Chloroquine fumardiamides as novel quorum sensing inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127336

Quorum sensing inhibitors (QSIs) that specifically interfere with bacterial cell-to-cell communication are considered as an alternative approach to conventional antibacterial therapy. In our study, a set of twenty-six fumardiamides with a quinoline head-group were evaluated as potential QSIs. Two strains of Gram-negative Chromobacterium violaceum (violacein-producing strain ATCC31532 and violacein-negative, mini-Tn5 mutant derivative CV026) were used as QS reporters for testing anti-QS and bactericidal activity of various quinoline fumardiamides. The initial screening of eighteen fumardiamides with primaquine, mefloquine and chloroquine scaffolds identified chloroquine derivatives as the most promising QSIs. Tail-group optimization of chloroquine fumardiamides led to the most active compounds 27, 29 and 30 bearing aminoethyl or piperidine moieties. At 400 μM concentration, these compounds inhibited the QS of C. violaceum strains in a manner similar to quercetin (the model QSI), while at the 40 μM concentration their inhibitory effect was twice less than that of quercetin. As none of the compounds displayed a bactericidal effect and that the QS inhibition was specific to the CV026 strain, our findings indicate that the structurally optimized chloroquine derivatives could function as quorum quenching (QQ) agents with a potential to block the signaling without entering the cell. In conclusion, our finding provides an important step toward the further design of agents targeting cell-to-cell communication.

Full text of DOI:10.1016/j.bmcl.2020.127336

Journal Pre-proofs
Chloroquine fumardiamides as novel quorum sensing inhibitors
Maja Beus, Kirsi Savijoki, Jayendra Z. Patel, Jari Yli-Kauhaluoma, Adyary
Fallarero, Branka Zorc
PII:
S0960-894X(20)30447-9
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127336
BMCL 127336
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
1 April 2020
1 June 2020
6 June 2020
Please cite this article as: Beus, M., Savijoki, K., Patel, J.Z., Yli-Kauhaluoma, J., Fallarero, A., Zorc, B.,
Chloroquine fumardiamides as novel quorum sensing inhibitors, Bioorganic & Medicinal Chemistry Letters
(2020), doi: https://doi.org/10.1016/j.bmcl.2020.127336
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Chloroquine fumardiamides as novel quorum sensing inhibitors
a,*
b,*
c
c
b
Maja Beus , Kirsi Savijoki , Jayendra Z. Patel , Jari Yli-Kauhaluoma , Adyary Fallarero , Branka
Zorca,**
a
University of Zagreb, Faculty of Pharmacy and Biochemistry, HR-10000 Zagreb, Croatia
b
Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy,
University of Helsinki, FI-00014 Helsinki, Finland
c
Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of
Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland
*
*
These authors equally contributed to the paper.
* Corresponding author.
E-mail address: bzorc@pharma.hr (B. Zorc).
Keywords:
Quinoline derivatives
Chloroquine
Fumardiamide
Quorum sensing inhibition
Quorum quenching
A B S T R A C T
Quorum sensing inhibitors (QSIs) that specifically interfere with bacterial cell-to-cell communication
are considered as an alternative approach to conventional antibacterial therapy. In our study, a set of
twenty-six fumardiamides with a quinoline head-group were evaluated as potential QSIs. Two strains
of Gram-negative Chromobacterium violaceum (violacein-producing strain ATCC31532 and
violacein-negative, mini-Tn5 mutant derivative CV026) were used as QS reporters for testing anti-
QS and bactericidal activity of various quinoline fumardiamides. The initial screening of eighteen
fumardiamides with primaquine, mefloquine and chloroquine scaffolds identified chloroquine
derivatives as the most promising QSIs. Tail-group optimization of chloroquine fumardiamides led
to the most active compounds 27, 29 and 30 bearing aminoethyl or piperidine moieties. At 400 µM
concentration, these compounds inhibited the QS of C. violaceum strains in a manner similar to
1

quercetin (the model QSI), while at the 40 µM concentration their inhibitory effect was twice less
than that of quercetin. As none of the compounds displayed a bactericidal effect and that the QS
inhibition was specific to the CV026 strain, our findings indicate that the structurally optimized
chloroquine derivatives could function as quorum quenching (QQ) agents with a potential to block
the signaling without entering the cell. In conclusion, our finding provides an important step toward
the further design of agents targeting cell-to-cell communication.
Most microbial pathogens exploit adherent growth on abiotic/biotic surfaces (i.e., biofilm
1
formation) as a default growth mode to escape the action of antibiotics and host immune cells. A
cell-to-cell communication, termed quorum sensing (QS), plays a key role in coordinating the biofilm
2
formation, secondary metabolite production, bioluminescence and virulence. QS enables the
microbial cells to detect and respond to cell population density via the production and release of a
chemical signal molecule called autoinducer (AI). Many bacteria possess several interacting QS
modules organized into regulatory hierarchies employing multiple signal molecules from the same or
3
different chemical classes. It is considered that molecules or compounds able to interrupt QS pose
4
less selective pressure on the microbes than the conventional antibiotics. Strategies that interrupt the
QS-communication outside the cell, also called quorum quenching (QQ), are highly promising since
the communication system can be blocked without entering the cell.5,6 Thus, search for novel anti-
QS compounds preventing the development of specific group behavior and causing chemical
attenuation of biofilm formation is considered the method of choice to combat biofilm infections
without boosting the resistance.5,7 However, there are currently no approved therapeutic treatment
options that target machinery critical to biofilm formation or biofilm maintenance.8
Our studies have focused on the derivatization of the quinoline drug primaquine (PQ) with
9
the aim to get novel biologically active agents. A literature survey revealed a number of reports on
quinoline derivatives as effective antagonists of PqsR, the receptor of pqs system of the human
pathogen Pseudomonas aeruginosa, which controls the expression of various virulence factors and is
involved in the biofilm formation. Based on the scaffold of the quinolone signal molecule PQS (2-
heptyl-3-hydroxy-4(1H)-quinolone) and its precursor HHQ (2-heptyl-4-hydroxyquinoline), several
classes of quinoline and quinazolinone PqsR antagonists were developed.10–12 Furthermore,
Abouelhassan et al. have reported on quinoline small molecules with potent dispersal activity against
methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis biofilms.13
Based on these facts, we have evaluated the potential of PQ derivatives synthesized in-house as
antibiofilm agents. We have shown that ureidoamides with PQ and amino acid moieties exerted strong
activity in a biofilm eradication assay involving Escherichia coli, P. aeruginosa and Candida
2

1
4
albicans as the biofilm models. Asymmetric PQ-halogenoaniline fumardiamides 1–6 have also
1
5
demonstrated a significant antibiofilm activity. Susceptibility of the tested microorganisms towards
PQ derivatives showed both a compound- and species-dependent patterns. The most active
compounds were fluoro derivatives 1 and 2 (Fig. 1), whereas the most susceptible microorganisms
were Enterococcus faecalis and S. aureus, followed by E. coli, Streptococcus pneumoniae and P.
aeruginosa. Our compound library was further enriched with analogous derivatives bearing
mefloquine (MQ) (7–12) or chloroquine (CQ) pharmacophores (13–18) (Fig. 1).16,17
O
R
NH
O
H
N
N
N
H
O
1–6
Cl
CF3
N
R
R
O
N
O
H
H
N
N
CF3
N
N
H
N
N
H
H
H
O
O
7
–12
13–18
1
,7,13 2,8,14 3,9,15 4,10,16 5,11,17 6,12,18
R: m-F p-F m-Cl p-Cl
m-CF3 p-CF3
Fig. 1. Chemical structures of primaquine- (1–6), mefloquine- (7–12) and chloroquine-based
fumardiamides (13–18).
However, the potential of quinoline derivatives 1–18 as quorum sensing inhibitors (QSIs) has
not been explored until now. Chromobacterium violaceum has shown promise in screening
compounds with QSI activities, as changes in QS can be directly detected and quantified by
1
8
monitoring the QS-dependent formation of a colored indicator/pigment, termed violacein. We have
previously established a miniaturized 96-well based screening platform based on the C. violaceum
reporter strains (ATCC31532 and CV026), which allow the distinction of QSIs from QQs that
sequester/inhibit the AI signal (C6-HSL), as well as the exclusion of compounds having bactericidal
effects.19,20 Here, the further-improved screening method, in which the growth conditions were
optimized to avoid bottlenecks interfering the maximal synthesis of violacein, was used to screen the
synthesized quinoline derivatives 1–18 and four newly prepared CQ derivatives 27–30, designed after
the initial screenings.
PQ, MQ and CQ derivatives 1–18 were prepared according to procedures reported in our
previous papers.15–17 The initial screening of these eighteen quinoline fumardiamides pointed
chloroquine derivatives as the most promising QS inhibitors. Based on the obtained results, we have
designed and prepared four novel CQ derivatives 27–30, in which halogenoaniline moiety was
replaced by scaffolds bearing primary or secondary amino group as the key feature (Scheme 1). The
reaction pathway leading to the title compounds included several synthetic steps: i) preparation of N1-
3

(7-chloroquinolin-4-yl)butane-1,4-diamine (19) from 4,7-dichloroquinoline and 1,4-diaminobutane;
ii) synthesis of (E)-ethyl 4-chloro-4-oxobut-2-enoate (monoethyl fumaroyl chloride, 20) from
monoethyl fumarate and thionyl chloride; iii) reaction of compounds 19 and 20 to obtain amido-ester
2
1; iv) basic hydrolysis of product 21 to amido-carboxylic acid 22; v) coupling reaction between 22
and the corresponding N-protected diamine in the presence of 1-[bis(dimethylamino)methylene]-1H-
,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) and N,N-
diisopropylethylamine (DIEA); vi) the deprotection step: removing of t-butyloxycarbonyl group
Boc) from intermediates 23–26. Compounds 23 and 24 were prepared using Boc-monoprotected 1,4-
1
(
diaminoethane or 1,4-benzenediamine in the coupling step, whereas compounds 25 and 26 were
obtained from two related 4-aminopiperidines, one bearing Boc protecting group on piperidine
nitrogen atom (4-amino-1-Boc-piperidine or t-butyl-4-aminopiperidine-1-carboxylate) and the
second one with Boc-protected primary amino group, i.e. 4-(N-Boc-amino)piperidine or t-butyl
piperidin-4-ylcarbamate. It is worth to mention that the coupling reaction between compound 22 and
p-nitroaniline failed in our hands, so fumardiamide 31 bearing CQ scaffold on one side and p-
nitroaniline motif on the other side was not obtained. We have also tried to prepare compound 31 in
the opposite direction: first to achieve the amide bond between monoethyl fumaroyl chloride and p-
nitroaniline and then the amide bond between CQ derivative, but this experiment was also
unsuccessful. The final CQ-based fumardiamides 27–30 were obtained by treating of precursors 23–
1
4
2
6 with trifluoroacetic acid (TFA). The following compounds were prepared: N -(2-aminoethyl)-N -
1
4
[4-[(7-chloroquinolin-4-yl)amino]butyl]fumaramide
(27),
N -(4-aminophenyl)-N -[4-[(7-
1
4
chloroquinolin-4-yl)amino]butyl]fumaramide (28), N -[4-[(7-chloroquinolin-4-yl)amino]butyl]-N -
(piperidin-4-yl)fumaramide (29) and (E)-4-(4-aminopiperidin-1-yl)-N-[4-[(7-chloroquinolin-4-
yl)amino]butyl]-4-oxobut-2-enamide (30).
4

Cl
NO2
Cl
N
O
H
N
Cl
Cl
N
N
H
H
31
O
N
O
H
O
N
NH2
TEA, DCM
N
H
N
H
N
N
O
Cl
O
H
27
23
24
28
O
NH2
N
N
N
O
Cl
O
H
H
19
20
21
O
H2N
NO2
Cl
TFA DCM
R2
HATU, DIEA, DMF
X =
N
O
N
LiOH MeOH/H2O
H
N
H
N
O
1
N
O
N
H
N
H
R HN
NH2-X-NHBoc
H
N
O
O
HATU, DIEA, DMF
HATU, DIEA, DMF
N
H
OH
Cl
R1 = H, R2 = Boc
R1 = Boc, R2 = H
22
O
X =
Cl
Cl
H
N
O
O
N
O
H
N
O
N
O
N
O
N
O
N
H
N
H
H
H
N
N
O
N
N
H
N
N
O
Cl
H
H
25
O
26
30
O
Cl
Cl
N
O
TFA DCM
H
NH2
TFA DCM
TFA DCM
N
O
H
N
N
O
NH
N
O
N
H
N
H
H
N
H
N
O
N
H
N
H
N
H
N
29
O
O
NH2
Scheme 1. Synthesis of novel chloroquine-based fumardiamides 27–30.
The C. violaceum ATCC31532 and CV026 strains were used as QS reporters for testing anti-
QS and bactericidal activities of the indicated quinoline fumardiamides, using quercetin (Q) as the
positive control for QS and azithromycin (AZ) as the positive control for bactericidal activity (cell
viability). Parallel assays monitoring both the violacein production (indication of induced QS
signaling) and the reduction of resazurin to resorufin (indication of viable cells) in both reporter
strains grown with/without the compound allow screening of a genuine anti-QS agent from
compounds with bactericidal effect. The initial evaluation was performed for 18 compounds: six PQ
derivatives (1–6), six MQ derivatives (7–12) and six CQ derivatives (13–18) (Table 1, Fig. 2).
Inhibitory effects were tested at 400 M concentration and for the most potent QSIs also at 40 M.
Five tested compounds, namely 13–16 and 18, all bearing the CQ scaffold, were able to reduce the
violacein production in ATCC31532 from 75 to 87%. Compounds 13–16 decreased cell viability of
the same strain from 27 to 57% in comparison to cells with 2% DSMO (solvent used in sample
preparation). These five compounds were further tested at 40 M concentration both on the wild-type
and the mutant cells, and the results indicated that the compounds were able to inhibit QS only at a
higher concentration (400 M). None of the tested compounds displayed QQ activity.
Table 1
Quorum sensing inhibition of quinoline fumardiamides 1–18 at 400 µM concentration.
Chromobacterium violaceum strain
Cmpd.
Chemical structure
ATCC31532
CV026
5

Bactericidal
effect (%)
Bactericidal
effect (%)
QSI (%)
ne
QQ (%)
nt
O
F
1
NH
O
ne
nt
H
N
N
O
N
H
O
F
2
3
4
5
6
NH
O
ne
ne
ne
ne
ne
24.2 ± 0.3
17.9 ± 4.1
ne
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
H
N
N
O
N
H
O
Cl
NH
NH
NH
O
H
N
O
N
N
H
O
Cl
O
H
N
N
N
H
O
O
CF3
O
16.4 ± 0.7
31.2 ± 6.7
H
N
O
N
N
H
O
CF3
NH
O
H
N
N
N
H
O
F3C
F
N
O
7
8
H
16.9 ± 16.6 ne
29.1 ± 11.5 ne
nt
nt
nt
nt
N
F3C
N
H
N
H
O
F3C
N
F
O
H
N
F3C
N
H
N
H
O
F3C
N
Cl
O
9
1
1
H
22.3 ± 6.1
22.3 ± 0.9
nt
nt
nt
nt
nt
nt
N
F3C
N
H
N
H
O
F3C
N
Cl
O
0
1
H
N
29.9 ± 12.7 ne
F3C
N
H
N
H
O
F3C
N
CF3
O
H
22.3 ± 6.1
ne
13.8 ± 0.1
N
F3C
N
H
N
H
O
F3C
N
CF3
O
1
1
1
2
3
4
H
ne
nt
nt
N
F3C
N
N
H
H
O
Cl
Cl
Cl
F
N
O
79.4 ± 0.4
87.9 ± 1.8
75.3 ± 2.8
85.0 ± 1.3
ne
27.8 ± 12.2
39.7 ± 7.7
ne
ne
ne
ne
H
N
N
N
H
H
O
F
N
O
H
N
N
N
H
H
O
Cl
1
1
1
1
5
6
7
8
N
O
45.8 ± 8.2
56.9 ± 10.1
ne
ne
ne
nt
ne
ne
nt
H
N
N
H
N
H
O
Cl
Cl
Cl
Cl
N
N
N
O
H
N
N
N
H
H
O
CF3
O
H
N
N
H
N
H
O
CF3
O
85.7 ± 2.2
27.7 ± 5.4
ne
ne
H
N
N
H
N
H
O
6

PQ
Q
ne
77.8 ± 10.1 ne
ne 94.8 ± 3.6
ne
ne
ne
71.7 ± 6.6
94.4 ± 0.4
ne
AZ
67.2 ± 1.5
QSI – quorum sensing inhibition; QQ – quorum quenching; PQ – primaquine; Q – quercetin; AZ – azithromycin; nt –
not tested; ne – no effect
Fig. 2. Comparison of violacein production (A) and cell viability of (B) ATCC31532 (left panel) and CV026 (right panel)
cultures with and without the indicated compounds. PDYT, wells containing only the culture medium; Cells, cultures
containing 2% DMSO (ATCC31532) or 2% DMSO and 0.5 µM C6-HSL (CV026). Error bars, ± SD (n = 3–4). No
inhibition, cells with maximal violacein production or maximal viability; Max inhibition, inhibition achieved with
quercetin (Q) or with bactericidal drug azithromycin (A). Red arrows indicate the most potent non-bactericidal
compounds with QSI activity.
After the initial head-group studies, CQ moiety was viewed as an attractive platform for the
synthesis of a focused small molecule library. We have designed CQ-fumardiamides with a primary
amino group attached to a flexible carbon chain (27), benzene ring (28) or piperidine heterocycle
(30), and one piperidine derivative with a secondary amino group (29). The amino motif was
chosen based on literature reports, which pointed out amine-containing small molecules as
compounds most likely to cross cellular membranes of Gram-negative pathogens and accumulate in
7

2
1
the cell cytoplasm, whereas replacing amine with other functional groups (hydroxyl, carboxyl,
amide, ester, azide, nitrile) or alkylation resulted in markedly reduced accumulation of the
compound. It is also accepted that promising Gram-negative antibacterial agents are amphiphilic,
rigid, less than 600 Da in size, and have low globularity.21,22 With their higher polarity and higher
molecular weight, they have been considered as an exception to the Lipinski’s rule of five.22
We have evaluated the effectiveness of amino CQ-fumardiamides 27–30, their chemical
precursors 23–26 with Boc-protected amino groups and the parent drug CQ as potential QSIs. Our
results revealed that compound 27 with the amino group attached to a short aliphatic chain and
piperidine derivatives 29 and 30, inhibited QS by approx. 46% in both the wild-type and the mutant
strains in comparison to the same strains grown in the presence of 2% DMSO, a solvent used in
dissolving the compounds. These compounds were almost as effective as quercetin that inhibited
the violacein production by 56% in comparison to violacein production in the wild-type with 2%
DMSO (Table 2, Fig. 3A). The cell viability staining indicated that among the series 23–30, only
two compounds, 24 and 26, displayed some bactericidal activity. On the other hand, the remaining
six compounds, including those with the highest QS inhibition, showed a negligible bactericidal
effect in both strains ranging from 6.5 to 16.2% compared to the same strains with 2% DMSO
(Table 2, Fig. 3B).
Table 2
Quorum sensing inhibition of chloroquine fumardiamides 23–30 at 400 µM concentration.
Chromobacterium violaceum strains
ATCC31532
CV026
Bactericidal
Cmpd.
Chemical structure
Bactericidal QQ (%)
QSI (%)
effect (%)
effect (%)
Cl
23
24
25
N
O
23.0 ± 1.7
10.7 ± 0.4
nt
nt
nt
nt
H
N
H
N
O
N
H
N
H
O
O
Cl
Cl
Cl
H
N
O
N
O
ne
ne
43.7 ± 4.0
8.3 ± 0.6
nt
nt
H
N
O
N
H
N
H
O
O
N
O
N
O
H
N
N
H
N
H
O
N
O
2
6
H
N
9.1 ± 0.3
44.6 ± 3.0
nt
nt
N
H
N
O
O
N
O
H
Cl
2
2
7
8
N
O
46.3 ± 0.9
4.7 ± 0.6
12.1 ± 0.5
17.5 ± 1.5
53.3 ± 4.7 10.3 ± 0.2
H
N
NH2
N
H
N
H
O
Cl
NH2
N
O
nt
nt
H
N
N
H
N
H
O
8

Cl
2
3
9
0
N
O
NH
45.9 ± 6.7
47.2 ± 5.0
6.5 ± 0.6
61.6 ± 6.7 10.3 ± 0.3
62.6 ± 7.5 13.4 ± 0.2
H
N
N
N
H
H
O
Cl
N
O
H
16.2 ± 1.5
N
N
N
H
O
NH2
CQ
Q
13.2 ± 1.8
55.8 ± 7.4
10.1 ± 0.9
14.7 ± 0.9
nt
nt
66.8 ± 1.4 17.1 ± 0.3
AZ
90.6 ± 10.6
85.6 ± 7.7
86.3 ± 12.6 85.9 ± 9.7
QSI – quorum sensing inhibition; QQ – quorum quenching; CQ – chloroquine; Q – quercetin; AZ – azithromycin; nt –
not tested; ne – no effect
Fig. 3. Comparison of violacein production (A) and cell viability (B) of ATCC31532 (left panel) and CV026 (right panel)
cultures with and without the indicated compounds. PDYT, wells containing only the culture medium; Cells, cultures
containing 2% DMSO (ATCC31532) or 2% DMSO and 0.5 µM C6-HSL. Error bars, ± SD (n = 3–4). Violacein was
detected at 595 nm and the reduction of resazurin to resorufin using λex = 560 nm and λem = 590 nm. No inhibition, cells
with maximal violacein production or viability. Max inhibition, QS inhibition achieved with quercetin (Q) or inhibition
of viability with bactericidal drug azithromycin (AZ). Red arrows indicate the most efficient QSIs/QQs.
9

The QS inhibition of the most potent compounds 27, 29 and 30 was further evaluated against
both reporter strains at 10-fold lower concentration (40 µM). The results showed that all three
compounds displayed a higher QS inhibition in CV026 than in ATCC31532 strain, with no detectable
bactericidal effects (Table 3, Fig. 4). In CV026 strain, compounds 27 and 29 showed more than 50%
of quercetin’s inhibitory activity, while compound 30 was less effective (approx. 30%). Due to
significant differences in QS inhibition profiles between the two reporters, we suggest that none of
the compounds interacted with CviR. This is supported by the finding that the final violacein levels
reached by both strains without the compound are comparable (Fig. 4A), which also suggests similar
CviR levels in both strains. In view of this, we propose two alternative mechanisms of action. In the
first proposal, the active compounds bind the externally added AHL molecules (C6-HSL), hamper
their diffusion into the cells and prevent the induction of QS (Fig. 5A). The tested compounds might
change the membrane permeability for AHLs as well. In the second proposal, the formed hybrid
molecules reach the cytoplasm, but due to steric hindrances are not able to interact with the CviR
receptor (Fig. 5B). Thus, the interaction between the compound and the AHL occurs outside the C.
violaceum cells, which suggests that compounds 27, 29 and 30 act as QQs.
Table 3
Quorum sensing inhibition of chloroquine fumardiamides 27, 29 and 30 at 40 µM concentrations.
QSI in C. violaceum
strains ATCC31532 (%)
ne
5.4 ± 3.8
7.0 ± 0.1
QQ in C. violaceum strain
CV026 (%)
52.5 ± 3.2
42.8 ± 1.9
39.9 ± 0.6
Cmpd.
2
2
3
7
9
0
Q
55.8 ± 7.4
66.8 ± 1,41
AZ
90.6 ± 10.6
86.3 ± 12.6
a
quercetin concentration c = 400 µM; QSI – quorum sensing inhibition;
QQ – quorum quenching; CQ – chloroquine; Q – quercetin; AZ – azithromycin
1
0

Fig. 4. Comparison of violacein production (A) and cell viability (B) of ATCC31532 (left panel) and CV026 (right panel)
cultures with and without the indicated compounds at 400 and 40 µM concentration. PDYT, wells containing only the
culture medium; Cells, cultures containing 2% DMSO (ATCC31532) or 2% DMSO and 0.5 µM C6-HSL. Error bars, ±
SD (n = 3–4). No inhibition, cells with maximal violacein production or maximal viability. Max inhibition, QS inhibition
achieved with quercetin (Q) or inhibition of viability with bactericidal drug azithromycin (AZ). Red arrows indicate the
most efficient QSIs/QQs.
1
1

Fig. 5. Proposed mechanisms-of-actions behind the 27-, 29-, 30-mediated inhibition of QS in C. violaceum. (A) In C.
violaceum ATCC 31532, the cviI synthase gene produces the AHL (C6-HSL) molecule that is recognized by the
cytoplasmic receptor CviR. When bound to AHL, CviR dimerizes and binds DNA, leading to the expression of QS-
regulated genes, including the vioABCDE operon genes coding for the colored pigment, violacein. (B) In CV026, the
QS is activated by the addition of AHL (C6-HSL) that after diffusion into the cells and interaction with the CviR leads
to the activation of the vio operon genes. Compounds 27, 29 and 30 (yellow objects) are likely to bind the AHL, which
either impairs the diffusion of the hybrid-molecule into the cell or prevents the interaction with CviR. O, operator
sequence harboring the CviR binding site.
Calculation of the physicochemical parameters of compounds 23–30 perfectly suits with the
biological findings and the above-mentioned requirements for Gram-negative bacteria. The most
potent compounds 27, 29 and 30 are very polar with low log P (0.409 or 1.05) and negative
distribution coefficient at pH 7.4 (log D –1.38 and –1.73, respectively), whereas the inactive
7
.4
aromatic amino compound 28 and N-protected compounds 23–26 are less polar (log P = 2.101–
4
.335 and log D = 1.86–4.09) and less basic (Table 4).
7.4
Table 4
Properties of novel compounds 23–30 calculated with Chemicalize.org program.23
Cmpd.a
Molecular
formula
Number
of
MW
log P
log
HBD HBA Lipinski
MR
(cm /mol)
TPSA
(Å )
b
3
2
D
7.4
score
atoms
2
2
2
2
2
2
2
3
3
4
5
6
7
8
9
0
C
C
C
C
C
C
C
C
24
28
27
27
19
23
22
22
H
H
H
H
H
H
H
H
32ClN
32ClN
36ClN
36ClN
24ClN
24ClN
28ClN
28ClN
5
5
5
5
5
5
5
5
O
O
O
O
O
O
O
O
4
4
4
4
2
2
2
2
66
70
73
73
51
55
58
58
489.21 2.101 1.86
537.21 4.335 4.09
529.25 2.333 2.09
529.25 2.333 2.09
389.88 0.409 –1.38
437.16 2.595 2.35
429.19 1.05 –1.73
429.19 1.05 –1.73
4
4
3
3
4
4
4
4
5
6
5
5
5
5
5
5
4
3
3
3
4
4
4
4
133.33
152.41
145.72
145.72
108.51
126.82
120.72
120.72
121.45
121.45
112.66
112.66
109.14
109.14
95.15
95.15
a
Physicochemical data of compounds 1–18 are reported previously.15–17 MW – molecular weight; log P – partition
coefficient; log D7.4 – distribution coefficient at pH 7.4; HBD – number of H-bond donors; HBA – number of H-bond
b
acceptors; MR – molecular refractivity; TPSA – topological polar surface area; out of four.
We demonstrated that CQ-fumardiamides 13–16 and 18 are promising QSIs. Optimization
of their tail domain led to more effective QS inhibition in the C. violaceum reporter strains. At 400
1
4
µM concentrations, three compounds, namely N -(2-aminoethyl)-N -[4-[(7-chloroquinolin-4-
1
4
yl)amino]butyl]fumaramide (27), N -[4-[(7-chloroquinolin-4-yl)amino]butyl]-N -(piperidin-4-
yl)fumaramide (29) and (E)-4-(4-aminopiperidin-1-yl)-N-[4-[(7-chloroquinolin-4-yl)amino]butyl]-
4
-oxobut-2-enamide (30), i.e. fumardiamides bearing CQ head-group and aminoethyl or piperidine
motifs, demonstrated excellent QS inhibition on both C. violaceum reporter strains. At 40 µM
concentrations, the differences in the QS inhibition profiles between the wild-type and the mutant
1
2

reporters occurred, indicating that QS communication might be interrupted outside the cells. Since
compounds 27, 29 and 30 had no bactericidal effect on the C. violaceum cells, we considered them
as new QQs able to block QS without inducing natural selection pressure as traditional
antimicrobial agents. Our results could help in further design of agents targeting cell-to-cell
communication and may open new avenues for the combat against drug-resistant bacteria.
Acknowledgments
The study was supported by the Croatian Science Foundation through the research project IP-09-
2
014-1501 and the Jane and Aatos Erkko Foundation (Helsinki, Finland). The work of doctoral
student M. Beus has been fully supported by the Young researcher’s career development project –
training of doctoral students of the Croatian Science Foundation founded by the European Union
from the European Social Fund. JZP proposed original idea of synthesizing terminal amino group-
containing compounds disclosed in this study.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
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Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that
could have appeared to influence the work reported in this paper.
Corresponding Author
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4

Professor Branka Zorc, PhD
Chloroquine fumardiamides as novel quorum sensing inhibitors
a,*
b,*
c
c
b
Maja Beus , Kirsi Savijoki , Jayendra Z. Patel , Jari Yli-Kauhaluoma , Adyary Fallarero ,
Branka Zorc a,**
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5