5-Arylideneimidazolones with amine at position 3 as potential antibiotic adjuvants against multidrug resistant bacteria

Article abstract of DOI:10.3390/molecules24030438

Searching for new chemosensitizers of bacterial multidrug resistance (MDR), chemical modifications of (Z)-5-(4-chlorobenzylidene)-2-(4-methylpiperazin-1-yl)-3H-imidazol-4(5H)-one (6) were performed. New compounds (7–17), with fused aromatic rings at posit

Full text of DOI:10.3390/molecules24030438

molecules
Article
5-Arylideneimidazolones with Amine at Position 3 as
Potential Antibiotic Adjuvants against Multidrug
Resistant Bacteria
Aneta Kaczor 1, Karolina Witek 1,2,3, Sabina Podlewska 1,4 , Joanna Czekajewska 2,
1
5
3
˙
Annamaria Lubelska , Ewa Zesławska
, Wojciech Nitek 6, Gniewomir Latacz 1
˙
,
Sandrine Alibert 3 , Jean-Marie Pagès , Elzbieta Karczewska , Katarzyna Kiec´-Kononowicz
and Jadwiga Handzlik 1,
2
1
*
1
Department of Technology and Biotechnology of Drugs, Jagiellonian University, Medical College,
Medyczna 9, 30-688 Cracow, Poland; aneta.kaczor@student.uj.edu.pl (A.K.); karolina.witek@uj.edu.pl (K.W.);
smusz@if-pan.krakow (S.P.); annamarialubelska@outlook.com (A.L.); glatacz@cm-uj.krakow.pl (G.L.);
mfkonono@cyf-kr.edu.pl (K.K.-K.)
Department of Pharmaceutical Microbiology; Jagiellonian University, Medical College, Medyczna 9,
30-688 Cracow, Poland; j.czekajewska@uj.edu.pl (J.C.); elzbieta.karczewska@uj.edu.pl (E.K.)
Aix Marseille Univ, INSERM, SSA, MCT, FAC PHARM, 27 Bd Jean Moulin, 13005 Marseille, France;
sandrine.alibert@univ-amu.fr (S.A.); Jean-Marie.PAGES@univ-amu.fr (J.-M.P.)
Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences, Sme˛tna 12,
31-343 Cracow, Poland
2
3
4
5
6
˙
Department of Chemistry, Institute of Biology, Pedagogical University of Cracow, Podchora˛zych 2,
30-084 Cracow, Poland; ewa.zeslawska@up.krakow.pl
Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Cracow, Poland;
nitek@chemia.uj.edu.pl
*
Correspondence: j.handzlik@uj.edu.pl; Tel.: +48-12-620-55-84
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 3 January 2019; Accepted: 21 January 2019; Published: 26 January 2019
Abstract: Searching for new chemosensitizers of bacterial multidrug resistance (MDR), chemical
modifications of (Z)-5-(4-chlorobenzylidene)-2-(4-methylpiperazin-1-yl)-3H-imidazol-4(5H)-one (
were performed. New compounds ( 17), with fused aromatic rings at position 5, were designed and
synthesized. Crystallographic X-ray analysis proved that the final compounds ( 17) were substituted
6
)
7–
7–
with tertiary amine-propyl moiety at position 3 and primary amine group at 2 due to intramolecular
Dimroth rearrangement. New compounds were evaluated on their antibiotic adjuvant properties
in either Gram-positive or Gram-negative bacteria. Efflux pump inhibitor (EPI) properties towards
the AcrAB-TolC pump in Enterobacter aerogenes (EA289) were investigated in the real-time efflux
(RTE) assay. Docking and molecular dynamics were applied to estimate an interaction of compounds
6–
17 with penicillin binding protein (PBP2a). In vitro ADME-Tox properties were evaluated for
compound . Most of the tested compounds reduced significantly (4-32-fold) oxacillin MIC in highly
9
resistant MRSA HEMSA 5 strain. The anthracene-morpholine derivative (16) was the most potent
(32-fold reduction). The tested compounds displayed significant EPI properties during RTE assay
(37–97%). The naphthyl-methylpiperazine derivative
9 showed the most potent “dual action” of both
oxacillin adjuvant (MRSA) and EPI (E. aerogenes). Molecular modeling results suggested the allosteric
mechanism of action of the imidazolones, which improved binding of oxacillin in the PBP2a active
site in MRSA.
Keywords: multi-drug resistance; Efflux pump; MRSA; Staphylococcus aureus; Enterobacter aerogenes;
5-arylideneimidazolones; microdilution assays; RTE studies; allosteric modulation; ADMET in vitro
Molecules 2019, 24, 438; doi:10.3390/molecules24030438
www.mdpi.com/journal/molecules

Molecules 2019, 24, 438
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1. Introduction
Multidrug resistance (MDR) is a non-susceptible phenotype of a given microorganism to
antimicrobial drugs that belong to different chemical classes and have different mechanisms of
action [
infectious diseases. Amongst Gram-positive bacteria that pose a particular threat to human health is
Staphylococcus aureus [ ]. Of great concern is the increased prevalence of methicillin-resistant S. aureus
(MRSA) strains that lack susceptibility to nearly all -lactams, which are considered the most relevant
class of antibiotics due to their bactericidal activity and excellent safety profile [ ]. Moreover,
1,2]. Bacterial MDR is nowadays a global problem that severely affects the therapy of
3
β
4–6
MRSA strains have an extraordinary versatility to develop resistance to macrolides, lincosamides,
streptogramin B, and many other classes of antibacterial drugs, including last resort antibiotics such
as vancomycin, linezolid and daptomycin [
are often difficult to treat and are associated with an increased risk of treatment failure [
MRSA is listed by the World Health Organization as one of the most problematic bacterial pathogens,
for which available treatment options are constantly decreasing [ ]. MDR strains use various ways
5
,7
]. Consequently, infections caused by MRSA strains
8]. Indeed,
9
to circumvent the harmful effects of antibacterial agents, including modification of the drug target,
production of enzymes which degrade the antibiotic molecules, over-expression of efflux pumps, and
reduction of envelope permeability [
Protein) to PBP2a (also called PBP20), a target for
resistance mechanism in S. aureus [12 13]. In Gram-negative bacteria, such as Enterobacter aerogenes
1
,
10,
11]. Nevertheless, the modification of PBP (Penicillin Binding
β-lactam antibiotics, seems to be the most common
,
and Escherichia coli, the reduction of antibiotic susceptibility is often reported with the overproduction
of efflux pumps that expel toxic substances out of bacterial cell. This mechanism is associated with
decreased membrane permeability, reducing the intracellular concentration of antimicrobial drugs and
promoting bacterial survival [10,14]. The tripartite system AcrAB-TolC is the well-characterized efflux
pump that belongs to resistance-nodulation-cell division (RND) transporters in Gram-negative bacteria.
This system is of special clinical importance as its modulation rejuvenates the effectiveness of multiple
antibacterial drugs, e.g., β-lactams, fluoroquinolones, and tetracyclines [14,15]. According to “the state
of art”, various strategies to combat MDR bacterial strains were proposed, e.g., chemical modifications
in order to improve the structure-activity relationship (SAR) of available antibiotics and the search
for so-called antibiotic adjuvants [13,16]. Antibiotic adjuvants, or chemosensitizers, are compounds
that have the ability to restore the antibacterial activity of antibiotics towards resistant strains of
bacterial pathogens. These molecules are supposed to potentiate antibiotics efficacy and display
a negligible intrinsic antibacterial action [17–19]. The expected advantage of pairing an antibiotic
with an adjuvant is a low rate of new emerging bacterial resistance in such combinations. A classic
example of the strategy, based on the application of antibiotic potentiators in the therapy of infectious
diseases, is the combination of
β-lactam antibiotic amoxicillin with
β-lactamase inhibitor clavulanic
acid, used in clinical treatment since 1981 [20
,
21]. Taking into consideration increasing prevalence of
MDR strains and the scarcity of new antibiotic classes, the search for new compounds able to block
bacterial mechanisms of resistance seems to be a promising strategy that could help to extend the life
span of these life-saving drugs [22,23].
Regarding MDR staphylococci, there are numerous reports describing structurally unrelated
compounds possessing adjuvant-like properties, i.e., quinolone (1) and indole derivatives (2), chalcones,
citral amides and alkenamides (3, Figure 1) [11,24].
N
O
O
O
CN
N
N
HN
H
OH
2
3
O
1
Figure 1. Chemical variety of compounds able to restore antibiotic efficiency in staphylococci (1–3).

Molecules 2019, 24, 438
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On the grounds of similarity of hydantoin core to a part of PA
βN, which is a well-known
efflux pump inhibitor, modifications in this group were carried out to obtain potential adjuvant
activity. Further studies indicate imidazolone derivatives with amphiphilic properties as the most
promising [10].
In our previous research on 5-arylideneimidazolone derivatives, unsubstituted piperazine at
position 2, displayed high potency to restore the efficacy of
ciprofloxacin in MDR strains of S. aureus (4a and 4b, Figure 2) [25]. Furthermore, the compounds with
arylidene moiety at position 5 and amine at position 2 (4a 4b ) were able to re-sensitize MDR strains
of Gram-negative bacteria for selected antibiotics and displayed strong efflux pump inhibitory (EPI)
properties towards AcrAB-TolC. Compound caused the highest reduction (32-fold) of rifampin MIC
β-lactams and fluoroquinolone antibiotic
,
, 5
5
(minimal inhibitory concentration) and an 8-fold reduction of a few antibiotics MIC values: oxacillin,
chloramphenicol, linezolid and clarytromycin [15].
O
O
O
N
NH
N
NH
N
NH
N
4a
N
N
4b
5
N
H
N
H
N
H
Figure 2. Active 5-arylideneimidazolones (4a, 4b, 5) found previously.
On the basis of those interesting results obtained for 5-arylideneimidazolones with an
unsubstituted piperazine moiety, we decided to explore their methylpiperazine analogues starting
from p-chlorobenzylideneimidazolone
6 (Table 1). The compound 6 gave yellow crystals and was used
for X-ray crystallographic analysis, as described earlier [26].
Table 1. Structure of investigated compounds: lead 6 and its modifications (7–17).
B
A
H
O
O
Cl
N
X
R
O
NH
N
N
NH
N
X
N
N
Lead 6
N
X
R
N
N
7'–17'
7–17
NH2
Expected lead modifications
Real lead modifications
Cpd
6
R
X
Cpd
12
R
X
-
-
N-CH3
N-CH3
O
7
N-CH3
N-CH3
N-CH3
13
14
15
Cl
8
N-CH3
N-CH3
H3CO
9
10
N-CH3
N-CH3
16
17
O
O
11
O

Molecules 2019, 24, 438
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Taking into consideration comprehensive structural data for the compound
6
, which also initiated
a new group of methylpiperazine derived 5-arylideneimidazolones, the compound was selected as a
lead structure for further modifications in search for new antibiotic adjuvants performed within this
study. The modifications aimed to introduce a spacer between imidazolone and the methylpiperazine at
position 2 as well as an extension of aromatic area at position 5 of imidazolone (70
in respect to the chemical structure of favorable compounds (4a 4b and
However, the structures of the new desired compounds turned out to be unexpectedly different (
part B, Table 1). In this context, the lead structure and its derivatives 17 were the main subject
–
170, part A, Table 1),
) found previously [15 25].
17
,
5
,
7
–
,
7–
of this study. Thus, synthesis of the new series and X-ray structural consideration was performed.
Final products were tested in microbiological assays for their antibiotic adjuvant potency as well as
in molecular modelling to determine the potential mechanism of action towards PBP2a. For selected
compounds, EPI properties were examined. One of the most active compounds (9) was selected
for ADME-Tox assays in vitro. Based on the results obtained, structure-activity relationship (SAR)
was discussed.
2. Results and Discussion
2.1. Chemical Synthesis
Synthesis of compounds
were described elsewhere [26]. Compounds
corresponding initial steps to obtain intermediates 18
first step (Scheme 1a), Knoevenagel condensation between thiohydantoin and aromatic aldehydes
was performed to give 5-arylidene-2-thiohydantoins 18 27. Then, the S-methylation of 18 27 using
iodomethane in basic sodium ethylate condition provided S-methyl intermediates (28 37). In the
next step, solvent-free reactions of S-methylimidazolones (28 37) with proper commercial amines
were carried out in melting condition to give different substitution at imidazolone ring, depending on
amine properties. In the case of the secondary amine (N-methylpiperazine), the product of simple
6–
17 was performed according to the Scheme 1. Details of synthesis for
17 were synthesized within three stages, starting from
27 and 28 37, respectively (Scheme 1). In the
6
7–
–
–
–
–
–
–
6
replacement of S-methyl group at position 2 was a result. In contrast, the similar process performed by
the use of primary amines caused a Dimroth rearrangement (Scheme 1b) [27] to give the final products
(7
–
17) with primary amine moiety at position 2, and an aminealkyl substituent at position 3 of the
5-arylideneimidazolone pattern (7–17).
Scheme 1. Cont.

Molecules 2019, 24, 438
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Scheme 1. Synthesis route for compounds 6–17; (a) (i), CH3COONa, CH3COOH, reflux, 7 h;
(ii) CH3I, EtONa, rt, 24 h; (iii) proper amine, EtOH, 120–150 ◦C, 15 min (iv) Dimroth rearrangement;
(b) Mechanism of Dimroth rearrangement.
2.2. Crystallographic Studies
The overall shape of molecule
7 with atom numbering is presented in Figure 3. The 5-(p-
chlorobenzylidene)-imidazolone fragment is almost planar. The angle between the planes of these
two rings is 12.36(5)◦. The molecule possesses a double bond C5=C14 and can form two geometric
isomers (Z or E). In the crystal structure of
in other crystal structures, determined earlier, containing 5-(p-chlorobenzylidene)-imidazolone
moiety [26 28]. We have conducted a search of the Cambridge Structural Database [29] and in
7 the Z isomer is observed. This isomer also occurred
,
all crystal structures containing 5-arylideneimidazolone fragments only the Z isomer was noted.
The molecule of the investigated compound adopts a bent conformation, with the torsion angles of
linker: N3-C6-C7-C8 =
−
60.3(2)◦, C6-C7-C8-N2 =
−
51.7(2)◦. This conformation is imposed by the
intramolecular hydrogen bond N5-H5B···N2 (Table 2). The piperazine ring has chair conformation
with equatorial positions of substituents at nitrogen atoms.
Figure 3. Atom numbering scheme of compound 7. Displacement ellipsoids are drawn at the 30%
probability level.

Molecules 2019, 24, 438
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Table 2. Parameters of intramolecular and intermolecular interactions for compound 7.
◦
D-H···A ( )
Symmetry Code
D-H (Å)
H···A (Å)
D···A (Å)
N5-H5B···N2
N5-H5A···O1
0.97(2)
0.90(2)
0.96
1.96(2)
1.98(2)
2.87
2.894(2)
2.846(2)
3.576(3)
160(2)
162(2)
131.0
x, −y+1/2, z+1/2
C13-H13B···CL1
−x+1, y−1/2, −z+3/2
The crystal network in the studied structure can be characterized by N5-H5A···O1 intermolecular
hydrogen bonds, creating a chain C(6) along [001] direction [30]. Furthermore, the C13-H13B···Cl1
interactions between the chains, which lead to the formation of layers (Figure 4), are observed (Table 2).
Figure 4. Partial packing view of compound 7 along [100] direction. Dashed lines indicate hydrogen
bonds: orange—intramolecular, purple—intermolecular.
Results of the crystallographic studies for compound
7
allowed us to identify the Dimroth
rearrangement and real structure of the series of compounds (7–17). It was difficult to recognize this
structure only on the basis of the results of both, NMR and LC-MS analyses, since the molecular
mass and number of protons is the same in the expected compound 70 and the real Dimroth product
(
7
). Before the crystallography results, we had identified the bright singlet (7.80 ppm) occurring in
1H-NMR spectrum of basic form of
7
(see Supplementary) as that coming from 2H of guanidine-like
fragment formed by N1 (in position 1), C2-NH- (position 2) and N3-H (position 3) of the imidazolone
ring. These two acid protons could give one bright singlet due tautomerism. The X-ray results have
undermined that wrong hypothesis, showing that this peak of two protons comes from the NH2 group
placed at position 2. This peak occurred in the basic form, while it was exchanged into a very bright
(almost plane) singlet at 9–10 ppm (typical for N1-H protons of 5-arylidene-2-thiohydantoin), which
indicated a probable transformation of NH2 (at C2) into the tautomeric form of N1-H and C2 = NH (see
Supplementary) in the case of the hydrochloric form of 7. Based on the results of X-ray analysis for 7,
it is also distinctly seen that the triplet at ~3.50 ppm comes from N3-CH2 protons because similar shifts
were observed for N3-CH2 protons of N3-propyl-substituted hydantoin derivatives [25], whereas this
shift is too high for any propyl-CH2 protons in the case of an unrearranged compound (70). Thus,
the crystallography studies allowed us to right assign 1H-NMR peaks to the suitable protons of the
Dimroth rearrangement product. Similar trends that occurred in the 1H-NMR spectra of the rest of the
series confirm the Dimroth rearrangement structure for the rest of the compounds (8–17), although the

Molecules 2019, 24, 438
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hydrochloric form and more extended aromatic areas disturb the detection of such clear and regular
peaks as those for the phenyl symmetric-substituted 7 in its basic form.
2.3. Biological Assays
All compounds were investigated on their adjuvant action in the reference and MDR
Gram-positive Staphylococcus aureus strains. Selected compounds (9–13, 16, 17) were also tested in
Gram-negative E. aerogenes strains, by employing both the microdilution and the real-time efflux (RTE)
assays. In the first step of the study, direct antibacterial activity of compounds against aforementioned
bacteria was evaluated. Next, the influence of compounds (at the concentrations corresponding to
25% of their intrinsic MICs) on MICs of antibiotics was investigated. Finally, real-time efflux (RTE)
assays were performed in order to determine efflux pump inhibitory properties of compounds towards
AcrAB-TolC in E. aerogenes.
2.3.1. Direct Antibacterial Activity
Gram-positive S. aureus
Initially, 12 arylideneimidazolone derivatives were evaluated in vitro for their intrinsic
antibacterial effect against two strains of S. aureus: the reference strain S. aureus ATCC 25923 and
extremely-drug resistant (XDR) MRSA HEMSA 5 clinical isolate. This step of the study was necessary
for: (i) elucidation whether molecules tested are devoid of antistaphylococcal activity and thus cannot
become antimicrobial agents by themselves; and (ii) determination of the concentrations of compounds
suitable for the further assay on their antibiotic adjuvant potency. In addition, the antistaphylococcal
efficacy of oxacillin, which was paired with compounds tested, in the following assays was also
assessed (Table 3).
Table 3. Intrinsic antibacterial activity of compounds tested against S. aureus strains used in the study.
MIC Value [mM]
Compound.
MRSA HEMSA 5
S. aureus ATCC 25923
6
7
8
1
1
>2
2
0.5–1
>2
9
0.5
>0.125
0.5
0.25–0.5
0.125
0.25
1–2
>0.125
0.25
0.5
>0.125
0.5
10 **
11
12
13
14
15
16 **
17
0.25
0.0625–0.125
0.12–0.25
1
>0.125
0.25
Oxacillin
1.21 (512) *
0.00059 (0.25) *
* Activity of oxacillin was also evaluated in µg/mL concentration; ** compounds which precipitated after addition
of bacterial suspension in cation-adjusted Mueller-Hinton (MH II) broth.
The results obtained for arylideneimidazolone derivatives 6–17 have demonstrated that the
compounds did not exhibit any notable antibacterial activity against S. aureus strains used in the study.
Among all compounds tested, the lowest MIC value was determined for the compound 13, which
inhibited the growth of the reference strain ATCC 25923 and drug-resistant strain MRSA HEMSA 5 at
the concentrations of 0.0625–0.125 mM (28.25–56.5
in the range of 0.25 mM to more than 2 mM.
µg/mL). The MICs of remaining compounds were

Molecules 2019, 24, 438
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Gram-Negative E. aerogenes
Then, MIC values of the compounds were evaluated with Gram-negative E. aerogenes.
Three following strains of this bacterial species were employed in the studies: (i) the clinical isolate
Ea-289 overexpressing the AcrAB-TolC efflux pump and exhibiting a porin-deficient phenotype; (ii) the
CM-64 strain which also overexpresses the AcrAB-TolC efflux pump but has no changes in porin
content; (iii) the Ea-294 and Ea-298 that are Ea-289 strain derivatives which are devoid of AcrAB and
TolC, respectively (Table 4). For most of the compounds, precipitation was observed after the addition
of bacterial suspension in MH II broth (10, 11, 16, 17). For these molecules, the MICs could not be
determined precisely.
Table 4. MIC values obtained for compounds tested, PA
βN and selected antibiotics against E. aerogenes.
MIC [mM]
Compound
Ea-294, Ea-298
Ea-289
CM-64
9
10 *
11 *, 17 *
12
1
0.5
0.5
1
>2
1
>2
2
>2
1
>2
2
13
16*
PaβN
0.25
2
0.125
0.5
>2
0.5
0.5
>2
0.5
Chloramphenicol
Erythromycin
Doxycycline
0.2 (64)
0.005 (4)
0.005 (2)
0.05 (16)
3.2 (1024)
0.35 (256)
0.018 (8)
0.2 (64)
1.6 (512)
0.7 (512)
0.072 (32)
0.006(2)
Norfloxacin
* Compounds, which precipitated after addition of bacterial suspension in MH II broth. Values in brackets represent
concentrations of antibiotics in µg/mL.
As summarized in Table 4, none of the compounds tested exhibited remarkable antibacterial
effects against the bacterial strains used in the study (MIC from 0.25 mM to >2 mM). Since imidazolone
derivatives 10, 12 and 13 did not show any significant differences when assayed in efflux pump
overexpressing strains (CM-64 and Ea-289) and efflux pump deficient strains (Ea-294 and Ea-308),
these compounds were not considered as substrates for the AcrAB-TolC transporter. Furthermore, the
absence of porins in Ea-289, Ea-298 and Ea-294 has no significant effect on the activity. Compounds 11
and 17 influenced the strains growth in corresponding manner to that of known EPI, PA
MICs at least in 4-fold for Ea-289 and CM-64 in comparison to Ea-298 and Ea-294. Similar trends can
be expected for and 16, but it was impossible to determine it exactly due lack of growth inhibition
βN, increasing
9
for Ea-289 and CM-64 caused by both compounds at their highest tested concentration.
2.3.2. Influence on Antibiotic Efficacy
Influence on Antibiotic Susceptibility in S. aureus Strains
The lack of a direct antibacterial activity of arylideneimidazolone derivatives tested allowed
analyzing their anti-resistance potency in combination with the
β-lactam antibiotic, oxacillin.
Compounds studied were examined at the concentrations corresponding to 25% of their respective
MIC values or at the highest concentrations, at which they did not precipitate. The potential of
compounds to become antibiotic adjuvants was evaluated by comparing the drug effectiveness in the
presence and absence of compounds tested, thereby, by determining activity gain parameter A (Table 5).
Most of the imidazolone derivatives (6–16) were able to significantly improve the susceptibility of
MRSA to oxacillin. At the same time, compounds did not exert any activity against the reference
S. aureus strain (A < 4). The strongest chemosensitizing effect was demonstrated by compounds 16 and
9
, which, at a concentration of 0.125 mM, reduced the MIC of oxacillin in 16- to 32-fold against MRSA
HEMSA 5. Slightly lower adjuvant-like activity was determined for the imidazolone derivatives
7

Molecules 2019, 24, 438
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and 10 (A = 8–16). The efficacy of the remaining compounds was slightly less marked, however, also
significant. Although compounds tested improved noticeably the antibacterial activity of oxacillin
against MRSA clinical isolate, none of them restored the activity of the antibiotic (MIC < 2
against this highly resistant strain.
µg/mL)
Table 5. Effect of imidazolidine derivatives on the susceptibility of S. aureus strains to oxacillin.
MRSA HEMSA 5
S. aureus ATCC 25923
Conc. of
Compound
[mM]
Conc. of
Compound
[mM]
Range of
Reduction
[µg/mL]
Cpd
Activity
Gain [A]
Range of Reduction
Activity
Gain [A]
[µg/mL]
6
7
8
9
10
11
12
13
14
15
16
17
0.25
0.25
0.5
0.125
0.125
0.125
0.0625
0.0313
0.0625
0.25
8
16
8
16
8–16
8
8
4–8
8
from 512 to 64
from 512 to 32
from 512 to 64
from 512 to 32
from 512 to 32-64
from 512 to 64
from 512 to 64
from 512 to 64-128
from 512 to 64
from 512 to 128
from 512 to 16-32
from 512 to 128-256
0.5
0.125
0.5
0.125
0.125
0.125
0.0625
0.0156
0.0313
0.25
2
1
1
2
1
2
2
1
2
1
1
2
from 0.25 to 0.125
no effect
no effect
from 0.25 to 0.125
no effect
from 0.25 to 0.125
from 0.25 to 0.125
no effect
from 0.25 to 0.125
no effect
4
0.125
0.0625
16–32
2–4
0.125
0.0625
no effect
from 0.25 to 0.125
Influence on Antibiotic Susceptibility in E. aerogenes
Since the compounds were found to be deprived of an intrinsic antibacterial effect in E. aerogenes
strains, their ability to enhance the activity of several antibiotics was assessed. The following four
antibiotics were selected for the assays: chloramphenicol, erythromycin, doxycycline, and norfloxacin.
1
The compounds were tested at the concentration corresponding to of their MIC. For compounds
9,
4
11
,
16 and 17, for which determination of the exact intrinsic MIC value was not possible (MIC > 2 mM,
N was used as
Table 4), the concentration of 0.5 mM was chosen. As in previous studies [10], PA
β
reference molecule in the assays. Results of the assays indicated that none of the tested compounds
significantly increased the effectiveness of chloramphenicol, erythromycin, doxycycline and norfloxacin
against both AcrAB-TolC-overexpressing strains and AcrAB-TolC-deficient strains (A
≤
2). The A value
obtained for PAβN combined with the aforementioned antibiotics coincided with data found in the
literature [31].
2.3.3. Efflux Pump Inhibitory Properties
Taking into account the structural similarity of the explored group (
6
–
17) to previously found
13 16 and
potent efflux pump inhibitors for AcrAB-TolC (4a
,
4b and
5
), the representative structures (
9–
,
17) were also evaluated for their ability to inhibit the activity of AcrAB-TolC pump in E. aerogenes by
measuring the efflux of a dye-substrate in the real-time efflux assay (RTE). The RTE assay is a dynamic
tool by which the ability of a potential EPI to inhibit substrate transport in a drug efflux pump can be
measured as a function of time and energy. Moreover, the test cannot discriminate for a possible outer
membrane-destabilizing action of compounds. The dye, 1,20-dinaphthylamine (1,20-dNA), a substrate
of the AcrAB-TolC efflux pump, is a sensitive to highly lipophilic environment where it is fluorescent,
but it is almost non-fluorescent in an aqueous solution [32]. The experiment was performed in the
E. aerogenes strain overexpressing AcrAB-TolC transporter (Ea-289).
Results have demonstrated that majority of the tested compounds caused a decrease in the
fluorescence of 1,20-dNA in the treated bacterial cell. The effect observed was most probably
due to molecular interactions between fluorophore and compounds tested that result in increasing
fluorescence quenching [33,34]. In order to analyze and quantitatively compare the ability of tested
compounds to inhibit the efflux of 1,20-dNA in the RTE assay, the inhibition efficiency (IE) of each
compound was calculated. The IE parameters have indicated that all the tested compounds promoted

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0
the accumulation of 1,2 -dNA through the inhibition of its efflux outside bacterial cells overexpressing
AcrAB-TolC (Figure 5).
RTE studies in E.aerogenes
97
100
80
80
60
40
20
0
64
61
60
44
37
9
10
11
12
13
16
17
Compound
Figure 5. Comparison of the inhibitory activity of imidazolone derivatives towards AcrAB-TolC efflux
pump in E. aerogenes Ea-289 strain.
The highest EPI property was detected for the compound 13, which at the concentration of
100
transporter. Slightly lower effectiveness was found for the compound
µ
M almost completely blocked 1,20-dNA efflux from the bacteria overexpressing the AcrAB-TolC
9
(80% of efflux inhibition).
By contrast, the weakest EPI activity among all compounds tested was determined for the compound
11 which blocked the efflux of the dye in 37% in the Ea-289 strain.
2.4. Studies in silico
2.4.1. Docking Studies
In order to estimate potential molecular mechanism of the significant oxacillin adjuvant action
of the tested imidazolones in MRSA strains, molecular modeling was applied. The attempts to
explain the mechanism were based on the verification if compounds tested had the capacity to
interact with the main proteins conferring
β-lactam resistance to MRSA strains. For this purpose,
all the compounds tested ( 17) were docked to the crystal structure of PBP2a protein. Moreover,
6
–
the arylidenemidazolones were subjected to molecular dynamics simulation analysis, which offers an
important insight into the molecular behavior of a molecule in its immediate microenvironment.
The results of docking studies are presented in the form of two groups of docking poses, in
respect to the active (Figure 6) and allosteric (Figure 7) sites of PBP2a. Detailed schemes with particular
amino acid residues interacting with compounds
(Supporting Information). Due to the relatively high number of considered compounds, they are
grouped in pictures according to their structural similarity, therefore compounds 11 13 15 are
6–17 are presented in ligand interaction diagrams
6–
8,
9
–
,
–
presented together, and the docking pose of compound 12 is presented together with compounds 16
and 17. Despite small structural differences occurring within the analyzed groups of compounds, there
are quite significant variations in their docking poses (Figure 6).

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Figure 6. Docking poses of the studied compounds in the active site of PBP2a (grid centered at S403).
When compounds
6
–
8
are considered (among which compound
7
displayed the highest activity,
and
), the most pronounced difference is connected with the orientation of the phenyl ring, which for
reducing MIC of oxacillin by 16-fold in comparison to 8-fold reduction observed for compounds
6
8
compound 7 is located the closest to the serine at position 403 of PBP2a. Moreover, for compound 7
and
glutamic acid at position 447 and 460 and hydrogen bond of amine group with serine at position 461),
which do not occur for compound (see ligand interaction diagrams in the Supporting Information).
Compounds 11, which are analyzed together, also displayed relatively high activity in terms of
restoring oxacillin activity, with the most active compound (16-fold reduction of MIC of oxacillin).
All those compounds are differently oriented in the active site of PBP2a in comparison to compounds
, and all of them form hydrogen bond with oxygen from imidazolone ring; however, different
residues from the side of protein are involved in this type of interaction: N464 for compound , T600
for compound 10, and S403 for compound 11. Compounds 13 15, for which the docking poses to the
8, a set of hydrogen bonds is observed (charged assisted hydrogen bond of piperazine ring with
6
9–
9
6–8
9
–
active site of PBP2a were analyzed together, displayed slightly lower activity. Compound 14, reducing
the MIC of oxacillin by 8-fold, oriented its piperazine part rather outside of the protein and oxygen
from imidazolone ring formed hydrogen bond with E602 and Q521. Compounds 13 and 15 were
located in different part of the PBP2a active site than compound 14, with aromatic rings forming pi–pi
interactions with Y446 and H583, respectively, and hydrogen bonds of oxygen in imidazolone moiety
with N464 and S462, respectively. The last group of analyzed compounds was the most diversified in
terms of activity; there was the most active compound 16 (16–32 fold reduction of MIC of oxacillin),
compound 12, which displayed an 8-fold MIC reduction, and compound 17, which was not effective in

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restoring oxacillin activity (2-fold MIC reduction). Compound 16 is differently oriented in comparison
to compounds 12 and 17. It seems that the clue for activity of this compound lies in the position of
aromatic rings of anthracene, located closely to S403 residue. The network of hydrogen bonds is similar
for all of these compounds, as they all form this type of interaction with S461 and S463.
Figure 7 presents docking results to the allosteric pocket of PBP2a with the compounds grouped
in an analogous way as previously.
Figure 7. Docking poses of the studied compounds in the allosteric site of PBP2a (grid centered at S240).
For compounds
depicted in green is flipped in comparison to poses of compound
most active compound
occurs for compounds
the flipped orientation of compound 10 (which has similar activity to compound
6
–
8
, there are high variations in docking poses, as the pose of compound
and , and the docking pose of the
is only slightly different from less active compound . An analogous situation
11, where molecules are differently docked to the allosteric site of PBP2a, but
and 11) is difficult
to correlate with observed activity relationships (an analogous situation occurs for compounds 13 15).
The last group of analyzed compounds (12 16 17) with activity decreasing in the following way:
16 12 17 adopted significantly different docking poses. The most active compound 16 displays an
extended network of ligand–protein interactions, including those that occur for the anthracene moiety:
lysine at position 148, glutamic acid at position 150 and arginine at position 151. For compound 16
6
7
8
7
9
8
–
9
–
,
,
>
>
,
a set of hydrogen bonds that is not observed for other compounds in the analyzed subgroup is present,
which take part in the interactions with serine and threonine from positions 240 and 238, respectively.

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2.4.2. Molecular Dynamic Studies
As docking captures the compound orientation only in one moment, molecular dynamic
simulation studies were carried out in order to explain more comprehensively the observed activity
relationships. Changes in the ligand–protein interactions in time that were observed during simulations
are presented in Figures 8 and 9 for dockings to the active and allosteric site of PBP2a, respectively.
For simplicity, we have presented the results for only selected compounds; all data is available in the
Supporting Information.
Figure 8. Ligand–protein interaction diagrams obtained for the PBP2a active site during molecular
dynamic simulations for compounds 16, 10, 15 and 17.
When simulations of compounds in the PBP2a active site are analyzed (Figure 8), the most
striking observation is the change in orientation of the most active compound (16), approximately
after 50 ns of simulation, which resulted in the loss of interaction with Q637 and M641 and the
formation of interaction with S643, Y644, K647, I648 and K651. On the other hand, a slightly less
active compound (10) preserved interaction with Y446 (which was lost by compound 16 at the
beginning of the simulation), Q637 and M641, although the last two interactions are not very frequent
despite being present from time to time during the whole simulation. Similar changes in compound
orientation occurred for compound 15 (4-fold reduction of MIC of oxacillin), which was observed
after approximately 40 ns of simulation and was connected with the loss of a set of interactions (e.g.,
T444, Y446, N464, Q521, and E602), the formation of interactions with S437, N510 and I512, and later
also with K506, N507, and L513. Additionally, inactive compound 17 moved from its initial docking
orientation and it happened the fastest out of all tested compounds (after approximately 20 ns). It led

Molecules 2019, 24, 438
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to the loss of interactions e.g., with E447, S461, N464, and K597 and formation of interactions with
S403, Q521, L525, T600, E602, and Q613.
On the other hand, when the outcome of molecular dynamic simulations within the allosteric
site of PBP2a is analyzed (Figure 9), the most visible observation from the ligand interaction diagrams
is the significantly greater perseverance for all of the considered compounds. For the most active
compound (16), the very strong interaction with R241 occurs for almost 100% of the simulation time,
and although interactions with other protein residues are less frequent, they are also present during
the whole simulation. The slightly different pose of compound 10 led to a different set of present
interactions, the most frequent of which was with E239. Compound 15 interacted more frequently
with K215 and T216, although the latter interaction appears after about 20 ns of simulation. For the
inactive compound 17, there is no such interaction that occurs for almost 100% of simulation time,
although the compound pose in the allosteric site of PBP2a is also rather preserved during the whole
molecular dynamic studies.
Figure 9. Ligand–protein interaction diagrams obtained for PBP2a allosteric site during molecular
dynamic simulations for compounds 16, 10, 15 and 17.
The observed dependencies in docking poses and changes in ligand–protein interactions during
molecular dynamic simulation studies suggest the allosteric mechanism of action of the studied
compounds that is an interaction with PBP2a in the allosteric site, improving binding of oxacillin in
the PBP2a active site.
2.5. ADMET Studies
Compound
9, as the most active agent considering both oxacillin adjuvant- and AcrAB-TolC
inhibitor properties, was chosen to be tested on ADME-Tox properties in vitro.

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2.5.1. Membrane Permeability
The permeability of compound
9
was estimated by the commercially available Pre-coated
PAMPA (Parallel Artificial Membrane Permeability Assay) Plate System Gentest
previously described protocols and formulas [35 36]. The calculated permeability coefficient for
(Pe = 0.72 0.27
10−6 cm/s) was comparable to the Pe value obtained for the low permeable
™ according to
,
9
±
×
reference norfloxacin and much lower than that for the highly permeable caffeine (Table 6). Thus,
the passive movement of compound 9 across the cell membranes was determined as low.
Table 6. PAMPA results for compound
9
and the references: well-permeable caffeine (CFN) and
low-permeable norfloxacin (NFX).
−6
Comp.
Pe (10 cm/s) ± SD
CFN
NFX
9
15.1 ± 0.4
0.56 ± 0.1
0.72 ± 0.27
2.5.2. Safety Assays In Vitro
The bacterial and eukaryotic in vitro cell growths were used to get results of potential mutagenicity
and hepatotoxicity of compound 9.
The mutagenicity was evaluated using Ames microplate fluctuation protocol (MPF) performed
with Salmonella typhimurium TA100 strain, which is able to detect base pair substitutions [37].
Calculation of medium control baseline (MCB) was performed basing on mean number of revertants
in standard medium control in addition with one standard deviation. According to the manufacturer’s
protocol, the mutagen alert is determined as 2-fold of medium control baseline (2 x MCB), which in
this study was calculated as 24 revertants. For the control mutagen nonyl-4-hydroxyquinoline-N-oxide
(NQNO, 0.5
maximum 11 revertants occurred (Table 7). The obtained data indicates no mutagenicity potential
of the tested compound. However, the Binomial B-value = 0.0093, calculated for at the 10
concentration, showed possible cytotoxic effect of 9 against S. typhimurium TA100 (Table 7).
µM) more than 40 revertants was observed, whereas in the presence of tested compound
9
9
µM
Table 7. The results of mutagenicity assay for
N-oxide (NQNO).
9 and the reference mutagen nonyl-4-hydroxyquinoline-
Cpd 9
Concentration (µM)
Mean of
Revertants
Fold Increase Over
Baseline **
Binomial
B-Value ***
n *
SD
0
1
10
9
3
3
9
9.00
9.33
5.33
3.00
2.08
4.73
4.50
0.78
0.44
3.75
0.6331
0.0093
1.0000
4-NQNO (0.5 µM)
45.00
* number of replicates; ** compound is considered to be mutagenic if data points with fold increase
B-value ≤ 0.01 may be the result of compound’s cytotoxic effect.
≥
2; *** Binomial
Determination of potential hepatotoxicity was performed with hepatoma HepG2 cells by standard
colorimetric MTS procedure. Compound was diluted into the cell culture media in the following
concentrations: 0.1, 1, 10 and 100 M, added to the cells and incubated for 72 h at 37 C in the presence
of 5% CO2. The cytostatic drug doxorubicin (DX) was used at 1 M, as the reference. The obtained
results showed that compound caused statistically significant decrease of the HepG2 cells viability
at the concentrations 10 and 100 M (Figure 10). However, in comparison to the result obtained for
9
µ
º
µ
9
µ
the reference DX the hepatotoxicity potential of tested compound is moderate, as its decreased cells’
viability to ~74% of control at 10 µM, whereas DX to ~15% of control at 1 µM (Figure 10).

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Figure 10. The viability of HepG2 cell line after incubation with compound 9 for 72 h. DX—doxorubicin.
Control—1% DMSO in the cell culture media. The statistical significance was evaluated by a one-way
ANOVA, followed by Bonferroni’s Comparison Test (*** p < 0.001 compared with control).
2.6. SAR Discussion
All synthesized compounds (
7
–17) belong to the group of 5-arylideneimidazolones with amine
moiety at position 2 ( ) and after Dimroth rearrangement (Scheme 1) also at position 3 (7–
6
17). A variety
of arylidene moieties at position 5 and distinct position and kind of amines give an opportunity to
evaluate impact of these fragments for the antibiotic adjuvant properties considered. Most of the tested
compounds displayed significant (at least four) reduction of oxacillin MIC in MRSA HEMSA 5 strain
(Table 5). In previous tests of 2-amine-5-arylideneimidazolones, only two compounds displayed a
significant reduction of oxacillin MIC in the same bacterial strain. However, the decrease of antibiotic
MIC was higher (64- to 128-fold) [25]. Additionally, a synergistic effect with oxacillin was not observed
in the case of the susceptible strain (ATCC), what indicated an ability to selectively block mechanisms of
resistance occurring in the MDR pathogen. The outcome seems to be more profitable for imidazolones
with condensed aromatic rings at the position 5 (8–32-fold activity gain for
The same conclusion was made for previously tested 5-arylideneimidazolones with unsubstituted
piperazine at position 2 [25]. Comparing the 2-naphthyl derivative ( ) and 1-naphthyl (15), the first
one ( ) was more potent. There was no difference between the adjuvant potency of biphenyl (14) and
4-phenoxyphenyl (12) derivatives. In the case of mono-aromatic 5-benzylideneimidazolones, chlorine
) in para position was more profitable than MeO. Compound 16 with an anthracene moiety and the
9–11, 13, 15 and 16).
9
9
(7
morpholine-terminated fragment at position 3 showed the highest ability to restore oxacillin activity,
and also demonstrated more potent ligand–protein interactions in the allosteric site in molecular
modelling studies. The similar anthracene derivative of methylpiperazine (11) was less active, thus
indicating a predominant role for morpholine. Additionally, the 5-anthracylmethylideneimidazolone
derivative with a 2-piperazine moiety was active in the same MRSA strain [25]. On this account, this
relationship seems to be aryl-dependent. In the case of phenoxybenzylidene derived imidazolones,
morpholine derivative (17) were less active than the methylpiperazine analogue (12). Considering the
topology of an amine moiety, the presence of aminoalkyl fragment at position 3 of imidazolone (7)
seems to be significantly more favourable than the presence of the tertiary amine substituted directly
at imidazolone position 2 (6).
Compounds 9–13, 16 and 17 were additionally tested for their activity in E. aerogenes strains.
Although 5-arylideneimidazolone derivatives did not display antibiotic adjuvant activity in assays
on their synergistic effects with chloramphenicol, erythromycin, doxycycline and norfloxacin, these
compounds were able to block AcrAB-TolC efflux activity in RTE assay. The highest inhibitory efficiency
(97%) demonstrated the fluorene derivative (13), while imidazolones with three fused aromatic rings,
antracene (11, 16) and phenanthrene (10), showed moderate ability to block efflux pumps (37–60%).

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These results confirmed the previous findings about EPI’s action in AcrAB-TolC pump in different
E. coli strains in the group of imidazolones [15]. In turn, the presence of condensed aromatic rings at
position 5 was beneficial either for MRSA or for E. aerogenes MDR reversal action. In previous studies,
the 2-piperazine derivative of 5-(2-naphthyl)imidazolone was the most potent (128-fold reduction
of oxacillin MIC) in the MRSA HEMSA 5 strain [25]. Although, taking into account results of both,
the adjuvant potency in MRSA and efflux pump inhibitory action in E. aerogenes, the 2-naphthyl and
methylpiperazine derivative (9) was found as the most potent “dual agent”. Hence, the compound was
selected as representative structure for primary ADMET assays in vitro. The obtained results allowed
us to classify this compound as lowly permeable (Table 6), non-mutagenic but cytotoxic for prokaryotic
cells (Table 7), and displaying slight hepatotoxic effects (Figure 10). This rather moderate “drugability”
found did not exclude a better drug-like property for other active members of the investigated
imidazolones, but indicated a strong need of further studies within this interesting chemical group.
Thus, new chemical modifications as well as an extension of biological- and “drugability” screening
will be intentional for this new family of 3-substituted 5-arylideneimidazolones in the near future.
3. Experimental
3.1. Chemistry
Reagents were purchased from Alfa Aesar (Karlsruhe, Germany) or Sigma Aldrich (Darmstadt,
Germany). Reaction progress was verified using thin layer chromatography (TLC), which was
carried out on 0.2 mm Merck silica gel 60 F254 plates. Spots were visualized by UV light. Melting
points (m.p.) were determined using the MEL-TEMP II apparatus (LD Inc., Long Beach, CA, USA)
1
and are uncorrected. The H-NMR and 13C-NMR spectra were obtained on a Mercury-VX 300
1
Mz spectrometer (Varian, Palo Alto, CA, USA) in DMSO-d6. Chemical shifts in H-NMR spectra
were reported in parts per million (ppm) on the
δ scale using the solvent signal as an internal
standard. Data are reported as follows: Chemical shift, multiplicity (s, singlet; br.s, broad singlet;
d, doublet; d def.-doublet deformated; t, triplet; t def.-triplet deformated; qui, quintet; m, multiplet;
taut.-tautomerism), coupling constant J in Hertz (Hz), number of protons, proton’s position (Ar-
aromatic moiety at position 5, Ph—phenyl, Pip—piperazine, Mor—morpholine). Mass spectra were
recorded on a UPLC-MS/MS system consisted of a Waters ACQUITY®UPLC® (Waters Corporation,
Milford, MA, USA) coupled to a Waters TQD mass spectrometer (electrospray ionization mode
ESI-tandem quadrupole). Chromatographic separations were carried out using the Acquity UPLC
BEH (bridged ethyl hybrid) C18 column; 2.1
Acquity UPLC BEH C18 VanGuard precolumn (Waters Corporation, Milford, MA, USA); 2.1
and 1.7
m particle size. The column was maintained at 40 ◦C and eluted under gradient conditions
from 95% to 0% of eluent A over 10 min, at a flow rate of 0.3 mL
min−1. Eluent A: water/formic acid
(0.1%, v/v); eluent B: acetonitrile/formic acid (0.1%, v/v). Chromatograms were made using Waters e
×
100 mm, and 1.7
µ
m particle size, equipped with
×
5 mm,
µ
·
λ
PDA detector. Spectra were analyzed in the 200–700 nm range with 1.2 nm resolution and sampling
rate 20 points/s. MS detection settings of Waters TQD mass spectrometer were as follows: source
temperature 150 ◦C, desolvation temperature 350 ◦C desolvation gas flow rate 600 L −1, cone gas
h
·
flow 100 L h
−1, capillary potential 3.00 kV, cone potential 40 V. Nitrogen was used for both nebulizing
·
and drying gas. The data were obtained in a scan mode ranging from 50 to 1000 m/z in time 0.5 s
intervals. Data acquisition software was MassLynx V 4.1 (Waters Corporation, Milford, MA, USA).
The UPLC/MS purity of all the final compounds was confirmed to be 95% or higher. Retention times
(tR) are given in minutes. The UPLC/MS purity of all final compounds was determined (%). Synthesis
of compounds 6, 18–20, 22, 25–30, 32, 35 and 36 were described earlier [15,25,26,38–42].
3.1.1. General Procedure to Obtain 5-Arylidenethiohydantoin (21, 23 and 24)
Thiohydantoin (2.90–5.80 g, 25–50 mmol), acetic acid (25–50 ml), sodium acetate (8.33–16.67 g,
100–200 mmol) with appropriate arylidene aldehyde (25–50 mmol) in flat-bottom flask were heated in

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boiling point for 4–6 h and then mixed for 20 h. Reaction was controlled by TLC-chloroform/ethyl
acetate: 1/1. If necessary, purification was performed using crystallization from acetone or acetic acid.
(Z)-5-(phenanthren-9-ylmethylene)-2-thioxoimidazolidin-4-one (21) Phenanthrene-9-carbaldehyde (30.5 mmol,
6.29 g) and thiohydantoin (30.5 mmol, 3.54 g) were used. Yellow solid. Yield 99%; mp 283–285 ◦C.
1
C18H12N2OS MW 304.37. LC/MS
±
: purity 96.05% tR = 6.42, (ESI) m/z [M+H] 305.04. H-NMR
δ
[ppm]: 8.94–8.63 (m, 3H, N1-H, Ar-4,5-H), 8.30 (m, 1H, Ar-1-H), 8.03–7.87 (m, 1H, Ar-8-H), 7.80–7.61
(m, 4H, Ar-2,3,6,7-H), 7.12 (s, 1H, Ar-10-H), 6.76 (s, 1H, C = CH).
(Z)-5-(4-phenoxybenzylidene)-2-thioxoimidazolidin-4-one (23) 4-Phenoxybenzaldehyde◦(50 mmol, 9.91 g)
and thiohydantoin (50 mmol, 5.81 g) were used. Yellow solid. Yield 81%; mp 233–235 C. C16H12N2O2S
1
MW 296.34. LC/MS
±
: purity 99.11% tR = 6.52, (ESI) m/z [M+H]+ 297.07. H-NMR
δ
[ppm]: 12.00
(br.s, 1H, N3-H), 7.80–7.77 (d def., 2H, Ph-2,6-H), 7.41–7.39 (d def., 2H, Ph-3,5-H), 7.20–6.97 (m, 5H,
Ph’-2,3,4,5,6-H), 6.42 (s, 1H, C = CH), 3.52 (br. s, 1H, taut. N1-H<->SH).
(Z)-5-((9H-fluoren-2-yl)methylene)-2-thioxoimidazolidin-4-one (24) 9H-Fluorene-2-carbaldehyde (25 mmol,
◦
4.86 g) and thiohydantoin (25 mmol, 2.90 g) were used. Yellow solid. Yield 86%; mp 276–278 C.
C17H12N2OS MW 292.35. LC/MS
±
: purity 94.88% tR = 6.45, (ESI) m/z [M+H]+ 293.01. 1H-NMR
δ
[ppm]: 12.38 (br.s, 1H, N3-H), 12.38 (br.s, 1H, N3-H), 12.22 (br.s, 1H, N1-H), 8.04 (s, 1H, Ar-1-H),
7.96–7.92 (m, 2H, Ar-4,5-H), 7.71 (d, J = 7.95 Hz, 1H, Ar-8-H), 7.60 (d, J = 6.92 Hz, 1H, Ar-3-H), 7.43–7.32
(m, 2H, Ar-6,7-H), 6.57 (s, 1H, CH = C), 3.96 (s, 2H, Ar-9-CH2).
3.1.2. General Procedure to Obtain 2-Methylthio-5-Arylidenethiohydantoins (31, 33, 34 and 37)
Sodium (0.48–0.87 g, 21.00–37.77 mmol) was put into ethanol (21.00–37.77 mL). Sodium ethoxide
mixed with appropriate 5–arylidenethiohydantion (21.00–37.77 mmol) in flat–bottom flask for 3 min.
Then, iodomethane (2.98–5.36 g, 21.00–37.77 mmol) was added and whole were mixed for 5–24 h.
Reaction was controlled by TLC-chloroform/ethyl acetate: 1/1. Purification was performed using
crystallization from acetone wherever necessary.
(Z)-2-(methylthio)-4-(phenanthren-9-ylmethylene)-1H-imidazol-5(4H)-one
(31
)
(Z)-5-(Phenanthren-9-
ylmethylene)-2-thioxoimidazolidin-4-one (21) (26.00 mmol, 7.91 g) with iodomethane (26.00 mmol,
◦
3.69 g) was used. Yellow solid. Yield 99%; mp 271–274 C. C19H14N2OS MW 318.39. LC/MS
±
: purity
1
97.70% tR = 6.77, (ESI) m/z [M+H]+ 319.06. H-NMR
δ
[ppm]: 9.24–9.10 (m, 1H, N3-H), 8.90–8.81 (m,
2H, Ar-4,5-H), 8.36–8.29 (m, 1H, Ar-8-H), 8.02–7.99 (m, 1H, Ar-1-H), 7.67–7.40 (m, 5H, Ar-2,3,6,7,10-H),
6.87 (m, 1H, C = CH), 2.71 (s, 3H, S-CH3).
(Z)-4-(4-phenoxybenzylidene)-2-(methylthio)-1H-imidazol-5(4H)-one (33) (Z)-5-(4-Phenoxybenzylidene)-2-
thioxoimidazolidin-4-one (23) (37.00 ◦mmol, 10.96 g) with iodomethane (37.00 mmol, 5.25 g) was used.
Yellow solid. Yield 92%; mp 177–179 C. C17H14N2O2S MW 310.37. LC/MS : purity 96.15% tR = 7.39,
±
(ESI) m/z [M+H]+ 311.09. 1H-NMR
δ [ppm]: 11.80 (br.s, 1H, N3-H), 8.29–8.18 (d def., 2H, N3-H,
Ph-2,6-H), 7.44–7.38 (t def., 2H, Ph-3,5-H), 7.21–7.13 (m, 1H, Ph’-4-H), 7.09–6.99 (m, 4H, Ph’-2,3,5,6-H),
6.71 (s, 1H, C = CH), 2.62 (s, 3H, S-CH3).
(Z)-5-((9H-fluoren-2-yl)methylene)-2-(methylthio)-3H-imidazol-4(5H)-one
(
34)
(Z)-5-((9H-Fluoren-2-
yl)methylene)-2-thioxoimidazolidin-4-one (24) (21.00 ◦mmol, 6.14 g) with iodomethane (21.00 mmol,
2.98 g) was used. Yellow solid. Yield 90%; mp 240–242 C. C18H14N2OS MW 306.38. LC/MS
±
: purity
1
91.26% tR = 7.58, (ESI) m/z [M+H]+ 307.10. H-NMR
δ
[ppm]: 8.39 (s, 1H, Ar-1-H), 8.27–8.18 (d def.,
1H, Ar-8-H), 7.99–7.87 (m, 2H, Ar-4,5-H), 7.64–7.54 (d def., 1H, Ar-3-H), 7.45–7.20 (m, 3H, Ar-6,7-H),
6.80 ( s, 1H, CH = C), 3.96 (s, 2H, Ar-9-CH2), 2.70 (s, 3H, SCH3).
(Z)-5-(3-phenoxybenzylidene)-2-(methylthio)-3H-imidazol-4(5H)-one (37) (Z)-5-(3-Phenoxybenzylidene)-2-
thioxoimidazolidin-4-one (27) (37.77 ◦mmol, 11.19 g) with iodomethane (37.77 mmol, 5.36 g) was used.
Yellow solid. Yield 95%; mp 214–216 C. C17H14N2O2S MW 310.37. LC/MS
±
: purity 95.24% tR = 7.32,
1
(ESI) m/z [M+H]+ 311.02. H-NMR
δ [ppm]: 11.80 (s, 1H, N3-H), 8.06 (s, 1H, Ph-2-H), 7.64–7.61 (d def.,

Molecules 2019, 24, 438
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1H, Ph-6-H), 7.43–7.38 (m, 3H, Ph-5-H, Ph’-3,5-H), 7.19–7.05 (m, 4H, Ph-4-H, Ph’-2,4,6-H), 6.67 (s, 1H,
C = CH), 2.35 (s, 3H, S-CH3).
3.1.3. General Procedure for Synthesis of Final Products (7–17)
(Z)-2-(methylthio)-5-arylidene-3H-imidazol-4(5H)-one (2–10 mmol) with appropriate amine
(3–15 mmol) derivative in 50 mL flat-bottom flask were heated in oil bath with controlled temperature
(120–130 ◦C) for 15 min. Then, ethanol (15-30 ml) was added and mixture was heated for 5–7 h and
mixed for the next 20 h. Then, compounds were converted into hydrochloride forms by gaseous
hydrochloride acid obtained from reaction between sodium chloride and sulphuric acid. Purification
was performed using crystallization from ethanol wherever necessary.
(Z)-5-(4-chlorobenzylidene)-2-amino-3-(3-(4-methylpiperazin-1-yl)propyl)-3H-imidazol-4(5H)-one hydrochloride
(7); (Z)-5-(4-Chlorobenzylidene)-2-(methylthio)-3H-imidazol-4(5H)-one (28) (10 mmol, 2.53 g) and
3-(4-methylpiperazin-1-yl)propan-1-amine (15 mmol, 2.36 g) were used. White solid. Yield 7.5%; mp
294 ◦C. C18H24ClN5Ox3HClxH2O MW 486.25. LC/MS
±
: purity 100.00% tR = 3.34, (ESI) m/z [M+H]+
1
362.26. H-NMR
δ
[ppm]: 11.82 (br. s, 1H, NH+), 9.22 (br. s, 2H, NH2-taut.: N1-H, C2 = NH), 7.84 (d,
J = 8.46 Hz, 2H, Ar-2,6-H), 7.49 (d, J = 8.72 Hz, 2H, Ar-3,5-H), 6.77 (s, 1H, CH = C), 3.78 (t, J = 6.41 Hz,
2H, N3-CH2), 3.48 (br. s, 8H, Pip), 3.19 (br. s, 2H, Pip-CH2), 2.80 (br. s, 3H, CH3), 2.02 (br. s, 2H,
N-CH2-CH2).
(Z)-5-(4-Methoxybenzylidene)-2-amino-3-(3-(4-methylpiperazin-1-yl)propyl)-3H-imidazol-4(5H)-one hydrochloride
(8); (Z)-5-(4-Methoxybenzylidene)-2-(methylthio)-3H-imidazol-4(5H)-one (29) (10 mmol, 2.46 g) and
3-(4-methylpiperazin-1-yl)propan-1-amine (15 mmol, 2.36 g) were used. Yellow solid. Yield 15%; mp
◦
262 C. C19H27N5O2x3HCl
·
0.5H2O MW 475.93. LC/MS
: purity 100.00% tR = 2.58, (ESI) m/z [M+H]+
±
1
358.34. H-NMR
δ
[ppm]: 11.8 (br. s, 1H, NH+), 9.5 (br. s, 2H, NH2-taut.: N1-H, C2 = NH), 7.76 (d,
J = 8.72 Hz, 2H, Ar-2,6-H), 7.01 (d, J = 8.72 Hz, 2H, Ar-3,5-H), 6.84 (s, 1H, CH = C), 3.81 (s, 3H, O-CH3),
3.70–3.20 (m, 12H, Pip, Pip-CH2, N3-CH2), 2.79 (s, 3H, CH3), 2.00 (br. s, 2H, N-CH2-CH2).
(Z)-2-Amino-3-(3-(4-methylpiperazin-1-yl)propyl)-5-(naphthalen-2-ylmethylene)-3H-imidazol-4(5H)-one
hydrochloride (9) (Z)-2-(methylthio)-4-(naphthalen-2-ylmethylene)-1H-imidazol-5(4H)-one (30) (5 mmol,
1.34 g) and 3-(4-methylpiperazin-1-yl)propan-1-amine (10 mmol, 1.57 g) were used. Yellow solid. Yield
◦
74%; mp 215-217 C. C22H28ClN5O MW 413.94. LC/MS
±
: purity 100.00% tR = 3.34, (ESI) m/z [M+H]+
1
378.28. H-NMR
δ
[ppm]: 8.39–8.34 (m, 2H, Ar-5,8-H), 7.85–7.78 (m, 5H, NH2, Ar-1,3,4-H), 7.47–7.45
(m, 2H, Ar-6,7-H), 6.50 (s, 1H, C = CH), 3.54 (t, J = 6.40Hz, N3-CH2), 2.45–2.20 (m, 10H, Pip, Pip-CH2),
2.09 (s, 3H, CH3), 1.68 (qui, J = 6.40Hz, 2H, N-CH2-CH2).
(Z)-2-Amino-3-(3-(4-methylpiperazin-1-yl)propyl)-5-(phenanthren-9-ylmethylene)-3H-imidazol-4(5H)-one
hydrochloride (10) (Z)-2-(Methylthio)-5-(phenanthren-9-ylmethylene)-3H-imidazol-4(5H)-one (31
(2 mmol, 0.62 g) and 3-(4-methylpiperazin-1-yl)propan-1-amine (3 mmol, 0.47 g) were used. Yellow
solid. Yield 17%; mp 266–268 ◦C. C26H30ClN5O MW 464.00. LC/MS
: purity 99.00% tR = 4.03, (ESI)
m/z [M+H]+ 428.19. 1H-NMR
[ppm]: 8.89–8.86 (m, 2H, Ar-1,10-H), 8.80–8.78 (m, 2H, Ar-4,7-H),
)
±
δ
8.24–8.21 (m, 1H, Ar-6-H), 7.89 (s, 2H, NH2), 7.73–7.69 (m, 2H, Ar-2,5-H), 7.68–7.64 (m, 2H, Ar-3,8-H),
6.93 (s, 1H, C = CH), 3.60–3.20 (m, 2H, N3-CH2), 2.45–2.15 (m, 10H, Pip, Pip-CH2), 2.09 (s, 3H, CH3),
1.75 (m, 2H, N-CH2-CH2).
(Z)-2-Amino-5-(anthracen-10-ylmethylene)-3-(3-(4-methylpiperazin-1-yl)propyl)-3H-imidazol-4(5H)-one
hydrochloride (11) (Z)-5-(Anthracen-10-ylmethylene)-2-(methylthio)-3H-imidazol-4(5H)-one (32
)
(4 mmol, 1.71 g) and 3-(4-methylpiperazin-1-yl)propan-1-amine (7.5 mmol, 1.18 g) were used. Yellow
solid. Yield 61%; mp 246-248 ◦C. C26H30ClN5O MW 464.00. LC/MS
±
: purity 100.00% tR = 3.89, (ESI)
1
m/z [M+H]+ 428.26. H-NMR
δ
[ppm]: 8.61 (br. s, 1H, Ar-5-H), 8.11-8.01 (m, 6H, Ar-1,4,6,9-H, NH2),
7.53–7.52 (m, 4H, Ar-2,3,7,8-H), 7.00 (s, 1H, C = CH), 3.40–3.15 (m, 2H, N3-CH2), 2.56–2.06 (m, 13H,
Pip, Pip-CH2, CH3), 1.58–1.52 (m, 2H, N-CH2-CH2).

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(Z)-5-(4-Phenoxybenzylidene)-2-amino-3-(3-(4-methylpiperazin-1-yl)propyl)-3H-imidazol-4(5H)-one
(12)
(Z)-5-(4-Phenoxybenzylidene)-2-(methylthio)-1H-imidazol-5(4H)-one (33) (4 mmol, 1.19 g) and
3-(4-methylpiperazin-1-yl)propan-1-amine (5 mmol, 0.78 g) were used. Yellow solid. Yield 68%; mp
220–230 ◦C. C24H30ClN5O2 MW 455.98. LC/MS : purity 100.00% tR = 4.1, (ESI) m/z [M+H]+ 420.28.
±
1H-NMR
δ [ppm]: 7.62–7.57 (m, 3H, NH2, Ph’-4-H), 7.46–7.36 (m, 4H, Ph-2,6-H, Ph’-3,5-H), 7.15–6.95
(m, 4H, Ph-3,5-H, Ph’-2,6-H), 6.72 (s, 1H, C = CH), 3.77 (br. s, 2H, N3-CH2), 3.70–3.14 (m, 10H, Pip,
Pip-CH2), 2.79 (s, 3H, CH3), 1.99 (br. s, 2H, N-CH2-CH2). 13C-NMR (DMSO-d6, ppm):
130.56, 124.01, 118.93, 40.77, 40.49, 40.20, 39.92, 39.64, 39.36, 39.08.
δ 157.23, 156.98,
(Z)-5-((9H-Fluoren-2-yl)methylene)-2-amino-3-(2-(4-methylpiperazin-1-yl)ethyl)-3H-imidazol-4(5H)-one
hydrochloride (13) (Z)-5-((9H-Fluoren-2-yl)methylene)-2-(methylthio)-3H-imidazol-4(5H)-one (34
)
(3.5 mmol, 1.09 g) and 3-(4-methylpiperazin-1-yl)propan-1-amine (5 mmol, 0.78 g) were used. Orange
solid. Yield 69.27%; mp 265–267 ◦C. C25H30ClN5O MW 451.99. LC/MS
±
: purity 99% tR = 3.92, (ESI)
1
m/z [M+H]+. H-NMR
δ
[ppm]: 12.00 (br. s, 1H, NH+), 9.35 (br. s, 1H, NH2-taut: N1-H), 8.32–7.70
(m, 4H, NH2-taut: C2 = NH, Ar-1,4,5-H), 7.67–7.55 (m, 2H, Ar-3,8-H), 7.49–7.24 (m, 2H, Ar-6,7-H),
6.82 (br. s, 1H, CH = C), 4.08 (s, 2H, Ar-9-CH2), 3.90–3.01 (m, 12H, N3-CH2, Pip, Pip-CH2), 2.90 (s, 3H,
CH3), 2.06 (m, 2H, N-CH2-CH2). 13C-NMR (DMSO-d6, ppm): δ 144.20, 143.96, 127.42, 126.88, 125.74,
125.51, 121.18, 121.00, 40.94, 40.89, 40.52, 40.24, 39.68, 39.39, 39.24, 38.95.
(Z)-2-Amino-5-(biphen-4-ylmethylene)-3-(2-(4-methylpiperazin-1-yl)ethyl)-3H-imidazol-4(5H)-one
hydrochloride (14) (Z)-5-(Biphen-4-ylmethylene)-2-(methylthio)-3H-imidazol-4(5H)-one (35) (2.5 mmol,
0.72 g) and 3-(4-methylpiperazin-1-yl)propan-1-amine (4 mmol, 0.63 g) were used. Yellow solid. Yield
49%; mp 258–260 ◦C. C24H30ClN5O MW 439.98. LC/MS
1H-NMR
±
: purity 96.39% tR = 3.78, (ESI) m/z [M+H]+.
δ
[ppm]: 12.00 (br. s, 1H, NH+), 8.24–8.05 (t def., 1H, Ph’-4-H), 7.80 (br. s, 2H, NH2), 7.79–7.61
(m, 4H, Ph’-3,5-H, Ph-2,6-H), 7.52–7.30 (m, 4H, Ph’-2,6-H, Ph-3,5-H), 6.83 (br. s, 1H, CH = C), 4.26–2.97
(m, 12H, Pip, Pip-CH2, N3-CH2), 2.80 (s, 3H, CH3), 2.04 (br. s, 2H, N-CH2-CH2). 13C-NMR (DMSO-d6,
ppm): δ 129.51, 127.11, 40.77, 40.49, 40.21, 39.92, 39.64, 39.36, 39.08.
(Z)-2-Amino-3-(3-(4-methylpiperazin-1-yl)propyl)-5-(naphthalen-1-ylmethylene)-3H-imidazol-4(5H)-one
hydrochloride (15) (Z)-2-(Methylthio)-5-(naphthalen-1-ylmethylene)-3H-imidazol-4(5H)-one (36
)
(5 mmol, 1.34 g) and 3-(4-methylpiperazin-1-yl)propan-1-amine (7 mmol, 1.09 g) were used. Yellow
solid. Yield 24%; mp 187–191 ◦C. C22H28ClN5O MW 413.94. LC/MS
±
: purity 99.07% tR = 3.21, (ESI)
1
m/z [M+H]+ 378.21. H-NMR
δ
[ppm]: 8.83 (br. s, 1H, NH+), 8.19 (br. s, 2H, taut NH2), 7.96–7.88 (d
def., 1H, Ar-8-H), 7.85–7.75 (d def., 2H, Ar-4,5-H), 7.61–7.44 (m, 4H, Ar-2,3,6,7-H), 6.98 (br. s, 1H,
CH = C), 3.32 (br. s, 2H, N3-CH2), 2.47–2.17 (m, 10H, Pip, Pip-CH2), 2.12 (s, 3H, CH3), 1.70 (br. s,
2H, N-CH2-CH2). 13C-NMR (DMSO-d6, ppm):
δ 133.79, 131.93, 131.62, 129.16, 128.53, 127.90, 126.89,
126.19, 126.05, 123.27, 55.09, 53.02, 46.12, 40.81, 40.52, 40.24, 39.96, 39.68, 39.20, 39.11.
(Z)-2-Amino-5-(anthracen-10-ylmethylene)-3-(3-morpholinopropyl)-3H-imidazol-4(5H)-one hydrochloride (16
)
(Z)-5-(Anthracen-10-ylmethylene)-2-(methylthio)-3H-imidazol-4(5H)-one (33) (4 mmol, 1.22 g) and
3-morpholinopropan-1-amine (5 mmol, 0.72 g) were used. Orange solid. Yield 70%; mp 244–246 ◦C.
1
C25H27ClN4O2 MW 450.96. LC/MS
±
: purity 98.71% tR = 4.19, (ESI) m/z [M+H]+ 415.17. H-NMR
δ
[ppm]: 11.25 (br. s, 1H, NH+), 10.21 (br. s, 1H, NH2-taut: N1-H), 9.39 (br. s, 1H, NH2-taut: C2 = NH),
8.72 (s, 1H, Ar-9-H), 8.25–7.95 (m, 4H, Ar-1,4,5,8-H), 7.68–7.43 (m, 5H, Ar-2,3,6,7-H, CH = C), 4.16–2.69
(m, 10H, Mor, N3-CH2), 2.12 (br.s, 2H, Mor-CH2), 1.80 (s, 2H, N-CH2-CH2). 13C-NMR (DMSO-d6,
ppm):
δ 131.49, 129.42, 129.28, 126.77, 125.96, 66.60, 55.99, 53.71, 40.79, 40.50, 40.22, 39.94, 39.66,
39.37, 39.09.
(Z)-5-(3-phenoxybenzylidene)-2-amino-3-(3-morpholinopropyl)-3H-imidazol-4(5H)-one hydrochloride (17
)
(Z)-5-(3-Phenoxybenzylidene)-2-(methylthio)-3H-imidazol-4(5H)-one (37) (5 mmol, 1.55 g) and
3-morpholinopropan-1-amine (7 mmol, 1.01 g) were used. Yellow solid. Yield 41%; mp 227–230 ◦C.
1
C23H27ClN4O3 MW 442.94. LC/MS
±
: purity 96.49% tR = 4.37, (ESI) m/z [M+H]+ 407.19. H-NMR
δ
[ppm]: 11.35 (br. s, 1H, NH+), 9.45 (br. s, 1H, NH2-taut: N1-H), 7.82-7.27 (m, 6H, NH2-taut: C2 = NH,

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Ph-5,6-H, Ph’-3,4,5-H), 7.26–6.85 (m, 4H, Ph-2,4-H, Ph’-2,6-H), 6.74 (br. s, 1H, CH = C), 4.03 (br. s, 4H,
Mor-OCH2), 3.66–2.65 (m, 8H, N3-CH2, Mor-NCH2, Mor-CH2), 2.16–1.88 (t def., 2H, N-CH2-CH2).
13C-NMR (DMSO-d6, ppm):
39.97, 39.68, 39.40, 39.12.
δ 157.67, 157.41, 130.55, 124.13, 118.97, 63.54, 51.44, 40.81, 40.53, 40.25,
3.2. Crystallographic Studies
Crystals suitable for an X-ray analysis were obtained by slow evaporation of the solvent at room
temperature from acetonitryl solution. Diffraction data for single crystal were collected at 293 K
using Oxford Diffraction SuperNova four circle diffractometer, equipped with the Cu (1.54184 Å)
K
α radiation source and graphite monochromator. The structure was solved by direct methods
using SIR-97 [43]. All non-hydrogen atoms were refined anisotropically using weighted full-matrix
least-squares on F2. The hydrogen atoms bonded to carbons were included in the structure at idealized
positions and were refined using a riding model with Uiso(H) fixed at 1.2 Ueq of C and 1.5 Ueq for
methyl groups. Hydrogen atoms attached to nitrogen atom were found from the difference Fourier
map and refined without any restraints. Refinement and further calculations were carried out using
SHELXL [44]. For molecular graphics ORTEP [45] and MERCURY [46] programs were used.
Crystallographic data: C18H24ClN5O, Mr = 361.87, 0.36
×
0.48
×
0.54 mm3, orthorhombic, space
group Pccn, a = 9.381(5) Å, b = 31.114(5) Å, c = 13.119(5) Å, V = 3829(3) Å3, Z = 8, T = 293(2)K,
50,215 reflections collected, 3377 unique reflections (Rint = 0.0364), R1 = 0.0410, wR2 = 0.1110 [I > 2
σ(I)],
R1 = 0.0499, wR2 = 0.1197 (all data).
The supplementary for these crystallographic data can be obtained free of charge from the
Cambridge Crystallographic Data Centre, deposited as CCDC 1,886,697.
3.3. Microbiological Assay
S. aureus and E. aerogenes bacteria were maintained and grown on Columbia agar (bioMérieux,
Marcy-l’Étoile, France) or Trypticase Soy Agar II (TSA II; Becton Dickinson, Franklin Lakes, NJ, USA)
supplemented with 5% sheep blood, respectively. Cation-adjusted Mueller-Hinton (MH II) broth used
in the microbiological assays was obtained from bioMérieux. Oxacillin, norfloxacin, chloramphenicol,
doxycycline, PAβN, and INT were purchased from Sigma Aldrich (St. Louis, MI, USA), whereas
erythromycin lactobionate was purchased from Amdipharm (United Kingdom). The compounds
tested were dissolved in DMSO (Merc, Stuttgart, Germany) and the solutions obtained were stored at
◦
−
20 C until used. Furthermore, the following chemical compounds were used to perform RTE assay:
K2HPO4 (Sigma Aldrich) and MgCl2 (Sigma Aldrich) to prepare potassium phosphate buffer (PPB),
CCCP (Sigma Aldrich), 1,20-dNA (TCI-Europe, Zwijndrecht, Belgium), glucose (Sigma Aldrich).
3.3.1. Susceptibility Testing
Susceptibility testing was performed by the 2-fold standard microdilution method in MH II broth
following the Clinical and Laboratory Standards Institute (CLSI) and the Comité de l’Antibiogramme
de la Société Française de Microbiologie (CA-SFM) recommendations [47,48]. MIC values were
detected in an Infinite M200 pro Tecan microplate reader (Tecan® France, SA-Lyon, France) after
overnight incubation at 37 ◦C. Experiments were carried out in triplicate and the resulting medians
were presented.
First, minimum inhibitory concentration (MIC) values of a series of imidazolidine derivatives were
examined against S. aureus and E. aerogenes strains. Then, MICs of selected antibiotics were determined
in the absence and in the presence of compounds in order to measure the adjuvant-like effect of these
structures. In the latter case, compounds tested were evaluated for the ability to enhance antibacterial
activity of β-lactam antibiotic oxacillin in methicillin-resistant and methicillin-susceptible S. aureus
(MRSA and MSSA) strains as well as chloramphenicol, erythromycin, doxycycline, and norfloxacin in
AcrAB-TolC-overexpressing and AcrAB-TolC-deficient mutants of E. aerogenes. The concentrations of
imidazolidine derivatives used in the MIC reduction assay were not greater than 25% of their respective

Molecules 2019, 24, 438
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MICs, therefore, cell viability was not affected by the direct antibacterial activity of compounds.
For compounds for which precipitation was observed after the addition of bacterial suspension in
MH II broth, the highest concentration at which they did not precipitate was chosen. The final
concentration of DMSO in the assays never exceed 2.5% v/v and had no influence on bacteria. In tests
with E. aerogenes, a well-known efflux pump inhibitor, PAβN, was used as reference compound.
The MICs of imidazolidine derivatives and antibiotics selected for experiments were recorded as the
lowest concentrations of compounds inhibiting the growth of bacteria after 18-h incubation at 37 ◦C.
Due to the heterogeneity of MRSA strains, the incubation of compounds with oxacillin was extended
to 24 h. The growth of bacterial cells was visualized by iodonitrotetrazolium chloride (INT). The results
of the MIC reduction assay were presented by employing activity gain parameter (A) calculated as
the ratio of the MIC value of an antibiotic alone to its MIC in the presence of the compound analyzed
(Equation (1)). All MIC determinations were repeated in at least two independent experiments
!
MICAnt
MICAnt+Comp
A =
(1)
3.3.2. Real-Time Efflux Assay
In the first step, bacteria were loaded with 1,20-dNA, a fluorescent membrane probe which is
a substrate of the AcrAB-TolC efflux pump, in the presence of a well-known EPI carbonyl cyanide
m-chlorophenylhydrazone (CCCP) that inactivates the pump in the pump energy-collapsing mode
(H+-consuming). After the addition of compounds, at the final concentration of 100
µ
M, an efflux was
initiated by automated injection of glucose that provides pump energy (H+-donating compound) to the
final concentration of 50 M. The fluorescence intensity was measured using a microplate reader (Tecan)
with an excitation wavelength of = 370 nm and an emission wavelength of = 420 nm. In order
µ
λ
ex
λ
em
to analyze and quantitatively compare the EPI activity of compounds tested, the pre-energization
fluorescence intensity was adjusted to 100 relative fluorescence units and the inhibition efficiency (IE)
of each compound was calculated according to Equation (2):
∆i1
∆i2
IE [%] =
× 100%
(2)
where
∆i1 corresponds to the difference between the fluorescence of the dye in the presence and
absence of compound tested after the addition of a source of energy for the pump (glucose) and i2 is
∆
the difference between the fluorescence of the dye in the presence and absence of compound tested
before the addition of a source of energy for the pump.
3.4. In Silico Studies
The three-dimensional conformations of compounds and respective protonation states (for pH
7.0 +/
the crystal structure of PBP2a protein (PDB code: 3ZFZ [50]). Following the suggested mechanism of
interaction of compounds via the allosteric modulation of this target [50 51], the studied compounds
were docked to the active and allosteric sites of the protein (with grids were centered at S403 and
−
2.0) were generated with the use of LigPrep [49]. At first, all the compounds were docked to
,
S240, respectively; the docking was performed in Glide [52
extra precision).
,53], and the compounds were docked in
The poses with the best Glide docking score were use as starting points for molecular dynamic
(MD) simulations. MD simulations were performed in Desmond [54
model [56] and lasted 100 ns.
,55], using TIP3P solvent
The interactions between ligands and PBP2a protein were analyzed manually and with the use of
the Simulation Interaction Diagram from the Schrodinger Suite.

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3.5. ADMET Studies
The references used in ADMET studies included caffeine (CFN), norfloxacin (NFX), doxorubicin
(DX), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), nonyl-4-hydroxyquinoline-N-oxide
(NQNO) and were provided by Sigma-Aldrich (St. Louis, MO, USA). HepG2 (ATCC® HB-8065
™)
hepatoma cell line was kindly donated by the Department of Pharmacological Screening, Jagiellonian
University Medical College. Salmonella typhimurium TA100 strain with base pair substitution (hisG46
mutation, which target is GGG) was purchased from Xenometrix, Allschwil, Switzerland.
3.5.1. Membrane Permeability
Pre-coated PAMPA Plate System Gentest
™
was purchased from Corning (Tewksbury, MA, USA).
M) were prepared first in PBS buffer (pH = 7.4)
The tested compound and the references (200
9
µ
and added to the donor wells of PAMPA Plate System. PBS was added to the acceptor wells.
The plate was incubated for 5 h at room temperature. The compounds’ as well as references’
concentrations in acceptor and donor wells were estimated by the UPLC-MS analyses with use
of internal standard. The permeability coefficients (Pe, cm/s) were calculated according to formulas
provided by the manufacturer.
3.5.2. Safety
Ames microplate fluctuation protocol (MPF) assay was obtained from Xenometrix AG (Allschwil,
Switzerland). The occurrence of mutagen-induced or spontaneous reversion events to histidine
prototrophy was determined as a growth of S. typhimurium TA100 in the indicator medium without
histidine after 72 h incubation at 37 ◦C temperature. NQNO (0.5
Compound was tested in triplicate at the final concentrations 1 and 10
µ
M) was used as a positive control.
9
µM. The revertants’ growth
induced the colour change of medium which was analysed next colourimetrically with a microplate
reader (EnSpire, PerkinElmer, Waltham, MA, USA) at 420 nm.
The HepG2 cells were seeded in 96-well plates at a concentration of 1
incubated for 24 h at 37 C in 5% CO2 atmosphere to reach 50% of confluence. Compound
×
104 cells/well and
was
M.
µM, respectively, and the cells were
◦
9
diluted into fresh growth medium and added to the cells at the final concentrations 0.1
The positive controls DX and CCCP were added at 1 M and 10
µM–100 µ
µ
incubated for 72 h. The MTS reagent (CellTiter 96® AQueous One Solution Cell Proliferation Assay,
Promega, Madison, WI, USA) was added next to the each well and incubated for 4 h. The absorbance
was measured next using a microplate reader (EnSpire, PerkinElmer, Waltham, MA USA) at 490 nm to
determine cells’ viability.
4. Conclusions
In order to search for successful antibiotic adjuvants, useful against both Gram-positive
and Gram-negative MDR pathogens, the performed chemical modifications of the lead structure
(Z)-5-(4-chlorobenzylidene)-2-(4-methylpiperazin-1-yl)-3H-imidazol-4(5H)-one (6) have provided a
series of new, and even unexpected, chemical structures due to Dimroth rearrangements within the
imidazolone ring. Most of those original compounds significantly improved oxacillin activity in MRSA,
while they did not influence the activity of oxacillin in the reference S. aureus strain, suggesting an
impact on MDR mechanisms associated with PBP2a. Docking and molecular dynamic simulations
have confirmed this hypothesis, indicating the ability of the most active compounds to interact
with the allosteric site of PBP2a, and to enhance the binding of oxacillin to the active site. On the
other hand, results of the RTE assay for the new 5-arylideneimidazolones towards AcrAB-TolC in
E. aerogenes (EA289) have confirmed their potent capacity to inhibit this important MDR efflux system.
The performed SAR analysis indicated the anthracene-morpholine derivative (16) as the most active
oxacillin adjuvant in the MRSA, and the naphthalene-methylpiperazine imidazolone (
9) as the most
potent “dual-action” compound displaying both oxacillin potentiating action in MRSA in the range of

Molecules 2019, 24, 438
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compound 16 and efflux pump inhibitory properties in E. aerogenes. The primary ADMET screening
in vitro for showed a rather moderate “drugability” profile for this active compound. In the context
of the obtained results, further comprehensive studies for this interesting chemical family are needed.
Overall, compound , and then 16, seem to be new lead structures for further modifications in order
9
9
to discover therapeutically useful adjuvants, able to restore the effectiveness of common antibiotics
against MDR pathogens.
Supplementary Materials: The following are available online, Spectral data for compounds, 1H
NMRs Compounds.
Author Contributions: A.K. performed most of syntheses; J.H. supervised and partly contributed in the syntheses;
K.W. and J.C. performed biological assays with S. aureus; K.W. performed biological studies in E. aerogenes; A.K.,
˙
A.L. and G.L. performed ADMET studies in vitro; S.P. performed molecular modelling; E.Z. and W.N. performed
crystallographic studies; E.K. supervised microbiological assays with S. aureus; S.A. and J.-M.P. supervised
biological studies in E. aerogenes; K.K.-K. supervised ADMET studies in vitro; A.K., K.W., S.P. and J.H. have
written the MS.
Funding: This research was partly financed from budget funds for science in 2017–2020, as a research project
within “Diamond Grant” no 0169/DIA/2017/46 and Polish Statutory research program K/ZDS/007886.
Acknowledgments: This work was also supported by Aix-Marseille Univ. and Service de Santé des Armées.
Authors thank Andrzej J. Bojarski for his scientific patronage in molecular modeling studies. Authors thank Maria
Kaleta and ex-master student, Beata Mastek, for their irreplaceable support in the synthesis works.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2019 by the authors. 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 (http://creativecommons.org/licenses/by/4.0/).