Phenylpiperazine 5,5-dimethylhydantoin derivatives as first synthetic inhibitors of Msr(A) Efflux Pump in Staphylococcus epidermidis

Article abstract of DOI:10.3390/molecules25173788

Herein, 15 phenylpiperazine 3-benzyl-5,5-dimethylhydantoin derivatives (1-15) were screened for modulatory activity towards Msr(A) efflux pump present in S. epidermidis bacteria. Synthesis, crystallographic analysis, biological studies in vitro and structure-activity relationship (SAR) analysis were performed. The efflux pump inhibitory (EPI) potency was determined by employing ethidium bromide accumulation assay in both Msr(A) efflux pump overexpressed (K/14/1345) and deficient (ATCC 12228) S. epidermidis strains. The series of compounds was also evaluated for the capacity to reduce the resistance of K/14/1345 strain to erythromycin, a known substrate of Msr(A). The study identified five strong modulators for Msr(A) in S. epidermidis. The 2,4-dichlorobenzyl-hydantoin derivative 9 was found as the most potent EPI, inhibiting the efflux activity in K/14/1345 at a concentration as low as 15.63 μM. Crystallography-supported SAR analysis indicated structural properties that may be responsible for the activity found. This study identified the first synthetic compounds able to inhibit Msr(A) efflux pump transporter in S. epidermidis. Thus, the hydantoin-derived molecules found can be an attractive group in search for antibiotic adjuvants acting via Msr(A) transporter.

Full text of DOI:10.3390/molecules25173788

molecules
Article
Phenylpiperazine 5,5-Dimethylhydantoin Derivatives
as First Synthetic Inhibitors of Msr(A) Efflux Pump in
Staphylococcus epidermidis
Karolina Witek 1,2, Gniewomir Latacz 1 , Aneta Kaczor 1, Joanna Czekajewska 2,
3
˙
Ewa Zesławska
, Anna Chudzik 1, Elz˙bieta Karczewska 2, Wojciech Nitek 4,
Katarzyna Kiec´-Kononowicz 1 and Jadwiga Handzlik 1,
*
1
Department of Technology and Biotechnology of Drugs, Jagiellonian University, Medical College,
ul. Medyczna 9, 30-688 Krakow, Poland; karolina.witek@uj.edu.pl (K.W.); glatacz@cm-uj.krakow.pl (G.L.);
aneta.kaczor@doctoral.uj.edu.pl (A.K.); ankachudzik@yahoo.com (A.C.); mfkonono@cyf-kr.edu.pl (K.K.-K.)
Department of Pharmaceutical Microbiology, Jagiellonian University, Medical College, ul. Medyczna 9,
30-688 Krakow, Poland; j.czekajewska@uj.edu.pl (J.C.); elzbieta.karczewska@uj.edu.pl (E.K.)
Institute of Biology, Pedagogical University of Krakow, ul. Podchora˛z˙ych 2, 30-084 Krakow, Poland;
ewa.zeslawska@up.krakow.pl
2
3
4
Faculty of Chemistry, Jagiellonian University, ul. Gronostajowa 2, 30-387 Krakow, Poland;
nitek@chemia.uj.edu.pl
*
Correspondence: j.handzlik@uj.edu.pl; Tel.: +48-12-620-55-84
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Stefano Sabatini and Tommaso Felicetti
Received: 22 July 2020; Accepted: 15 August 2020; Published: 20 August 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Herein, 15 phenylpiperazine 3-benzyl-5,5-dimethylhydantoin derivatives (
1–
15) were
screened for modulatory activity towards Msr(A) efflux pump present in S. epidermidis bacteria.
Synthesis, crystallographic analysis, biological studies in vitro and structure–activity relationship
(SAR) analysis were performed. The efflux pump inhibitory (EPI) potency was determined by
employing ethidium bromide accumulation assay in both Msr(A) efflux pump overexpressed
(K/14/1345) and deficient (ATCC 12228) S. epidermidis strains. The series of compounds was also
evaluated for the capacity to reduce the resistance of K/14/1345 strain to erythromycin, a known
substrate of Msr(A). The study identified five strong modulators for Msr(A) in S. epidermidis.
The 2,4-dichlorobenzyl-hydantoin derivative
activity in K/14/1345 at a concentration as low as 15.63
9
was found as the most potent EPI, inhibiting the efflux
M. Crystallography-supported SAR analysis
µ
indicated structural properties that may be responsible for the activity found. This study identified
the first synthetic compounds able to inhibit Msr(A) efflux pump transporter in S. epidermidis. Thus,
the hydantoin-derived molecules found can be an attractive group in search for antibiotic adjuvants
acting via Msr(A) transporter.
Keywords: Staphylococcus epidermidis; efflux pump; Msr(A); phenylpiperazine 5,5-dimethylhydantoins;
efflux pump inhibitors; EPIs; X-ray crystallography
1. Introduction
The Gram-positive bacteria Staphylococcus epidermidis belong to coagulase-negative staphylococci
(CoNS) [1]. The bacteria are highly abundant on the skin and mucous membranes of humans and
compete with other potentially harmful microorganisms, such as S. aureus, to maintain healthy
skin microflora. For this reason, in the past, S. epidermidis was considered to be an important
non-pathogenic commensal organism. However, it became evident that this natural skin colonizer
is a true opportunistic pathogen, particularly in critically ill immunocompromised patients and
those with indwelling foreign devices [2]. Indeed, S. epidermidis has been recognized as the leading
Molecules 2020, 25, 3788; doi:10.3390/molecules25173788
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3788
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causative agent of implanted medical device-related infections (e.g., intravascular catheter or prosthetic
joint infections) in the hospital settings. Nosocomial infections caused by S. epidermidis have gained
considerable attention in recent years due to an increase of resistance of the bacterium to antimicrobial
drugs [
the world are reported to be methicillin-resistant [
most -lactam antibiotics, such as penicillins, cephalosporins (except for the fifth generation drugs),
carbapenems and monobactams, useless in the therapy of staphylococcal infections [ ]. In the presence
of -lactam resistance and in patients allergic to penicillin, the first-line alternative treatment is
macrolide-lincosamide-streptogramin B (MLSB) group of antibiotics [ ]. Although possessing
2,3
]. Currently, between 75% and 90% of S. epidermidis strains isolated in hospitals around
1,2]. Acquisition of methicillin-resistance renders
β
4
β
5–7
different chemical structures, the mechanism of action of these antibacterials is similar. They act as
protein synthesis inhibitors through interaction with the 23S rRNA component of the large 50S ribosomal
subunit that is involved in the regulation of translation accuracy [
and streptogramins B share the same binding site, conformational changes in the ribosome lead to the
development of cross-resistance of bacteria to these three groups of antibiotics [ ]. The widespread use
8,9]. Since macrolides, lincosamides
9
of MLSB antibiotics over the last twenty years resulted in the number of staphylococcal strains acquiring
resistance to these drugs [10]. The lack of susceptibility of staphylococci to MLSB antibacterials is mostly
attributed to the following mechanisms: (i) target site modification; (ii) active efflux; and (iii) enzymatic
inactivation. Depending on the occurrence of the aforementioned mechanisms, different resistance
phenotypes are expressed. The most prevalent ones are MLSB, which, in S. aureus and CoNS, arise from
the methylation of adenine within 23S rRNA ribosomal subunits mediated by the erythromycin
resistance methylase genes ermA and ermC [11]. The expression of MLSB phenotype can be either
constitutive (cMLSB) and confer a high level of cross-resistance to all MLSB antibiotics or inducible
(iMLSB) occurring when the production of methylases is a result of the induction of erm genes that
consequently leads to the lack of susceptibility of bacteria to macrolides with a 14- and 15-membered
lactone ring (e.g., erythromycin, azithromycin or clarithromycin) [12]. The active efflux of antibiotics is
determined by msr genes (predominantly msrA) and is associated with the MSB phenotype. It confers
resistance only to 14- and 15-membered ring macrolides and streptogramins B [13]. Since 16-membered
ring macrolides (josamycin and spiramycin) and lincosamides are neither inducers nor substrates for
the pump, these antibiotics remain active. The msrA resistance determinant was reported for the first
time in S. epidermidis, and, since then, it has also been found in S. aureus and other staphylococcal
species [14]. The gene encodes Msr(A) efflux pump that is a member of the ATP-binding-cassette (ABC)
transporter superfamily comprising important mediators of drug resistance in microorganisms [13].
Notably, the study conducted by Eady et al. indicated that the presence of msr(A) was associated with
erythromycin resistance in 36.4% clinical CoNS [15]. Given the clinical importance of efflux pumps in
bacterial drug resistance, attempts have been made to identify potent EPIs of Msr(A) transporter in
staphylococci. By means of fluorometric assays, it has been demonstrated that carnosic acid, an abietane
diterpene presented in the popular herbs rosemary (Rosmarinus officinalis) and sage (Salvia officinalis),
is able to modulate Msr(A) transporter in Msr(A)-overexpressing S. aureus strain RN4220. Furthermore,
it has been shown that this natural compound is a good potentiator of erythromycin activity in the
resistant to this antibiotic S. aureus RN4220 [16]. However, to the best of our knowledge, no synthetic
compound that displays EPI effect on Msr(A) exporter in S. epidermidis has been described so far.
During the course of efforts to discover novel efflux pump inhibitors with antibiotic adjuvant
activity, our research group has found that some phenylpiperazine 5,5-dimethylhydantoin derivatives
act as inhibitors of intrinsic and overexpressed efflux transporters present in S. aureus strains [17].
Based on that interesting outcome, seven phenylpiperazine 5,5-dimethylhydantoins, which were
previously tested for their EPI activity in S. aureus pathogen ( ), were selected for this approach
1
–
7
(Table 1). Thus, the compounds ( ) and their new derivatives synthesized within this study (8–15,
1–7
Table 1) were evaluated for the ability to block the Msr(A) efflux pump in S. epidermidis K/14/1345
clinical isolate harboring MSB resistance phenotype.

Molecules 2020, 25, 3788
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Table 1. Structures of phenylpiperazine 5,5- dimethylhydantoin derivatives (1–15) investigated in
the study.
Cpd
Group
R1
n
R2
1
2
3
4
5
6
7
8
9
A
A
A
A
A
A
A
A
A
A
A
H
H
H
H
H
5
5
5
5
5
5
5
6
5
5
5
2,4-diF
H
2-MeO
3-MeO
2-F
4-F
2,4-diF
2-F
4-F
4-F
4-Cl
2,4-diCl
2,4-diCl
2,4-diCl
2-F
4-F
2,4-diF
10
11
12
13
14
B
B
B
2,4-diCl
2,4-diCl
2,4-diCl
-
-
-
15
B
2,4-diCl
-
Moreover, the effect displayed by the series of derivatives in Msr(A)-overexpressing S. epidermidis
K/14/1345 was compared to that assessed in Msr(A)-deficient S. epidermidis strain ATCC 12228. The EPI
activity of the 5,5-dimethylhydantoins was detected by the aid of the real-time ethidium bromide (EtBr)
accumulation method. Antibiotic adjuvant properties of the compounds have been verified in the MIC
reduction assay, in which their capacity to reduce or reverse the resistance of S. epidermidis K/14/1345
to erythromycin, a well-known substrate of Msr(A) transporter, was assessed. For representative
compounds, crystallographic analyses were performed to enrich SAR discussion. Thus, the study was
focused on synthesis, X-ray crystallography, comprehensive biological assays and SAR analysis for the
series of phenylpiperazine hydantoins 1–15 given in Table 1.
2. Results
2.1. Chemistry
Synthesis of compounds
1
–
15 was performed according to Scheme 1. Details of synthesis for
17 19 21 23 were described elsewhere [18]. The synthesis
compounds and intermediates 16
1–
7
,
,
– ,
route consists of three steps: (i) N3-alkylation; (ii) N1-alkylation; and (iii or iv) N-alkylation using
1-phenylpiperazine derivatives. Firstly, N3-alkylation (i) between 5,5-dimethylhydantoin and benzyl
chloride derivatives was performed. In this reaction, 3-benzyl-5,5-dimethylimidazolidine-2,4-diones
(
16
performed with dibromo-alkanes (20
respectively. This reaction provided intermediates 20
–
19) were obtained. The intermediates (16
23, Group A) or 2-(chloromethyl)oxirane (24, Group B),
24. In the last step (iii or iv), N-alkylations
–19) were used for N1-alkylation (ii), which was
–
–

Molecules 2020, 25, 3788
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of 1-phenylpiperazine derivatives were performed, using either a suitable bromoalkyl intermediate
20 23) or the oxirane derivative 24. In the first case, the process proceeding in basic conditions
gave products 11 (Group A), while the reaction with 24 occurred via oxirane ring opening under
microwave irradiation in solvent-free conditions to give 12–15 (Group B, Scheme 1).
(
–
1–
Scheme 1. Synthesis route for compounds 1–15: (i) K2CO3, TEBA, acetone, reflux, 4 h; (ii) K2CO3,
TEBA, acetone, rt, 120–144 h; (iii) K2CO3, acetone, reflux, 4–11 h; and (iv) mw, 300–450 W, 3–7 min.
2.2. X-ray Crystallographic Studies
For two representative compounds, displaying EPI action (6) and inactive one (15), X-ray
crystallographic analysis was carried out. The molecular geometry in the crystal structures of
together with the atom numbering schemes is shown in Figure 1.
6 and 15
(a)
(b)
Figure 1. The molecular geometry with the atom numbering schemes of: (a) 6; and (b) 15. Hydrogen
atoms have been omitted for clarity. Displacement ellipsoids are drawn at the 50% probability level.
Orange dashed lines show hydrogen bonds between the chloride anion and protonated N2 atom for
both structures and between the chloride anion and hydroxy group in 15.

Molecules 2020, 25, 3788
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Both molecules contain 3-benzyl-5,5-dimethylhydantoin moiety with different substituents in the
aromatic ring (4-F for
rings is 80.52(5)◦ for
6
6
and 2,4-di-Cl for 15). The angle between the planes of aromatic and hydantoin
and 71.56(7)◦ for 15. The introduction of the second substituent in position
2 reduced the value of this angle. None of these halogen atoms is involved in intermolecular halogen
bonds. The investigated compounds differ in the linker between hydantoin and piperazine rings.
The linker of
6 consists of five methylene units and adopts an extended conformation, while the linker
of 15 has only three methylene units with hydroxy group located at the second carbon atom. In this
molecule, the linker adopts bent conformation.
In the presented crystal structures, the piperazine ring adopts chair conformation with an
equatorial position of substituents at N2 and N4 atoms. The angle between the planes of aromatic
and the carbon atoms of piperazine rings in
6
is 26.22(8)◦. Similar values of this angle have been
also observed in other crystal structures containing 4-(4-fluorobenzene)piperazine moiety deposited
in Cambridge Structure Database [19]. In 15, diphenylmethyl substituent at piperazine ring adopts
conformation, which is similar to other crystal structures containing this moiety [19].
In both structures, the chlorine anion is involved in charge assisted hydrogen bond with protonated
N2 atom (Figure 1). In the crystal of 15, the chlorine ion is also engaged in interaction with oxygen
atom of hydroxy group. The intermolecular interactions in the crystals are dominated by N-H···Cl,
C-H···Cl and C-H···O contacts. Additionally, C-H···N and C-H···F in and O-H···Cl in 15 contacts are
6
observed. The parameters of these interactions are listed in Table 2.
Table 2. Parameters of intermolecular interactions for 6 and 15.
Cmp.
6
Interaction
H···A (Å) D···A (Å) D-H···A (◦)
Symmetry Code
N2-H2N···Cl1
C14-H14A···Cl1
C20-H20···Cl1
C28-H28C···Cl1
C10-H10A···O2
C12-H12B···O4
C21-H21B···O4
C13-H13B···N4
C29-H29C···F1
2.14
2.72
2.85
2.84
2.36
2.50
2.66
2.57
2.54
3.023(1)
3.558(1)
3.753(1)
3.735(2)
3.187(2)
3.046(2)
3.385 (2)
3.531(2)
3.500(2)
176
142
159
152
141
114
131
163
166
−x + 1, −y + 1, −z + 1
−x + 1, −y + 1, −z + 1
−x, −y + 1, −z + 1
−x, −y + 1, −z + 1
x + 1, −y + 1/2, z + 1/2
−x − 1, y + 1/2, −z + 1/2
−x + 1, −y, −z + 1
−x + 1, −y, −z + 1
15
N2-H2N···Cl1
2.07
2.12
2.66
2.91
2.95
2.59
2.36
2.58
2.64
3.067(2)
3.049(2)
3.645(2)
3.638(2)
3.755(2)
3.550(3)
3.325(3)
3.463(3)
3.358(3)
174
168
179
135
140
163
166
156
131
O1-H1···Cl1
C9-H9A···Cl1
C16-H16···Cl1
C21-H21C···Cl1
C6-H6B···O4
C9-H9A···O1
C18-H18···O2
C21-H21A···O1
X − 1, y, z
X − 1, y, z
−x + 2, −y + 1, −z + 1
x + 1, y, z
−x + 2, −y + 1, −z + 1
−x + 1, −y + 2, −z + 1
X − 1, y, z
2.3. Biological Assays
2.3.1. Direct Antibacterial Activity
In the first step of the biological assays, direct antibacterial activity of 15 phenylpiperazine
5,5-dimethylhydantoin derivatives ( 15) was determined against S. epidermidis K/14/1345 and
1–
S. epidermidis ATCC 12228 strains to select the concentrations of the compounds at which their antibiotic
adjuvant effect should be tested. The macrolide antibiotic, erythromycin, was used as reference in the
assay. For most of the compounds (1, 2, 4–6 and 8–15), precipitation in MH II broth was observed at the
highest concentrations tested. For these molecules, MICs could not be established precisely but was
approximated as a higher value than the highest possible concentration at which the growth of bacteria
was still observed. MIC values of the series of phenylpiperazine 5,5-dimethylhydantoin derivatives

Molecules 2020, 25, 3788
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are shown in Table 3. The results of the study indicate that phenylpiperazine 5,5-dimethylhydantoin
derivatives tested exerted very weak ( and 12 14) to moderate ( 11 and 15) antibacterial activity
1–8
–
9–
against S. epidermidis strains selected for the study. Among the 15 derivatives, the most remarkable
antibacterial effect was found for compound 15, which affected bacterial growth at the concentration
<10 µM (<5 µg/mL) in the case of reference strain (Table 3). The significant difference in MIC values of
erythromycin against S. epidermidis strains K/14/1345 and ATCC 12228 was in high accordance with the
resistant and susceptible phenotypes of these strains, respectively.
Table 3. Antibacterial activity of phenylpiperazine 5,5-dimethylhydantoin derivatives and erythromycin
against S. epidermidis K/14/1345 and S. epidermidis ATCC 12228 strains.
MIC [µM] (µg/mL)
Compounds
S. epidermidis K/14/1345
S. epidermidis ATCC 12228
>250 (130.26) 1
>250 (121.27) 1
1000 (515.09)
>125 (64.39) 1
>500 (251.53) 1
>250 (130.26) 1
125 (67.38)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
250 (130.26)
>250 (121.27) 1
250 (128.77)
62.5 (32.19)
250 (125.96)
250 (130.26)
250 (134.76)
62.5 (34.38)
31.25 (17.88)
31.25 (17.88)
15.63 (9.22)
62.5 (35)
>62.5 (34.38) 1
>62.5 (35.74) 1
>31.25 (17.88) 1
>31.25 (18.44) 1
>125 (70) 1
>125 (69.99) 1
>125 (72.24) 1
>31.25 (19.76) 1
0.087 (64)
125 (69.99)
62.5 (36.12)
7.81 (4.94)
Erythromycin
0.00068 (0.5)
1 The highest possible concentration of a compound (due to precipitation) at which growth of bacteria was observed.
2.3.2. Inhibitory Action on Msr(A) Efflux Pump
To establish whether compounds tested have the EPI activity on the Msr(A) efflux transporter,
accumulation assay, which employed ethidium bromide (EtBr), the dye-substrate of the pump,
was performed. For this task, EtBr at a final concentration of 1 µg/mL and compounds tested at a
final concentration corresponding to 1/4 or 1/2 of their MIC values (the highest possible concentration
that did not inhibit bacterial growth in the case of precipitating compounds) were added to the
bacterial suspensions. The experiment was performed under conditions that allow maximum efflux
(the presence of glucose, which is a source of energy for the pump, and the temperature of 37 ◦C).
The data obtained in accumulation assay enabled identifying five strong EPIs (1, 6, 7, 9 and 14)
of Msr(A) efflux transporter. The level of accumulation of EtBr in the absence and presence of active
compounds in Msr(A)-overexpressing S. epidermidis strain K/14/1345 and Msr(A)-deficient S. epidermidis
strain ATCC 12228 is shown in Figures 2 and 3.
As shown in Figures 2 and 3, the addition of compounds 1, 6, 7, 9 and 14 caused a statistically
significant rise of the fluorescence intensity in Msr(A)-overexpressing S. epidermidis strain K/14/1345.
The increase in the intracellular concentrations of the dye in combination with the aforementioned
compounds was dose-dependent with the most prominent effect observed at the concentration of 1/2
of their MIC values. To compare the modulatory effect of new hydantoin-derived EPIs towards Msr(A)
protein, relative fluorescence index (RFI) was calculated using the relative final fluorescence (RFF) at
20-min time point of the EtBr accumulation curve. The RFF parameter is an indicator of the capacity of
a compound to modulate the efflux pump activity of bacterium. The greater is the RFF, the greater
is the degree of inhibition of an efflux pump transporter and, therefore, the more potent is the EPI
activity of a given compound tested [17].

Molecules 2020, 25, 3788
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a)
b)
S. epidermidis K/14/1345
S. epidermidis ATCC 12228
1000
EtBr c = 1 g/ml
1000
500
EtBr c = 1 g/ml
EtBr c = 1 g/ml +
cpd 1 c = 31.25 M
EtBr c = 1 g/ml +
cpd 1 c = 62.5 M
EtBr c = 1 g/ml +
cpd 1 c = 31.25 M
500
0
EtBr c = 1 g/ml +
cpd 1 c = 62.5 M
0
0
10
20
20
20
20
20
0
10
20
Time [min]
Time [min]
c)
dd) )
S. epidermidis K/14/1345
S. epidermidis ATCC 12228
1000
500
1000
500
EtBr c = 1 g/ml
EtBr c = 1 g/ml
EtBr c = 1 g/ml +
cpd 6 c = 62.5 M
EtBr c = 1 g/ml +
cpd 6 c = 62.5 M
EtBr c = 1 g/ml +
cpd 6 c = 125 M
EtBr c = 1 g/ml +
cpd 6 c = 125 M
0
0
0
10
0
10
20
Time [min]
Time [min]
e)
f)
S. epidermidis K/14/1345
S. epidermidis ATCC 12228
1000
500
0
EtBr c = 1 g/ml
EtBr c = 1 g/ml
EtBr c = 1 g/ml +
cpd 7 c= 62.5 M
EtBr c = 1 g/ml +
cpd 7 c= 125 M
1000
500
0
EtBr c = 1 g/ml +
cpd 7 c = 31.25 M
EtBr c = 1 g/ml +
cpd 7 c = 62.5 M
0
10
0
10
20
Time [min]
Time [min]
g)
h)
h)
S. epidermidis K/14/1345
S. epidermidis ATCC 12228
1000
500
1000
500
EtBr c = 1 g/ml
EtBr c = 1 g/ml
EtBr c = 1 g/ml +
cpd 9 c= 7.81 M
EtBr c = 1 g/ml +
cpd 9 c= 15.63 M
EtBr c = 1 g/ml +
cpd 9 c = 15.63 M
EtBr c = 1 g/ml +
cpd 9 c = 31.25 M
0
0
0
10
0
10
20
Time [min]
Time [min]
i)
j)
S. epidermidis K/14/1345
S. epidermidis ATCC 12228
1000
1000
500
0
EtBr c = 1 g/ml
EtBr c = 1 g/ml
EtBr c = 1 g/ml +
cpd 14 c= 31.25 M
EtBr c = 1 g/ml +
cpd 14 c = 15.63 M
500
0
EtBr c = 1 g/ml +
cpd 14 c= 62.5 M
EtBr c = 1 g/ml +
cpd 14 c = 31.25 M
0
10
0
10
20
Time [min]
Time [min]
Figure 2. Influence of compounds
1
(a,
b),
6
(c,d
),
7
(e,
f
),
9
(
g
,
h) and 14
(
i
,
j) tested at the concentrations
corresponding to 1/4 and 1/2 of their MIC values on the accumulation of EtBr (1
µg/mL) in
Msr(A)-overexpressing S. epidermidis strain K/14/1345 and Msr(A)-deficient S. epidermidis strain ATCC
12228. Each data point expresses the mean ± standard deviation (SD) from four replicates.

Molecules 2020, 25, 3788
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S. epidermidis K/14/1345
1500
EtBr c = 1 g/mL
****
EtBr c = 1 g/ml +
cpd c = 1/4 MIC
****
****
****
1000
****
****
**
****
EtBr c = 1 g/ml +
cpd c = 1/2 MIC
**
***
500
0
cpd 7
cpd 1
cpd 6
cpd 9
g/mL) after addition of compounds 1, 6, 7, 9 and 14 at
cpd 14
Figure 3. Fluorescence intensity of EtBr (1
µ
20-min time point in S. epidermidis K/14/1345. Statistical significance was calculated using a one-way
analysis of ANOVA, followed by the multiple comparison test (** p < 0.05, *** p < 0.001, **** p < 0.0001).
Since EPI potency of 5,5-dimethylhydantoin derivatives depended on the concentration of
compounds used in the study, specific activity (SA) of the inhibition of an efflux pump was determined.
The parameter was calculated by dividing the RFI by the number of micromoles of a molecule evaluated
in the accumulation assay [17]. The results of the analysis performed on the EPI effect of the hydantoin
derivatives are given in Table 4. As in the case of RFF and RFI, the higher is the SA of a compound
tested, the greater is the inhibitory effect on a given efflux pump transporter.
Table 4. Specific activity (SA) of active 5,5-dimethylhydantoin derivatives tested at the concentration
corresponding to 1/2 and 1/4 of their MIC values on the accumulation of EtBr in S. epidermidis strain
K/14/1345 and ATCC 12228.
SA
SA
S. epidermidis K/14/1345
RFI/µmol
Compound
S. epidermidis ATCC 12228
RFI/µmol
1/2 MIC 1
1/4 MIC 1
Compound
1/2 MIC
1/4 MIC
1
6
7
9
14
12.22
6.02
22.16
24.82
14.32
13.31
4.59
23.77
25.66
13.04
1
6
7
9
14
1.48
0.87
0.65
4.29
1.9
0.094
0.49
0.23
1.52
−5.27
1
The highest possible concentration that did not inhibit bacterial growth in case of precipitating compounds 1, 6, 9
and 14.
The data obtained show that, among all identified EPIs of Msr(A) efflux transporter, the most
prominent effect was observed for the member of Group A, compound . The compound increased
significantly the accumulation of EtBr in S. epidermidis K/14/1345 overproducing Msr(A) efflux pump
at the lowest concentrations (15.63, 25 and 31.25 M) tested (SA = 24.82). The effectiveness of was
followed by the one of compound (SA = 22.16). After 20 min of incubation, compound , tested at
the concentration of 62.5 M, increased more than two-fold the EtBr accumulation in S. epidermidis
strain K/14/1345. The influence of the compound on the EtBr accumulation at the concentration of
31.25 M was less pronounced, but also significant. Compound 14 was the most active EPI of Msr(A)
found in Group B. The compound showed high Msr(A) inhibitory efficacy at the concentration of both
62.5 and 31.25 M. The strength of the inhibitory effect of and 14 was followed by the activity of
two members of Group A: compounds 1 and 6.
9
µ
9
7
7
µ
µ
µ
7, 9

Molecules 2020, 25, 3788
9 of 18
Remarkably, compounds
1,
6
,
7,
9
and 14 did not influence EtBr accumulation in Msr(A)-deficient
S. epidermidis strain ATCC 12228. Remaining 5,5-dimethylhydantoin derivatives, including 2–5, 8, 10–13
and 15, were devoid of inhibitory activity on the extrusion of the dye from both Msr(A)-overexpressing
and Msr(A)-deficient strains (Figures S1–S20). At the same time, the addition of compound 15 to EtBr
decreased the fluorescence of the dye when tested in both S. epidermidis K/14/1345 and S. epidermidis
ATCC 12228. The effect might be due to molecular interactions between fluorophore and 15 that
resulted in increased quenching of fluorescence. Importantly, additional controls performed indicated
that none of compounds tested shown its own fluorescence and, thus, did not interfere with the EtBr
fluorescence measured in the accumulation assay.
It should be noted that the increase in fluorescence intensity of EtBr in the absence and presence of
compounds tested in S. epidermidis K/14/1345 isolate was relatively slower than that noted for the ATCC
strain (Figure 2 and Figures S1–S20). This observation suggests that the rate of EtBr accumulation was
less pronounced in the strain that overexpresses the Msr(A) efflux pump and agrees with the general
premise that the higher is the level of efflux, the lower is the accumulation of a substrate of an efflux
pump inside the bacterial cell [20].
2.3.3. Influence on the Activity of Erythromycin
The ability of the series of phenylpiperazine 5,5-dimethylhydantoin derivatives (1–15) to enhance
the antibacterial activity of erythromycin against both S. epidermidis K/14/1345 and S. epidermidis ATCC
12228 was investigated. The compounds were tested at the sublethal concentrations of 1/4 their
respective MICs. In each experiment, the control of the efficacy of erythromycin against S. epidermidis
strains in the absence of compounds tested was also performed. Results obtained in the assay show that
none of the phenylpiperazine 5,5-dimethylhydantoins could strengthen the effect of Msr(A) substrate,
erythromycin, against resistant to this antibiotic S. epidermidis K/14/1345 clinical isolate. Similarly, the
compounds did not influence the activity of erythromycin in susceptible S. epidermidis ATCC 12228
(Table S1).
3. Discussion
In the present work, the influence of 15 phenylpiperazine 5,5-dimethylhydantoin derivatives
on Msr(A) efflux pump transporter in S. epidermidis strain K/14/1345 possessing MSB phenotype was
evaluated. The capacity of compounds to reduce the extrusion of EtBr through the inhibition of the
pump was analyzed by the use of the real-time accumulation assay in which 5,5-dimethylhydantoin
derivatives were tested at the doses nontoxic for the bacteria. The results of the assay show that five
derivatives (
1
,
6
,
7
,
9
and 14) significantly increase the accumulation of EtBr in Msr(A)-overexpressing
, containing fluorine
S. epidermidis isolate. The most potent EPI activity was noted for compound
9
atom at positions ortho of the phenylpiperazine moiety connected with hydantoin core by pentyl linker
and 2,4-dichlorobenzyl moiety at hydantoin position 3. This molecule at the concentrations as low as
15.63 and 31.25
K/14/1345 overproducing Msr(A) protein. A similar modulatory effect, but at a higher concentration
tested, was observed for derivative , which possesses two fluorine atoms at positions 2 and 4 of the
phenylpiperazine moiety linked to 5,5-dimethylhydantoin skeleton by pentyl chain. Compound at
the concentration of 62.5 M reduced efflux activity of Msr(A) more than two-fold in S. epidermidis
K/14/1345 strain. Potent EPI activity of was already confirmed by the previous study of Amaral et
µM highly effectively inhibited the extrusion of the fluorescent dye from S. epidermidis
7
7
µ
7
al. where the compound displayed high effectiveness in inhibiting overproduced Qac transporter
present in S. aureus strain HPV-107 and constitutively expressed efflux system of the wild-type S. aureus
strain ATCC 25923 [17]. The fact that
7 has the capacity to interact with different types of efflux
pumps expressed in staphylococci indicates that the compound possesses a multitarget specificity for
these membrane-related transporters. The EPI property towards Msr(A) exporter amongst remaining
phenylpiperazine 5,5-dimethylhydantoin derivatives was shown to decrease as follows: 14
> 1 > 6.

Molecules 2020, 25, 3788
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Remarkably, it has been demonstrated that the identified inhibitors of Msr(A) transporter protein did
not influence the accumulation of EtBr in S. epidermidis strain ATCC 12228.
It is intriguing that the found EPIs structurally differ from each other more significantly than
from some inactive members of the series (1–15). Considering the three modified structural moieties
(Table 1), some structure–activity effects may be noted. For the influence of the phenylpiperazine
moiety substituents, the following EPI-activity order can be seen: 2-F > 2,4-diCl > 2,4-diF > 4-F. On the
other hand, 2,4-diCl-, 4-F- and lack of substituents were beneficial in the case of benzyl moiety at
the hydantoin position 3. Most of the active EPIs contain 5-carbon linker (1, 6, 7 and 9). Although
one exception, i.e., the hydoxypropyl derivative 14, is placed in the exact middle of this range, other
compounds with the hydroxypropyl linker were totally inactive. This can suggest a beneficial role of the
C5-linker, predominant for EPI properties in comparison to either the shorter branched hydroxypropyl
linker (12–15) or the longer one (C6, compound 8).
Despite the relevant EPI activity of five phenylpiperazine 5,5-dimethylhydantoin derivatives
in the EtBr accumulation assay, the compounds tested were not able to increase the effectiveness of
erythromycin against resistant to this drug S. epidermidis strain K/14/1345. The reason for discrepancies
in the behavior of the compounds in the MIC reduction assay and the EtBr accumulation assay might
be explained by the significant differences in the experimental conditions used in the MIC reduction
assay and in the real-time accumulation assay. The latter is carried out in a minimal volume of medium
and over a short period of time, while the test that is based on the determination of the capacity of
molecules to increase the susceptibility of a strain to a given antibiotic is performed in a complete
medium and over 18 h of incubation. Bearing in mind that during the MIC reduction assay bacteria are
exposed to an antibiotic and a compound for an extended period of time, the test provides favorable
conditions for the inducement of genes responsible for efflux pumps overexpression. Ultimately,
an antibiotic or a compound selected for the experiment may promote the production of efflux pump
units leading to the reduction of the efficacy of a potential EPI under investigation [17,21].
The analysis of the obtained results does not give an evident SAR. In general, two aromatic ends
with hydrophobic halogen substituents seem to favor the EPI properties for Msr(A). This remains the
structural traits required for modulators of Pgp that also represents ABC transporter family. Within
the considered series (1–15), there is a “black swan” 15 that does not possess phenylpiperazine, but the
benzhydrylpiperazine moiety. This compound stood out from the rest with its significant antibacterial
activity and may constitute a lead structure for a new separate research direction, which can be
supported by 3D-structure details coming from the X-ray analysis performed within the present study.
4. Experimental
4.1. 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 MEL-TEMP II apparatus (LD Inc., Long Beach, CA, USA) and are
uncorrected. The 1H-NMR and 13C-NMR spectra were obtained on a Mercury-VX 300 Mz spectrometer
1
(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; q def., quarted deformated qui, quintet; m,
multiplet), coupling constant J in Hertz (Hz), number of protons, proton’s position (Pp-Ph, phenyl
connected to piperazine; Ph, phenyl of benzyl fragments; Pp, piperazine). 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

Molecules 2020, 25, 3788
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ethyl hybrid) C18 column, 2.1
×
100 mm, and 1.7
µ
m particle size, equipped with Acquity UPLC BEH
C18 VanGuard precolumn (Waters Corporation, Milford, MA, USA), 2.1 × 5 mm, and 1.7 µm 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
B: acetonitrile/formic acid (0.1%, v/v). Chromatograms were made using Waters e
·
min−1. Eluent A: water/formic acid (0.1%, v/v); eluent
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
·
h
−1, cone gas flow 100 L −1, capillary
h
·
potential 3.00 kV and 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 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 (%). IR spectra (for compounds 9–11 to
confirm their hydrochloride forms at the absence of NH+ proton peak in 1H-NMR) were recorded on a
Jasco FT/IR-410 apparatus using KBr pellets and are reported in cm−1. Syntheses under microwave
irradiation were performed in household microwave oven Samsung MW71B. Synthesis of compounds
1–7, 16–17, 19–21 and 23 was described earlier [18].
4.1.1. 3-(4-Chlorobenzyl)-5,5-dimethylimidazolidine-2,4-dione (18)
5,5-Dimethylhydantoin (120 mmol, 15.37 g), potassium carbonate (34.73 mmol, 48.0 g),
TEBA (15.8 mmol, 3.6 g) and acetone (480 mL) were heated in boiling point for 20 min. Then,
1-chloro-4-(chloromethyl)benzene (120 mmol, 19.32 g) dissolved in acetone (80 mL) was added.
Mixture was heated in boiling point for 4 h and then mixing for 20 h. Filtration was performed and
distilled water was added to filtrate. Product appeared as solid and another filtration was performed.
◦
White solid. Yield 67.0%; mp 108–110 C. C12H13ClN2O2 MW 252.07. LC/MS
±
: (ESI) m/z [M+H]
1
253.25. H-NMR
δ
(ppm): 1.28 (s, 6H, 2
×
5-CH3), 4.50 (s, 2H, N3-CH2), 7.22–7.25 (d def., 2H, Ph-2,6-H),
7.37–7.40 (d def., 2H, Ph-3,5-H), 8.38 (s, 1H, N1-H).
4.1.2. 3-(4-Chlorobenzyl)-1-(6-bromohexyll)-5,5-dimethylimidazolidine-2,4-dione (22)
3-(4-Chlorobenzyl)-5,5-dimethylimidazolidine-2,4-dione (18) (40 mmol, 10.1 g), potassium
carbonate (115.77 mmol, 16.0 g), TEBA (5.27 mmol, 1.2 g) and acetone (80 mL) was heated for
30 min. Then, 1,6-dibromohexane (60 mmol, 14.63 g) dissolved in acetone (40 mL) was added. It was
mixed for 144 h. Filtration was performed. Filtrate was evaporated, dichloromethane was added
and mixture was extracted three times with 1% sodium hydroxide and twice with distilled water.
Organic fraction was dried under sodium sulphate anhydrous, then filtrated and evaporated. To obtain
pure product, column chromatography was performed in dichloromethane:acetone/10:1. Colourless
oil. Yield 25.0%. C18H24BrClN2O2 MW 414.07. LC/MS
(ppm): 1.22–1.25 (t def., 1H, N3-CH2-CH2-CH2-CH2a), 1.28 (t def., 1H, N3-CH2-CH2-CH2-CH2b), 1.31
(s, 6H, 2 5-CH3), 1.37–1.41 (m, 2H, N3-CH2-CH2-CH2), 1.49–1.59 (m, 2H, N3-CH2-CH2), 1.73–1.82
±
: (ESI) m/z [M + H] 415.03. 1H-NMR
δ
×
(m, 2H, N3-CH2-CH2-CH2-CH2-CH2), 3.19–3.24 (t, J = 7.6 Hz, 2H, N1-CH2), 3.48–3.53 (t def., 2H,
N3-CH2-CH2-CH2-CH2-CH2-CH2), 4.52 (s, 2H, N3-CH2), 7.22–7.24 (d def., 2H, Ph-2,6-H), 7.37–7.40
(d def., 2H, Ph-3,5-H).
4.1.3. 3-(2,4-Dichlorobenzyl)-5,5-dimethyl-1-(oxiran-2-ylmethyl)imidazolidine-2,4-dione (24)
3-(2,4-Dichlorobenzyl)-5,5-dimethylimidazolidine-2,4-dione (19) (100 mmol, 28.71 g), potassium
carbonate (289.4 mmol, 40.0 g), TEBA (13.17 mmol, 3.0 g) and acetone (200 mL) were heated for
30 min. Then, 2-(chloromethyl)oxirane (110 mmol, 10.18 g) dissolved and acetone (96 mL) was added.
Mixture was mixing in room temperature for 120 h. Filtration was performed. Filtrate was evaporated.
To obtain pure product, column chromatography was performed in dichloromethane:acetone/10:1.
Colourless oil. Yield 39.0%. C15H16Cl2N2O3 MW 342.05. LC/MS : purity 93.35% tR = 6.60, (ESI) m/z
±

Molecules 2020, 25, 3788
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1
[M + H] 344.21. H-NMR
δ
(ppm): 1.34–1.40 (s, 6H, 2
×
5-CH3), 2.61–2.64 (m, 1H, N1-CH2-CH-CH2),
2.75–2.78 (m, 1H, N1-CH2-CH-CH2), 3.04–3.09 (m, 1H, N1-CH2-CH), 3.17–3.24 (m, 1H, N1-CH2a),
3.61–3.68 (m, 1H, N1-CH2b), 4.62 (s, 2H, N3-CH2), 7.17–7.20 (d def., 1H, Ph-6-H), 7.38–7.42 (d def., 1H,
Benz-5-H), 7.62–7.63 (d def., 1H, Ph-3-H).
4.1.4. General Procedure to Obtain Final Products (8–11)
1-Phenylpiperazine derivatives (3–4 mmol), potassium carbonate (10–13 mmol, 1.35–1.80 g),
acetone (11–15 mL) and 3-((di)chlorobenzyl)-1-bromoalkyl-5,5-dimethylimidazolidine-2,4-dione
derivatives (22–23) (4.8–5.8 mmol) dissolved in acetone (10–12.5 mL) were heated in boiling point for
4–11 h. Filtration of inorganic compound was performed. Filtrate was evaporated, dichloromethane
was added and mixture was extracted 2–3 times with 1% hydrochloric acid (20 mL). Organic fraction
was dried under sodium sulphate anhydrous, then filtrated and evaporated. For compounds 9–11,
crystallization using methanol was performed. Solid products were obtained with Method A or B.
Method A: The pure product precipitated after saturation with gaseous hydrochloric acid. When Method
A did not provide a precipitate of an expected hydrochloric salt, Method B was further involved,
according to the following procedure: the solution was evaporated, a residue dissolved in anhydrous
ethanol. The cold diethyl ether (0 ◦C) was added until a precipitation appeared, followed by mixing in
a cork flask for 30 min. The mixture was cooled at <
10 ◦C for 48–168 h, filtered off and the precipitate
−
washed with 2–5 mL of cold ether or acetone to give the pure product of a desirable arylpiperazine
hydantoin derivative.
3-(4-Chlorobenzyl)-1-(6-(4-(2-fluorophenyl)piperazin-1-yl)hexyl)-5,5-dimethylimidazolidine-2,4-dione
hydrochloride (8)
1-(2-Fluorophenyl)piperazine (3 mmol, 0.54 g) and the 6-bromohexyl derivative 22 (5.8 mmol,
2.43 g) were used. Product was obtained with Method B. White solid. Yield 5.5%; mp 172–174 ◦C.
1
C28H37Cl2FN4O2 MW 550.23. LC/MS
±
: purity 95.44% tR = 5.92, (ESI) m/z [M + H] 515.56. H-NMR
δ
(ppm): 1.30–1.35 (m, 4H,N1-CH2-CH2-CH2-CH2), 1.32 (s, 6H, 2
×
5-CH3,), 1.50–1.65 (d def.,
2H, N1-CH2–CH2-CH2-CH2-CH2), 1.72 (s, 2H, N1-CH2-CH2), 3.03–3.25 (m, 8H, Pp-CH2, Pp-2,6-H,
N1-CH2), 3.43–3.53 (m, 4H, Pp-3,5-H), 4.53 (s, 2H, N3-CH2), 7.01–7.17 (m, 4H, Pp-Ph-3,4,5,6-H),
7.22–7.24 (d def., 2H, Ph-2,6-H), 7.37–7.40 (d def., 2H, Ph-3,5-H), 10.91 (br. s, 1H, NH+).
3-(2,4-Dichlorobenzyl)-1-(5-(4-(2-fluorophenyl)piperazin-1-yl)pentyl)-5,5-dimethylimidazolidine-2,4-
dione hydrochloride (9)
1-(2-Fluorophenyl)piperazine (4 mmol, 0.72 g) and the 5-bromopentyl derivative 23 (4.8 mmol,
2.00 g) were used. Product was obtained with Method B. White solid. Yield 6.0%; mp 172–174 ◦C.
1
C27H34Cl3FN4O2 MW 570.17. LC/MS
±
: purity 98.68% tR = 6.09, (ESI) m/z [M + H] 535.14. H-NMR
δ
(ppm): 1.22 (s, 2H, Pp-CH2-CH2-CH2), 1.37 (s, 6H, 2
×
5-CH3), 1.60–1.73 (m, 4H, Pp-CH2-CH2,
N1-CH2-CH2), 3.06–3.14 (m, 6H, Pp-CH2, Pp-2,6-H), 3.24–3.29 (m, 2H, N1-CH2), 3.43–3.58 (m, 4H,
Pp-3,5-H), 4.60 (s, 2H, N3-CH2), 7.02–7.17 (m, 5H, Pp-Ph-3,4,6-H, Ph-5,6-H), 7.39–7.43 (m, 1H,
Pp-Ph-5-H), 7.63–7.64 (d def., 1H, Ph-3-H). IR (KBr) (cm−1): 3449 (NH), 3047 (CH Ar), 2933 (CH),
2358 (NH+), 1764.24 (C2=O), 1704 (C4=O), 1448 (C=C Ar).
3-(2,4-Dichlorobenzyl)-1-(5-(4-(4-fluorophenyl)piperazin-1-yl)pentyl)-5,5-dimethylimidazolidine-2,4-
dione hydrochloride (10)
1-(4-Fluorophenyl)piperazine (4 mmol, 0.72 g) and the 5-bromopentyl derivative 23 (4.8 mmol,
2.00 g) were used. Product was obtained with Method A. White solid. Yield 42.0%; mp 187–189 ◦C.
1
C27H34Cl3FN4O2 MW 570.17. LC/MS
±
: purity 96.22% tR = 6.06, (ESI) m/z [M + H] 535.14. H-NMR
δ
(ppm): 1.22 (s, 2H, Pp-CH2-CH2-CH2), 1.36 (s, 6H, 2
×
5-CH3), 1.60–1.73 (m, 4H, Pp-CH2-CH2,
N1-CH2-CH2), 3.02–3.10 (m, 6H, Pp-CH2, Pp-2,6-H), 3.24–3.29 (m, 2H, N1-CH2), 3.51–3.72 (dd def.,
4H, Pp-3,5-H), 4.60 (s, 2H, N3-CH2), 6.98–7.11 (m, 4H, Pp-Ph-2,3,5,6-H), 7.15–7.19 (d def., 1H, Ph-5-H),

Molecules 2020, 25, 3788
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7.39–7.43 (m, 1H, Ph-5-H), 7.63–7.64 (d def., 1H, Ph-3-H). IR (KBr) (cm−1): 3464 (NH), 3057 (CH Ar),
2982 (CH), 2444 (NH+), 1768 (C2=O), 1706 (C4=O), 1515 (C=C Ar).
3-(2,4-Dichlorobenzyl)-1-(5-(4-(2,4-difluorophenyl)piperazin-1-yl)pentyl)-5,5-dimethylimidazolidine-
2,4-dione hydrochloride (11)
1-(2,4-Difluorophenyl)piperazine (4 mmol, 0.79 g) and the 5-bromopentyl derivative 23 (5 mmol,
2.18 g) were used. Product was obtained with Method A. White solid. Yield 41.0%; mp 158–160 ◦C.
1
C27H33Cl3F2N4O2 MW 588.16. LC/MS
±
: purity 98.38% tR = 6.08, (ESI) m/z [M + H] 553.14. H-NMR
δ
(ppm): 1.22 (s, 2H, Pp-CH2-CH2-CH2), 1.37 (s, 6H, 2
×
5-CH3), 1.57–1.72 (m, 4H, Pp-CH2-CH2,
N1-CH2-CH2), 3.07–3.13 (m, 6H, Pp-CH2, Pp-2,6-H), 3.24–3.29 (t def., 2H, N1-CH2), 3.37–3.55 (m, 4H,
Pp-3,5-H), 4.60 (s, 2H, N3-CH2), 7.00–7.05 (m, 1H, Pp-Ph-6-H), 7.15–7.17 (d def., 2H, Ph-6-H, Pp-Ph-5-H),
7.21–7.29 (m, 1H, Pp-Ph-3-H), 7.39–7.43 (m, 1H, Ph-5-H), 7.63–7.64 (d def., 1H, Ph-3-H). IR (KBr) (cm−1):
3463 (NH), 3050 (CH Ar), 2987 (CH), 2359 (NH+), 1768(C2=O), 1705(C4=O), 1447 (C=C Ar).
4.1.5. General Procedure to Obtain Final Products (12–15)
Piperazine derivatives (3 mmol) and the 1-oxiran-2-ylmethyl derivative 24 (3 mmol) were
dissolved in acetone (15–20 mL). Mixture was evaporated and the solvent-free residue was irradiated
in household microwave oven (300–450 W) for 3–7 min, under TLC control. The resulted glassy
mass was dissolved in anhydrous ethanol and saturated with gaseous HCl to give a pure product in
the hydrochloride form (Method A). When Method A did not provide a precipitate of an expected
hydrochloric salt, the following procedure was further involved (Method B): the solution (from Method
A) was evaporated, a residue dissolved in anhydrous ethanol. The cold diethyl ether (0 ◦C) was added
until a precipitation appeared, followed by mixing in a cork flask for 30 min. The mixture was cooled
at <
10 ◦C for 48–168 h, filtered off and the precipitate washed with 2–5 mL of cold ether or acetone to
−
give the desirable product.
3-(2,4-Dichlorobenzyl)-1-(3-(4-(2-fluorophenyl)piperazin-1-yl)-2-hydroxypropyl)-5,5-
dimethylimidazolidine-2,4-dione hydrochloride (12)
1-(2-Fluorophenyl)piperazine (3 mmol, 0.54 g) and the 1-oxiran-2-ylmethyl derivative 24 (3 mmol,
1.03 g) were used. Parameters of electromagnetic radiation: 300 W for 2 min., 450 W for 1 min.
and 450 W for 1 min. Product was obtained with Method B. White solid. Yield 61.0%; mp 99–101 ◦C.
1
C25H30Cl3FN4O3 MW 558.14. LC/MS
(ppm): 1.40 (s, 6H, 2
N1-CH2, Pp-3,5-H2b), 3.67–3.69 (d def., 1H, C
±
: purity 98.76% tR = 5.70, (ESI) m/z [M + H] 524.43. H-NMR
δ
×
5-CH3), 3.06–3.39 (m, 9H, Pp-3,5-HaH1b, Pp-2,6-H, Pp-CH2), 3.44–3.55 (m, 3H,
-OH), 4.36–4.38 (d, J = 7.7 Hz, 1H, OH), 4.61 (s, 2H,
H
N3-CH2), 6.99–7.25 (m, 5H, Pp-Ph-3,4,5,6-H, Ph-6-H), 7.39–7.42 (m, 1H, Ph-5-H), 7.62–7.63 (d def., 1H,
Ph-3-H), 10.69 (br. s, 1H, NH+).
3-(2,4-Dichlorobenzyl)-1-(3-(4-(4-fluorophenyl)piperazin-1-yl)-2-hydroxypropyl)-5,5-
dimethylimidazolidine-2,4-dione hydrochloride (13)
1-(4-Fluorophenyl)piperazine (3 mmol, 0.54 g) and the 1-oxiran-2-ylmethyl derivative 24 (3 mmol,
1.03 g) were used. Parameters of electromagnetic radiation: 300 W for 3 min. Product was obtained
with Method B. White solid. Yield 90.0%; mp 186–188 ◦C. C25H30Cl3FN4O3 MW 558.14. LC/MS
±
:
1
purity 100.0% tR = 5.57, (ESI) m/z [M + H] 524.43. H-NMR
δ
(ppm): 1.40 (s, 6H, 2
×
5-CH3), 3.08–3.34
(m, 10 H, Pp-2,3,5,6-H, Pp-CH2), 3.68 (s, 3H, CH-OH, N1-CH2), 4.36 (s, 1H, OH), 4.61 (s, 2H, N3-CH2),
6.97–7.11 (m, 4H, Pp-Ph-2,3,5,6-H), 7.22–7.25 (d, 1H, Ph-6-H), 7.39–7.42 (m, 1H, Ph-5-H), 7.62–7.63 (d, J
= 1.8 Hz, 1H, Ph-3-H), 10.62 (br. s, 1H, NH+).

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3-(2,4-Dichlorobenzyl)-1-(3-(4-(2,4-difluorophenyl)piperazin-1-yl)-2-hydroxypropyl)-5,5-
dimethylimidazolidine-2,4-dione hydrochloride (14)
1-(2,4-Difluorophenyl)piperazine (3 mmol, 0.59 g) and the 1-oxiran-2-ylmethyl derivative 24
(3 mmol, 1.03 g) were used. Parameters of electromagnetic radiation: ◦300 W for 3 min. Product
was obtained with Method B. White solid. Yield 37.0%; mp 179–181 C. C25H29Cl3F2N4O3 MW
1
576.13. LC/MS
±
: purity 98.42% tR = 5.63, (ESI) m/z [M + H] 542.42. H-NMR
δ
(ppm): 1.40 (s, 6H, 2
×
5-CH3), 3.09–3.24 (m, 6H, Pp-2,6-H, Pp-CH2), 3.34–3.38 (m, 4H, Pp-3,5-H), 3.51–3.65 (m, 3H, CH-OH,
N1-CH2), 4.34 (s, 1H, OH), 4.62 (s, 2H, N3-CH2), 6.98–7.20 (m, 2H, Pp-Ph-3,5-H), 7.22–7.27 (d, 2H,
Ph-6-H, Pp-Ph-6-H), 7.39–7.42 (m, 1H, Ph-5-H), 7.62–7.63 (d, J = 2.0 Hz, 1H, Ph-3-H), 10.52 (br. s,
1H, NH+).
3-(2,4-Dichlorobenzyl)-1-(3-(4-benzhydrylpiperazin-1-yl)-2-hydroxypropyl)-5,5-dimethylimidazolidine
-2,4-dione hydrochloride (15)
1-(Diphenylmethyl)piperazine (3 mmol, 0.882 g) and the 1-oxiran-2-ylmethyl derivative 24
(3 mmol, 1.03 g) were used. Parameters of electromagnetic radiation: 450 W for 2 min, then 300 W
for 1 min and 450 W for 4 min. Product was obtained with Method A. White solid. Yield 74.0%;
mp 147–149 ◦C. C32H37Cl3N4O3 MW 630.19. LC/MS
±
: purity 100.0% tR = 6.47, (ESI) m/z [M + H]
1
596.56. H-NMR
δ
(ppm): 1.38 (s, 6H, 2
×
5-CH3), 2.37–2.45 (m, 2H, Pp-CH2), 2.76–2.80 (d def., 2H,
-OH, N1-CH2), 4.27 (br. s, 1H, OH), 4.43 (s,
Pp-2,6-Ha), 2.96–3.53 (m, 9H, Pp-2,6-Hb, Pp-3,5-H, C
H
1H, CH(Ph)2), 4.60 (s, 2H, N3-CH2), 7.17–7.24 (m, 3H, Pp-Ph-5,50-H, Ph-6-H), 7.27–7.32 (t def., 4H,
Pp-Ph-3,4,30,40-H)), 7.38–7.43 (m, 5H, Pp-Ph-2,6,20,60-H, Ph-5-H), 7.62–7.63 (d, J = 2.0 Hz, 1H, Ph-3-H),
10.33 (br. s, 1H, NH+).
4.2. Crystallographic Studies
Crystals suitable for an X-ray structure analysis were grown from butyl acetate for 6 and acetonitryl
for 15, by slow evaporation of the solvent at room temperature.
Data for single crystals of
diffractometer equipped with a Mo (0.71069 Å) K
were collected at 130 K using the Oxford Diffraction SuperNova four circle diffractometer equipped
with the Mo (0.71073 Å) K radiation source, graphite monochromator. Positions of all non-hydrogen
atoms were determined by direct methods using SHELXS-97 [22] for and SIR-2014 for 15 [23]
6
were collected at 100 K on the Bruker-Nonius Kappa CCD four circle
α
radiation source, while for single crystals of 15
α
6
programs. Refinement and further calculations were carried out using SHELXL-2018 program [24].
All non-hydrogen atoms were refined anisotropically using weighted full-matrix least-squares on F2.
The hydrogen atoms attached to nitrogen and oxygen atoms were identified on difference Fourier
maps, whereas all hydrogen atoms bonded to carbon atoms 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. For molecular graphics, MERCURY [25] program was used.
Compound
6
: C27H35F2 O2N4+ Cl−, Mr = 521.04, crystal size = 0.24
×
0.26
×
0.38 mm3, monoclinic,
space group P21/c, a = 13.5070(3) Å, b = 8.1930(2) Å, c = 26.3923(6) Å,
β
= 113.387(2)◦, V = 2680.7(1) Å3,
Z = 4, T = 100(2) K, 13924 reflections collected, 5882 unique reflections (Rint = 0.0251), R1 = 0.0376,
wR2 = 0.0877 [I > 2σ(I)] and R1 = 0.0475, wR2 = 0.0940 [all data].
Compound 15: C32H37Cl2O3N4+ Cl−, Mr = 632.00, crystal size = 0.07
×
0.24
×
0.41 mm3, monoclinic,
space group P21/c, a = 7.3343(2) Å, b = 16.1643(3) Å, c = 28.7674(6) Å,
β
= 110.934(2)◦, V = 3185.4(1) Å3,
Z = 4, T = 130(2) K, 42439 reflections collected, 6171 unique reflections (Rint = 0.0795), R1 = 0.0447,
wR2 = 0.1152 [I > 2σ(I)] and R1 = 0.0558, wR2 = 0.1262 [all data].
CCDC 2015564-2015565 contain the supplementary crystallographic data. These data can be
obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.

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4.3. Microbiological Studies
S. epidermidis K/14/1345 was isolated from the blood of a patient admitted to University Children’s
Hospital in Krakow, Prokocim. S. epidermidis ATCC 12228 was purchased from GRASO Biotech
(Poland). Bacteria were maintained and grown on Trypticase Soy agar supplemented with 5% sheep
blood (TSA II; Becton Dickinson, Germany). Cation-adjusted Mueller-Hinton (MH II) broth and Tryptic
Soy broth (TSB), used in susceptibility testing and EtBr accumulation assays, were obtained from Difco,
Becton Dickinson (Spain) and bioMérieux (France), respectively. The culture media were prepared by
dissolving appropriate dry culture media powders in distilled water. The liquid media obtained were
sterilized by autoclaving at 121 ◦C for 15 min. Compounds tested and erythromycin (Sigma-Aldrich
Chemie GmbH, Darmstadt, Germany) used in microbiological assays were dissolved in dimethyl
sulfoxide (DMSO; Merck, Germany) and 70% ethanol, respectively. EtBr, which was used to evaluate
the efflux pump inhibitory activity of compounds tested, was obtained from Sigma-Aldrich Chemie
GmbH (Munich, Germany). Stock solution of the dye at 2 µg/mL was freshly prepared in phosphate
buffered saline (PBS) with 0.4% glucose (Sigma-Aldrich Quimica, Madrid, Spain) before use. PBS in
tablets (0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4)
was dissolved in distilled, sterile water and autoclaved. Glucose (Chempur, Poland) was dissolved in
distilled, sterile water and stored at 4 ◦C.
4.3.1. Susceptibility Testing
Susceptibility testing was carried out according to the standard microdilution method in MH II
broth following the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) [26].
MIC values were detected through the measurement of optical density at 600 nm in Tecan Sunrise reader.
Initially, minimal inhibitory concentration (MIC) values of 15 phenylpiperazine 5,5-dimethylhydantoin
derivatives were determined against two strains of S. epidermidis, the reference strain ATCC 12228
and the clinical isolate K/14/1345 whose overexpression of Msr(A) transporter is associated with
MSB phenotype. Next, the adjuvant-like effect of 5,5-dimethylhydantoins was evaluated by the
determination of erythromycin MICs in the absence and in the presence of compounds tested.
The concentrations of compounds employed in the MIC reduction assay were not greater than 1/2
of their MIC values, therefore, cell viability was not influenced by the intrinsic antibacterial activity
of these compounds. The final concentration of DMSO in broth medium never exceeded 2.5% (v/v)
and did not affect bacterial viability, which was confirmed by conducting additional assays. The MIC
values of 5,5-dimethylhydantoins and erythromycin were recorded as the lowest concentrations of
compounds/antibiotic at which no growth of bacteria was detected (OD600 < 0.07) after 18-h incubation
at 37 ◦C [26]. The results obtained in the MIC reduction assay were expressed by employing activity
gain parameter (A) calculated as the MIC of antibiotic tested alone to its MIC tested in combination
with compound examined (Equation (1)) [21
,
27]. All MIC evaluations were performed in at least two
independent experiments.
!
MICant
MICant + MICcpd
A =
(1)
4.3.2. Ethidium Bromide Accumulation Assay
The methodology is commonly used for the expedite detection and qualification of the efflux
capabilities in bacteria as well as for the search of new efflux pump inhibitors that could overcome
bacterial drug resistance. It enables direct detection of EtBr accumulation inside bacterial cells on a
real-time basis following the monitoring of the fluorescence of the dye over a period of time [28,29].
EtBr has been shown to be particularly suitable for this method since it fluoresces weakly in aqueous
solution (extracellularly) and becomes strongly fluorescent in the hydrophobic environment, especially
when it penetrates the bacterial cell wall and binds to cellular components such as DNA. Consequently,
the lower is the level of efflux of EtBr, the higher is the fluorescence of this dye within bacterial

Molecules 2020, 25, 3788
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cells [20,30]. The EtBr accumulation assay is performed under conditions that promote efflux, i.e.,
the presence of a source of energy (glucose) and the temperature of 37 ◦C. The results obtained in the
experiment are illustrated by the comparison of accumulation of EtBr in the absence and presence of
compounds tested in efflux pump overexpressing strain. The changes in the fluorescence of EtBr are
measured at excitation and emission wavelengths of 530 and 585 nm, respectively [29,30]. Prior to the
main experiment, MICs of EtBr against S. epidermidis strain K/14/1345 which overexpresses Msr(A)
efflux pump as well as in the reference strain S. epidermidis ATCC 12228 were evaluated according
to the protocol described above. Moreover, the optimal concentration of EtBr that was within the
capability of the bacteria to extrude from the cytoplasm was established. For the main accumulation
assay in which the EPI properties of compounds were determined, bacteria were grown in TSB at 37 ◦C
with shaking at 150 rpm until they reached an OD600 of 0.6. Bacterial cultures were then centrifuged
at 12,500 g for 3 min, the supernatant was discarded and the remaining pellet washed twice in
×
PBS. Thereafter, the pellet was resuspended in PBS pH 7.4 containing 0.4% glucose and the resulting
bacterial suspension was adjusted to OD600 = 0.6. Forty-five microliters of bacterial suspension were
treated with 1
The level of EtBr accumulation was established using a microplate reader (EnSpire, PerkinElmer,
Waltham, MA, USA), with an excitation wavelength of ex = 530 nm and an emission wavelength of
em = 585 nm at a constant temperature of 37 ◦C for 30 min. The modulatory activity of compounds
µg/mL EtBr and compounds tested at the concentration of 1/4 and 1/2 of their MICs.
λ
λ
on the efflux pump of interest was evaluated in at least three independent experiments (biological
replicates). The results were presented on graphs using GraphPad Prism 5.0 (GraphPad Software
Inc., San Diego, CA, USA). Each value of the experimental accumulation curve was corrected by
subtracting the fluorescence of the background corresponding to the initial EtBr concentration in PBS.
To compare the inhibitory activity of the potent EPIs against overexpressed Msr(A) exporter, the relative
fluorescence index (RFI) and the specific activity (SA) of compounds on the accumulation of EtBr
by S. epidermidis K/14/1345 clinical isolate were calculated. The modulatory effect of the compounds
towards transporter protein in S. epidermidis K/14/1345 was compared with the one determined in the
reference S. epidermidis strain ATCC 12228. The RFI parameter was calculated using the relative final
fluorescence (RFF) of the last time point (minute 20) of the EtBr accumulation curve according to the
following Equations (2) and (3) [17]:
RFF = RFtreated − RFuntreated
(2)
(3)
RFF
RFI =
RFuntreated
where RFtreated corresponds to the relative fluorescence at the last time point of the EtBr retention curve
in the presence of a compound tested and RFuntreated is the relative fluorescence at the last time point
of the EtBr retention curve in the absence of a compound tested.
Dividing RFI value by the number of micromoles of 5,5-dimethylhydantoin derivative used in
the assay provided a specific activity of inhibition of the Msr(A) efflux pump present in S. epidermidis
bacterium (Equation (4)) [17].
RFI
SA =
(4)
n [µmol]
5. Conclusions
Herein, we identified first synthetic compounds able to inhibit Msr(A) efflux pump transporter
present in S. epidermidis bacterium. Design, chemical synthesis, comprehensive microbiological
assays and X-ray supported deeper insight into structural properties of two compounds with
different activity. As results, five compounds with significant potency to inhibit the dye-substrate
of the ABC transporter, Msr(A), were found. This finding may contribute to form the basis
for further search for potent EPIs among hydantoin derivatives in order to enhance their
inhibitory activity towards Msr(A) efflux pump system. The most active EPI identified in

Molecules 2020, 25, 3788
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this study, i.e., 3-(2,4-dichlorobenzyl)-1-(5-(4-(2-fluorophenyl)piperazin-1-yl)pentyl) derivative of
5,5-dimethylhydantoin ( ), seems to be a good lead structure for further pharmacomodulations. In a
9
therapeutic context, this could open new perspectives for rational design and synthesis of antimicrobial
resistance breakers that are able to restore the activity of macrolide antibiotics against S. epidermidis
strains, in the case of the membrane-associated resistance mechanisms.
Supplementary Materials: The following are available online. Table S1: Influence of phenylpiperazine
5,5-dimethylhydantoin derivatives on the susceptibility of S. epidermidis strains to erythromycin, Figures S1a–S20a:
Influence of different compounds on accumulation of EtBr in S. epidermidis K/14/1345 and S. epidermidis ATCC
12228. Figures S1b–S20b: Fluorescence intensity at last time point of the EtBr retention curve in the presence of
different compounds.
Author Contributions: K.W. optimized EtBr accumulation assay in S. epidermidis K/14/1345 and S. epidermidis
˙
ATCC 12228; K.W., J.C., and A.C. performed susceptibility testing and EtBr accumulation assays; E.Z. and W.N.
performed crystallographic studies, J.H. designed the compounds and supervised the syntheses; G.L., K.K.-K.,
and E.K. supervised microbiological assays; K.W., A.K. and J.H. wrote the MS; and J.H. performed the tasks of
corresponding author. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Jagiellonian University Medical College grant numbers N42/DBS/000027
and N42/DBS/000165.
Acknowledgments: The authors thank master students Małgorzata Pycin´ska, Aleksandra Janik, and Maria Glomb
for their participation in the synthesis work. Syntheses were partly performed within Student Scientific Group of
Medicinal Chemistry (Studenckie Koło Chemii Medycznej UJ CM).
Conflicts of Interest: The authors declare no conflict of interest.
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©
2020 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/).