Aurone derivatives as promising antibacterial agents against resistant Gram-positive pathogens

Article abstract of DOI:10.1016/j.ejmech.2019.01.022

A set of variously substituted aurones was synthesized and evaluated against Methicillin-Resistant S. aureus (MRSA) and P. aeruginosa. Several analogues were found active against MRSA, but no effect was recorded against P. aeruginosa. Compounds 27, 30 and 33 showed low cytotoxicity, and were tested against a full range of bacterial (Gram-positive and Gram-negative) and fungal species, including resistant strains. These aurones displayed a selective inhibition of Gram-positive bacteria with excellent Therapeutic Index values, while showing no significant action on several Gram-negative strains, H. pylori and V. alginolyticus being the only susceptible strains among the Gram-negative bacteria tested. A permeabilization assay showed that the antibacterial activity of at least some of the aurones could be linked to alterations of the bacterial membrane. Overall, this study endorses the use of the aurone scaffold for the development of new potent and selective antibacterial agents.

Full text of DOI:10.1016/j.ejmech.2019.01.022

Accepted Manuscript
Aurone derivatives as promising antibacterial agents against resistant Gram-positive
pathogens
Hamza Olleik, Samir Yahiaoui, Brayan Roulier, Elise Courvoisier-Dezord, Josette
Perrier, Basile Pérès, Akram Hijazi, Elias Baydoun, Josette Raymond, Ahcène
Boumendjel, Marc Maresca, Romain Haudecoeur
PII:
S0223-5234(19)30032-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.022
EJMECH 11029
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 October 2018
Revised Date: 7 January 2019
Accepted Date: 10 January 2019
Please cite this article as: H. Olleik, S. Yahiaoui, B. Roulier, E. Courvoisier-Dezord, J. Perrier, B. Pérès,
A. Hijazi, E. Baydoun, J. Raymond, Ahcè. Boumendjel, M. Maresca, R. Haudecoeur, Aurone derivatives
as promising antibacterial agents against resistant Gram-positive pathogens, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.01.022.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Aurone derivatives as promising antibacterial agents against
resistant Gram-positive pathogens
Hamza Olleik,a Samir Yahiaoui,1,b Brayan Roulier,b Elise Courvoisier-Dezord,a Josette
Perrier,a Basile Pérès,b Akram Hijazi,c Elias Baydoun,d Josette Raymond,e Ahcène
Boumendjel,b Marc Maresca,a,* Romain Haudecoeurb,*
aAix Marseille Univ., CNRS, Centrale Marseille, iSm2, Marseille, France.
E-mail: m.maresca@univ-amu.fr
bUniv. Grenoble Alpes, CNRS, DPM UMR 5063, F-38041, Grenoble, France.
E-mail: Romain.Haudecoeur@univ-grenoble-alpes.fr
cLebanese University, Doctoral School of Science and Technology, PRASE, Beirut, Lebanon.
dAmerican University of Beirut, Department of Biology, Beirut 1107 2020, Lebanon.
eUniversité Paris 5, Hôpital Cochin, Service de bactériologie.
1Present address: Department of Drug Design and Optimization, Helmholtz Institute for
Pharmaceutical Research Saarland (HIPS), Campus E8.1, 66123 Saarbrücken, Germany.
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Graphical abstract
Abstract
A set of variously substituted aurones was synthesized and evaluated against Methicillin-
Resistant S. aureus (MRSA) and P. aeruginosa. Several analogues were found active against
MRSA, but no effect was recorded against P. aeruginosa. Compounds 27, 30 and 33 showed
low cytotoxicity, and were tested against a full range of bacterial (Gram-positive and Gram-
negative) and fungal species, including resistant strains. These aurones displayed a selective
inhibition of Gram-positive bacteria with excellent Therapeutic Index values, while showing
no significant action on several Gram-negative strains, H. pylori and V. alginolyticus being
the only susceptible strains among the Gram-negative bacteria tested. A permeabilization
assay showed that the antibacterial activity of at least some of the aurones could be linked to
alterations of the bacterial membrane. Overall, this study endorses the use of the aurone
scaffold for the development of new potent and selective antibacterial agents.
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Keywords
Aurones, antibacterial, resistance, MRSA, Gram-positive, bacterial membrane
1. Introduction
Flavonoids are a large and diverse class of natural products, including widespread flavones,
chalcones, flavanones and derivatives, which provided promising antibacterial activities in the
past decades [1]. Among flavonoids, aurones constitute a minor subclass occurring in a very
limited number of advanced plant species, where they play a variety of roles, such as flower
pigments, nectar guides or antioxidants [2], while being increasingly studied in medicinal
chemistry contexts [3,4]. Among the hundred-plus acknowledged natural derivatives of this
molecular subclass, three particular compounds, cephalocerone, hispidol and hispidol-4’-O-β-
D-glucoside (Figure 1), have been identified as phytoalexins, produced by the taxa
Cephalocereus senilis [5,6] and Medicago truncatula [7] in response to elicitation. In the first
case, cephalocerone has been found to inhibit the growth of Erwinia cacticida, a Gram-
negative plant pathogenic bacteria responsible for common cactus rot infections. In the second
case, hispidol and its glucoside derivative showed significant inhibition of Phoma
medicaginis, a fungal pathogen causing spring black stem and leaf spot disease in several
Fabaceae species. Based on these natural antimicrobial properties, and since the related
medicinal chemistry works reported so far mainly focused on heavy modifications of the
aurone scaffold itself (e.g. prenylated aurones and corresponding cyclized analogues [8], 5-
nitrofuran and 5-nitroimidazole aurones [9,10], ferrocene-based derivatives [11], 2,2-
bisaminomethyl analogues [12], fused aurone–chromanones and aurone–quinolones [13,14],
3

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see Figure 1), we thought that further exploring the diversity of aurone substituents could
provide more active compounds against a broader spectrum of pathogens.
Indeed there is still a crucial need for novel antimicrobial agents, as the emergence of drug-
resistant bacteria is progressively dismissing current standard antibiotics, leading to an
elevating mortality rate of infectious diseases. Methicillin-resistant Staphylococcus aureus
(MRSA) is of particular concern, as it was recently estimated that ~80 000 individuals are
inflected in the United States annually, with ~11 000 deaths, making this strain the most
deadly antibiotic-resistant bacteria [15]. Moreover, several other Gram positive and Gram
negative pathogens have become resistant at a worrisome level, such as Pseudomonas
aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae or Enterococcus species. In this
context, nature’s strategies for fighting microbial invasions could be a valuable source of
inspiration for discovering new therapeutic options. We thus proposed to synthesize a series
of aurones, variously substituted at positions 4, 6, 7, 2’, 3’, 4’, 5’ and/or 6’ (Figure 1), and to
engage them in antimicrobial and cytotoxicity assays.
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Figure 1. Structure of the aurone scaffold, natural antimicrobial aurones, and examples of synthetic,
heavily modified analogues.
2. Chemistry
The aurone derivatives were prepared starting from substituted benzofuran-3(2H)-ones,
through an aldol condensation with various benzaldehyde derivatives, either in basic or acidic
conditions, depending of the substitution pattern of the compounds (Scheme 1). While
methoxy- and polymethoxyaurones were obtained either by reaction with KOH/H2O in
MeOH at room temperature or by neutral Al2O3-mediated condensation, the hydroxy and
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polyhydroxy counterparts needed stronger base- or acid-mediated conditions, or an additional
deprotection step from the methoxy analogues with boron tribromide in dichloromethane [16].
Scheme 1. General pathways for the synthesis of aurone derivatives. Reagents and conditions: (a)
KOH/H2O, MeOH or EtOH, 25–80 °C, 5 min to 18 h; (b) Al2O3, CH2Cl2, 25 °C, 18 h; (c) BBr3,
CH2Cl2, rt, 24–72 h.
All these methods required the prior synthesis of various benzofuran-3(2H)-ones, as only
benzofuran-3(2H)-one, 6-hydroxybenzofuran-3(2H)-one and 7-methoxybenzofuran-3(2H)-
one were commercially available (Scheme 2). Compound 1 was obtained from the
corresponding diacetoxyacetophenone, after a α-bromination / deprotection / cyclization
sequence. The 4,6-dihydroxy analogue 2 was prepared from phloroglucinol, through a
Houben-Hoesch reaction using chloroacetonitrile and zinc chloride in HCl / Et2O, followed by
one-pot hydrolysis and cyclization in refluxed H2O. The methoxy derivatives 3 and 4 were
synthesized from the corresponding hydroxy analogues, using Me2SO4 as the methyl donor
agent.
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Scheme 2. Synthesis of benzofuran-3(2H)-one derivatives 1–4. Reagents and conditions: (a)
trimethylphenylammonium tribromide, THF, rt, 6 h; (b) KOH/H2O, MeOH, 65 °C, 3 h; (c) ClCH2CN,
HCl, ZnCl2, Et2O, 0 °C, 3 h; (d) HCl, H2O, 100 °C, 1 h; (e) Me2SO4, K2CO3, dimethoxyethane, 80 °C,
3 h; (f) Me2SO4, K2CO3, acetone, rt, 4 h.
3. Results & discussion
Antimicrobial activity of the aurone derivatives was first tested using MRSA (Methicillin-
Resistant S. aureus, Gram positive) and P. aeruginosa (Gram negative) with the
determination of MIC values as detailed in the Experimental section. Bacteria were exposed
to increasing concentrations of compounds 5–38 (from 0 to 500 µM) and MICs were
determined as the lowest concentration of the molecule able to prevent bacterial growth
(Table 1). On the 34 derivatives tested, 15 were found active (i.e. compounds 8–10, 15, 16,
23–25, 27–33) against MRSA (MICMRSA <250 µM) but not against P. aeruginosa, suggesting
a specific activity against Gram positive bacteria. The determination of the Minimal
Bactericidal Concentration (MBC) on MRSA showed that for all active derivatives, MBC and
MIC were similar, suggesting a bactericidal mechanism of action as previously demonstrated
for other aurones (data not shown) [11].
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Table 1. Structure and antibacterial activity of aurone derivatives against MRSA (MICMRSA) and P.
aeruginosa (MICPA). >500 µM indicates that MIC is higher than the highest dose tested (i.e. 500 µM).
MICMRSA
(µM)
MICPA
(µM)
Cd
R4
R6
R7
R2’
R3’
R4’
R5’
R6’
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
5
6
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
>500
7
H
H
H
H
OH
OH
Et
H
8
125–250
125–250
62.5–125
>500
H
H
H
OH
H
H
9
H
H
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
H
H
H
H
i-Pr
n-Bu
t-Bu
Me
H
H
H
H
H
H
>500
H
H
H
H
H
>500
H
H
Me
OMe
OMe
H
H
H
>500
H
H
H
H
OMe
OMe
H
250
H
H
H
OMe
N-MePpz
OH
H
250
H
H
H
>500
OH
OH
OH
OH
H
H
H
H
H
>500
H
H
H
OH
H
H
>500
H
H
OH
H
H
H
>500
H
H
H
N-MePpz
H
H
>500
H
OH
OH
OH
OH
OH
H
H
H
H
>500
H
H
H
OH
OH
OH
H
OH
H
H
125–250
250
H
H
OH
H
H
H
H
OH
CO2H
H
H
125–250
>500
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
H
OH
OH
OH
OH
OH
OH
OH
OMe
OMe
OMe
OMe
OMe
H
H
H
15.6–31.25
62.5–125
125–250
7.8–15.6
15.6–31.25
7.8
H
H
H
OH
H
H
H
H
OH
H
H
H
H
F
H
H
H
H
n-Bu
c-Hex
OBn
OMe
H
H
H
H
H
H
H
H
OMe
OMe
H
H
15.6
H
H
H
>500
OMe
OMe
OMe
OMe
H
OH
Me
H
H
>500
H
H
Me
H
>500
H
H
n-Bu
OMe
H
>500
H
OMe
H
H
>500
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The five most active derivatives (i.e. compounds 27 and 30–33 with MICSA = 7.8–31.25 µM)
were selected for further evaluation. Their innocuity was evaluated using normal human cells
as explained in the Experimental section. The five derivatives displayed a dose-dependent
reduction of human cell viability after 48 h of incubation (Supporting Information). Results
showed that IC50 (the inhibitory concentrations reducing 50% of the cell viability) values for
BEAS-2B and IMR-90 cells were similar for each derivative (Table 2). Compounds 27, 30
and 33 were found as the least toxic ones (IC50 = 191–623 µM) whereas compounds 31 and
32, bearing alkyl groups at position 4’, showed more toxicity (IC50 = 31–87 µM).
Table 2. Evaluation of the toxicity of selected aurone derivatives against normal human cells (IC50
values). Human normal lung epithelial (BEAS-2B) and fibroblast (IMR-90) were tested.
Compounds
BEAS-2B
IMR-90
27 (µM)
280 ± 50
250 ± 50
30 (µM)
590 ± 90
620 ± 90
31 (µM)
90 ± 20
80 ± 20
32 (µM)
33 ± 8
33 (µM)
190 ± 30
340 ± 70
31 ± 6
The spectrum of antimicrobial activity of the three most active and least toxic derivatives (i.e.
compounds 27, 30, 33) was further tested using a larger panel of microorganisms (Table 3).
Results showed that compounds 27, 30 and 33 were active against all tested Gram positive
bacteria, however with differences in terms of sensitivity. Thus, compound 27 was more
active against C. perfringens whereas compound 33 was found more active on B. subtilis. On
the other hand, E. faecalis was more sensitive to compounds 27 and 33 than compound 30,
whereas Methicillin-resistant S. aureus, P. acnes, L. lactis or A. gandavensis gave similar
sensitivity for the three compounds. Compounds 27, 30 and 33 were found inactive against
most of the Gram negative strains tested, except H. pylori that was found sensitive to
compounds 27 and 33 and V. alginolyticus that was found sensitive to compounds 27, 30 and
33. No activity was detected against M. smegmatis. In order to extend our investigation to
other infectious pathogens, we evaluated their antifungal activity on various unicellular and
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filamentous fungi causing infection in animals, humans or plants. Compound 27 was found
active against A. niger and F. graminearum. Compound 33 was only found active against C.
albicans whereas compound 30 was found inactive in all anti-fungal assays. The
determination of MBC values demonstrated that the antimicrobial effect of compounds 27, 30
and 33 was due to a bactericidal action since their MBC and MIC values were identical (data
not shown).
Table 3. Spectrum of activity of the most active aurone derivatives 27, 30 and 33 on various selected
microorganisms (MIC values). >500 µM indicates that MIC is higher than the highest dose tested (i.e.
500 µM).
Compounds
27 (µM)
31.25
62.5–125
31.25
500
30 (µM)
15.6
33 (µM)
31.25
7.8
A. gandavensis
B. subtilis
62.5
B. subtilis (Nisin-resistant)
C. botulinum
C. coccoides
C. difficile
31.25
500
3.9
62.5
>500
500
250
62.5
62.5
31.25
31.25–62.5
15.6–31.25
31.25
31.25
15.6
Gram positive
bacteria
C. perfringens
E. faecalis
7.8
31.25–62.5
125–250
15.6
15.6
L. lactis
15.6
P. acnes
31.25
15.6–31.25
31.25
>500
>500
>500
>500
>500
>500
>500
>500
15.6
31.25
7.8–15.6
31.25
>500
S. aureus MRSA
S. thermophilus
A. baumannii
A. baumannii MRS
B. thetaioataomicron
C. farmer
125
>500
>500
>500
>500
>500
>500
>500
>500
15.6
>500
>500
>500
C. rodentium
E. coli
>500
>500
Gram negative
bacteria
E. coli MRS
E. coli EHEC K88
H. pylori
>500
>500
>500
K. pneumoniae
K. variicola
>500
>500
>500
>500
>500
>500
>500
>500
S. enterica
>500
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S. flexneri
>500
>500
125
>500
3.9
V. alginolyticus
V. diabolicus
M. smegmatis
A. flavus
31.25
>500
>500
>500
>500
62.5
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
125
Mycobacterium
A. ochraceus
A. niger
C. albicans
>500
31.25
>500
>500
>500
Fungi
500
F. graminearum
F. oxysporum
>500
>500
>500
F. verticillioides
P. verrucosum
S. chartarum
>500
>500
>500
Finally, the Therapeutic Index (T.I) of compounds 27, 30 and 33 was determined by
comparing their antimicrobial and cytotoxic dose-effects. The T.I was determined by dividing
the IC50 value found with one compound on the more sensitive normal human cells by the
MIC value found with the same compound on the more sensitive microorganism. Using this
equation, T.Is of compounds 27, 30 and 33 were found equal to 32, 76 and 49, respectively
(Figure 2).
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Figure 2. Evaluation of the Therapeutic Index (T.I) of compounds 27 (A), 30 (B) and 33 (C). T.I was
calculated by dividing IC50 values obtained from cytotoxicity curves (closed circles) by MIC values
obtained from antimicrobial curves (open squares). Data used were: (a) for compound 27 (A): MIC on
C. perfringens and IC50 on IMR90 cells, (b) for compound 30 (B): MIC on MRSA and IC50 on BEAS-
2B cells and (c) for compound 33 (C): MIC on B. subtilis and IC50 on BEAS-2B cells.
Overall, if some 4-hydroxy (8–10, 15, 16) and 7-hydroxyaurones (23–25) showed residual
activity, the five most active compounds against MRSA were all 4,6-dihydroxyaurones (27,
30–33), while strongly varying in terms of B-ring substitution. Such a substitution pattern at
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the A-ring appeared as a crucial feature, since even closely related analogues were completely
inactive. As an illustrative example, compound 31 showed a MICMRSA value of 15.6–31.25
µM, versus the 6-deoxy analogue 12 and the dimethoxy counterpart 37 having MICMRSA >500
µM. The same trend was recorded when comparing compound 27 with analogues 5 and 7
(MICMRSA = 15.6–31.25 µM versus >500 µM, respectively). This is reminiscent of
antibacterial activities measured from other natural and synthetic flavonoids, which often
share the same resorcinol-type hydroxylation pattern at the A-ring [1]. However in some cases
the aurone scaffold afforded better results. For example, compound 27 yielded a MICMRSA
value of 15.6–31.25 µM, while the analogous chalcone, flavone and flavanone (respectively
2’,4’-dihydroxychalcone, chrysin and pinocembrin) were reported earlier as far weaker
inhibitors (MICMRSA = 131, 407 and 488 µM, respectively) [17]. In addition, as direct B-
hydroxylated analogues 28 and 29 did not share the potency of 27 and 30–33, a hydrophobic
environment could be mandatory at the B-ring for reaching decent antibacterial activity, an
interesting observation that runs contrary to previous global structure-relationship studies
regarding flavonoids, which highlighted a beneficial effect of OH groups at B-rings of
chalcones, flavonols or flavanols [1]. In terms of global Gram positive antibacterial potency,
aurones 27 and 33 share similar MIC values with isolupalbigenin or licochalcone A, which
were ranked among the most potent antibacterial flavonoids [18]. Finally, none of the
evaluated molecules were inhibitors of Gram negative strains growth (with the notable
exceptions of H. pylori and V. alginolyticus), indicating a good selectivity towards Gram
positive strains. Again, this finding seems surprising, as overall reported flavonoid derivatives
often showed similar activities against Gram positive and Gram negative strains (e.g. the
flavonol and flavanols analogues of 27, i.e. galangin or pinocembrin) [19].
To date, the mechanism of action of aurones and derivatives described as antimicrobial is
uncharacterized. Classically, antibiotics act either through an inhibition of macromolecules
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biosynthesis and/or through pore-forming activity. More specifically, flavonoids were often
associated with the targeting of cytoplasmic membrane, nucleic acid synthesis, and energy
metabolism of bacteria [18]. Thus, both three hypotheses constitute plausible mechanisms of
action for aurones. The fact that MIC and MBC values of compounds 27, 30 and 33 were
similar suggests that they possess bacteriolytic activity, potentially through pore-forming
effect. Thus, the influence of compounds 27, 30 and 33 on membrane permeability was
evaluated on C. perfringens, since it was found as one of the most sensitive bacteria in the
antimicrobial assay (Table 3). Membrane integrity was measured using propidium iodide
assay, as documented in the Experimental section (Figure 3). Results showed that only
compound 33 caused a partial permeabilization of the bacterial membrane (around 30% after
240 min), whereas compounds 27 and 30 did not affect membrane integrity, suggesting these
two compounds have target(s) distinct from the bacterial membrane.
Figure 3. Evaluation of the impact of compounds 27, 30 or 33 on the membrane integrity of C.
perfringens. C. perfringens were exposed to compounds 27, 30 or 33 or positive control of membrane
permeabilization (CTAB or Nisin) in the presence of the cell-impermeable DNA probe propidium
iodide. Fluorescent signal (excitation at 530 nm and emission at 590 nm) corresponding to membrane
permeabilization and propidium iodide cell entry was measured over 240 min at 37 °C in anaerobic
condition. Results corresponding to means ± SD (n = 3) were fitted using Graph Pad Prism software.
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4. Conclusion
In conclusion, we synthesized 34 compounds through substitution of the naturally occurring
aurone scaffold. Among the derivatives possessing preliminary antimicrobial activity, mainly
directed against Gram-positive bacteria, three compounds were selected based on
innocuousness assays. These molecules shared structural similarity, such as a 4,6-dihydroxy
substitution pattern. Interestingly, although they were active against a large number of Gram-
positive bacteria, possibly through alterations of the bacterial membrane (as suggested by
preliminary membrane permeabilization assays), two of them (27 and 33) showed an
unexpected potency against Gram-negative human pathogens H. pylori and V. alginolyticus. It
is worth noting that the aurone scaffold itself bears the activity herein, unlike some reported
derivatives incorporating already known antibacterial moieties embedded on aurones (e.g.
ferrocene, nitroimidazole, nitrofuran) [9–11]. In addition, far better T.Is were obtained (T.I =
32–76 for 27, 30 and 33), as those from the literature were crippled by collateral high
cytotoxicity values (e.g. T.I ≤ 0.72 for ferrocene-substituted aurones) [11,12]. Taken together,
our results demonstrate that the aurone scaffold itself is a well-suited platform for
antibacterial agents design, and that diversifying the substitution pattern could be an efficient
way to produce new antibiotics active against pathogenic bacteria and safe for human cells.
5. Experimental
5.1. Chemistry
1H and 13C NMR spectra were recorded on an AC-400 instrument (Bruker, 400 MHz for 1H,
13
100 MHz for C). Chemical shifts are reported in ppm relative to the solvent in which the
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spectrum was recorded [1H: δ (d6-DMSO) = 2.50 ppm, δ (CDCl3) = 7.27 ppm; 13C: δ (d6-
DMSO) = 39.52 ppm, δ (CDCl3) = 77.16 ppm]. Electrospray ionization (ESI) high resolution
mass spectra were acquired at the ICMG PSM Platform on a LTQ Orbitrap XL Thermo
Scientific instrument. Combustion analyses were performed at the analysis facilities of DCM;
all compounds had purity higher than 95%. IR spectra were recorded with a Perkin Elmer
Spectrum Two FT-IR spectrometer using an ATR unit Silica gel F-254 plates (0.25 mm;
Merck) were used for thin-layer chromatography (TLC), and silica gel 60 (200–400 mesh;
Merck) was used for flash chromatography. Unless otherwise stated, reagents were obtained
from commercial sources and were used without further purification. The synthesis and
characterization of compounds 1–3, 6–20, 22, 24–32 and 35–37 have already been reported
elsewhere [16,20–23].
5.1.1. 6-Methoxybenzofuran-3(2H)-one (4)
To a suspension of 6-hydroxybenzofuran-3(2H)-one (1.00 g, 6.66 mmol) in acetone (5 mL)
were added K2CO3 (1.38 g, 9.99 mmol) and Me2SO4 (690 µL, 7.28 mmol), and the mixture
was stirred at room temperature for 4 h. Solvents were removed under reduced pressure, H2O
was added, and the mixture was extracted with CH2Cl2. The combined organic layers were
washed with H2O and brine, dried over MgSO4, filtered, and the filtrate was concentrated
under reduced pressure. The residue was washed with cold MeOH, filtered and dried to afford
4 (735 mg, 68%) as a pale yellow solid. 1H NMR (CDCl3) δ 7.47 (d, 1H, J = 8.5 Hz), 6.57 (d,
J = 8.5 Hz), 6.47 (s, 1H), 4.55 (s, 2H), 3.81 (s, 3H). 13C NMR (CDCl3) δ 197.5, 176.5, 168.2,
125.0, 114.3, 111.7, 96.3, 75.5, 55.9.
5.1.2. (Z)-2-Benzylidenebenzofuran-3(2H)-one (5)
To a solution of benzofuran-3(2H)-one (300 mg, 2.24 mmol) in anhydrous CH2Cl2 (25 mL)
was added benzaldehyde (0.272 mL, 2.69 mmol) and Al2O3 (8.0 g), and the suspension was
stirred at room temperature for 18 hours. The solid was removed by filtration, and the filtrate
16

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was concentrated under reduced pressure. The residue was washed with MeOH, filtered and
dried to afford 5 (231 mg, 47%) as a pale yellow solid. 1H NMR (CDCl3) δ 7.94 (m, 2H), 7.83
(m, 1H), 7.67 (ddd, 1H, J1 = 8.6 Hz, J2 = 7.3 Hz, J3 = 1.4 Hz), 7.45 (m, 3H), 7.34 (d, 1H, J =
13
8.3 Hz), 7.23 (m, 1H), 6.91 (s, 1H). C NMR (CDCl3) δ 185.0, 166.3, 147.0, 137.1, 132.4,
131.7, 130.0, 129.0, 124.8, 123.6, 121.8, 113.2, 113.1. IR (ATR) υ 1712, 1596, 1475, 1459,
1299, 1091, 1024, 857, 754, 697, 554 cm-1. HRMS (ESI) calc. for C15H11O2 [M + H]+
223.0754, found 223.0748.
5.1.3. (Z)-2-(4-(4-Methylpiperazin-1-yl)benzylidene)-6-hydroxybenzofuran-3(2H)-one
(21)
To a solution of 6-hydroxybenzofuran-3(2H)-one (73 mg, 0.49 mmol) in MeOH (10 mL)
were added an aqueous solution of KOH (50%, 0.75 mL) and 4-(4-methylpiperazin-1-
yl)benzaldehyde (100 mg, 0.49 mmol), and the mixture was refluxed for 3 hours. After
cooling, solvents were removed under reduced pressure, then the residue was diluted with
H2O and an aqueous solution of HCl (1 N) was added to adjust the pH to 6–7. The resulting
precipitate was filtered, washed with Et2O and dried to afford the hydrochloride salt of 21
1
(182 mg, 100%) as an orange solid. H NMR (d6-DMSO) δ 11.23 (s, 1H), 10.95 (br s, 1H,
NH+), 7.86 (d, 2H, J = 8.4 Hz), 7.60 (d, 1H, J = 8.4 Hz), 7.11 (d, 2H, J = 8.4 Hz), 6.77 (m,
13
3H), 4.02 (m, 2H), 3.46 (m, 2H), 3.25 (m, 4H), 2.81 (s, 3H). C NMR (d6-DMSO) δ 181.1,
167.4, 166.3, 150.1, 146.0, 132.7, 125.7, 122.8, 115.2, 113.2, 112.9, 111.2, 98.6, 51.8, 44.3,
41.9. IR (ATR) υ 3368 (br), 1603, 1568, 1517, 1456, 1393, 1341, 1293, 1252, 1144, 1123,
922, 818, 804, 770, 701, 665, 612, 550, 505 cm-1. HRMS (ESI) calc. for C20H21O3N2 [M +
H]+ 337.1547, found 337.1540.
5.1.4. (Z)-2-(3,4-Dihydroxybenzylidene)-7-hydroxybenzofuran-3(2H)-one (23)
To a solution of 7-methoxybenzofuran-3(2H)-one (150 mg, 0.914 mmol) in anhydrous
CH2Cl2 (25 mL) was added 3,4-dimethoxybenzaldehyde (183 mg, 1.10 mmol) and Al2O3 (3.4
17

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g), and the suspension was stirred at room temperature for 2 hours. The solid was removed by
filtration, and the filtrate was concentrated under reduced pressure. The residue was washed
with MeOH, filtered and dried to afford (Z)-2-(3,4-dimethoxybenzylidene)-7-
methoxybenzofuran-3(2H)-one (113 mg, 40%) as a yellow solid. 1H NMR (CDCl3) δ 7.64 (d,
1H, J = 2.0 Hz), 7.52 (dd, 1H, J1 = 8.4 Hz, J2 = 2.0 Hz), 7.40 (dd, 1H, J1 = 6.8 Hz, J2 = 2.0
Hz), 7.16 (m, 2H), 6.97 (d, 1H, J = 8.4 Hz), 6.91 (s, 1H), 4.03 (s, 3H), 4.00 (s, 3H), 3.96 (s,
3H). 13C NMR (CDCl3) δ 184.7, 155.7, 151.1, 149.3, 146.2, 146.1, 126.3, 125.5, 123.9, 123.5,
118.7, 116.1, 114.3, 114.0, 111.4, 56.7, 56.1, 56.0. To a solution of (Z)-2-(3,4-
dimethoxybenzylidene)-7-methoxybenzofuran-3(2H)-one (100 mg, 0.320 mmol) in
anhydrous CH2Cl2 (20 mL) was added BBr3 (0.618 mL, 6.40 mmol) at 0 °C, and the mixture
was stirred at room temperature for 3.5 hours. Cold H2O was then added, and the resulting
mixture was extracted with AcOEt. The combined organic layers were washed with H2O and
brine, dried over MgSO4, filtered, and the filtrate was concentrated under reduced pressure.
The residue was purified by flash chromatography on silica gel (eluent AcOEt/cyclohexane)
to afford 23 (60 mg, 69%) as a red solid. 1H NMR (d6-DMSO) δ 10.51 (br s, 1H), 9.81 (br s,
1H), 9.33 (br s, 1H), 7.48 (d, 1H, J = 2.1 Hz), 7.39 (dd, 1H, J1 = 8.3 Hz, J2 = 2.1 Hz), 7.22
(dd, 1H, J1 = 7.7 Hz, J2 = 1.2 Hz), 7.18 (dd, 1H, J1 = 7.7 Hz, J2 = 1.2 Hz), 7.09 (t, 1H, J = 7.7
Hz), 6.87 (d, 1H, J = 8.3 Hz), 6.78 (s, 1H). 13C NMR (d6-DMSO) δ 183.6, 153.9, 148.6,
145.6, 144.8, 143.6, 125.2, 124.2, 123.3, 122.9, 122.8, 118.6, 116.1, 114.0, 113.8. IR (ATR) υ
3382 (br), 3167 (br), 1644, 1579, 1506, 1289, 1209, 1158, 1137, 1057, 1024, 909, 862, 791,
746, 732, 691, 620, 573, 557, 524 cm-1. HRMS (ESI) calc. for C15H11O5 [M + H]+ 271.0601,
found 271.0601.
5.1.5. (Z)-2-(4-Benzyloxy-3-methoxybenzylidene)-4,6-dihydroxybenzofuran-3(2H)-one
(33)
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To a solution of 4,6-dihydroxybenzofuran-3(2H)-one (100 mg, 0.602 mmol) in EtOH (2 mL)
were added an aqueous solution of KOH (50%, 3 mL) and 4-benzyloxy-3-
methoxybenzaldehyde (3 mL), and the mixture was refluxed for 2 hours. After cooling,
solvents were removed under reduced pressure, then the residue was diluted with H2O and an
aqueous solution of HCl (1 N) was added to adjust the pH to 2–3. The resulting mixture was
extracted three times with AcOEt. The combined organic layers were washed with H2O and
brine, dried over MgSO4, filtered, and the filtrate was concentrated under reduced pressure.
The residue was recrystallized from MeOH to afford 33 (150 mg, 64%) as a yellow solid. 1H
NMR (d6-DMSO) δ 10.92 (s, 1H), 10.85 (s, 1H), 7.32–7.54 (m, 7H), 7.14 (d, 1H, J = 8.5 Hz),
6.59 (s, 1H), 6.23 (d, 1H, J = 1.7 Hz), 6.08 (d, 1H, J = 1.7 Hz), 5.15 (s, 2H), 3.83 (s, 3H). 13C
NMR (d6-DMSO) δ 179.0, 167.6, 167.1, 158.2, 149.0, 148.8, 146.6, 136.7, 128.4, 128.0,
127.9, 125.3, 124.3, 114.2, 113.5, 108.8, 102.7, 97.7, 90.6, 69.8, 55.5. IR (ATR) υ 3541 (br),
3059 (br), 1603, 1586, 1509, 1456, 1272, 1243, 1231, 1145, 1075, 1003, 885, 818, 741, 697,
679, 635, 626, 562, 541, 519 cm-1. HRMS (ESI) calc. for C23H19O6 [M + H]+ 391.1176, found
391.1172.
5.1.6. (Z)-2-(3,4-Dimethoxybenzylidene)-6-methoxybenzofuran-3(2H)-one (34)
To a solution of 6-methoxybenzofuran-3(2H)-one (58 mg, 0.354 mmol) in MeOH (5 mL)
were added an aqueous solution of KOH (50%, 0.30 mL) and 3,4-dimethoxybenzaldehyde (59
mg, 0.354 mmol), and the mixture was stirred at room temperature for 2 hours. The solid was
then filtered, washed with cold MeOH and dried. The crude product was recrystallized from
MeOH to afford 34 (25 mg, 23%) as a yellow solid. 1H NMR (CDCl3) δ 7.72 (d, 1H, J = 8.3
Hz), 7.51 (d, 1H, J = 1.9 Hz), 7.48 (dd, 1H, J1 = 8.3 Hz, J2 = 1.9 Hz), 6.94 (d, 1H, J = 8.3 Hz),
6.80 (s, 1H), 6.77 (dd, 1H, J1 = 8.3 Hz, J2 = 2.1 Hz), 6.76 (s, 1H), 3.99 (s, 3H), 3.95 (s, 3H),
3.94 (s, 3H). 13C NMR (CDCl3) δ 183.0, 168.4, 167.4, 150.8, 149.2, 147.0, 126.0, 125.9,
125.6, 115.3, 113.9, 112.6, 112.2, 111.4, 96.8, 56.3, 56.2, 56.2. IR (ATR) υ 1596, 1516, 1442,
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1424, 1292, 1283, 1261, 1169, 1145, 1122, 1091, 1043, 1015, 949, 896, 862, 835, 816, 771,
700 cm-1. HRMS (ESI) calc. for C18H17O5 [M + H]+ 313.1071, found 313.1065.
5.1.7. (Z)-2-(2,4-Dimethoxybenzylidene)-4,6-dimethoxybenzofuran-3(2H)-one (38)
To a solution of 4,6-dimethoxybenzofuran-3(2H)-one (245 mg, 2.06 mmol) in MeOH (30
mL) were added an aqueous solution of KOH (50%, 2 mL) and 2,4-dimethoxybenzaldehyde
(315 mg, 3.09 mmol), and the mixture was stirred at room temperature for 2 hours. The solid
was then filtered, washed with cold MeOH and dried. The crude product was recrystallized
1
from MeOH to afford 38 (394 mg, 91%) as a pale yellow solid. H NMR (CDCl3) δ 8.19 (d,
1H, J = 8.7 Hz), 7.27 (s, 1H), 6.57 (dd, 1H, J = 8.7 Hz, J = 2.3 Hz), 6.45 (d, 1H, J = 2.3 Hz),
6.36 (d, 1H, J = 1.7 Hz), 6.11 (d, 1H, J = 1.7 Hz), 3.94 (s, 3H), 3.90 (s, 3H), 3.87 (s, 3H), 3.86
13
(s, 3H). C NMR (CDCl3) δ 180.6, 168.5, 168.4, 162.2, 160.1, 159.2, 146.8, 132.8, 114.6,
105.6, 105.5, 105.3, 97.9, 93.7, 89.0, 56.1, 56.0, 55.5, 55.4. IR (ATR) υ 1591, 1566, 1426,
1318, 1240, 1208, 1157, 1088, 1042, 1030, 945, 916, 883, 845, 806, 783, 696, 675, 550, 507
cm-1. HRMS (ESI) calc. for C19H19O6 [M + H]+ 343.1176, found 343.1170.
5.2. Biology
5.2.1. Microorganism strains and growth conditions
Bacterial strains used in this study were reference strains and were obtained from either the
American Type Culture Collection (ATCC), the German Leibniz Institute (DSMZ) or the
French Pasteur Institute (CIP). Gram negative bacterial strains used were: Acinetobacter
baumannii (CIP 110431), Acinetobacter baumannii multi resistant strain (MRS) (CIP
110431), Bacteroides thetaioataomicron (DSM 2255), Citrobacter farmeri (ATCC 51631),
Citrobacter rodentium (ATCC 51116), Escherichia coli (ATCC 8739), Escherichia coli multi
resistant strain (MRS) (DSM 22314), Enterohemorrhagic E. coli -EHEC (K88), Helicobacter
pylori (ATCC 43504), Klebsiella pneumoniae (DSM 26371), Klebsiella variicola (DSM
15968), Pseudomonas aeruginosa (CIP 107398), Salmonella enterica (CIP 80.39), Shigella
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flexneri (ATCC 12022), Vibrio alginolyticus (DSM 2171), Vibrio diabolicus (generous gift
from Dr Aurélie Tasiemski from EEP UMR 8198). Gram positive bacterial strains used were:
Arthrobacter gandavensis (DSM 2446), Bacillus subtilis (ATCC 6633), Nisin-resistant B.
subtilis (DSM 347), Clostridium botulinum (DSM 1985), Clostridium coccoides (DSMZ
935), Clostridium difficile (DSMZ 1296), Clostridium perfringens (ATCC 13124),
Enterococcus faecalis (DSM 13591), Lactococcus lactis (MG1363), Propionibacterium acnes
(ATCC 6919), Streptococcus thermophilus (LMD-9), and Methicillin Resistant S. aureus
strain MRSA USA300 (ATCC BAA-1717 USA 300 CA-MRSA). In addition to Gram
negative and Gram positive strains, Mycobacterium smegmatis (mc2155, ATCC 700084) was
also used as model of Mycobacterium species. Bacteria were cultured as previously described
[24–26]. Briefly, all bacterial strains (except B. thetaioataomicron, C. botulinum, C.
coccoides, C. difficile, C. perfringens, E. faecalis, P. acnes, M. smegmatis and S.
thermophilus) were grown on Luria Bertani (LB) agar plates and in LB broth at 37 °C in
aerobic condition. M. smegmatis was cultured in Middlebrook 7H10 agar plate and
Middlebrook 7H10 broth at 37 °C in aerobic condition. B. thetaioataomicron, C. botulinum,
C. coccoides, C. difficile, C. perfringens, E. faecalis, P. acnes, M. smegmatis and S.
thermophilus were cultured in Brain Heart Infusion (BHI) agar plates and BHI broth
supplemented or not with fetal calf serum (10%) at 37 °C in an anaerobic cabinet (Coy
Laboratory Products, Grass Lake, MI). Fungi used in this study were Aspergillus flavus
(DSMZ 1959), A. niger (ATCC 9142), A. ochraceus (DSMZ 824), Candida albicans (DSM
10697), Fusarium graminearum (DSMZ 1095), F. oxysporum (DSMZ 62316),
F. verticillioides (DSMZ 62264), Penicillium verrucosum (DSMZ 12639), Stachybotrys
chartarum (DSMZ 2144). C. albicans was grown on LB agar plates and in liquid RMPI
medium (buffered with MOPS of final concentration 0.165 M for pH 7.0 from Thermofisher).
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All other fungal strains were grown on Potatoes Dextrose agar and RMPI medium at room
temperature for 2 to 5 days.
5.2.2. Antimicrobial activity assay
Antimicrobial activity of aurone derivatives was evaluated using determination of the minimal
inhibitory concentration (MIC) using two-fold serial dilutions of antimicrobial peptides in
bacterial liquid media following the National Committee of Clinical Laboratory Standards
(NCCLS, 1997) as previously described [24–26]. Briefly, single colonies of the different
bacterial strains cultured on specific agar plates were used to inoculate 3 mL of Mueller-
Hinton (MH) broth for all species except for B. thetaioataomicron, C. botulinum, C.
coccoides, C. difficile, C. perfringens, E. faecalis, P. acnes and S. thermophilus that were
cultured in 3 mL of BHI or M. smegmatis that was cultured in 3 mL of Middlebrook 7H10
broth. Tubes were then incubated overnight (for approximately 16 h) at 37 °C under stirring
(200 rpm). The next day, optical density (OD) of the bacterial suspensions were read at 600
nm, adjusted to 1 with medium before bacteria were diluted 1/100 in 3 mL of fresh medium
and incubated at 37°C, 200 rpm until bacteria reached log phase growth (OD600nm around 0.6).
Stock solutions of aurone derivatives at 200 mM were prepared in DMSO. Bacteria were
diluted in medium to reach bacterial density around 105 bacteria / mL. 100 µL per well of
bacterial suspension were then added into sterile polypropylene 96 well microplates (Greiner
BioOne) already containing 100 µL of medium with increasing concentrations of aurone
derivatives obtained by serial dilution (from 0 to 500 µM, 1:2 dilution). Volume of DMSO
corresponding to the highest dose of aurone derivatives tested (0.2 % DMSO final
concentration) was used as negative control and was found inactive. Plates were incubated at
37 °C for 18–24 h for all bacteria except C. perfringens and M. smegmatis that where
incubated for 24 and 72 hours respectively. All bacterial strains were tested in aerobic
conditions except B. thetaioataomicron, C. botulinum, C. coccoides, C. difficile, C.
22

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perfringens, P. acnes, M. smegmatis and S. thermophilus that were tested at 37 °C in an
anaerobic cabinet (Coy Laboratory Products, Grass Lake, MI). H. pylori and E. faecalis were
tested in anaerobic conditions generated using micro-anaerobic BD GasPack system. For C.
albicans, plates were incubated at 35 °C during 24 hours before reading. A. flavus, A.
ochraceus, F. graminearum, F. oxysporum, F. verticillioides, P. verrucosum and S. chartarum
plates were incubated at room temperature for 2 to 5 days before reading. Finally, for A.
niger, plates were incubated at 35 °C during 48 hours before reading. At the end of the
incubation, OD600nm was measured using microplate reader (Synergy Mx, Biotek). The MIC
was defined as the lowest concentration of drug that inhibited visible growth of the organism.
Experiments were conducted in independent triplicate (n = 3). Antimicrobial activity of
aurone derivatives was also evaluated by determination of the minimal bactericidal
concentration (MBC), i.e. the minimum concentration of the molecule killing 99.9% of the
bacteria [24–26]. MBCs were measured by streaking 10 µL of wells with no growth of
previously prepared MIC plates on proper agar plates. After incubation in the proper
condition, numbers of colonies were counted. Concentrations where ≤ 1 colony grew were
considered the MBC.
5.2.3. Cytotoxic assay on human cells
The impact of aurone derivatives on the viability of normal human cells was evaluated using
Resazurin assay with some modifications [27]. Normal human cells were preferred to cell
lines (i.e. cancer cells) as cancer cells and normal cells may display different sensitivity to
drugs [28]. Normal human cells used were BEAS-2B (ATCC CRL-9609) and IMR-90
(ATCC CCL-186) corresponding respectively to human normal lung epithelial and lung
fibroblast cells. Cells were cultured in Dulbecco's modified essential medium (DMEM)
supplemented with 10% fetal calf serum (FCS), 1% L-glutamine and 1% antibiotics (all from
Invitrogen) and were seeded onto 25 cm2 flasks maintained in a 5% CO2 incubator at 37 °C.
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Cells grown on 25 cm2 flasks were detached using trypsin–EDTA solution (from
Thermofisher), counted using Mallasez counting chamber and seeded into 96-well cell culture
plates (Greiner bio-one) at approximately 104 cells per well. The cells were left to grow for
48–72 h at 37 °C in a 5% CO2 incubator until they reached confluence. Plates were then
aspirated and increasing concentrations of aurone derivatives were added to the cells for 48 h
at 37 °C in a 5% CO2 incubator, DMSO (0.2% DMSO final concentration) being used as
negative control. At the end of the incubation, wells were emptied and cell viability was
evaluated using Resazurin based in vitro toxicity assay kit (from Sigma-Aldrich) following
manufacturer’s instructions. Briefly, Resazurin stock solution was diluted 1:100 in sterile PBS
containing calcium and magnesium (PBS++, pH 7.4) and empty wells were filled with 100 µL
of the diluted solution. After 4 h incubation at 37 °C, fluorescence intensity was measured
using microplate reader (Synergy Mx, Biotek) with an excitation wavelength of 530 nm and
an emission wavelength of 590 nm. The fluorescence values were normalized by the controls
(DMSO treated cells) and expressed as percent viability. The IC50 values of aurone
derivatives on cell viability (i.e. the concentration of derivative causing a reduction of 50% of
the cell viability) were calculated using GraphPad® Prism 7 software. t-Test and two way
ANOVA analyses were used to address the significant differences between mean values with
significance set at p < 0.05.
5.2.4. Membrane permeability assay
Bacterial membrane permeabilization was evaluated using the cell-impermeable DNA/RNA
probe propidium iodide as previously explained [24,29]. Logarithmic growing bacterial
suspension was prepared from overnight bacterial suspension by 1 in 10 dilution. After 3 h
incubation at 37 °C, 200 rpm, bacterial suspensions were centrifuged for 5 min at 3000×g.
Cell pellets were then resuspended in sterile PBS at about 109 bacteria/mL. Propidium iodide
(solution at 1 mg/mL from Sigma Aldrich) was then added to the suspension at a final
24

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concentration of 60 µM. 100 µL of this suspension were then transferred into 96-well black
plates already containing 100 µL of medium with increasing concentrations of aurone
derivatives obtained by serial dilution (1:2 dilution). Volume of DMSO (vehicle)
corresponding to the highest doses of derivatives was used as negative control and was found
inactive. Kinetics of fluorescence variations (excitation at 530 nm and emission at 590 nm)
were then recorded over 240 min of incubation at 37 °C in anaerobic condition using a
microplate reader (Biotek, Synergy Mx), cetyl trimethylammonium bromide (CTAB) at 300
µM and Nisin at 5-times its MIC value being used as positive controls giving 100%
permeabilization. Results were expressed in % of permeability. All experiments were done in
triplicate.
Notes
The authors declare no competing financial interests.
Acknowledgements
This work has been partially supported by Labex ARCANE and CBH-EUR-GS (ANR-17-
EURE-0003). We thank the AVB platform (iSm2 CNRS UMR 7313, Marseille) for its
support in growing fungal strains used in this study.
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