7.32 (d, 2H, J = 8 Hz), 4.15 (s,
2H), 3.56 (t, 2H, J = 6 Hz), 3.42 (t, 2H, J = 6 Hz), 1.36 (s, 18H). 13
NMR (CD3OD): 168.9, 157.5, 152.1, 141.8, 140.2, 132.5, 127.5,
127.1, 126.4, 120.7, 45.9, 40.7, 38.5, 34.7, 30.2. HRMS-ESI:
C25H38N5O3S [M+H]+ calcd: 488.2695, found: 488.2676; mp 193-
195 C (decomp.).
C
2-(4-((3,5-Di-tert-butylphenyl)sulfonamido)phenyl)-N-(2-
guanidinoethyl)acetamide hydrochloride (H10) 1H NMR (CD3OD):
7.63 (s, 1H), 7.56 (s, 2H), 7.18 (d, 2H, J = 8.4 Hz), 7.05 (d, 2H, J =
8.4 Hz), 3.46 (s, 2H), 3.37-3.25 (m, 4H), 1.28 (s, 18H). 13C NMR
(CD3OD): 173.4, 157.5, 151.8, 138.8, 136.7, 131.9, 129.4, 126.4,
121.6, 120.9, 41.5, 40.6, 38.2, 34.6, 30.1. HRMS-ESI: C25H38N5O3S
[M+H]+ calcd: 488.2695, found: 488.2690; mp > 135-137 C
(decomp.).
Compounds G4 and I12 were synthesized in a similar manner
(Scheme 1).
N-(3-Aminopropyl)-4-((3,5-di-tert-butyl-N-
methylphenyl)sulfonamido)benzamide hydrochloride (G4) 1H NMR
(CD3OD): 7.85 (d, 2H, J = 8.4 Hz), 7.74 (s, 1H), 7.29 (s, 2H), 7.25
(d, 2H, J = 8.4 Hz), 3.52 (t, 2H, J = 6.4 Hz), 3.16 (s, 2H), 3.01 (t,
2H, J = 6.4 Hz), 1.99 (p, 2H, J = 6.4 Hz), 1.28 (s, 18H). 13C NMR
(CD3OD): 168.2, 152.0, 144.9, 135.0, 131.9, 127.6, 127.0, 125.5,
121.5, 36.9, 36.6, 36.1, 34.6, 30.1, 27.6. HRMS-ESI: C25H38N3O3S
[M+H]+ calcd: 460.2634, found: 460.2612; mp 121-124 C
(decomp.).
3-((3,5-Di-tert-butylphenyl)sulfonamido)-N-(2-
guanidinoethyl)benzamide hydrochloride (I13) 1H NMR (CD3OD):
7.70 (t, 1H, J = 1.6 Hz), 7.64 (t, 1H, J = 1.6 Hz), 7.58 (d, 2H, J = 1.6
Hz), 7.58-7.53 (m, 1H), 7.34 (t, 1H, J = 8 Hz), 7.25-7.19 (m, 1H),
3.55 (t, 2H, J = 6 Hz), 3.41 (t, 2H, J = 6 Hz), 1.28 (s, 18H). 13
C
N-(2-Aminoethyl)-3-((3,5-di-tert-butyl-N-
methylphenyl)sulfonamido)benzamide hydrochloride (I12) 1H NMR
(CD3OD): 7.85 (d, 1H, J = 8 Hz), 7.75 (s, 1H), 7.69 (s, 1H), 7.47 (t,
1H, J = 8 Hz), 7.29 (s, 2H), 7.26 (d, 1H, J = 8 Hz) 3.67 (t, 2H, J = 6
Hz), 3.18 (t, 2H, J = 6 Hz), 3.16(s, 3H), 1.29 (s, 18H). 13C NMR
(CD3OD): 168.5, 152.0, 142.2, 134.9, 134.2, 129.5, 128.8, 127.1,
125.8, 125.7, 121.7, 39.6, 37.4, 36.9, 34.6, 30.1. HRMS-ESI:
C24H36N3O3S [M+H]+ calcd: 446.2477, found: 446.2462; mp > 95-
97 C (decomp.).
NMR (CD3OD): 168.7, 157.5, 151.9, 138.6, 138.5, 134.8, 128.9,
126.6, 124.4, 122.8, 120.9, 120.4, 40.6, 38.6, 34.6, 30.1. HRMS-ESI:
C24H36N5O3S [M+H]+ calcd: 474.2539, found: 474.2531; mp 140-
143 C (decomp.).
3-((3,5-Di-tert-butyl-N-methylphenyl)sulfonamido)-N-(2-
guanidinoethyl)benzamide hydrochloride (I14) 1H NMR (CD3OD):
7.78 (dd, 1H, J1 = 8 Hz, J2 = 1.6 Hz), 7.73 (t, 1H, J = 1.6 Hz), 7.64
(t, 1H, J = 1.6 Hz), 7.436 (t, 1H, J = 8 Hz), 7.27 (d, 2H, J = 1.6 Hz),
7.23 (dd, 1H, J1 = 8 Hz, J2 = 1.6 Hz) 3.53 (t, 2H, J = 6 Hz), 3.40 (t,
2H, J = 6 Hz), 3.13(s, 3H), 1.26 (s, 18H). 13C NMR (CD3OD): 168.2,
157.5, 152.0, 142.2, 134.8, 134.4, 129.6, 128.8, 127.1, 125.7, 125.4,
121.7, 40.7, 38.6, 36.9, 34.6, 30.1. HRMS-ESI: C25H38N5O3S
[M+H]+ calcd: 488.2695, found: 488.2684; mp 77-79 C (decomp.).
2-((3,5-Di-tert-butylphenyl)sulfonamido)-N-(2-
4-((3,5-Di-tert-butylphenyl)sulfonamido)-N-(2-
guanidinoethyl)benzamide hydrochloride (G2) was synthesized as
previously described in Igumnova et al.3 Briefly, N-(2-aminoethyl)-
4-((3,5-di-tert-butylphenyl)sulfonamido)benzamide (200 mg, 0.5
mmol) was dissolved in DMF (0.5 ml) and 1-amidino-1H-1,2,4-
triazole hydrochloride (74 mg, 0.5 mmol) was added to the reaction
mixture. The reaction vessel was sealed with a septum, and the
reaction mixture stirred for 18 h at room temperature. The reaction
mixture was filtered, Et2O (10 ml) was added to the filtrate, and the
mixture was stirred for 1 h at room temperature. The ether layer was
decanted off, a new portion of Et2O (10 ml) was added, and the
mixture stirred for 10 h at room temperature. Et2O was removed
from the solid precipitate. Obtained 4-((3,5-di-tert-
butylphenyl)sulfonamido)-N-(2-guanidinoethyl)benzamide
hydrochloride was purified by recrystallization from MeOH/Et2O in
91% yield (216 mg). 1H NMR (CD3OD): 7.74 (d, 2H, J = 8.8 Hz),
7.62 (s, 3H), 7.19 (d, 2H, J = 8.8 Hz), 3.51 (t, 2H, J = 6 Hz), 3.38 (t,
2H, J = 6 Hz), 1.27 (s, 18H). 13C NMR (CD3OD): 168.6, 157.5,
152.0, 141.5, 138.9, 129.0, 128.1, 126.6, 120.8, 119.3, 40.6, 38.5,
34.6, 30.1. HRMS-ESI: C24H36N5O3S [M+H]+ calcd: 474.2539,
found: 474.2538; mp 247-249 C (decomp.).
guanidinoethyl)benzamide hydrochloride (J17) 1H NMR (CD3OD):
7.66 (d, 1H, J = 8 Hz), 7.61 (t, 1H, J = 1.6 Hz), 7.55 (d, 1H, J = 8
Hz), 7.53 (d, 2H, J = 1.6 Hz), 7.43 (d, 1H, J = 8 Hz), 7.14 (t, 1H, J =
8 Hz), 3.47 (t, 2H, J = 6 Hz), 3.36 (t, 2H, J = 6 Hz), 1.24 (s, 18H).
13C NMR (CD3OD): 169.6, 157.5, 152.0, 138.7, 138.3, 132.2, 128.0,
126.7, 123.9, 122.0, 121.7, 120.9, 40.4, 38.4, 34.6, 30.1. HRMS-ESI:
C24H36N5O3S [M+H]+ calcd: 474.2539, found: 474.2535; mp > 103-
105 C (decomp.).
3-((3,5-Di-tert-butylphenyl)sulfonamido)-N-(2-guanidinoethyl)-2-
naphthamide hydrochloride (J18) 1H NMR (CD3OD): 8.26 (s, 1H),
7.87 (d, 1H, J = 8 Hz), 7.86 (s, 1H), 7.77 (d, 1H, J = 8 Hz), 7.57 (t,
1H, J = 8 Hz), 7.55 (s, 1H), 7.54 (s, 2H), 7.48 (t, 1H, J = 8 Hz), 3.54
(t, 2H, J = 6 Hz), 3.44 (t, 2H, J = 6 Hz), 1.15 (s, 18H). 13C NMR
(CD3OD): 171.1, 158.9, 153.3, 139.2, 136.1, 135.5, 131.1, 130.8,
129.9, 129.7, 129.7, 128.1, 128.1, 124.1, 122.6, 120.9, 41.9, 39.9,
35.9, 31.4. HRMS-ESI: C28H38N5O3S [M+H]+ calcd: 524.2695,
found: 524.2696; mp > 76-79 C (decomp.).
Compounds G5, G6, H9, H10, I13, I14, J17, J18, K19, K20,
K21, K22, L23, L24, L25, and L26 were synthesized in similar
manner (Scheme 1).
4-((3,5-Di-tert-butylphenyl)sulfonamido)-N-(2-guanidinoethyl)-3-
methylbenzamide hydrochloride (K19) 1H NMR (CD3OD): 7.66 (t,
1H, J = 1.6 Hz), 7.62 (dd, 1H, J1 = 8 Hz, J2 = 1.6 Hz), 7.61 (d, 2H, J
= 1.6 Hz), 7.48 (d, 2H, J = 1.6 Hz), 7.33 (d, 2H, J = 8 Hz), 3.52 (t,
4-((3,5-Di-tert-butyl-N-methylphenyl)sulfonamido)-N-(2-
guanidinoethyl)benzamide hydrochloride (G5) 1H NMR (CD3OD):
7.82 (d, 2H, J = 8.8 Hz), 7.72 (t, 2H, J = 1.6 Hz), 7.28 (d, 2H, J = 1.6
Hz), 7.23 (d, 2H, J = 8.8 Hz), 3.55 (t, 2H, J = 6 Hz), 3,41 (t, 2H, J =
6 Hz), 3.14 (s, 3H), 1.26 (s, 18H). 13C NMR (CD3OD): 168.3, 157.5,
152.0, 145.0, 135.0, 131.8, 127.6, 127.0, 125.5, 121.5, 40.7, 38.6,
36.6, 34.6, 30.0. HRMS-ESI: C25H38N5O3S [M+H]+ calcd: 488.2695,
found: 488.2685; mp > 98-102 C (decomp.).
2H, J = 6 Hz), 3.39 (t, 2H, J = 6 Hz), 1.91 (s, 3H), 1.25 (s, 18H). 13
NMR (CD3OD): 168.6, 157.5, 152.0, 139.4, 138.7, 133.3, 131.1,
129.6, 126.5, 125.6, 125.1, 120.7, 40.7, 38.5, 34.6, 30.1, 16.4.
HRMS-ESI: C25H38N5O3S [M+H]+ calcd: 488.2695, found:
488.2678; mp > 169-172 C (decomp.).
C
4-((3,5-Di-tert-butyl-N-methylphenyl)sulfonamido)-N-(3-
guanidinopropyl)benzamide hydrochloride (G6) 1H NMR (CD3OD):
7.83 (d, 2H, J = 8.8 Hz), 7.74 (s, 1H), 7.29 (s, 2H), 7.24 (2, 2H, J =
1-((3,5-Di-tert-butylphenyl)sulfonyl)-N-(2-
guanidinoethyl)piperidine-4-carboxamide hydrochloride (K20) 1H
NMR (CD3OD):7.78 (s, 1H), 7.60 (s, 2H), 4.83-4.76 (m, 2H), 3.39-

11
3.24 (m, 4H), 3.38-3.25 (m, 2H), 3.25-3.14 (m, 1H), 1.94-1.82 (m,
2H), 1.82-1.65 (m, 2H), 1.39 (s, 18H). 13C NMR (CD3OD): 176.5,
157.5, 152.3, 135.5, 126.9, 121.4, 45.5, 41.5, 40.6, 38.0, 34.7, 30.2,
27.9. HRMS-ESI: C23H40N5O3S [M+H]+ calcd: 466.2852, found:
466.2847; mp > 184-186 C (decomp.).
Difco Laboratories, USA) adjusted to approximately 2.5-3 x 104
cells/ml, was added to flat-bottom microplates (Nunc, Roskilde,
Denmark) in a ratio 1:1 with serial dilutions of the test compounds.
The solutions of the compounds were prepared with up to 100%
DMSO, but the final DMSO concentration in the test wells did not
exceed 1%. After the plates were incubated at 35 °C for 48 h, the
MIC value defined as the lowest concentration of compound
resulting in no bacterial growth, was determined by OD600
measurement.
Antimicrobial screening against clinical isolates was performed
as previously described,3 with approximately 1-1.2 x 106 cells/ml and
24 h of incubation time. MIC90 was defined as the MIC value that
inhibited ≥90% of the isolates.46
(S)-1-((3,5-Di-tert-butylphenyl)sulfonyl)-N-(2-
guanidinoethyl)pyrrolidine-2-carboxamide hydrochloride (K21) 1H
NMR (CD3OD):7.83 (s, 1H), 7.71 (s, 2H), 4.08-4.00 (m, 1H), 3.66-
3.56 (m, 1H), 3.56-3.35 (m, 4H), 3.26-3.16 (m, 1H), 2.02-1.73 (m,
3H), 1.64-1.52 (m, 1H), 1.40 (s, 18H). 13C NMR (CD3OD): 174.4,
157.5, 152.7, 135.6, 127.4, 121.5, 62.5, 49.4, 40.5, 38.1, 34.7, 30.7,
30.2, 24.1. HRMS-ESI: C22H38N5O3S [M+H]+ calcd: 452.2695,
found: 452.2690; mp > 82-84 C (decomp.).
Hemolytic assay: The lytic activity against human RBCs was
determined as described before.3 Briefly, blood from a healthy adult
was collected in heparinized tubes. Washed RBC suspension in
phosphate-buffered saline (PBS, pH 7.4), was adjusted to 10% and
incubated in a ratio 9:1 with serial dilutions of the compounds at 37
°C for 1 h. After centrifugation at 450×g for 10 min, the supernatants
were transferred to flat-bottom microplates (Nunc, Roskilde,
Denmark) and OD550 was measured. Incubation of RBCs in 50%
PBS and 0.5% DMSO gave baseline hemolysis, while incubation in
50% PBS and 0.05% Triton X-100 gave complete (100%)
hemolysis. EC50 was defined as the concentration of compound
giving 50% lysis.
Determination of cytotoxicity against MRC-5 cells: The test
compounds were screened for cytotoxicity at two-fold concentrations
up to 100 μg/ml against normal human lung fibroblast (MRC-5,
ATCC CCL-171™) cells.47 MRC-5 cells were cultured and assayed
in Dulbecco’s modified Eagle’s medium (D-MEM, Thermo Fisher
Scientific) supplemented with 10% fetal bovine serum (FBS, Merck)
and 5 ml of L-alanyl-Lglutamine (200 mM, Merck) and incubated in
5% CO2 at 37 °C. Briefly, the cells were seeded in 96-well microtiter
plates (Thermo Fisher Scientific) at 4000 cells/well and incubated for
24 h to allow the cells to adhere before the cell media was replaced
and the compounds (50 µl) were added. The plates were then
incubated for 72 h. Subsequently, 10 μl of MTS solution (Cell Titer
96 Aqueous One Solution Reagent, Promega) was added to each
well, and the cells were incubated for 1 h at 37 °C. The absorbance
was measured at 485 nm using a DTX multimode detector (Beckman
Coulter). The negative control was defined as cells assayed with their
respective cell media with 1% DMSO (1% was the highest DMSO
concentration used in wells with compound), and positive control as
cells assayed with 0.5% Triton X-100 (Sigma-Aldrich). Cell survival
was calculated as follows: Cell survival (%) = (absorbance treated
wells − absorbance positive control)/(absorbance negative
control − absorbance positive control) × 100. EC50 values for
cytotoxicity against MRC-5 cells refer to the concentration (μg/ml)
causing 50% inhibition of cell survival.
Membrane integrity assay: The membrane integrity of B. subtilis
168 transformed with the plasmid pCSS962 according to Virta et al.,
29 was assayed as previously described.3 Briefly, aliquots of bacterial
suspension with an OD600=0.1 containing 1 mM D-luciferin
potassium-salt (pH 7.4, SynChem Inc, IL, USA) were automatically
injected to black microplates (Nunc, Roskilde, Denmark) with
dilutions of the compounds. The luminescence emission was
monitored before and after the addition of the membrane-disruptive
control chlorhexidine acetate (Fresenius Kabi, Halden Norway).
Biofilm eradication assay: Eradication of mature biofilms of S.
epidermidis RP62A (MRSE) (ATCC 35984 / CCUG 31568) was
determined based on Klingenberg et al.48 and Christensen et al.49
Briefly, overnight cultures were prepared in Tryptic soy broth (TSB;
BD, USA) at 37 °C with agitation. The cultures were centrifuged for
5 min at 13 000 rpm and the pellets resuspended in 0.9% NaCl. The
suspension was filtered through a syringe filter with pore size of 5
µm (Acrodisc®, Pall Corporation, Neuquay Cornwall, UK) to
remove aggregates and adjusted to 2 McFarland in 0.9% NaCl,
before being diluted in TSB with 1% glucose (TSBglu) to a final
inoculum of approximately 6 x 106 CFU/ml. A volume of 50 µl of
3,5-Di-tert-butyl-N-(4-guanidinobutyl)benzenesulfonamide
hydrochloride (K22) 1H NMR (CD3OD): 7.70 (s, 3H), 3.17 (t, 2H, J
= 6.8 Hz), 2.86 (t, 2H, J = 6.8 Hz), 1.69-1.50 (m, 4H), 1.37 (s, 18H).
13C NMR (CD3OD): 158.6, 153.6, 141.2, 127.8, 122.0, 43.4, 42.0,
36.1, 31.6, 27.9, 27.0. HRMS-ESI: C19H35N4O2S [M+H]+ calcd:
383.2481, found: 383.2475; mp > 168-170 C (decomp.).
4-((3,5-Dimethylphenyl)sulfonamido)-N-(2-
guanidinoethyl)benzamide hydrochloride (L23) 1H NMR (CD3OD):
7.71 (d, 2H, J = 8.8 Hz), 7.43 (s, 2H), 7.21 (s, 1H), 7.19 (d, 2H, J =
8.8 Hz), 3.50 (t, 2H, J = 6 Hz), 3.37 (t, 2H, J = 6 Hz), 2.31 (s, 6H).
13C NMR (CD3OD): 168.7, 157.5, 141.4, 139.4, 139.1, 134.1, 128.7,
128.1, 124.2, 118.6, 40.6, 38.5, 19.7. HRMS-ESI: C18H24N5O3S
[M+H]+ calcd: 390.1600, found: 390.1602; mp 257-259 C
(decomp.).
4-((3,5-Bis(trifluoromethyl)phenyl)sulfonamido)-N-(2-
guanidinoethyl)benzamide hydrochloride (L24) 1H NMR (CD3OD):
8.30 (s, 2H), 8.25 (s, 1H), 7.78 (d, 2H, J = 8.8 Hz), 7.23 (d, 2H, J =
8.8 Hz), 3.51 (t, 2H, J = 6 Hz), 3.38 (t, 2H, J = 6 Hz). 13C NMR
(CD3OD): 168.4, 157.5, 142.5, 140.3, 132.4 (q, J = 34 Hz), 130.1,
128.5, 127.2 (q, J = 4 Hz), 126.3 (q, J = 4 Hz), 122.5 (q, J = 273 Hz),
119.8, 40.6, 38.5. HRMS-ESI: C18H18N5O3SF6 [M+H]+ calcd:
498.1035, found: 498.1032; mp 215-217 C (decomp.).
N-(2-Guanidinoethyl)-4-(naphthalene-2-sulfonamido)benzamide
hydrochloride (L25) 1H NMR (CD3OD): 8.41 (d, 1H, J = 1.6 Hz),
7.97 (d, 2H, J = 8.4 Hz), 7.92 (d, 1H, J = 8 Hz), 7.79 (dd, 1H, J1 = 8
Hz, J2 = 1.6 Hz), 7.68 (d, 2H, J = 8.8 Hz), 7.66-7.58 (m, 2H), 7.23
(d, 2H, J = 8.8 Hz), 3.47 (t, 2H, J = 6 Hz), 3.34 (t, 2H, J = 6 Hz). 13
NMR (CD3OD): 168.7, 157.4, 141.3, 136.4, 134.9, 131.9, 129.1,
129.0, 128.8, 128.7, 128.3, 128.1, 127.5, 127.3, 121.8, 118.9, 40.6,
38.5. HRMS-ESI: C20H22N5O3S [M+H]+ calcd: 412.1443, found:
412.1423; mp > 157-159 C (decomp.).
C
4-((3,5-Di-tert-butyl-4-methoxyphenyl)sulfonamido)-N-(2-
guanidinoethyl)benzamide hydrochloride (L26) 1H NMR (CD3OD):
7.74 (d, 2H, J = 8.8 Hz), 7.67 (s, 2H), 7.19 (d, 2H, J = 8.8 Hz), 3.66
(s, 3H), 3.51 (t, 2H, J = 6 Hz), 3.36 (t, 2H, J = 6 Hz), 1.35 (s, 18H).
13C NMR (CD3OD): 168.6, 163.26, 157.5, 144.8, 141.6, 133.3,
128.9, 128.1, 125.4, 119.2, 64.0, 40.7, 38.5, 35.6, 30.7. HRMS-ESI:
C25H38N5O4S [M+H]+ calcd: 504.2645, found: 504.2642; mp 247-
249 °C (decomp.).
4.4 Biological test methods
The initial antimicrobial screening included bacterial reference
strains listed under Table 1. The expanded screenings were
performed against the collection of 275 clinical isolates deposited at
the Norwegian Organization for Surveillance of Resistant
Microorganisms (NORM) (2012-2014), and the collection of 30
multidrug-resistant clinical isolates from the The Norwegian
National Advisory Unit on Detection of Antimicrobial Resistance.8-10
Bacteria included are denoted in the respective tables (Table 2, Table
3). The collection of Enterococcus spp. contained isolates of both E.
faecium and E. faecalis.3 Multidrug resistance was defined as
resistance to at least one class of antimicrobial agents.44
Minimum inhibitory concentration (MIC) assay: To determine
the MIC, a microdilution susceptibility test was performed according
to CLSI M07-A945 with modifications described previously.3
Briefly, the bacterial inoculum in Mueller-Hinton broth (MHB,

12
the bacterial suspension was added into flat-bottom microplates
(Nunclon™ Surface, Nunc, Roskilde, Denmark). The plates were
sealed, incubated for 24 ±2 h at 37 °C without shaking and then
washed 2x with PBS. Dilutions of test compounds in 50% TSBglu
were added to the biofilms. Untreated samples containing 50%
TSBglu were run on the same plates. Following 24 ±2 h of incubation
at 37 °C without shaking, the plates were washed 3x and dried
overnight at room temperature or for 1 hour at 55 °C before staining
with 0.1% crystal violet for 5 min. The plates were washed 3x with
tap water, and crystal violet was solubilized with 70% ethanol. OD600
was measured after a brief agitation. All compounds and
15. Tadesse, M.; Svenson, J.; Jaspars, M.; Strøm, M.B.;
Abdelrahman, M.H.; Andersen, J.H.; Hansen, E.; Kristiansen,
P.E.; Stensvag, K.; Haug, T. Tetrahedron Lett. 2011, 52, 1804.
16. Yakubu, D.; Konstanidis, P.; Gray, M.; O´Shaughnessy, A.;
Harburn, J.J.; Kottakota, S. 13th International Electronic
Conference on Synthetic Organic Chemistry. 2009, Nov.
(Accessed May 6. 2018).
17. Thirionet, I.; Daloze, D.; Braekman, J.C.; Willemsen, P. Nat.
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Acknowledgments
The study was funded by the Research Council of Norway (grant
no. 174885/I30) and UiT-The Arctic University of Norway as part of
the Centre for Research-Based Innovation on Marine Bioactives and
Drug Discovery (MabCent-SFI). Screenings against clinical bacterial
isolated was supported by MABIT grant no. BS0068. The authors
express their gratitude to professor Ørjan Samuelsen (University
Hospital of North Norway, UNN) and associate professor Kristin
Hegstad (UiT) for access to the multi-drug resistant clinical isolates,
researcher Dr. Espen Hansen (Marbio - UiT) for MS analysis,
engineer Kirsti Helland (Marbio - UiT) for MRC-5 screening, and
senior engineer Dr. Martina Havelkova (UiT) for technical assistance
with chemical analysis. Authors also thank department engineers
Alena Didriksen (UNN) and Hege Devold (UiT) for their technical
assistance with antimicrobial and hemolytic activity testing.
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A. Supplementary data
Supplementary data (1H-NMR spectra of synthesized compounds)
associated with this article can be found, in the online version, at…