Antimicrobial activity of a library of thioxanthones and their potential as efflux pump inhibitors

Article abstract of DOI:10.3390/ph14060572

The overexpression of efflux pumps is one of the causes of multidrug resistance, which leads to the inefficacy of drugs. This plays a pivotal role in antimicrobial resistance, and the most notable pumps are the AcrAB-TolC system (AcrB belongs to the resistance-nodulation-division family) and the NorA, from the major facilitator superfamily. In bacteria, these structures can also favor virulence and adaptation mechanisms, such as quorum-sensing and the formation of biofilm. In this study, the design and synthesis of a library of thioxanthones as potential efflux pump inhib-itors are described. The thioxanthone derivatives were investigated for their antibacterial activity and inhibition of efflux pumps, biofilm formation, and quorum-sensing. The compounds were also studied for their potential to interact with P-glycoprotein (P-gp, ABCB1), an efflux pump present in mammalian cells, and for their cytotoxicity in both mouse fibroblasts and human Caco-2 cells. The results concerning the real-time ethidium bromide accumulation may suggest a potential bacterial efflux pump inhibition, which has not yet been reported for thioxanthones. Moreover, in vitro studies in human cells demonstrated a lack of cytotoxicity for concentrations up to 20 μM in Caco-2 cells, with some derivatives also showing potential for P-gp modulation.

Full text of DOI:10.3390/ph14060572

pharmaceuticals
Article
Antimicrobial Activity of a Library of Thioxanthones and Their
Potential as Efflux Pump Inhibitors
Fernando Durães 1,2 , Andreia Palmeira 1,2 , Bárbara Cruz 3, Joana Freitas-Silva 2,4 , Nikoletta Szemerédi 5
,
Luís Gales 6,7 , Paulo Martins da Costa 2,4 , Fernando Remião 3 , Renata Silva 3 , Madalena Pinto 1,2
,
Gabriella Spengler 5,
*
and Emília Sousa 1,2,
*
1
Laboratory of Organic and Pharmaceutical Chemistry, Department of Chemical Sciences, Faculty of
Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal;
fduraes5@gmail.com (F.D.); apalmeira@ff.up.pt (A.P.); madalena@ff.up.pt (M.P.)
2
3
CIIMAR-Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Novo Edifício
do Terminal de Cruzeiros do Porto de Leixões, Avenida General Norton de Matos, S/N,
4450-208 Matosinhos, Portugal; joanafreitasdasilva@gmail.com (J.F.-S.); pmcosta@icbas.up.pt (P.M.d.C.)
UCIBIO-REQUIMTE, Laboratory of Toxicology, Faculty of Pharmacy, University of Porto, Rua de Jorge
Viterbo Ferreira 228, 4050-313 Porto, Portugal; barbara_cruz96@hotmail.com (B.C.); remiao@ff.up.pt (F.R.);
rsilva@ff.up.pt (R.S.)
4
5
6
7
ICBAS–Institute of Biomedical Sciences Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira
228, 4050-313 Porto, Portugal
Department of Medical Microbiology, Albert Szent-Györgyi Health Center and Faculty of Medicine,
University of Szeged, Semmelweis utca 6, 6725 Szeged, Hungary; szemeredi.nikoletta@med.u-szeged.hu
Department of Molecular Biology, ICBAS–Instituto de Ciências Biomédicas Abel Salazar, University of Porto,
Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal; lgales@ibmc.up.pt
Bioengineering & Synthetic Microbiology, I3S–Instituto de Investigação e Inovação em Saúde, University of
Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal
Correspondence: spengler.gabriella@med.u-szeged.hu (G.S.); esousa@ff.up.pt (E.S.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Durães, F.; Palmeira, A.;
Cruz, B.; Freitas-Silva, J.; Szemerédi,
N.; Gales, L.; da Costa, P.M.; Remião,
F.; Silva, R.; Pinto, M.; et al.
*
Abstract: The overexpression of efflux pumps is one of the causes of multidrug resistance, which
leads to the inefficacy of drugs. This plays a pivotal role in antimicrobial resistance, and the most
notable pumps are the AcrAB-TolC system (AcrB belongs to the resistance-nodulation-division
family) and the NorA, from the major facilitator superfamily. In bacteria, these structures can also
favor virulence and adaptation mechanisms, such as quorum-sensing and the formation of biofilm. In
this study, the design and synthesis of a library of thioxanthones as potential efflux pump inhibitors
are described. The thioxanthone derivatives were investigated for their antibacterial activity and
inhibition of efflux pumps, biofilm formation, and quorum-sensing. The compounds were also
studied for their potential to interact with P-glycoprotein (P-gp, ABCB1), an efflux pump present in
mammalian cells, and for their cytotoxicity in both mouse fibroblasts and human Caco-2 cells. The
results concerning the real-time ethidium bromide accumulation may suggest a potential bacterial
efflux pump inhibition, which has not yet been reported for thioxanthones. Moreover, in vitro studies
Antimicrobial Activity of a Library of
Thioxanthones and Their Potential as
Efflux Pump Inhibitors.
Pharmaceuticals 2021, 14, 572. https://
doi.org/10.3390/ph14060572
Academic Editors: Eduarda M.
P. Silva and Diana Cláudia Pinto
Received: 31 May 2021
Accepted: 14 June 2021
Published: 15 June 2021
in human cells demonstrated a lack of cytotoxicity for concentrations up to 20
with some derivatives also showing potential for P-gp modulation.
µM in Caco-2 cells,
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Keywords: thioxanthones; antimicrobial resistance; efflux pumps; biofilm; quorum-sensing
1. Introduction
Copyright:
© 2021 by the authors.
Multidrug resistance (MDR), the ability to evade the activity of a wide range of
structurally diverse compounds, is a rising problem in treating a multiplicity of pathologies,
such as cancer and infectious diseases [1]. One of the reasons for this phenomenon is the
overexpression of efflux pumps, transmembrane structures present in every cell from
human to bacteria, which usually play an important part in detoxification pathways, but
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
are also responsible for the appearance of MDR [1,2]. In many cancers, overexpression
of P-glycoprotein (P-gp, ABCB1) contributes to the found multidrug resistance (MDR)
Pharmaceuticals 2021, 14, 572. https://doi.org/10.3390/ph14060572
https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021, 14, 572
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phenotype [
1
,2
]. P-gp has been an interesting target to reverse MDR in cancer, although
attempts to find successful therapies have been challenging [
resistance mechanism, resorting to the overexpression of efflux pumps, which leads to an
increased outward transport of drugs, rendering them ineffective [ ]. These are divided
into six families, and of these, the resistance-nodulation-division (RND) [ ] and the major
facilitator superfamily (MFS) [ ] are the most relevant. One of the most notable pumps
3]. Bacteria present a similar
4
5
6
in the RND family, well-distributed in Gram-negative bacteria, is the AcrAB-TolC efflux
system, comprised of three distinct parts: AcrA, the membrane fusion protein; AcrB, a
resistance-nodulation-division transporter; and TolC, a multifunctional outer membrane
channel [
4
]. The MFS efflux pumps are more associated with Gram-positive bacteria,
]. In bacteria, efflux
and the NorA pump acquires the most relevance in this family [
4
pumps are associated with many other mechanisms, such as resistance, adaptation, and
virulence mechanisms. When it comes to biofilm, the capacity of certain microorganisms to
form a complex association that latches onto surfaces, efflux pumps have been proven to
play a pivotal role [7]. They can be involved in the efflux of quorum-sensing molecules
and/or extracellular polymeric substances, which regulate bacteria communication and
the development of the biofilm matrix, respectively. These pumps are also responsible for
the efflux of harmful molecules and can influence the aggregation [8,9].
Thioxanthones are compounds that possess a dibenzo-γ-pyrone, tricyclic scaffold,
they are privileged structures [10] and they have previously been described as capable of
modulating the human efflux pump P-gp [11]. Thioxanthones have also demonstrated
their potential as antimicrobials and have proved to be effective in enhancing the activity
of antibiotics in resistant bacterial strains [12]. They are isosteres of xanthones, which
have recently been reported as bacterial efflux pump inhibitors [13]. Herein, a library of
thioxanthones as potential efflux pump inhibitors was designed and synthesized, and
characterized according to their antimicrobial activity, synergy with antibiotics, bacterial
efflux pump inhibition and related resistance mechanisms, such as inhibition of biofilm
formation and quorum-sensing. Additionally, studies on the ability of new thioxanthones
to modulate mammalian P-gp were also carried out. Docking studies were performed to
understand the interactions between the hit compounds with the best overall results and
putative efflux pump targets. Results obtained suggest the potential of these compounds
to tackle multiple bacterial resistance mechanisms.
2. Results and Discussion
2.1. Chemistry
The aim of this work was to build a library of thioxanthone derivatives with poten-
tial for bacterial efflux pump inhibition, based on previously found P-gp inhibitors [11].
Moieties involved in bacterial efflux pump inhibitors and antimicrobials were associ-
ated to a thioxanthone scaffold, previously explored by some of us to discover P-gp
modulators [11]. Firstly, with a preliminary virtual screening, docking studies were per-
formed to select the thioxanthones that preferably interacted with bacterial efflux pumps
(Section 2.3). Thirteen thioxanthones and four tetracyclic thioxanthenes were selected
and synthesized from commercially available 1-chloro-4-propoxy-9H-thioxanthen-9-one
(
1
, Table 1). The synthesis of compounds
scribed [11]. New 1-nitrogen substituted thioxanthones
in a similar way, via a copper-catalyzed Ullmann-type C–N coupling (procedure
selection of chemical ligands to couple in order to obtain the proposed derivatives included
primary or secondary aliphatic amines and sulfamides. Compound , a 1-methoxyl thiox-
anthone derivative, was a secondary product of the reaction in procedures and , further
included in the screening. A 1-bromine derivative (14, Table 1) was also synthesized,
based on the reaction described by Grossman et al. (2006), starting from (procedure
) [14], and was also used as a starting material for the coupling with sulfamides, which
were less reactive (procedure ). Four tetracyclic thioxanthenes (15 18, Table 1) were also
synthesized under the same conditions as compounds 13. These were obtained from
2
–
7
(Table 1) was performed as previously de-
13 (Table 1) were synthesized
). The
8–
a
4
a
b
1
c
b
–
8
–

Pharmaceuticals 2021, 14, 572
3 of 30
guanidine or urea derivatives leading to a quinazoline-thiochromene scaffold, as previously
described [15].
Table 1. Synthetic procedures and structures of the compounds. Reagents and conditions (a) X = Cl
◦
(1
), amine, K2CO3, CuI, MeOH, 100 C, 48 h, sealed flask; (b) X = Br (14), amine, K2CO3, CuI, MeOH,
◦
100 C, 48 h, sealed flask; (c) KBr, H3PO4, CuI, nitrobenzene, 48 h, reflux.
Compound
2
R
Procedure
a
3
4
a
a
5
6
a
a
7
8
a
a
9
a
a
10
11
a

Pharmaceuticals 2021, 14, 572
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Table 1. Cont.
Compound
R
Procedure
12
a, b
13
14
b
c
Br
Compound
R
Procedure
15
16
H
NH2
a
a
17
a
2.2. Structure Elucidation
The structures of the compounds were elucidated by nuclear magnetic resonance
(NMR), Fourier-transform infrared spectroscopy (FT-IR), high resolution mass spectrom-
etry (HRMS) and, in the case of compounds 13–16, X-ray crystallography. Figure S1
(Supplementary Data) presents the main 1H and 13C NMR chemical shifts of the signals for
the newly synthesized thioxanthones 8–14.
The newly synthesized thioxanthones
thioxanthone skeleton in the NMR spectra, previously described for compounds
The heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond
8–
14 displayed characteristic signals of the
2–7 [11].

Pharmaceuticals 2021, 14, 572
5 of 30
correlation (HMBC) spectra were also obtained. HMBC was particularly useful in the
quaternary carbons assignment, as illustrated in Figure S2 (Supplementary Data).
The structures of compounds 13 and 14 were determined by single crystal X-ray
diffraction (Figure 1). A general feature of xanthones [16], which is shared by 13 and
14, is the planarity of the three ring skeleton. The central pyranoid rings have partial
aromatic character, with the shortening of the C—S bonds showing the partial double-bond
character (ranging from 1.741(4) to 1.752(3) Å in 13 and 14, while C—S single bond lengths
in tetrahydrothio-γ-pyrone are 1.806(4) and 1.814(4)Å [17]).
Figure 1. ORTEP view of compounds 13 and 14.
2.3. Docking Studies
The library of 13 thioxanthone derivatives and four tetracyclic thioxanthenes was
investigated, to find their predicted binding affinities against relevant bacterial efflux
pumps. Due to the effect of xanthones on strains with these transporters [13], and due to
structurally related thioxanthenes reported as NorA inhibitors [18], we hypothesized that
the series of in-house thioxanthones
2
–
7, with P-glycoprotein inhibitory activity [11], as
well as the newly synthesized thioxanthones
8–
18 could be effective efflux pump inhibitors.
These derivatives were docked against the most relevant targets, the AcrAB-TolC efflux
system from the RND family [19], and the MFS pump NorA [20], and their predicted
affinity against these targets was ranked. To predict their selectivity for bacterial efflux
pumps, docking studies against mammalian P-glycoprotein were also performed. Both
NorA and human P-gp do not have a crystal structure deposited in the Protein Data Bank,
and homology models needed to be built. Reference compounds described as efflux pump
inhibitors were used as positive controls.
Docking studies were performed based on the crystal structures of the AcrB (4DX5),
AcrA (2F1M), and TolC (1EK9) portions of the AcrAB-TolC efflux system. For AcrB and
AcrA, these studies were performed in two different sites: the substrate-binding site (SBS)
and the hydrophobic trap (HT) for AcrB [21], and the helical hairpin (HH) and the lipoyl
domain (LD) for AcrA [19]. For TolC, only the lysine residues that interact with the
3,30-dithiobis(sulfosuccinimidyl proprionate) (DTSSP) bifunctional crosslinker [19] were
considered. For the NorA homology model, the sites used for docking of the compounds
were the binding core region (BCR) and the cytoplasmic side (CS), as described in [22].
Additionally, compounds 2–18 were docked into a homology model of the mammalian
efflux pump P-gp, in the transmembrane domain (TMD) and in the nucleotide binding
domain (NBD) [11]. The results are presented in Table 2.

Pharmaceuticals 2021, 14, 572
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Table 2. Docking results for the thioxanthones
2–
18 and reference compounds against bacterial and
mammalian efflux pumps.
Docking Scores
Compound
AcrB
AcrA
NorA
P-gp
TolC
SBS
HT
HH
LD
BCR
CS
TMD
NBD
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
−6.3
−6.1
−6.9
−6.5
−7.5
−7.4
−7.6
−8.1
−7.3
−7.2
−7.5
−8.2
−7.8
−6.3
−6.5
−7.4
−7.9
−8.5
−9.7
−8.9
−7.9
−8.7
−7.1
−8.7
17.5
−1.0
−5.0
−5.1
4.2
−5.4
−5.4
−5.6
−5.4
−6.2
−6.5
−5.7
−6.0
−5.8
−5.8
−6.1
−6.9
−6.5
−5.4
−5.7
−5.8
−6.9
−6.8
−6.2
−7.2
−7.9
−6.2
−5.8
5.6
−4.2
−5.4
−4.7
−5.2
−6.3
−6.6
−4.9
−5.2
−4.8
−5.3
−5.7
−6.4
−6.0
−5.3
−5.7
−6.1
−5.6
−5.7
−5.1
−5.6
−6.2
−5.4
−4.9
4.6
−6.2
−6.3
−6.8
−6.2
−7.5
−6.9
−7.7
−7.3
−6.9
−6.7
−7.2
−7.8
−7.1
−6.4
−6.6
−7.0
−9.0
−8.4
−7.4
−7.2
−7.7
−7.7
−7.1
−7.5
−5.2
−5.6
−3.9
−5.3
−7.8
−7.4
−2.3
−6.2
−4.0
−6.1
−6.1
−5.5
−6.2
−4.7
−5.8
−5.5
−5.2
4.0
−4.9
−4.9
−5.7
−5.3
−6.2
−5.3
−5.5
−5.0
−5.8
−5.7
−5.7
−5.7
−5.1
−5.7
−5.4
−5.6
−6.5
−7.4
-
−5.7
−7.2
−8.5
−7.1
−8.6
−9.2
−8.0
−9.2
−8.2
−7.2
−7.4
−8.9
−9.2
−7.4
−7.3
−8.0
−9.2
−10.4
-
−6.6
−5.2
−5.3
−5.7
−5.2
−6.3
−5.6
−6.3
−5.6
−5.7
−5.9
−6.6
−5.8
−5.5
−5.1
−6.7
−7.2
−6.0
-
−3.4
4.8
−2.5
−3.6
−5.7
1.6
−5.7
−4.8
−4.7
−5.6
−5.7
−6.2
32.9
26.5
15.4
2.9
18
D13-9001
Doxorubicin
MBX-3132
Minocycline
PAβN
Reserpine
trans(E)-
flupentixol
Verapamil
-
-
-
-
-
-
-
−8.3
−7.5
-
-
26.7
−4.7
10.9
−6.8
-
-
−6.3
-
-
−9.4
−5.3
1.0
−4.6
-
-
-
-
-
-
-
-
−3.2
-
−4.6
-
-
-
-
-
−7.1
−4.1
SBS: Substrate-binding site; HT: Hydrophobic trap; HH: Helical hairpin; LD: Lipoyl domain; BCR: Binding core
region; CS: Cytoplasmic side; TMD: Transmembrane domain; NBD: Nucleotide binding domain.
The results predicted thioxanthones
ported inhibitors, considering that they present comparable docking scores, and globally
they present better scores than the starting materials and 14. Among the portions of the
2–18 to bind with an affinity similar to the re-
1
AcrAB-TolC efflux system, compounds generally present the highest affinities towards
the SBS of AcrB portion, and AcrA would be the portion towards which the compounds
are predicted to present the least affinity, only with better docking scores than for the
hydrophobic trap of AcrB. In terms of docking into the NorA homology model, there is an
even distribution between both sites targeted. It is noteworthy that most of the compounds
presented the lowest docking scores for the P-gp model, compared to all the bacterial
pumps tested, suggesting that these compounds also act as mammalian efflux substrates
(with the exception of compounds 8 and 10). Considering that the compounds presented
docking scores similar to those previously reported as bacterial efflux pump inhibitors, this
library was selected to be investigated in vitro in resistant bacterial models.
2.4. Antibacterial Activity and Synergy with Antimicrobials
The referred library of thioxanthones was initially tested for their antimicrobial activity
against six bacterial strains: the Gram-negative Escherichia coli ATCC 25922, Pseudomonas
aeruginosa ATCC 27853 and Salmonella enterica serovar Typhimurium SL1344 (SE03), with
the acrA gene deleted, and the Gram-positive Staphylococcus aureus ATCC 29213, Enterococ-
cus faecalis ATCC 29212 and the methicillin and ofloxacin-resistant clinical isolate S. aureus
272123. For the synergy assay, two clinically relevant isolates were used: the extended-
spectrum β-lactamase (ESBL)-producing E. coli SA/2 [23] and the vancomycin-resistant E.
faecalis (VRE) B3/101 [23]. The antibiotics used were cefotaxime (CTX) and vancomycin
(VAN), respectively.

Pharmaceuticals 2021, 14, 572
7 of 30
The results are depicted in Table 3 and show that, out of all the compounds tested,
only the aminated derivative
the used strains, with a minimum inhibitory concentration (MIC) of 83
S. aureus ATCC 23213 and E. faecalis ATCC 23121, respectively, with 12 also active against
SE03 (MIC = 50 M). Curiously, the sulfamide precursor of 12, 4-sulfamoylbenzoic acid, did
2
and the sulfamide derivative 12 presented activity against
µ
M and 34 M for
µ
µ
not display an observable MIC for the concentrations tested (results not shown). Moreover,
sulfamide 12 does not contain the para-amino group essential for the antibacterial activity
of these p-aminobenzoic acid (PABA) antimetabolites [24]. Therefore, we can hypothesize
a distinct mechanism of action for this new antibacterial agent. Moreover, compound 12
was found to be more active than the previously described antibacterial thioxanthones [12],
and deserves to be further explored.
Table 3. Minimum inhibitory concentrations of the compounds in the antibacterial activity assay and synergy
with antibiotics.
Antibacterial Activity
Minimum Inhibitory Concentration (MIC) (µM)
Synergy with Antimicrobials
E. faecalis
E. coli SA/2
B3/101
Compound
E. coli
ATCC
25922
CTX MIC = 1124
(512 µg/mL)
VAN MIC = 707
(1024 µg/mL)
P. aeruginosa
ATCC 27853
E. faecalis
ATCC 29212
S. aureus
ATCC 29213
S. aureus
272123
SE03
CTX +
VAN +
Compound 1
Compound 2
83 (32
µg/mL)
83 (32
µg/mL)
2
-
-
>100
>100
-
-
3
4
5
6
7
-
-
-
-
-
-
-
-
-
-
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
281
(128 µg/mL)
1124
(512 µg/mL)
281
(128 µg/mL)
1124
(512 µg/mL)
70
(32 µg/mL)
1124
(512 µg/mL)
1124
(512 µg/mL)
1124
(512 µg/mL)
1124
(512 µg/mL)
1124
(512 µg/mL)
281
(128 µg/mL)
707
8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
50
(1024 µg/mL)
44
(64 µg/mL)
707
(1024 µg/mL)
707
(1024 µg/mL)
707
(1024 µg/mL)
707
(1024 µg/mL)
707
(1024 µg/mL)
707
(1024 µg/mL)
707
9
10
11
12
13
14
15
16
17
34
34
(16 µg/mL)
(16 µg/mL)
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
(1024 µg/mL)
707
(1024 µg/mL)
707
18
(1024 µg/mL)
1
All compounds were kept at the highest concentration tested in the antibacterial activity (64 µg/mL), considering that none showed a
2
direct inhibitory effect on E. coli. As compound 12 revealed antibacterial activity against E. faecalis, the synergy with vancomycin was
determined in the presence of the highest concentration that did not affect bacterial growth when the compound was used alone (8 g/mL).
All the other compounds were kept at the highest concentration tested in the antibacterial activity assay (64 g/mL). Compounds were
µ
µ
2–7
previously tested for their antimicrobial activity and synergy in these strains [12]. CTX: cefotaxime; VAN: vancomycin. SE03: Salmonella
enterica serovar Typhimurium SL1344 (SE03).
Concerning the synergy with antimicrobials, four new thioxanthones (
18) were able to decrease the MIC of CTX in E. coli SA/2 and one reversed the resistance to
vancomycin in E. faecalis B3/101. In fact, compounds 10 and 18 caused a 4-fold decrease
in the MIC of CTX, while compound 12 caused a 16-fold decrease. Regarding E. faecalis
8, 10, 12, and
8,

Pharmaceuticals 2021, 14, 572
8 of 30
B3/101, compound
9
can cause a 16-fold decrease of the MIC of VAN. As E. coli SA/2 is
an ESBL producer and E. faecalis B3/101 had the capacity to modify the peptidoglycan
synthesis pathway, it can be hypothesized that the compounds could interact with these
targets, but further studies are needed to elucidate the precise mechanisms.
2.5. Efflux Pump Inhibition Assay
To investigate the potential of these compounds as efflux pump inhibitors, a prelim-
inary screening of compounds 2–18 on two resistant Gram-positive and Gram-negative
bacterial strains was performed to investigate the compounds’ capability of modulating
the accumulation of the efflux pump substrate ethidium bromide (EB). S. aureus 272123 is a
clinical strain and was used to compare the activity of thioxanthones with xanthones which
were previously tested in the same model [13]. In this strain, the expressions of the mepA
and norA genes were studied in the presence of antimicrobials of natural origin, and the
norA expression levels did not change [25]. As AcrA was the target with the least binding
affinity (Section 2.3), Salmonella enterica serovar Typhimurium SE03, with a deletion of the
acrA gene, was selected as a Gram-negative strain.
All the compounds, except for compound 12, were tested at the concentration of
50
µM, since none of them showed antibacterial activity at this concentration. Compound
12 was tested at one-third of its MIC, 17
µ
M, for SE03, and at 50 M for S. aureus 272123.
µ
The relative fluorescence index (RFI) was calculated based on the means of relative fluo-
rescence units, and theses RFIs are depicted in Table 4. Reserpine and carbonyl cyanide
3-chlorophenylhydrazone (CCCP) were used as positive controls for S. aureus 272123 and
SE03, respectively, at the sub-MIC concentration of 25 µM.
Table 4. Relative fluorescence index (RFI) of tested derivatives.
RFI ± SD
Compound
S. aureus 272123
SE03
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
0.75 ± 0.08
1.50 ± 0.04
0.11 ± 0.26
−0.13 ± 0.04
0.09 ± 0.11
−0.10 ± 0.02
0.24 ± 0.07
0.14 ± 0.02
4.71 ± 0.78
0.89 ± 0.06
0.29 ± 0.19
−0.18 ± 0.05
1.13 ± 0.13
−0.07 ± 0.50
0.96 ± 0.27
9.69 ± 0.95
0.20 ± 0.03
0.50 ± 0.04
—
−0.02 ± 0.04
0.67 ± 0.01
−0.10 ± 0.05
−0.20 ± 0.01
−0.20 ± 0.01
0.49 ± 0.11
0.74 ± 0.08
1.54 ± 0.09
6.00 ± 0.80
1.37 ± 0.054
−0.21 ± 0.07
1.23 ± 0.18
0.87 ± 0.11
0.54 ± 0.11
7.52 ± 0.01
25.51 ± 2.93
0.20 ± 0.02
—
Reserpine
CCCP
0.23 ± 0.04
SD: Standard deviation; SE03: Salmonella enterica serovar Typhimurium SL1344; CCCP: carbonyl cyanide 3-
chlorophenylhydrazone.
From the analysis of Table 4, it can be noted that 2, 3, 7, 8, 9, 10, 11, 13, 14, 15, 16, and 17
can increase the fluorescence in comparison to the positive control. This can be attributed
to the inhibition of the efflux of EB, but the fluorescence emitted by the compound itself
can also interfere. Therefore, an analysis of the curves of the variation of fluorescence over
the course of the assay was performed (data not shown). Additionally, for compounds
2,
3,
9
,
10, 11, 14, 15, 16, and 17, which displayed a high/erratic fluorescence pattern
over the duration of the assay, an additional fluorescence investigation was performed

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(Figures S3–S11, Supplementary Data). Out of these compounds, fluorescence was not
a determinant factor for
2 and 3, along with compounds 7, 8 and 13, whose behavior
during the real-time EB accumulation assay did not suggest the compounds’ fluorescent
interference. It can be noticed that some compounds present negative RFI values. This
is attributed to the compound displaying a lower extent of EB efflux inhibition than the
control, and these compounds were considered ineffective for this purpose.
From the analysis of Figure 2, it can be highlighted that compound
than the positive controls in the inhibition of EB accumulation in both models tested.
Compounds , and 13 were shown to be more effective than CCCP against the efflux
systems of SE03, while was found to be more effective than the reference compound
3 is more effective
7, 8
2
reserpine against the efflux systems of the S. aureus 272123. None of these compounds
present MIC for the respective strain whose efflux they inhibit. This is considered an
advantage, as it has been described that an adequate efflux pump should preferably not
display antibacterial activity, as this may lead to the appearance of resistance [26].
Figure 2. Relative fluorescence index (RFI) of the thioxanthone derivatives
and Salmonella enterica serovar Typhimurium SL1344 (SE03, Bottom). Results are presented as mean
comparisons were performed using the t-test [* p < 0.05; ** p < 0.01; *** p < 0.001 vs. control (DMSO 1% v/v)].
2
,
3
,
7
,
8
, and 13 in S. aureus 272123 (Top
)
±
SD. Statistical

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The structural similarity of thioxanthones and phenothiazines, previously reported
as NorA inhibitors [18], reinforce the idea that thioxanthones may be potential inhibitors
towards this target. However, bacteria present multiple efflux pumps of varied families in
their membranes, and additional studies would clear the question of which specific pump
in being inhibited. To predict how these compounds would interact with their putative
targets, a visual inspection of the interactions that could be established was performed
using PyMOL. Firstly, compounds
2 and 3 were visualized in a homology model of the
NorA pump, particularly in the BCR binding site, as this was the site that was predicted
to be where they would bind with the highest affinity (Section 2.3). A general view
(Figure 3A) shows these to bind differently, in opposite positions relatively to one another.
For compound
Glu-222 and a
homology model. Compound
2
π-π
(Figure 3B), the amine directly bonded to C-1 forms a hydrogen bond with
stacking interaction between one of the aromatic rings and Phe-47 of the
(Figure 3C) is predicted to interact through the oxygen in
3
the propoxy chain with Tyr-292, establishing a hydrogen bond. The thioxanthene trans(E)-
flupentixol, already described as a NorA inhibitor [18], was also predicted to interact with
Tyr-292, but in a different manner, establishing a
hydrogen bonding.
π-π stacking, and with Trp-293, through
Figure 3. Molecular visualization of thioxanthones on targets in the BCR of the NorA homology model. (
compounds (blue) and (pink); ( ) Interaction between compound and the BCR; ( ) Interaction between compound
and the BCR; (D) in the SBS of AcrB, general view of compounds 3 (red), 7 (pink), 8 (green) and 13 (blue).
A) General view of
2
3
B
2
C
3
Compounds
3, 7, 8 and 13 were visualized in the SBS of AcrB. A general view (Figure 3D)
shows that the compounds are predicted to bind in the approximate site, but suggests they

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interact with different residues. Details of these interactions (Figure S12, Supplementary
Data), which could guide the future design of thioxanthones as efflux pump inhibitors, can
be found in Supplementary Data.
2.6. Biofilm Inhibition and Quorum Sensing Inhibition Assays
Efflux mechanisms are one of the factors that can lead to biofilm-mediated resistance,
along with slow growth and reduced penetration due to production of extracellular polysac-
charides [27]. It has been demonstrated that efflux pumps can influence the transport of
extracellular polymeric substances in biofilms, and regulate the expression of genes con-
cerning biofilm formation, as they have an impact in the way bacterial cells adhere and
aggregate to solid surfaces. Additionally, they can also modulate QS, through an influence
in the transport of QS signal molecules [28]. Thioxanthones 2–18 were tested for their
potential for inhibiting biofilm formation by S. aureus ATCC 29213 and the multi-resistant
S. aureus 272123. The biofilm inhibition, presented in percentage (%), was calculated based
on the mean of absorbance units. Reserpine, which has previously been described as
both an efflux pump inhibitor and an inhibitor of biofilm formation in Gram-positive
bacteria [27,28] was used as control in both strains. The results obtained are presented in
Table 5.
Table 5. Biofilm and quorum sensing inhibition of the thioxanthones tested.
Biofilm Inhibition (%) ± SD
Quorum Sensing Inhibition (mm) ± SD
Compound
S. aureus
S. aureus
272123
S. marcescens
wt85
EZF + CV026
ATCC 29213
0.53 ± 1.05 1
81.11 ± 1.18 3
93.49 ± 0.33 4
47.92 ± 1.08 4
0 4
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
93.35
±
0.86 2
31 ± 0.8
0.17 ± 0.83 2
0
0
0
0.37 ± 0.47 2
0 2
0 4
0 2
47.07 ± 7.54 4
30 ± 0.5
0 2
0 5
32 ± 0.1
5.18 ± 1.02 2
31.48 ± 8.99 5
55.17 ± 4.20 5
20.17 ± 4.00 4
96.50 ± 0.67 5
72.04 ± 6.66 4
2.46 ± 0.72 4
75.95 ± 2.06 4
73.59 ± 1.80 4
6.32 ± 4.32 4
1.62 ± 2.43 5
0
0
0
0
0 2
0 2
0
31 ± 0.8
7.81 2
0
0
0
53.32
±
0.42 ± 1.36 2
51 ± 0.1
0 2
0
0 2
32 ± 0.5
50 ± 0.1
29 ± 0.8
49 ± 0.5
0
0 2
0 2
0
0
0
19.68 ±7.46 2
3
90.34 ± 3.35
1
30.37
22.29
±
7.32
5.10
4
Reserpine
PMZ
ND
ND
ND
72.10 ± 1.54
2
±
5
93.76 ± 2.01
ND
ND
18 ± 0.8
40 ± 0.1
41 ± 0.5
1–5
The value of the positive control in each different assay; wt85: C. violaceum wild-type 85; EZF: Sphingomonas
paucimobilis Ezf 10–17; CV026: C. violaceum CV026; SD: Standard deviation. PMZ: Promethazine. ND: not
determined.
From the analysis of the results, it can be seen that compounds
as inhibitors of biofilm formation along with 15 and 16. Most are preferably inhibitors
on S. aureus 272123, with better results than reserpine and with compound being the
most active derivative. Compound also displayed a similar but potent inhibition (higher
2, 3 and 13 were active
3
3
that 90%) in the ATCC strain. It can be hypothesized that this compound may act in the
same mechanism of biofilm formation, and that may be unaltered in both strains. It is
noteworthy that compound
3 also presented inhibition of efflux pumps in S. aureus 272123

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(Figure 2), opening the possibility of these results being related. The antibacterial sulfamide
12 could also inhibit approximately 53% of biofilm formation in this strain.
Concerning the QS inhibition assay, the sensor strain Chromobacterium violaceum
CV026 and the AHL producer strain Sphingomonas paucimobilis Ezf 10-17 (EZF) were
inoculated as parallel lines, and the AHL producers Chromobacterium violaceum wild-type
85 (wt85) and Serratia marcescens AS-1 were inoculated as a single line. The interaction
between the strains and compounds 2–18 was evaluated as the reduction of pigment
production, in millimeters (mm) (Table 5) with promethazine (PMZ) as a positive control.
From the analysis of Table 5, it is noteworthy that, in general, the compounds tested
were more effective against the inhibition of QS in EZF+CV026, with seven compounds (
13 15 16, and 17) presenting an inhibition of pigment production. Two compounds
11 and 15) were active against the inhibition of QS in S. marcescens, and no compounds
3,
7,
8,
,
,
(
were active against wt85. The potential of thioxanthone derivatives as QS inhibitors should
not come as a surprise, due to their structural similarity to PMZ, which was used as a
positive control in this assay [29].
2.7. In Vitro Studies in Mammalian Cells
2.7.1. Cytotoxicity in Mouse Embryonic Fibroblast Cell Line
The cytotoxicity of the most promising derivatives, 3, 8, 12, 13, and 17, which presented
good results in the overall assays performed, such as antibacterial activity, synergy, efflux
pump inhibition assay plus biofilm inhibition and/or QS inhibition assays, was investigated
for toxicity using a mouse embryonic fibroblast cell line (NIH/3T3). The IC50 values of the
tested compounds were >100
µ
M, except for compound 12, with an IC50 of 64.47
±
4.54
µM. Doxorubicin was used as a positive control, with an IC50 value of 12.05
±
0.81 µM
(Table S1, Supplementary Data).
2.7.2. Compounds’ Cytotoxicity in Human Cells
For the newly synthetized thioxanthones 9–17 the potential for P-glycoprotein in-
hibition was also explored. Thus, the cytotoxicity of the thioxanthone derivatives 9–17
(0–20 µM) was initially evaluated, in Caco-2 cells, 24 h after exposure, using two different
cytotoxicity assays; the neutral red (NR) uptake and the sulforhodamine B (SRB) binding
assays, which are based on different principles to measure cellular dysfunction as previ-
ously studied for compounds
the modulation of P-gp expression and/or activity at the non-cytotoxic concentration of
20 M and after 24 h of exposure, which justifies the incubation time and concentration
2–7. In these studies, the tested derivatives show efficacy in
µ
range used [30]. As observed in Figure 4, no significant differences in NR uptake were ob-
served 24 h after exposure to compounds 9–12, and 14, at any tested concentration (10 and
20
impairment. For compounds 15
uptake was observed for the highest tested concentration (20
control cells (NR uptake significantly decreased to 92.2, 94.1 and 95.5%, 24 h after exposure
to compounds 15 16, and 17, respectively) (Figure 3). For compound 13, no significant
effect on NR uptake was detected 24 h after exposure to the highest tested concentration
(20 M), while a slight but significant decrease in the NR uptake was observed for the
10 M concentration (NR uptake significantly decreased to 95.5% over control cells, 24 h
after exposure to 10 µM compound 13).
µ
M), and when compared to control cells (0
16, and 17, a slight but significant decrease in the NR
M), and when compared to
µM), demonstrating the lack of lysosomal
,
µ
,
µ
µ

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Figure 4. Cytotoxicity of the thioxanthone derivatives
9–17 (0–20
µM), in Caco-2 cells, by the neutral red (NR) uptake assay,
24 h after exposure. The results are presented as mean
Statistical comparisons were performed using the one-way ANOVA parametric method, followed by Dunnett’s multiple
±
SD from four independent experiments, carried out in triplicate.
comparisons test [** p <0.01 vs. control (0 µM)].
In the SRB binding assay, and as observed in Figure 5, no significant differences were
detected in the ability of the dye to bind to the basic amino acids of cellular proteins,
24 h after exposure to compounds 9–16, at any tested concentration (10 and 20 µM),
demonstrating the lack of significant differences in the total protein mass and, consequently,
in the number of cells in culture. In what concerns compound 17, no significant differences
in SRB binding were detected 24 h after exposure to 10
but significant increase in SRB binding was observed for the highest tested concentration
(20 M), and when compared to control cells (SRB binding significantly increased to 105.8%
µM concentration, but a slight
µ
24 h after exposure to 20 µM compound 17).
Overall, the obtained results demonstrate the lack of relevant cytotoxicity of the thiox-
anthone derivatives towards Caco-2 cells, for concentrations up to 20 µM, and 24 h after
exposure. Furthermore, the high agreement between the results obtained in the NR uptake
and SRB binding assays should be highlighted, which provides a high confidence in the
obtained data. Based on these results, the non-cytotoxic 20
for the evaluation of the potential effects of compounds in P-gp expression and activity. In
fact, the ability of similar thioxanthone derivatives for P-gp induction/activation has
µM concentration was selected
2–7
already been reported, at the same concentration and using the same in vitro model, which
was the Caco-2 cells [30].

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Figure 5. Cytotoxicity of the thioxanthone derivatives
9–
17 (0–20
µM), in Caco-2 cells, by the sulforhodamine B binding
assay, 24 h after exposure. The results are presented as mean
triplicate. Statistical comparisons were performed using the one-way ANOVA parametric method, followed by Dunnett’s
±
SD from four independent experiments, carried out in
multiple comparisons test [** p <0.01 vs. control (0 µM)].
2.7.3. Effects on P-gp Expression and Activity
The effect of compounds (20 µM) on P-gp protein expression was evaluated, by
flow cytometry, 24 h after exposure, using the monoclonal UIC2-PE antibody, as was
previously studied for the other compounds 2–7. However, the ability of compounds 9–17
to interfere with the fluorescence of the UIC2-PE antibody was assessed first. For this
purpose, Caco-2 cells were incubated for 24 h, in the presence or absence of thioxanthone
derivatives (20
P-gp expression assay. A remarkable increase in the intracellular fluorescence of cells
exposed to compounds 10–13 and 15 17 was observed, making it impossible to assess the
µM), and subsequently analysed, by flow cytometry, as described for the
–
effect of these thioxanthone derivatives on P-gp expression. In fact, compounds 15–17 had
previously been reported as fluorescent compounds [15] and this characteristic limited
some of the studies reported herein. Only compounds
potential to modulate P-gp expression. As observed in Figure 6, no significant differences
in P-gp expression in cells pre-exposed to compound for 24 h were detected, when
compared to control cells (0 M). In contrast, compound 14 caused a significant reduction
in P-gp expression levels (to 83%), when compared to control cells (0 µM, 100%).
9 and 14 were tested further for their
9
µ

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Figure 6. Evaluation of the expression levels of P-glycoprotein (P-gp), by flow cytometry, in Caco-
2 cells exposed to compounds 9 and 14 (20 µM) for 24 h. The results are presented as mean ± SD of
three independent experiments (performed in triplicate). Statistical comparisons were made using the
one-way ANOVA parametric method, followed by Dunnett’s multiple comparisons test [*** p < 0.001
vs. control (0 µM)].
The effects of the compounds (20 µM) on P-gp activity were further assessed, by
fluorescence spectroscopy, using rhodamine (RHO) 123 as a P-gp fluorescent substrate
and zosuquidar (ZOS) as a specific third generation P-gp inhibitor. For that purpose, two
experimental designs were performed. In the first experimental approach, the accumulation
of the P-gp fluorescent substrate was assessed in Caco-2 cells pre-exposed to compounds
(20 µM) for 24 h, aiming to elucidate possible changes in P-gp activity resulting from
changes in P-gp expression, given the long period of incubation with the thioxanthone
derivatives. The combination of the evaluation of both P-gp expression and activity is
extremely important, as an increased P-gp protein expression may not be necessarily
accompanied by an increased activity of the pump [30
the potential interference of compounds’ autofluorescence in the analysis of P-gp activity
were also performed. Accordingly, compounds 11 15 16, and 17 were not tested for their
,31]. Preliminary studies to assess
,
,
ability to modulate P-gp activity, given their remarkable fluorescence in the experimental
conditions used to evaluate RHO 123 intracellular fluorescence. As observed in Figure 7,
none of the tested compounds significantly changed the P-gp activity 24 h after exposure.
Figure 7. P-glycoprotein (P-gp) activity assessed by fluorescence spectroscopy in Caco-2 cells
pre-exposed to the thioxanthone derivatives
9, 10, and 12–14 (20 µM) for 24 h. The results are
presented as mean SD of five independent experiments, performed in triplicate. Statistical compar-
±
isons were made using the one-way ANOVA parametric method, followed by Dunnett’s multiple
comparisons test.
Concerning the second experimental approach used to evaluate the effects of the
compounds in P-gp activity, Caco-2 cells were exposed to thioxanthone derivatives (20
µM)
only during the 90 min of incubation with the fluorescent substrate (RHO 123, 10 M). This
µ
experimental approach aims to evaluate the potential immediate effects of thioxanthone
derivatives on P-gp activity as a result of a direct inhibition or activation of the protein, i.e.,

Pharmaceuticals 2021, 14, 572
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not to change the protein expression. According to the obtained results, and as observed in
Figure 7, no significant differences in P-gp activity were detected in Caco-2 cells exposed
to compounds
12 and 13 caused a significant and immediate decrease in P-gp activity (to 82 and 85%,
respectively), when compared to control cells (0 M). On the other hand, the incubation
9 and 10, when compared to control cells (0 µM). In contrast, compounds
µ
of Caco-2 cells with compound 14 resulted in a slight, but significant, immediate increase
in P-gp activity (116%), when compared to control cells (100%). Therefore, compounds 12
and 13 demonstrated potential as P-gp inhibitors, while compound 14 showed itself to be
an activator of this important efflux protein, overall suggesting a potential new source of
new P-gp modulators. Furthermore, although compound 14 significantly reduced P-gp
expression in Caco-2 cells 24 h after exposure (Figure 6), this decrease did not translate into
a concomitant decrease in the protein activity (Figure 7). A possible explanation for this
result could be a possible contribution of the protein activation, mediated by compound
14, that could have remained intracellularly (after the 24-hour pre-exposure period), in
agreement with its P-gp activation potential, which was observed in the experimental
approach of evaluation of P-gp activity in Caco-2 cells incubated with compound 14, only
during the accumulation with the P-gp fluorescent substrate (Figure 8).
Figure 8. P-glycoprotein (P-gp) activity assessed by fluorescence spectroscopy in Caco-2 cells pre-
exposed to thioxanthone derivatives
9,
10, and 12
–
14 (20
µM) only during the 90 min of the incubation
period with the fluorescent substrate (RHO 123, 10
µ
M). The results are presented as mean
±
SD of four independent experiments, performed in triplicate. Statistical comparisons were made
using the one-way ANOVA parametric method, followed by Dunnett’s multiple comparisons test
[**** p < 0.0001 vs. control (0 µM)].
The results obtained with this assay may lead to some conclusions. Firstly, compound
12 presents both antibacterial activity and P-gp inhibition, which can pose as a limitation
for its use as an antibacterial since it can compromise detoxification pathways. Compound
13, on the other hand, presents activity regarding the inhibition of P-gp and can also inhibit
the efflux systems in SE03. This may lead to the assumption that this compound is a dual
inhibitor of human and bacterial efflux pumps. Further studies are needed to investigate
which efflux pump is being inhibited in SE03 specifically.
3. Materials and Methods
All reagents and solvents used for the synthesis were purchased from Sigma-Aldrich
(Sigma-Aldrich Co. Ltd., Gillinghan, UK) and no further purification process was imple-
mented. Solvents were evaporated using a rotary evaporator under reduced pressure,
Buchi Waterchath B-480. All reactions were monitored by thin-layer chromatography (TLC)
carried out on precoated plates with 0.2 mm of thickness using Merck silica gel 60 (GF254
)
with appropriate mobile phases. Compounds were easily detectable at 254 nm or 365 nm.

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Flash column chromatography using silica gel 60 (0.040–0.063 mm, Merck, Darmstadt,
Germany) was used in the purification of the synthesized compounds. Melting points (mp)
were measured in a Köfler microscope (Wagner and Munz, Munich, Germany) and are
1
uncorrected. H- and 13C-nuclear magnetic resonance (NMR) spectra were recorded at the
University of Aveiro, Department of Chemistry in CDCl3 or DMSO-d6 (Deutero GmbH,
Ely, UK) at room temperature on a Bruker Avance 300 spectrometer (300.13 MHz for 1H
and 75.47 MHz for 13C, Bruker Biosciences Corporation, Billerica, MA, USA). Chemical
shifts are expressed in
δ (ppm) values relative to tetramethylsilane (TMS) as an internal
reference. Coupling constants are reported in Hertz (Hz). 13C-NMR assignments were
made by bidimensional heteronuclear single quantum coherence (HSQC) and heteronuclear
multiple bond correlation (HMBC) NMR experiments (long-range C, H coupling constants
were optimized to 7 Hz) or by comparison with the assignments of similar molecules. High-
resolution mass spectroscopy (HRMS) spectra were measured on a Bruker FTMS APEX III
mass spectrometer (Bruker Corporation, Billerica, MA, USA) and recorded as electrospray
ionization (ESI) mode in Centro de Apoio Cientifico e Tecnolóxico á Investigación (CACTI,
University of Vigo, Pontevedra, Spain) or on a LTQ Orbitrap XL hybrid mass spectrometer
(Thermo Fischer Scientific, Bremen, Germany) at CEMUP, University of Porto, Portugal.
Quantitative and qualitative GC-MS analyses were performed on a Trace GC 2000 Se-
ries ThermoQuest gas chromatograph equipped with ion-trap GCQ Plus ThermoQuest
Finnigan mass detector (Austin, TX, USA). Chromatographic separation was achieved us-
ing a capillary column (30 m 0.25 mm 0.25 µm, cross-linked 5% diphenyl and 95% dimethyl
polysiloxane) from Restek® (Huntingdon, PA, USA) and high-purity helium C-60 as carrier
gas. An initial temperature of 80 ◦C was maintained for 1 minute, increased to 300 ◦C at
◦
10 C/min rate, and held for 5 min, giving a total run time of 45 min. The flow of the carrier
gas was maintained at 1.5 mL/min. The injector port was set at 280 ◦C.
The culture media used in the experiments were the following: cation-adjusted
Mueller-Hinton broth (MHB II; Sigma-Aldrich, St. Louis, MO, USA and Biokar Diag-
nostics, Allone, Beauvais, France), Luria-Bertani broth (LB-B; Sigma, St. Louis, MO, USA),
Tryptic Soy broth (TSB; Scharlau Chemie S. A., Barcelona, Spain), and Tryptic-Soy agar
(TSA; Biokar Diagnostics, Allone, Beauvais, France) were purchased. The modified Luria-
Bertani agar (LB*-A), used for the quorum sensing (QS) inhibition assays, was prepared
in-house, according to the formula: 1.0 g yeast extract (Merck, Darmstadt, Germany), 10.0 g
tryptone (Biolab, Budapest, Hungary), 10.0 g NaCl (Molar Chemicals, Halásztelek, Hun-
gary), 1.0 g K2HPO4 (Biolab, Budapest, Hungary), 0.3 g MgSO4 x 7H2O (Reanal, Budapest,
Hungary), 5 ml Fe-EDTA stock solution and 20.0 g of bacteriological agar (Molar Chemicals,
Halásztelek, Hungary) per 1 L of media.
Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose, neutral red
solution 0.33% (NR), Sulforhodamine B (SRB), rhodamine 123 (RHO 123), zosuquidar
(ZOS), dimethylsulfoxide (DMSO) and Trizma® base were obtained from Sigma (St. Louis,
MO, USA). Nonessential amino acids (NEAA), heat-inactivated fetal bovine serum (FBS),
antibiotic (10,000 U/mL penicillin, 10,000 µg/mL streptomycin), Hank’s Balanced Salt
Solution with or without calcium and magnesium (HBSS (+/+) and HBSS (-/-), respec-
tively) and 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA) were purchased
from Gibco Laboratories (Lenexa, KS, USA). TritonTM X100 was purchased from Thermo
Fisher Scientific (Waltham, MA, USA). Dulbecco’s Phosphate-Buffered Saline solution
without calcium, magnesium and phenol red (PBS (-/-)) was purchased from Grisp (Porto,
Portugal). P-gp monoclonal antibody (clone UIC2) conjugated with phycoerythrin (PE),
was purchased from Abcam (Cambridge, UK). Reagents used in flow cytometry, namely,
the cleaning solution, decontamination solution and flow cell extended clean solution were
purchased from BD Biosciences (San Jose, CA, USA).
Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), sodium dodecyl sulfate (SDS), phosphate-buffered saline (PBS; pH 7.4),
ethidium bromide (EB), reserpine, carbonyl cyanide 3-chlorophenylhydrazone (CCCP),
promethazine (PMZ) and crystal violet (CV) were purchased from Sigma-Aldrich Chemie

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GmbH (Steinheim, Germany). Doxorubicin 2 mg/mL was purchased from Teva Phar-
maceuticals, Budapest, Hungary. The antibiotic cefotaxime (CTX) was purchased from
Duchefa Biochemie (Haarlem, The Netherlands) and vancomycin (VAN) from Oxoid (Bas-
ingstoke, UK). Bacteria were purchased from ATCC, the cell line NIH/3T3 was purchased
from Sigma (Steinheim, Germany) and Caco-2 cells were obtained from the European
Collection of Cell Culture (ECACC, Salisbury, UK).
3.1. Chemistry
The syntheses of compounds
2–7 [11] and 15–18 [15] have been previously reported.
Compounds 8–13 were synthesized in the same way as 15–18, through a copper-catalyzed
Ullmann-type C–N coupling, starting from compound
were as follows.
1 or 14. The general procedures
3.1.1. General Procedure for the Synthesis of 1-Nitrogen Substituted Thioxanthones (8–13)
Compounds 13 were obtained by adding the appropriate amine or sulfamide to a
suspension of 1-chloro-4-propoxy-9H-thioxanthen-9-one ( , 0.250 mg, 0.8 mmol) (procedure
) or 1-bromo-4-propoxy-9H-thioxanthen-9-one (14, 0.250 mg, 0.7 mmol) (procedure b)
8
–
1
a
in methanol (25 mL), CuI (2 mg) and K2CO3 (0.1 mmol). The suspension was heated at
100 ◦C for 48 h, in a sealed flask.
1-[(4,6-Dimethylpyrimidin-2-yl)amino]-4-propoxy-9H-thioxanthen-9-one (
8): 3% as
red dust (procedure
a
). mp 179.7–181.7 ◦C (methanol). IR (KBr)max: 3445, 2966, 2919, 2878,
2851, 1594, 1575, 1505, 1475, 1465, 1443, 1401, 1384, 1335, 1291, 1267, 1240, 1226, 1166, 1116,
1
1086, 1063, 973, 890, 822, 789, 769, 629. H NMR (CDCl3, 300.13 MHz)
δ (ppm): 12.8 (1H, s,
NH), 9.01 (1H, d, J = 9.21, H2), 8.62 (1H, dd, J = 8.13 and 1.94 Hz, H8), 7.58 (2H, m, H5 and
H6), 7.45 (1H, ddd, J = 8.19, 5.10 and 3.14 Hz, H7), 7.22 (1H, d, J = 9.24 Hz, H3), 6.53 (1H, s,
H30), 4.12 (2H, t, J = 6.44 Hz, Ha), 2.42 (6H, s, H50 and H60), 1.93 (2H, st, J = 7.22 Hz, Hb),
1.14 (3H, t, J = 7.410Hz, Hc). 13C NMR (CDCl3, 75.48 MHz)
δ
(ppm): 184.0 (C9), 167.5 (C20
and C4 ), 159.9 (C1 ), 146.7 (C4), 139.9 (C1), 136.9 (C10a), 132.1 (C6), 130.2 (C8a), 129.9 (C8),
129.0 (C4a), 126.3 (C7), 126.0 (C5), 116.6 (C3), 116.4 (C9a), 115.6 (C2), 112.2 (C30), 71.8 (Ca),
24.2 (C50 and C60), 22.8 (Cb), 10.9 (Cc) (Figure S13, Supplementary Data).
0
4-(9-Oxo-4-p◦ropoxy-9H-thioxanthen-1-yl)piperazin-2-one (
9): 12% as red dust (proce-
dure a). mp >350 C (methanol). IR (KBr)max: 3435, 2963, 2922, 2852, 2360, 1604, 1508, 1457,
1430, 1395, 1348, 1320, 1291, 1261, 1232, 1170, 1106, 1061, 998, 967, 919, 809, 744, 732, 621,
1
575, 529, 472, 418. H NMR (DMSO, 500.13 MHz)
δ
(ppm): 8.55 (1H, s, NH), 7.90 (1H, dd,
J = 7.70 and 1.38 Hz, H8), 7.51 (1H, dd, J = 7.80 and 1.09 Hz, H5), 7.40 (1H, td, J = 7.98 and
1.57 Hz, H6), 7.35 (1H, td, J = 7.45 and 1.27 Hz, H7), 7.00 (1H, d, J = 9.00 Hz, H3), 6.91 (1H,
d, J = 8.90 Hz, H2), 3.98 (2H, t, J = 6.38 Hz, Ha), 3.86 (2H, t, J = 5.30 Hz, H10), 3.73 (2H, t,
J = 5.53 Hz, H20), 3.29 (2H, s, H40), 1.75 (2H, st, J = 7.32 Hz, Hb), 1.03 (3H, t, J = 7.40 Hz,
Hc). 13C NMR (DMSO, 75.48 MHz) δ (ppm): 171.8 (C30), 163.1 (C9), 146.6 (C4), 144.9 (C1),
134.9 (C8a), 131.7 (C10a), 128.8 (C6), 126.8 (C8), 126.3 (C7), 125.2 (C5), 122.5 (C4a), 121.0
(C9a), 117.3 (C2), 114.2 (C3), 70.3 (Ca), 60.7 (C10), 50.2 (C20), 57.7 (C40), 22.3 (Cb), 10.6 (Cc)
(Figure S14, Supplementary Data). HRMS (ESI+): m/z [C20H20N2O3S + H]+ calcd. for
[C20H21N2O3S]: 369.1273; found 369.1263 (Figure S15, Supplementary Data).
1-[(2-Hydroxyethyl)amino]-4-propoxy-9H-thioxanthen-9-one (10): 7% as red needles
(procedure
494, 458. H NMR (CDCl3, 300.13 MHz)
a
). mp 173.1–173.9 ◦C (methanol). IR (KBr)max: 3447, 2360, 2342, 1636, 669, 649,
1
δ (ppm): 10.1 (1H, s, OH), 8.49 (1H, td, J = 8.1 and
1.1 Hz, H8), 7.53 (2H, m, H5 and H7), 7.40 (1H, ddd, J = 8.2, 4.6 and 3.7 Hz, H6), 7.12 (1H, d,
J = 9.0 Hz, H3), 6.61 (1H, d, J = 9.1 Hz, H2), 4.01 (2H, t, J = 6.5 Hz, Ha), 3.96 (2H, t, J = 5.4 Hz,
H20), 3.46 (2H, t, J = 5.4 Hz, H10), 1.88 (2H, st, J = 7.4 Hz, Hb), 1.12 (3H, t, J = 7.4 Hz, Hc).
13C NMR (CDCl3, 125.77 MHz)
δ (ppm): 183.6 (C9), 148.5 (C1), 143.1 (C4), 136.9 (C10a),
132.6 (C5), 130.2 (C9a), 129.8 (C8a), 129. 3 (C8), 126.0 (C6 and C7), 119.3 (C3), 112.8 (C4a),
107.8 (C2), 72.7 (Ca), 61.3 (C20), 45.9 (C10), 23.0 (Cb), 10.9 (Cc) (Figure S16, Supplementary
Data). HRMS (ESI+): m/z calcd. for [C18H19NO3S]: 329.1086; found 329.1066 (Figure S17,
Supplementary Data).

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1-[(2-Morpholinoethyl)amino]-4-propoxy-9H-thioxanthen-9-one (11): 45% as orange
needles (procedure a
). mp 126.4–127.7 ◦C (methanol). IR (KBr)max: 3448, 3287, 2963, 2924,
2853, 2818, 2363, 1739, 1615, 1574, 1497, 1466, 1443, 1435, 1392, 1294, 1270, 1245, 1226, 1171,
1
1147, 1115, 1072, 1037, 794, 746, 722. H NMR (CDCl3, 500.13 MHz)
δ
(ppm): 9.90 (1H, t,
J = 4.50 Hz, NH), 8.52 (1H, dd, J = 8.01 and 1.86 Hz, H8), 7.52 (2H, m, H5 and H6),
7.40 (1H, ddd, J = 8.16, 4.79 and 3.44 Hz, H7), 7.13 (1H, d, J = 9.06 0Hz, H3), 6.52 (1H, d,
0
J = 9.06 Hz, H2), 4.01 (2H, t, J = 6.50 Hz, Ha), 3.79 (4H, t, J = 4.65 Hz, H3 and H4 ), 3.36 (2H, q,
J = 6.51 Hz, H10), 2.76 (2H, t, J = 6.59 Hz, H20), 2.57 (4H, t, J = 4.55 Hz, H30 and H60), 1.88
(2H, st, J = 7.32 Hz, Hb), 1.11 (3H, t, J = 7.40 Hz, Hc). 13C NMR (CDCl3, 75.48 MHz)
δ
(ppm):
183.4 (C9), 149.2 (C1), 142.6 (C4), 136.8 (C10a), 131.7 (C6), 130.4 (C8a), 129.8 (C4a), 129.3
0
0
(C8), 126.0 (C5), 125.9 (C7), 120.3 (C3), 113.2 (C10a), 106.5 (C2), 72.8 (Ca), 67.2 (C4 and C5 ),
57.2 (C20), 53.8 (C30 and C60), 40.6 (C10), 23.1 (Cb), 10.7 (Cc) (Figure S18, Supplementary
Data). HRMS (ESI+): m/z [C22H26N2O3S + H]+ calcd. for [C22H27N2O3S]: 399.1742; found
399.1749 (Figure S19, Supplementary Data).
4-[N-(9-Oxo-4-propoxy-9H-thioxanthen-1-yl)sulfamoyl]benzoic ◦acid (12): 3% (proce-
dure a) and 48% as orange powder (procedure b). mp 324.6–326.4 C (chloroform). IR
(KBr)max: 3440, 2967, 2879, 1595, 1554, 1460, 1437, 1383, 1357, 1292, 1266, 1205, 1165, 1133,
1087, 1061, 992, 832, 787, 751, 729, 699, 628, 604, 557. 1H NMR (DMSO, 300.13 MHz)
δ
(ppm): 12.7 (1H, s, OH), 8.44 (1H, dd, J = 8.28 and 1.44, H8), 7.88 (3H, d, J = 8.07 Hz, H5,
H30 and H50), 7.77 (1H, ddd, J = 8.22, 6.92 and 1.38 Hz, H6), 7.71 (2H, d, J = 8.01, H20 and
H60), 7.59 (1H, ddd, J = 8.25, 6.95 and 1.38 Hz, H7), 7.51 (1H, d, J = 8.94 Hz, H2), 7.43 (1H,
d, J = 8.94 Hz, H3), 4.10 (2H, t, J = 6.30 Hz, Ha), 1.78 (2H, st, J = 7.04 Hz, Hb), 1.02 (3H,
t, J = 7.38 Hz, Hc). 13C NMR (DMSO, 75.48 MHz)
δ
(ppm): 185.2 (C9), 168.1 (C70), 149.2
(C4), 145.2 (C40), 138,0 (C10), 136.1 (C10a), 134.2 (C1), 133.5 (C6), 129.6 (C30 and C50), 129.1
(C8), 128.8 (C4a), 128.0 (C9a), 127.2 (C7 and C8a), 126.8 (C5), 126.2 (C20 and C60), 115.9
(C2 and C3), 72.3 (Ca), 22.7 (Cb), 10.5 (Cc) (Figure S20, Supplementary Data). HRMS (ESI+):
m/z [C23H19NO6S2 + H]+ calcd. for [C23H20NO6S2]: 470.0732; found 470.0726 (Figure S21,
Supplementary Data).
4-Amino-N-(9-oxo-4-propoxy-9H-thioxanthen-1-yl)benzenesulfonamide (13): 28% as
orange crystals (procedure b
). mp 211.9–213.5 ◦C [dichloromethane (9):acetone (1)]. IR
(KBr)max: 3479, 3377, 2968, 2879, 1630, 1591, 1569, 1500, 1460, 1435, 1362, 1316, 1287, 1264,
1211, 1185, 1176, 1147, 1089, 1061, 1008, 984, 939, 829, 745, 722, 673, 659, 635, 563, 547, 527,
1
474, 459. H NMR (CDCl3, 300.13 MHz)
δ
(ppm): 12.4 (1H, s, NH), 8.51 (1H, dd, J = 8.18 and
1.97 Hz, H8), 7.66 (1H, d, J = 8.94 Hz, H3), 7.60 (4H, m, H5, H6, H20 and H60), 7.47 (1H,
ddd, J = 8.22, 6.05 and 2.21 Hz, H7), 7.06 (1H, d, J = 9.00 Hz, H2), 6.51 (2H, dt, J = 9.24 and
2.36 Hz, H30 and H50), 4.07 (2H, t, J = 6.36 Hz, Ha), 1.90 (2H, st, J = 7.37 Hz, Hb), 1.12 (3H,
t, J = 7.41 Hz, Hc). 13C NMR (CDCl3, 125.77 MHz)
δ (ppm): 184.2 (C9), 150.2 (C1), 149.2
(C4), 137.3 (C40), 137.2 (C10a), 136.2 (C8a and C9a), 132.7 (C6), 129.8 (C8), 129.6 (C20 and
C60), 129.3 (C10), 126.6 (C7), 126.4 (C4), 117.9 (C4a), 116.6 (C3), 115.0 (C2) 114.2 (C30 and
C50), 71.5 (Ca), 22.7 (Cb), 10.8 (Cc) (Figure S22, Supplementary Data). HRMS (ESI+): m/z
[C22H20N2O4S2 + Na]+ calcd. for [C23H19NO6S2Na]: 462.0683; found 462.2658 (Figure S23,
Supplementary Data).
3.1.2. General Procedure for the Synthesis of 1-Bromo-4-Propoxy-9H-Tioxanthen-9-One (14)
From a suspension of 1-chloro-4-propoxy-9H-tioxanthen-9-one (1) (400 mg, 1 mmol)
in nitrobenzene (10 mL), KBr (5 mmol), CuI (0.01 mmol) and H3PO4 (8 mmol) (procedure
c
), compound 14, a bromine derivative, was obtained. The reaction mixture stirred at
200 ◦C (reflux) for 48 h.
1-Bromo-4-propoxy-9H-thioxanthen-9-one (14): 54% as brown needles (procedure
◦
c
); mp 88-90 C (methanol). IR (KBr)max: 3442, 2961, 2874, 2360, 1637, 1592, 1580, 1560,
1460, 1427, 1375, 1307, 1254, 1173, 1114, 1053, 961, 911, 865, 819, 807, 787, 753, 745, 677, 642,
1
566, 478. H NMR (CDCl3, 300.13 MHz)
δ
(ppm): 8.45 (1H, dd, J = 8.01 and 0.83 Hz, H8),
7.66 (1H, d, J = 8.61 Hz, H3), 7.58 (2H, m, H5 and H6), 7.44 (1H, ddd, J = 8.13, 5.40 and
2.80 Hz, H7), 6.89 (1H, d, J = 8.61 Hz, H2), 4.11 (2H, t, J = 6.39 Hz, Ha), 1.94 (2H, st, J =

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7.35 Hz, Hb), 1.14 (3H, t, J = 7.41 Hz, Hc). 13C NMR (CDCl3, 75.48 MHz)
δ
(ppm): 180.5
(C9), 153.3 (C4), 135.7 (C8a), 133.2 (C3), 132.1 (C6), 130.6 (C1), 130.5 (C9a), 129.8 (C8),
127.4 (C10a), 126.7 (C7), 126.1 (C5), 113.7 (C4a), 113.2 (C2), 71.4 (Ca), 22.6 (Cb), 10.8 (Cc)
(Figure S24, Supplementary Data). MS (EI) m/z (%): 349.9 (9) [M + 2]+., 347.9 (10) [M]+.,
307.9 (32), 305.9 (32), 279.9 (20), 277.9 (20), 227.0 (65), 199.0 (57), 171.0 (100) (Figure S25,
Supplementary Data).
3.2. X-ray Crystallography
Diffraction data were collected at 291 K with a Gemini PX Ultra (Rigaku/Oxford, Neu-
Isenburg, Germany) equipped with CuKα radiation (λ = 1.54184 Å). The structures were
solved by direct methods using SHELXS-97 [32] and refined with SHELXL-97 [32]. Carbon,
oxygen, nitrogen, and sulfur atoms were refined anisotropically. Hydrogen atoms were
either placed at their idealized positions using appropriate HFIX instructions in SHELXL,
and included in subsequent refinement cycles, or were directly found from difference
Fourier maps and were refined freely with isotropic displacement parameters. Full details
of the data collection and refinement and tables of atomic coordinates, bond lengths and
angles, and torsion angles have been deposited with the Cambridge Crystallographic Data
Centre (CCDC).
4-Amino-N-(9-oxo-4-propoxy-9H-thioxanthen-1-yl)benzenesulfonamide (13): Crystal
was monoclinic, space group P21/n, cell volume 2018.0(11) Å3 and unit cell dimensions
a = 11.016(3) Å, b = 8.542(4) Å and c = 21.450(4) Å, and
β
= 91.252(17)◦ (uncertainties in
parentheses). The refinement converged to R (all data) = 7.92% and wR2 (all data) = 17.94%.
CCDC 2081536.
1-Bromo-4-propoxy-9H-thioxanthen-9-one (14): Crystal was triclinic, space group
P-1, cell volume 2018.0(11) Å3 and unit cell dimensions a = 11.3197(8) Å, b = 11.9631(8) Å
and c = 12.7932(10) Å, and cell angles
α β γ
= 108.678(6)◦, = 111.986(7)◦ and = 99.290(6)◦
(uncertainties in parentheses). The refinement converged to R (all data) = 11.96% and wR2
(all data) = 26.62%. CCDC 2081537.
3.3. Docking Studies
The crystal structure of the AcrB (PDB: 4DX5) [33], AcrA (PDB: 2F1M) [34], and TolC
(PDB: 1EK9) [35] portions of the AcrAB-TolC bacterial efflux system, downloaded from the
protein databank (PDB) [36], were used for this study. The known AcrAB-TolC inhibitors
D13-9001, doxorubicin, MBX-3132, minocycline, phenyl-arginyl-β-naphthylamide, reser-
pine, trans(E)-flupentixol, and verapamil, along with the tested compounds, were drawn
with ChemDraw (PerkinElmer Informatics) and minimized using ArgusLab. Docking was
carried out using AutoDock Vina (Scripps, CA, USA) [37], in the sites described in [19,21].
The NorA efflux pump does not have an available crystal structure, and a homology
model was prepared. The model was generated using the Swiss Model server [38] and the
sequence deposited in Uniprot (Q5HHX4) [39], using the EmrD pump from E. coli (PDB:
2GFP) as the homolog, as described in [22].
Regarding the P-glycoprotein (P-gp) efflux pump, the 3D structure of the ATP-bound
P-gp in the outward-facing conformation, which has a low affinity for substrates and
modulators, was established by cryo-electron microscopy [40]. Although the human P-gp
structure in the inward-facing conformation, which has higher affinity for substrates and
modulators, is still not described, there are several mice P-gp structures obtained by X-ray
structures [41] and by electron density maps already published [42,43]. Therefore, a human
P-gp homology model was built, using as a template the mice 3D P-gp with the highest
identity percentage and the availability of 3D coordinates with the best resolution, which
was 4Q9H mice P-gp, with an identity percentage of 89.0%, and a resolution of 3.4 Å [43].
After single sequence alignment, model creation, incorporation of missing loops to the
model, insertion of side chains, verification of overall geometry, addition of hydrogen
atoms, adding of charges, and final staged minimization performed using Sybyl (Tripos
Certara, MO, USA), the human P-gp model was obtained and validated with a single-point

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energy calculation and Ramachandran plot analysis (98.7%, i.e., 1179/1194 of all residues
were in the allowed regions). The top nine poses were collected for each molecule and
the lowest docking score value was associated with the most favorable binding conforma-
tion. PyMOL (Schrödinger, NY, USA) [44] was used for visual inspection of results and
graphical representations.
3.4. Bacterial Strains
As Gram-positive strains, Staphylococcus aureus American Type Culture Collection
(ATCC) 25923, Enterococcus faecalis ATCC 29212, methicillin and ofloxacin-resistant Staphy-
lococcus aureus 272123 clinical isolate, and environmental isolate vancomycin-resistant
enterococci (VRE) E. faecalis B3/101 [23] were used. As Gram-negative strains, Escherichia
coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, the acrA gene inactivated mutant
Salmonella enterica serovar Typhimurium SL1344 (SE03), and clinical isolates of the extended-
spectrum
β-lactamase producer (ESBL) E. coli SA/2 were investigated in
this study.
For the QS tests, all the strains used were Gram-negative. The bacteria used were Chro-
mobacterium violaceum wild type 85 (wt85) characterized by the acyl-homoserine-lactones
(AHL) signal molecule-mediated production of the purple violacein pigment, capable
of endogenous QS-signal molecule-production (N-hexanoyl-L-HSL), C. violaceum CV026
(CV026), a Tn5 transposase-mutant, AHL-signal molecule indicator strain (produces purple
violacein pigment in the presence of AHL), which is incapable of endogenous QS-signal
molecule-production, but is useful in the detection of external stimuli, Sphingomonas pauci-
mobilis Ezf 10-17 (EZF), AHL-producing-strain (used with C. violaceum CV026), and Serratia
marcescens AS-1, characterized by the production AHL signal molecule-mediated pro-
duction of the orange-red pigment prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosin),
capable of endogenous QS-signal molecule-production (N-hexanoyl- L-HSL) [45].
3.5. Antibacterial Assay and Synergy with Antimicrobials
The minimum inhibitory concentration (MIC) for each compound was determined
by broth microdilution method in a 96-well plate according to the Clinical and Laboratory
Standard Institute (CLSI) guidelines [46]. The medium used was MHB II. The concentra-
tions tested ranged from 100
10 mM in DMSO, for S. aureus 272123 and SE03; for the other strains, the concentrations
tested ranged from 64 g/mL to 4 g/mL and were prepared from a stock solution of
µM to 0.195 µM and were prepared from a stock solution of
µ
µ
10 mg/mL in DMSO. DMSO was used in subinhibitory concentrations (1% v/v). The MIC
was determined by visual inspection.
The combined effect of the compounds and clinically relevant antimicrobial drugs
was evaluated by determining the antibiotic’s MIC in the presence of each compound.
Briefly, the MIC of cefotaxime (CTX) (Duchefa Biochemie, Haarlem, The Netherlands)
and vancomycin (VAN) (Oxoid, Basingstoke, UK) for E. coli SA/2 and E. faecalis B3/101,
respectively, were determined in the presence of the highest concentration of each com-
pound tested that did not affect bacterial growth when the compound was used alone. The
antibiotic tested was serially diluted, whereas the concentration of each compound was
kept fixed. In the case of compound 12, the concentration used was 8
µg/mL. For all other
compounds, the concentration used was 64
described above.
µg/mL. Antibiotic MICs were determined as
3.6. Real-Time EB Accumulation Assay
The compounds were evaluated for their ability to inhibit the efflux of EB, an ef-
flux pump substrate, in Salmonella enterica serovar Typhimurium SE03 and S. aureus
272123 strains, through real-time fluorimetry, monitoring the intracellular accumulation
of EB, an efflux pump substrate. This was determined by the automated method using a
CLARIOstar Plus plate reader (BMG Labtech, Ortenberg, Germany). Reserpine and CCCP
were applied at 25 µM as positive controls, and the solvent DMSO was applied at 1% v/v.

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The bacterial strains were incubated in an appropriate culture media (TSB–S. aureus 272123;
◦
LB-B–SE03) at 37 C until they reached an optical density (OD) of 0.6 at 600 nm. The culture
was centrifuged at 13,000
phosphate buffered saline (PBS, pH 7.4). The suspension was centrifuged again in the same
conditions and resuspended in PBS.
×
g for 3 min, and the pellet was washed and resuspended with
Compound 12 was applied at a one-third MIC concentration, 17
applied at 50 M, in a solution of a non-toxic concentration of EB (1
50 L of this solution were transferred into a 96-well black microtiter plate (Greiner Bio-One
Hungary Kft, Mosonmagyaróvár, Ferto˝sor, Hungary), and 50 L of bacterial suspension
µM, and the rest was
µ
µg/mL) in PBS. Then,
µ
µ
(OD600 = 0.6) were added to each well. The plates were placed into the CLARIOstar plate
reader, and the fluorescence was monitored at excitation and emission wavelengths of
530 nm and 600 nm, respectively, every minute for one hour on a real-time basis. From the
real-time data, the activity of the compounds, namely the relative fluorescence index (RFI)
of the last time point (minute 60) of the EB accumulation assay, was calculated according to
the following equation:
RFtreated − RFuntreated
RFI =
(1)
RFuntreated
where RFtreated is the relative fluorescence (RF) at the last time point of EB accumulation
curve in the presence of the compound, and RFuntreated is the RF at the last time point
of the EB accumulation curve of the untreated control, having only the solvent (DMSO)
control [13]. The accumulation curves were designed using Microsoft Excel®. The samples
were tested in triplicate, and the RFI presented comes from the average of these three
values. The accumulation graphs present the mean of the RF at the last time point. The
standard deviation (SD) was calculated automatically and included in the RF graphs.
3.7. Inhibition of Biofilm Formation
The thioxanthone derivatives were tested for their ability to decrease the formation
of biofilm. The bacterial strains used were the Gram-positive S. aureus ATCC 25923 and
S. aureus 272123. The detection of the biofilm formation was carried out using the dye
crystal violet (CV; 0.1% v/v). The initial inoculum was incubated in TSB overnight, and
then diluted to an OD600 of 0.1. Then, the bacterial suspension was added to 96-well
microtiter plates and the compounds were added at a sub-MIC concentration. This meant
that compound 12 was added at 50
µM, while the rest of the compounds were added
at the concentration of 100 M. The final volume in each well was 200
µ
µL. Reserpine
was used as a positive control, as it was the same compound used in the efflux pump
inhibition assay and it has shown activity in the inhibition of biofilm formation in S. aureus
strains [27]. The plates were incubated at 30 ◦C for 48 h, with gentle stirring (100 rpm).
After this incubation period, the TSB medium was discarded, and the plates were washed
with tap water to remove unattached cells. Afterwards, 200 µL of a 0.1% v/v CV solution
were added to the wells and incubated for 15 min at room temperature. Then, the CV
solution was removed from the wells, and the plates were washed again with tap water,
and 200 µL of a 70% ethanol solution was added to the wells. The biofilm formation was
determined by measuring the OD600 using a Multiscan EX ELISA plate reader (Thermo
Labsystems, Cheshire, WA, USA). The anti-biofilm effect of the compounds was expressed
in the percentage (%) of decrease of biofilm formation.
3.8. Quorum Sensing Assay
The QS inhibitory effect of the compounds was examined on the EZF and the sensor
CV026 strains, on the wt85 strain, and on S. marcescens. The method used was the parallel
inoculation method, where pair combinations of the used sensor strain CV026 and the
N-acyl-homoserine lactone (AHL) producing strain EZF were inoculated directly onto the
LB*-A agar surface in parallel, at an approximate distance of 5 mm from each other. S.
marcescens AS-1 and wt85 were inoculated as a single line. Filter paper disks (7 mm in
diameter) were placed on the center of the inoculated line(s) and impregnated with 8 µL

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of a solution of 10 mM of the compounds. PMZ was used as positive control, as previous
results have demonstrated its activity as a QS inhibitor [29]. The agar plates were incubated
◦
at room temperature (20 C) for 24
−
48 h. The QS inhibition was accessed visually, through
the inhibition of pigment production. The discolored, but intact, bacterial colonies were
measured with a ruler [29].
3.9. In Vitro Studies in Mammalian Cells
3.9.1. Cytotoxicity in NIH/3T3 Cell Line
A 10.0 mM stock solut◦ion of each compound was prepared in DMSO. All stock
solutions were stored at
cell culture medium (ensuring that DMSO did not exceed 0.1% DMSO concentration of the
exposure media).
−
80 C and freshly diluted on the day of the experiment in a fresh
Mouse fibroblasts (NIH/3T3, ATCC CRL-1658TM) were cultivated in DMEM (Gibco
52100-039) and supplemented with 10% heat-inactivated fetal bovine serum (Biowest, VWR
International Kft, Debrecen, Hungary), 2 mM of L-glutamine, 1 mM Na pyruvate, 100 U/L
and 10 mg/L penicillin/streptomycin mixture (Sigma-Aldrich Chemie GmbH, Steinheim,
Germany), respectively, and 0.1% nystatin (8.3 g/L in ethylene glycol). The adherent cells
were detached using a combination of 0.25% Trypsin-Versene (EDTA) solution for 5 min
at 37 ◦C. Before each cytotoxicity assay using this cell line, cells were seeded in untreated
96-well flat-bottom microtiter plates, following a 4-hour incubation period in a humidified
atmosphere (5% CO2, 95% air) at 37 ◦C [47].
The cytotoxicity of
MTT assay. Prior to the assay, the cells were seeded for 4 h using 1
compounds were added by 2-fold serial dilutions to the cells distributed into 96-well flat
bottom microtiter plates starting with 100 M. The plates were incubated for 24 h, after
which a solution of MTT in PBS was added to each well and incubated for another 4 h.
After this, 100 L of SDS (10% in a 0.01 M HCl solution) was added to each well and
incubated overnight at 37 ◦C. Doxorubicin was used as a positive control. Cell growth
was determined in quadruplicate by measuring OD at = 540 nm (reference 630 nm) in a
3
,
11,
12
,
13, and 17 was assessed in NIH/3T3 cells, using the
×
104 cells/well. The
µ
µ
λ
Multiscan EX ELISA reader (Thermo Labsystems, Cheshire, WA, USA). The percentage of
inhibition of cell growth was determined according to the Equation (2).
ꢀ
ꢁ
ODsample − ODmedium control
100 −
× 100
(2)
ODcell control − ODmedium control
3.9.2. Cytotoxicity in Caco-2 Cell Line
A 20.0 mM stock solut◦ion of each compound was prepared in DMSO. All stock
solutions were stored at
−
80 C and freshly diluted on the day of the experiment in a fresh
cell culture medium (ensuring that DMSO did not exceed 0.1% DMSO concentration of the
exposure media).
Caco-2 cells were routinely cultured in 75 cm2 flasks using DMEM with 4.5 g/L
glucose supplemented with 10% FBS, 1% NEAA and 1% antibiotic. Cells were maintained
at 37 ◦C, in a 5% CO2 and 95% air atmosphere, and the cell culture medium was changed
every 2 3 days. Cultures were passaged weekly by trypsinization (0.25% trypsin/1 mM
−
EDTA). In all experiments, the cells were seeded at a density of 60,000 cells/cm2 and used
three days after seeding, when confluence was reached. The cells used in all experiments
were taken between the 55th and 64th passages.
The compounds’ (0 20 µM) cytotoxicity was evaluated by the neutral red (NR) uptake
−
and Sulforhodamine B (SRB) binding assays, 24 h after exposure. Caco-2 cells were seeded
onto 96-well plates at a density of 60,000 cells/cm2 and exposed, after reaching confluence
(three days after seeding), to the compounds (0–20
Triton™ X-100 (0.1%) was used as a positive control.
µM), in a fresh cell culture medium.

Pharmaceuticals 2021, 14, 572
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3.9.3. Neutral Red Uptake Assay
The NR assay is based on the ability of viable cells to incorporate and bind the
supravital dye NR into the lysosomes, thus providing a quantitative estimation of the
number of viable cells in a culture [48]. Twenty-four hours after exposure to the compounds,
the cell culture medium was removed, followed by the addition of fresh cell culture medium
containing 50 µ
g/mL NR, and incubation at 37 ◦C in a humidified 5% CO2 and 95% air
atmosphere, for 90 min. After this incubation period, the cell culture medium was removed,
the dye absorbed only by viable cells extracted with absolute ethyl alcohol/distilled water
(1:1) with 5% acetic acid, and the absorbance measured at 540 nm in a multiwell plate reader
(PowerWaveX BioTek Instruments, VT, USA). The percentage of NR uptake relatively to
that of the control cells (0 µM) was used as the cytotoxicity measure. Five independent
experiments were performed, in triplicate.
3.9.4. Sulforhodamine B Binding Assay
The SRB binding assay is based on the pH-dependent binding of SRB to basic amino
acids of cellular proteins under mild acidic conditions. The colorimetric evaluation of
bounded SRB thus provides an estimation of the total protein mass, which is related to
the number of cells in culture [49]. Twenty-four hours after exposure to the compounds,
the cell culture medium was removed, the cells were washed with HBSS (+/+) and fixed
◦
overnight, at
−
20 C, with a methanolic solution of 1% acetic acid (v/v). The fixation
medium was then removed, and the cells incubated with a 0.05% SRB solution (prepared
in 1% acetic acid) for 60 min, at 37 ◦C. After incubation, the SRB solution was removed
and the cells washed with 1% acetic acid (v/v) to remove the unbound dye. The bounded
SRB was then extracted with a Tris base solution (10 mM, pH 10.5) and the absorbance was
measured, at 540 nm, in a multi-well plate reader (PowerWaveX BioTek Instruments, VT,
USA). The percentage of SRB binding relatively to that of the control cells (0 µM) was used
as the cytotoxicity measure. Six independent experiments were performed, in triplicate.
3.9.5. Flow Cytometry Analysis of P-Glycoprotein Expression
The effect of the compounds on P-gp expression was evaluated by flow cytometry,
24 h after exposure, using a P-gp monoclonal antibody (UIC2 clone conjugated with
PE), as previously described [50]. Briefly, Caco-2 cells were seeded in 24-well plates at
a density of 60,000 cells/cm2 and exposed, three days after seeding, to a non-cytotoxic
concentration of the tested compounds (20 µM). After 24 h of exposure, the cell culture
medium was removed, the cells washed with HBSS (-/-) and harvested by trypsinization
(0.25% trypsin/1 mM EDTA) to obtain a cell suspension. The obtained cell suspension was
transferred into Eppendorf tubes, centrifuged (300 g for 5 min, at 4 ◦C) and re-suspended
in HBSS (+/+) buffer containing the P-gp antibody (the antibody dilution was applied
according to the manufacturer’s instructions for flow cytometry). The incubation with the
P-gp antibody was performed at 37 ◦C, in the dark and under gentle shaking, for 60 min.
The cells were then washed twice with HBSS (+/+), centrifuged (300 g for 5 min, at 4 ◦C)
and kept on ice until analysis. Cells were then re-suspended on ice-cold PBS (-/-) buffer
immediately before the cytometer analysis, which was performed using a Becton a BD
AccuriTM C6 flow cytometer (BD Biosciences, CA, USA), equipped with the FCS Express
analysis software. The fluorescence of the P-gp antibody (UIC2-PE) was measured by a
585
±
40 nm band-pass filter (FL2). Logarithmic fluorescence was recorded and displayed
as a single parameter histogram and based on the acquisition of data for at least 20,000 cells.
The parameter used for comparison was the mean fluorescence intensity (MFI), calculated
as a percentage of control cells (0 µM). The cells autofluorescence (unlabelled cells with or
without exposure to the tested compounds) was also evaluated to eliminate its potential
contribution to the analysed fluorescence signals. Given the high autofluorescence of
several of the tested compounds, only compounds
9 and 14 were able to be tested for their
potential effect on P-gp expression. Three independent experiments were performed, in

Pharmaceuticals 2021, 14, 572
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triplicate. Mouse IgG2a-PE was used as an isotype-matched negative control to estimate
nonspecific binding of the PE-labelled anti-P-gp antibody (UIC2-PE).
3.9.6. Evaluation of P-Glycoprotein Transport Activity
The effect of the tested compounds on P-gp activity was evaluated using rhodamine
123 (RHO 123) as a P-gp fluorescent substrate, and ZOS as a specific third-generation P-gp
inhibitor. Indeed, several studies report the use of RHO 123 accumulation/efflux assays
for the direct assessment of P-gp activity through the quantification of the intracellular
fluorescence of the P-gp substrate [30]. Caco-2 cells were seeded in 24-well plates at a
density of 60,000 cells/cm2 and, three days after seeding, and submitted to two different
protocols of RHO 123 accumulation: 1) RHO 123 accumulation in cells pre-exposed to
the compounds (20 µM) for 24 h; 2) RHO 123 accumulation in the presence of the tested
compounds (20 µM) for 90 min, as previously described [50].
The first protocol, given the longer pre-incubation with the tested compounds for
24 h, aimed to assess whether potential compounds-induced increases (or decreases) in
P-gp protein expression levels can be translated into potential increases (or decreases) in
the pump activity. For that purpose, 3 days after seeding, Caco-2 cells were exposed to
compounds (20 µM) in a fresh cell culture medium. Twenty-four hours after exposure, the
cell culture medium containing the compounds was removed, the cells washed with HBSS
(+/+) and incubated with RHO 123 in the absence of the tested compounds, and in the
presence or absence of the specific P-gp inhibitor, zosuquidar (ZOS), accordingly to the
following procedures:
RHO 123 accumulation under normal conditions (NA): pre-incubation with HBSS
(+/+), for 30 min at 37 ◦C, followed by incubation with RHO 123 (5
µM) for 90 min, at
37 ◦C, in a humidified 5% CO2 and 95% O2 air atmosphere.
RHO 123 accumulation in the presence of the P-gp inhibitor (IA): pre-incubation with
ZOS (5
M), prepared in HBSS (+/+), for 30 min at 37 ◦C, followed by incubation with
RHO 123 (5
M) for 90 min, at 37 ◦C, in a humidified 5% CO2 and 95% O2 air atmosphere.
After the 90 min of incubation with RHO 123, the cells were washed twice with HBSS
(-/-) and lysed with 1% Triton X100, for 30 min, in the dark, at room temperature. RHO
µ
µ
™
123 intracellular fluorescence was then measured at excitation/emission wavelengths of
485/528 nm, in a multi-well plate reader (PowerWave-X, BioTek Instruments, VT, USA)
and expressed as fluorescence intensity (FI). The ratio between the FI after inhibited RHO
123 accumulation (IA) and the FI of non-inhibited RHO 123 accumulation (NA) was the
parameter used for comparison, and the results were expressed as a percentage of control
cells, as presented in the following equation.
FI o f RHO123 accumulation under P − gp inhibition (IA)
Rho123 Accumulation =
(3)
FI o f RHO123 accumulation under normal conditions (NA)
Given the high autofluorescence of several of the tested compounds at the conditions
used to measure RHO 123 fluorescence, only compounds 10 12 13 and 14 were able to
9,
,
,
be tested for their potential effect on P-gp activity. Five independent experiments were
performed, in triplicate.
On the other hand, in the second protocol, the cells were exposed to the tested
compounds 30 min prior to addition of the P-gp fluorescent substrate, RHO 123, and
remained in contact with the cells only during the substrate accumulation period, thus
aiming to detect immediate effects of the tested compounds on P-gp activity as a result of
a direct P-gp activation or inhibition. For that purpose, three days after seeding, the cell
culture medium was removed, and the cells submitted to the following procedures:
RHO 123 accumulation under normal conditions (NA): pre-exposure to compounds
◦
(20
(5
cells were only exposed to RHO 123.
µ
M), prepared in HBSS (+/+), for 30 min at 37 C, followed by incubation with RHO 123
µ
M) for 90 min, at 37 ◦C, in a humidified 5% CO2 and 95% O2 air atmosphere. Control

Pharmaceuticals 2021, 14, 572
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RHO 123 accumulation in the presence of the P-gp inhibitor (IA): simultaneous pre-
exposure to compounds (20
µ
M) and ZOS (5
µ
M), both prepared in HBSS (+/+), for 30 min
at 37 ◦C, followed by incubation with RHO 123 (5
µ
M) for 90 min, at 37 ◦C, in a humid-
ified 5% CO2 and 95% O2 air atmosphere. Control cells were only exposed to ZOS and
RHO 123.
After the 90 min of incubation with RHO 123, the cells were washed twice with HBSS
(-/-) and lysed with 1% Triton
™ X100, for 30 min, in the dark, at room temperature. RHO
123 intracellular fluorescence was then measured at excitation/emission wavelengths of
485/528 nm, in a multi-well plate reader (PowerWave-X, BioTek Instruments, VT, USA)
and expressed as fluorescence intensity (FI). The ratio between the FI after inhibited RHO
123 accumulation (IA) and the FI of non-inhibited RHO 123 accumulation (NA) was the
parameter used for comparison, and the results were expressed as a percentage of control
cells (Equation (3)). Given the high autofluorescence of several of the tested compounds
at the conditions used to measure RHO 123 fluorescence, only compounds 9, 10, 12, 13,
and 14 were able to be tested for their potential immediate effect on P-gp activity. Four
independent experiments were performed, in triplicate.
3.9.7. Statistical Analysis
The results of the cytotoxicity in the NIH/3T3 cell line were expressed as the mean
±
standard deviation (SD), and the IC50 values were obtained by best fitting the dose-
dependent inhibition curves in GraphPad Prism 5.03 for Windows software.
Statistical analysis of the P-glycoprotein assays described in 3.9.2. through 3.9.6. was
performed using GraphPad Prism version 6.00 for Windows (GraphPad Software, San
Diego, CA, USA). The normality of data distribution was evaluated with three different
tests: KS normality test, D’Agostino and Pearson omnibus normality test and Shapiro–
Wilk normality test. For data with parametric distribution, one-way ANOVA was used to
perform the statistical comparisons, followed by the Dunnett’s multiple comparisons test.
Details of the performed statistical analysis are described in each figure legend. In all cases,
p values smaller than 0.05 were considered significant.
4. Conclusions
In this study, a set of thioxanthone derivatives showed their potential as antimicrobials
and as antibiotic adjuvants. From the tested library, three compounds, 2, 3 and 12, display
antibacterial activity against Gram-positive bacterial species, with 12 equally effective in
the Salmonella strain. Furthermore, when tested for their potential as adjuvants of clinical
important antibiotics towards resistant strains, compounds 10, 11, 12, and 18 were able to
decrease the MIC of cefotaxime in a clinical ESBL-producing E. coli, with 12 displaying a
16-fold reduction in the MIC of this third-generation cephalosporin, while not displaying
antimicrobial activity. Moreover, compound 9 showed a decrease in the same magnitude
in the VRE E. faecalis B3/101, and, as with compound 12, it did not show antibacterial
activity for this strain. This may suggest an underlying capacity to neutralize their specific
resistance mechanism, although further studies are needed to clarify this subject.
The compounds were also tested for their potential to inhibit the efflux of EB, which
may translate into the inhibition of bacterial efflux pumps. In this assay, five compounds,
2,
3, 7,
11, and 13, showed an increase in fluorescence compared to the positive controls, and
were shown not to be fluorescent themselves. These results could imply that these thioxan-
thones can act as bacterial efflux pump inhibitors. However, there are other mechanisms
behind the inhibition of EB efflux, such as membrane destabilization. The lack of MIC for
the concentrations tested may be a suggestion that these compounds do not interfere with
membrane stability, but studies on bacterial membrane depolarization, or gene and protein
expression, are warranted to clarify this issue.
Regarding the biofilm and QS assays, tolerance and virulence mechanisms related
to efflux pump inhibition, thioxanthones have also displayed promising results. It is
noteworthy that compound 3, which displayed a high biofilm inhibition in both tested

Pharmaceuticals 2021, 14, 572
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strains and QS inhibition in EZF+CV026, was previously highlighted in the EB efflux
inhibition in both tested strains. Adding to these activities, this compound was proven
to be non-cytotoxic in the tested cell line, emphasizing its potential to be employed as
an antimicrobial adjuvant, or to be further studied as an antimicrobial itself, as previous
studies have shown its activity against resistant strains [12].
The pyrimidine derivative 8 was also successful in inhibiting EB efflux in SE03 and
QS, while not displaying cytotoxicity. Its capability of decreasing the MIC of cefotaxime
in a resistant E. coli strain also makes it an interesting compound, due to difficulties in
reaching Gram-negative strains.
The sulfamide derivatives 12 and 13 also show potential with different profiles. Com-
pound 12 was effective as a Gram-positive antimicrobial and as a Gram-negative antimicro-
bial adjuvant; it was effective in inhibiting biofilm formation in the resistant S. aureus strain,
but it also displayed cytotoxicity, warranting further modifications to improve its safety
profile. Additionally, this compound displayed inhibition of P-gp (ABCB1). Compound
13, on the other hand, was effective at inhibiting EB efflux in the Salmonella strain tested,
and in inhibiting biofilm formation of the resistant strain, while not displaying cytotoxicity.
As with compound 12, it also showed inhibitory activity concerning P-gp. In contrast, the
1-bromo precursor 14 was found to be an activator of P-glycoprotein, with a slight but
immediate activity.
Previous studies with compounds 2–7 described
as non-competitive inhibitors [11]. P-gp belongs to the ATP-binding cassette family,
a family which is also present in bacteria, but does not acquire the relevance of the MFS
or RND families. However, the fact that compounds , and 13 present action in both
2 is a competitive inhibitor of P-gp
and 3–7
2, 3, 7
the inhibition of P-gp and EB efflux, can either mean that these compounds can act as dual
human and bacterial efflux pump inhibitors, or that they are inhibitors of ABC pumps,
which warrants further studies regarding the bacterial efflux pump inhibition.
Overall, these results show the interesting profile of thioxanthone derivatives, opening
the possibility of their use as antimicrobials or in the circumvention of resistance mecha-
nisms. So far, to the best of our knowledge, thioxanthones have not yet been described for
their potential in these activities, to the best of our knowledge. Future studies should focus
on the combination of these compounds, for potential synergy studies. The clarification on
the precise mechanisms through which they exert their action or to which, if any, efflux
pumps they bind to is an opportunity for further research. Docking results performed
suggest their favorable affinity towards the most relevant targets, AcrAB-TolC and NorA,
but deeper insights will allow to conclude with more certainty about the specific action of
these compounds.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/ph14060572/s1. Figure S1. Main 1H and 13C signals for the 1-nitrogen substituted thioxan-
thones 8–14; Figure S2. Main connectivities found in the HMBC for the thioxanthone scaffold used
in this work; Figure S3. Evaluation of the fluorescence of compound
with EB; Figure S4. Evaluation of the fluorescence of compound alone and in combination with EB;
Figure S5. Evaluation of the fluorescence of compound alone and in combination with EB; Figure S6.
2 alone and in combination
3
9
Evaluation of the fluorescence of compound 10 alone and in combination with EB; Figure S7. Evalua-
tion of the fluorescence of compound 11 alone and in combination with EB; Figure S8. Evaluation of
the fluorescence of compound 14 alone and in combination with EB; Figure S9. Evaluation of the
fluorescence of compound 15 alone and in combination with EB; Figure S10. Evaluation of the fluores-
cence of compound 16 alone and in combination with EB; Figure S11. Evaluation of the fluorescence
of compound 17 alone and in combination with EB; Figure S12. Molecular visualization in the SBS
of AcrB. (A) Interaction between compound 3 and the SBS; (B) and (C) Different perspectives of the
interactions between compound
between compound
13 and the SBS; Figure S13. H NMR and 13C NMR for compound
NMR for compound
7 and the SBS; (D) and (E) Different perspectives of the interactions
and the SBS; (G) and (H) Different perspectives of the interactions between
8
1
1
8
; Figure S14. H NMR and 13
C
1
9
; Figure S15. Electrospray ESI data for compound
9
; Figure S16. H NMR and
13C NMR for compound 10; Figure S17. Electrospray ESI data for compound 10; Figure S18. H NMR
1

Pharmaceuticals 2021, 14, 572
28 of 30
1
and 13C NMR for compound 11; Figure S19. Electrospray ESI data for compound 11; Figure S20. H
NMR and 13C NMR for compound 12; Figure S21. Electrospray ESI data for compound 12; Figure
1
S22. H NMR and 13C NMR for compound 13; Figure S23. Electrospray ESI data for compound 13
;
Figure S24. H NMR and 13C NMR for compound 14; Figure S25. GC-MS spectra of compound 14.
1
Author Contributions: E.S., G.S., P.M.d.C., and M.P. designed the experiment. F.D. and A.P. synthe-
sized, purified and elucidated the structures of the compounds. F.D., J.F.-S. and P.M.d.C. performed
the antibacterial and synergy assays; F.D. and N.S. performed the efflux pump inhibition, biofilm,
quorum sensing and cytotoxicity in mouse fibroblasts assays. L.G. performed the X-ray crystallogra-
phy. B.C., F.R. and R.S. performed the cytotoxicity in Caco-2 cells and the P-gp assays. F.D. performed
the docking studies. F.D. and E.S. wrote the manuscript, and all the authors made significant contri-
butions and revised the manuscript. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was supported by national funds through FCT (Foundation for Science and
Technology) within the scope of UIDB/04423/2020, UIDP/04423/2020 (Group of Natural Products
and Medicinal Chemistry—CIIMAR), and under the project PTDC/SAU-PUB/28736/2017 (reference
POCI-01–0145-FEDER-028736), co-financed by COMPETE 2020, Portugal 2020 and the European
Union through the ERDF and by FCT through national funds and structured program of R&D&I
ATLANTIDA (NORTE-01-0145-FEDER-000040), supported by NORTE2020, through ERDF, and
CHIRALBIO ACTIVE-PI-3RL-IINFACTS-2019.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: The acrA gene inactivated mutant Salmonella enterica serovar Typhimurium
SL1344 (SE03), was provided by Jessica Blair, Institute of Microbiology and Infection, College of
Medical and Dental Sciences, University of Birmingham, UK. The methicillin and ofloxacin-resistant
Staphylococcus aureus 272123 clinical isolate was provided by Leonard Amaral, Institute of Hygiene
and Tropical Medicine, Lisbon, Portugal. The bacteria used for the QS assay were kindly provided by
Erno˝ Szegedi, Institute of Viticulture and Enology, National Agricultural Research Center, Badacsony-
tomaj, Hungary. F.D. acknowledges FCT for his PhD grant (SFRH/BD/114681/2019). The authors
thank Sara Cravo, Gisela Adriano and Gábor Tóth for the technical support.
Conflicts of Interest: The authors declare no conflict of interest.
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