Search for new tools to combat Gram-negative resistant bacteria among amine derivatives of 5-arylidenehydantoin

Article abstract of DOI:10.1016/j.bmc.2012.10.053

A series of amine-alkyl derivatives of 5-arylidenehydantoin 3-21 was evaluated for their ability to improve antibiotic effectiveness in two strains of Gram-negative Enterobacter aerogenes: the reference strain (ATCC-13048) and the chloramphenicol-resistan

Full text of DOI:10.1016/j.bmc.2012.10.053

Bioorganic & Medicinal Chemistry 21 (2013) 135–145
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Search for new tools to combat Gram-negative resistant bacteria among
amine derivatives of 5-arylidenehydantoin
Jadwiga Handzlik a,c, , Ewa Szymanska , Sandrine Alibert b,c, Jacqueline Chevalier b, Ewa Otre˛bska a,c
,
⇑
a
´
a
b,c
a,c
_
´
Elzbieta Pe˛kala , Jean-Marie Pagès , Katarzyna Kiec-Kononowicz
a Department of Technology and Biotechnology of Drugs Jagiellonian University Medical College, Medyczna 9, PL 30-688 Kraków, Poland
b UMR-MD1, Transporteurs Membranaires, Chimiorésistance et Drug Design, Aix-Marseille Université/IRBA, Facultés de Médecine et de Pharmacie,
27 Bd Jean Moulin, 13385 Marseille cedex 05, France
c COST Action BM0701 (ATENS), Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of amine-alkyl derivatives of 5-arylidenehydantoin 3–21 was evaluated for their ability to
improve antibiotic effectiveness in two strains of Gram-negative Enterobacter aerogenes: the reference
strain (ATCC-13048) and the chloramphenicol-resistant derivative over-producing the AcrAB-TolC efflux
pump (CM-64). Three antibiotics, chloramphenicol, nalidixic acid and sparfloxacin were used as markers
of efflux pump activity. New compounds (5–16) were obtained within 3–4 step synthesis using
Knoevenagel condensation, Mitsunobu reaction and microwave aided N-alkylation. Molecular modeling
based structure–activity relationship (SAR) studies were performed. The most active compounds:
(Z)-5-(4-(diethylamino)benzylidene)-3-(2-hydroxy-3-(4-(2-hydroxyethyl)piperazin-1-yl)propyl)imidaz-
olidine-2,4-dione (14) and (Z)-5-(2,4-dimethoxybenzylidene)-3-(2-hydroxy-3-(isopropylamino)pro-
pyl)imidazolidine-2,4-dione (15) induced fourfold decrease of minimal inhibition concentration (MIC)
of all tested antibiotics in the strain CM-64 overexpressing the AcrAB-TolC pump.
Received 28 May 2012
Revised 30 October 2012
Accepted 31 October 2012
Available online 15 November 2012
Keywords:
Arylideneimidazolidine-2,4-dione
Arylidenehydantoin
Enterobacter aerogenes
Multidrug resistance
Efflux pump
AcrAB-TolC
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
an appropriate intracellular concentration of antibiotics are ur-
gently needed.3,5 To this aim, different ways have been proposed:
Multidrug resistance (MDR) is a serious problem in therapy of
bacterial-,1–9 fungal-10 and cancer diseases.11–13 In each case, one
of the main mechanisms of MDR involves efflux pumps, for exam-
ple, membrane transport proteins which are able to expel drugs or
antibiotics before they can reach their intracellular targets. The
MDR efflux pumps belong to transporter families, including two
main superfamilies: major facilitator superfamily (MFS) and the
ATP-binding cassette (ABC) superfamily8 playing an important role
in cancer MDR.11–13 Furthermore, two sub-classes of efflux trans-
port proteins are described: the small multidrug resistance family
(SMR) and the resistance/nodulation/division family (RND).7
The RND transporters are particularly widespread among Gram-
negative bacteria as it is illustrated by the tripartite efflux system
AcrAB-TolC in Escherichia coli or MexAB-OprM in Pseudomonas
aeruginosa (the active pump component being AcrB or MexB,
respectively).3,8
(i) inhibiting the activity of efflux pump by selective competition,
(ii) blocking the membrane channel by original plugs or (iii) col-
lapsing the energy driving force used by efflux mechanism.3,5 Dur-
ing last decade, new compounds, so called efflux pump inhibitors/
modulators (EPIs), have been described for structural-activity stud-
ies on MDR mechanism at the molecular level and for their capac-
ity to improve the activity of antibiotics and chemotherapeutics.3,5
During the search for new tools to combat MDR among bacterial
pathogens, a special attention is paid to Gram-negative bacteria
due to their double-membrane cells and the complex membrane
barrier for antibiotic penetration corresponding to outer mem-
brane structure. Among resistant Gram-negative bacteria, Entero-
bacter aerogenes is frequently described. This bacterium,
responsible for hospital-acquired respiratory tract infections,
exhibits a significant decrease of antibiotic susceptibility.2 Studies
on MDR mechanism in E. aerogenes indicated that tripartite efflux
pump AcrAB-TolC is a major MDR system in clinical isolates.
Recent lines of evidence3–6 have described several groups of
chemical compounds with EPI-properties in Gram-negative bacte-
ria, including peptidomimetics, arylpiperazines, quinolines or pyr-
idopyrimidines (Fig. 1a). For most of the compounds, however, a
therapeutic usage as ‘adjuvant’ is rather impossible because of
their toxicity and possible adverse side effects. Thus, a search for
With the continuous increase of reports describing MDR bacte-
ria and the involvement of efflux pumps in clinical resistant iso-
lates, strategies to annihilate the pump mechanism to preserve
⇑
Corresponding author. Tel.: +48 012 620 55 80; fax: +48 012 620 55 96.
E-mail address: jhandzli@cm-uj.krakow.pl (J. Handzlik).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.053

136
J. Handzlik et al. / Bioorg. Med. Chem. 21 (2013) 135–145
Pyridopyrimidine derivatives
N
Arylpiperazines
Peptidomimetics
(a)
NH
S
N
O
NBP
H2N
N
N
H
N
O
NH2
NH
Quinoline derivatives
N
HN
H
N
NH
N
O
N
O
N
O
N
D13-9001
PAβN (MC-207,110)
BG 905
(b)
O
CO2H
O
CO2H
N
N
N
HN
HN
O
N
O
O
H
N
NH2
NH
HO
N
N
N
H3CO
N
O
H3CO
NH2
2
PAβN
1
Figure 1. Structure of modulators of bacterial MDR-protein pump; a) potent efflux pump inhibitors; b) the recently found hydantoin derivatives with moderate activity (1
and 2); structural similarities to the reference inhibitor PAbN (peptide cores in bold style, marked aromatic and basic fragments).5,9
new EPIs in different chemical groups is a current question of
medicinal chemistry.
2. Results and discussion
2.1. Synthesis
On the other hand, hydantoin derivatives, that are in the area of
interest in our research group, display various biological actions,
particularly GPCR-ligand properties14–16 and/or hypotensive, anti-
arrhythmic or anticonvulsant activities in both in vitro and
in vivo assays.17–19 Our previous studies,9 focused on hydantoin
derivatives with amine-alkyl substituents at position N1, showed
that the hydantoin could be a new appropriate scaffold to investi-
gate relationships between molecular structures and their influ-
ence on antibiotic susceptibility. Indeed, the hydantoin skeleton
could be viewed as a cyclic peptide sequence similar to the open
sequence located in the central part of PAbN structure (Fig. 1b).
This semi-rigid structure allows to modify the nature and the posi-
tion of substituents and facilitates molecular modeling to guide ra-
tional synthesis. The 5,5-diphenylhydantoin compounds described
previously9 displayed weak or moderate ability to increase the
activity of nalidixic acid in E. aerogenes. Among 28 tested com-
pounds with the same scaffold, only two 2-methoxyphenylpipera-
zine derivatives with acetic acid motif (compounds 1 and 2, Fig. 1b)
displayed significant anti-MDR effect at a dose range close to that
of PAbN. On the basis of those not satisfying results, in the present
work we decided to introduce significant changes into the struc-
ture of hydantoins comparing to the initial series.9 In this context,
chemical modifications of the 5,5-diphenylhydantoins were de-
signed to improve activity by: (1) a replacement of 5,5-diphenyl-
substituents with 5-arylidene one, (2) an exchange of position of
amine-alkyl substituents from N1 into N3 or (3) an introduction
of carbonyl or hydroxyl motif into the alkyl chain (Table 1). Thus,
the present studies concern nineteen amine derivatives of 5-aryl-
idenehydantoin 3–21 (Table 1). Synthesis of new compounds and
microbiological assays to evaluate their anti-MDR properties in
Gram-negative bacteria were performed. Molecular modeling
aided structure–activity relationship analysis was carried out.
The presented groups of 5-arylidenehydantoin derivatives 3–21
were divided into three sub-groups, including (A) amide hydroxy-
ethylpiperazine derivatives 3 and 4, (B) derivatives of secondary-
or tertiary non-aromatic amine 5–16, and (C) derivatives of
phenylpiperazine 17–21 (Table 1). Synthesis routes of the
arylidenehydantoins (3–21) are shown in the Schemes 1 and 2.
Synthesis of compounds 3, 4 and 17–21 is described elsewhere.19,24
Compounds 5–9 and 11–16 were synthesized within three-step
synthesis starting from hydantoin (Scheme 1) that was condensed
with suitable aromatic aldehydes to give 5-arylidenehydantoins
23–29. The condensations went as typical Knoevenagel reactions
in basic conditions19,24–26 by the use of sodium acetate in acetic
acid (23–25, 27–29)19,24 or equimolar natrium carbonate and ala-
nine in water (26).26 The resulted 5-arylidenehydantoins (23–29)
occurred at their (Z)-configuration, which is preferable in the case
of Knoevenagel condensation between N1-unsubstituted hydan-
toin and the considered benzaldehydes.27–29 In the next step, the
arylidenehydantoins 23–29 were N-alkylated at position 3 within
Mitsunobu reactions to give oxiran derivatives 30–36. The reac-
tions were performed using racemate of oxiran-2-ylmethanol
(1.5 equiv), triphenylphosphine (1.0 equiv) and diethyl azodicar-
boxylate (1.0 equiv) in dry DMF24 (Scheme 1). In the last step,
the oxiran derivatives 31–35 were used as alkylating agents to re-
act with primary- or secondary amines. In the case of compound
33, the alkylation was performed in solvent-free conditions under
microwave irradiation14,18 (Scheme 2), what allowed to obtain 2-
hydroxypropyl amine derivatives 5–9 in 16–28 min. Synthesis of
compounds 11–16 was performed in methanol. The oxiranes 31,
32, 34 or 35 were refluxed in excess of a suitable amine (4.0 equiv)
for 3 h. Compound 10, possessing free piperazine terminated

J. Handzlik et al. / Bioorg. Med. Chem. 21 (2013) 135–145
137
Table 1
Structure of the tested derivatives of 5-arylidenehydantoin 3–21
R2
R1
R1
R1
O
N
O
N
O
OH
OH
N
O
Amine
N
HN
HN
N
HN
N
O
O
N
O
OH
Group C
Group A
Group B
Compound
Group
R1
R2
Amine
3
4
A
A
H
4-Br
—
—
—
—
5
6
7
B
B
B
4-MeO
4-MeO
4-MeO
—
—
—
N
N
N
CH3
N
HN
N CH3
N
N
C
O
8
B
4-MeO
—
9
B
B
4-MeO
4-MeO
—
—
N
N
O
10
NH
11
12
13
14
15
16
B
B
B
B
B
B
4-Cl
—
—
—
—
—
—
N
H
OH
4-Cl
N
N
N
N
N
N
4-Br
N
H
OH
OH
4-NEt2
2,4-diMeO
2,4-diMeO
N
H
17
18
19
20
21
C
C
C
C
C
H
4-Cl
4-Cl
3,4-diMeO
3,4-diMeO
2-MeO
2-MeO
2-EtO
2-MeO
H
—
—
—
—
—
fragment at position 3 of hydantoin, was obtained by acidic hydro-
weak direct antibacterial activities. The lowest MICs observed for
lysis of acetylpiperazine derivative 8 in 15% HCl (Scheme 2).
hydroxyethylpiperazine derivatives 3, 14 and 16 were in the range
of 125 lM whereas all derivatives of 4-methoxybenzylidenehyd-
2.2. Pharmacology
antoin (5–10) did not inhibit the growth of both strains of E. aerogenes
at 2 mM. This low intrinsic effect allowed us to test these
molecules in combination with usual antibiotics.
Compounds 3–21 were tested in microbiological assays for their
ability to increase antibiotics effectiveness in two strains of E. aer-
ogenes: a reference strain (ATCC-13048) and the derivative CM-64
which over-produces the AcrAB-TolC efflux pump.
2.2.2. Influence on antibiotic MIC
In the next step of the microbiological assays, the compounds
3–21 were tested for their ability to influence the activity of the
three following antibiotics: nalidixic acid (NAL), sparfloxacin
(SFX) and chloramphenicol (CHL) in the tested bacteria strains.
All the compounds have been assayed at a concentration that have
no intrinsic effect, usually 1 4 of their respective MIC (Table 2), in com-
bination with usual antibiotics and a control was systematically
2.2.1. Intrinsic antibacterial activity
In the first step of the biological assays, direct antibacterial
activity of the hydantoin derivatives was evaluated. Minimal inhib-
itory concentration (MIC) of the compounds 3–21 for both strains
was determined (Table 2). The tested compounds displayed very

138
J. Handzlik et al. / Bioorg. Med. Chem. 21 (2013) 135–145
R1
O
HN
N
R1
R1
CH2COOC2H5
37, 38
O
i
i
i
O
CHO
, i
O
NH
HN
NH
HN
HO
O
O
23, 30, 37: R1= H
O
R1
22
23-29
,
i
i
24, 31, 38: R1=4-Br
25, 32: R1= 4-Cl
O
N
HN
26, 33: R1= 4-MeO
27, 34: R1= 4-NEt2
28, 35: R1= 2,4-di MeO
29, 36: R1= 3,4-diMeO
O
O
30-36
Scheme 1. Synthesis of intermediates 23–38: (i) Knoevenagel condensation CH3COONa/CH3COOH19,24 or Na2CO3/alanine/H2O26; (ii), DIAD or DEAD, TPP, dry DMF, 0 °C;24 (iii)
ClCH2COOEt, K2CO3, TEBA, acetone.14,18
OH
N
R1
R1
R1
O
N
O
N
N
H
O
, v
O
iv
HN
HN
N
CH2COOH
HN
N
CH2COOC2H5
37, 38
N
O
OH
O
O
39, 40
3, 37, 39: R1= H
3, 4
(Group A)
4, 38, 40: R1=4-Br
R1
O
N
R1
N
O
C
N
NH
O
N
H3C
O
N
Amine-H, vi or vii
HO
HN
O
NH
8
O
HN
O
N
OH
Amine
O
30-36
O
R2
OCH3
viii
5-7, 9, 11-16
N
HN
(Group B)
R2
R1
H3CO
O
O
OH
NH
OH
N
N
HN
N
N
HN
N
x HCl
O
O
10
17-21
(Group C)
(Group B)
Scheme 2. Synthesis of products 3–21: (iv) EtOH/H2O, NaOH; (v) TEA, DMF, BOP;19 (vi) microwave irradiation; (vii) MeOH, boiling temp, 3 h; (viii) H2O, H+.
carried out without antibiotic. For most of the compounds, the
used concentration (63 M, 6MIC/4) was in the range of the
active concentration used for PAbN (50 M). The concentration
‘activity gain’ (A)9 was calculated according to the equation pre-
sented in Figure 2. The tested compounds showed moderate or
low chemosensitizing properties when conjointly added to each
antibiotic, displaying the highest activity gains at the range of 4.
In the case of nalidixic acid assay (Fig. 2a), only twofold decrease
of MIC values was observed for relatively most efficient compounds
(6, 14, 15, 17, 18 and 21) in reference strain of E. aerogenes, whereas
eight arylidene hydantoins (3, 4, 12, 14–16, 18 and 19) decreased
l
l
was established for the tested compounds as a suitable one to com-
pare their activities to those of previous hydantoins tested at the
concentration of 63
compounds 17–21 were tested at their concentration (100–
200 M) which ensured a lack of a direct antibacterial effect. The
l
M, as well.9 Weakly active phenylpiperazine
l

J. Handzlik et al. / Bioorg. Med. Chem. 21 (2013) 135–145
139
Table 2
MIC of this antibiotic in fourfold in the efflux over-producer
CM-64. A slight increase of efficacy of nalidixic acid (A = 2) in
CM-64 was observed for eight arylidenehydantoin derivatives
5–8, 11, 17, 20 and 21, and four compounds (4, 9, 10 and 13) did
not cause any increase. In the case of assays with sparfloxacin, four
compounds (6, 11, 12 and 15) significantly decreased MIC of this
antibiotic in the reference strain (ATCC 13048). Particularly, com-
pounds 12 and 15 caused effect higher than that of PAbN in the
same strain (Fig. 2b). Compounds 11, 15 and the diethylaminoben-
zylidene derivative 14 showed fourfold decrease of MIC of spar-
floxacin in the efflux over-producer CM-64. Twofold increase of
sparfloxacin efficacy was observed in the presence of hydantoin
derivatives 3, 5, 10, 16 or 19. The rest of the tested compounds
(6, 8, 9, 12, 17, 18, 20 and 21) did not influence the MIC of sparflox-
acin during the assay. Results for the assays with chloramphenicol
(Fig. 2c) indicated that only two compounds (6 and 19) slightly re-
duced MIC of this antibiotic in the reference strain, but four com-
pounds (11, 14–16) caused fourfold- and six compounds (3, 6,
Intrinsic antibacterial activity for compounds 3–21, tested in two strains of E.
aerogenes, ATCC 13048 (reference) and the derivative strain CM-64 (over-producing
AcrAB-TolC)
Compound
MIC (mM) EA ATCC 13048
MIC (mM) EA CM-64
4, 13
5–10
20
17–19
21
>1.25
>2
1
0.5
0.5
0.25
0.125
0.008
6 Â 10À5
0.004
5
>1.25
>2
1
1
0.5
0.25
0. 25
0.128
0.001
0.256
5
11, 12, 15
3, 14, 16
NALa
SFXb
CHLc
PAbN
a
b
c
Nalidixic acid.
Sparfloxacin.
Chloramphenicol.
(a) Nalidixic acid
Activity gains (A) for PAβN, at concentration of 50 μM, were as follows: 64 (ATCC 13048), 128 (CM-64).
(b) Sparfloxacin
Activity gains (A) for PAβN, at concentration of 50 μM, were as follows: 8 (ATCC 13048), 32 (CM-64).
(c) Chloramphenicol
Activity gains (A) for PAβN, at concentration of 50 μM, were as follows: 2 (ATCC 13048), 64 (CM-64).
MIC of antibiotic in absence of a compound (3-21)
A =
MIC of antibiotic in presence of the compound (3-21)
Figure 2. The influence of the compounds 3–21 on minimal inhibitory concentration (MIC) of (a) nalidixic acid (NAL), (b) sparfloxacin (SFX) and (c) chloramphenicol (CHL),
⁄
tested in two strai⁄n⁄s of E. aerogenes, ATCC 13048 (reference) and CM-64 (with over-production of AcrAB-TolC). Compounds tested at concentration of 63
l
M; compound
tested at 200 lM, compound tested at 100 lM.

140
J. Handzlik et al. / Bioorg. Med. Chem. 21 (2013) 135–145
Table 3
Effect of hydantoin derivatives (3–21) on the antibiotic susceptibility of E. aerogenes ATCC-13048 and CM-64 strains
Compounda
EA ATCC-13048
EA CM-64
SFX MIC ( g/mL)
NAL MIC (
lg/mL)
SFX MIC (
0.06
lg/mL)
CHL MIC (
l
g/mL)
NAL MIC (
lg/mL)
l
CHL MIC (lg/mL)
No
3
4
8
8
8
4
8
128
32
32
64
1
512
0.06
0.5
256
b
b
b
8
b
b
5
8
0.03
0.5
6
7
4
8
0.015
2
64
64
1
256
b
b
b
b
8
9
8
8
8
8
8
8
4
4
8
4
4
32
16
4
0.03
0.06
0.06
0.007
4
8
8
8
8
4
8
8
8
8
8
2
8
8
64
1
1
0.5
0.25
512
512
512
128
128
128
64
32
128
32
32
32
64
32
10
11
12
13
14
15
16
17
18
19
20
21
0.003
1
512
b
b
b
0.06
0.003
0.06
0.06
0.06
0.06
0.06
0.06
0.25
0.25
0.5
1
1
0.5
1
128
128
128
256
256
512
256
256
32
64
64
1
a
Compounds concentrations according to the Figure 2.
Compounds which induced aggregates of bacteria in the presence of sparfloxacin or chloramphenicol. These aggregates did not allow MICs measurement.
b
17, 18, 20, 21) caused twofold reduction of chloramphenicol MIC in
the efflux over-producer CM-64.
inactive compounds showing no effect in the efflux pump over-
producing strains (9 and 13). From the effect obtained on CM-64
(Table 3), we can conclude that 14 and 15 were the more attractive
molecules.
The results obtained during the assays with nalidixic acid and
chloramphenicol indicated that ability of the hydantoin derivatives
(3–21) to improve antibiotic efficacy is distinctly higher in CM-64,
the strain of E. aerogenes which over-produces AcrAB-TolC efflux
pump, than that in the reference strain (Table 3). This suggests that
their way to modulate the bacterial resistance is connected to the
tripartite efflux pump action. Four compounds (6, 11, 12 and 15)
involve some additional anti-MDR mechanism distinct from Ac-
rAB-TolC pump as they cause decrease of MIC of sparfloxacin sig-
nificantly higher in the reference strain of E. aerogenes.
Particularly, it applies to compound 12 while the chemosensitizer
potency of compounds 11 and 15 in CM-64 is noticeable as well.
An analysis of direct MIC of the tested compounds in compari-
son to their properties to enhance antibiotics indicate that the
most active compounds 14 and 15 displayed relatively higher
2.3. Molecular modeling
Analysis of structural features and structure–activity relation-
ship for the set of the obtained compounds was based on their
low energy conformations, calculated for a free base form of 3–
21. On the basis of various lines of evidence including crystallo-
graphic studies for our previous 5-arylidenehydantoins,20–24,27–29
only (Z)-configuration was considered for whole investigated pop-
ulation. Our previous investigations performed for the arylpipera-
zines 17–21 showed that in case of molecules at the global
minimum energy point the absolute configuration of the secondary
alcohol carbon almost did not influence on calculated distances
between crucial pharmacophoric elements.24 Thus, this time we
limited our investigations to one enantiomer with the (S)-configu-
ration. The obtained lowest energy conformations for compounds
3–21 are presented in Figure 3. The superimposition of one of
the most active chemosensitizer 14 with the rest of the tested
population (3–13 and 15–21) is shown in respect to the three
structural categories A–C.
From the structural point of view, all the studied molecules con-
sist of a flat moiety of hydantoin, linked by methylidene bridge
with a (un)substituted phenyl ring from one side, and by flexible
hydroxypropyl or ethanone chain with a secondary or tertiary
amine group from the other. In the energetic global minimum all
the compounds adopted an extended conformation with the ary-
lidene phenyl ring rotated either by c.a. 57° or À57° with reference
to the imidazol ring plane, regardless of the amine fragment
(Fig. 3). It should be noticed here that both positions of the phenyl
ring were almost equivalent energetically and differed from each
other with not more than 0.05 kJ/mol.
intrinsic antibacterial action (MIC = 250 lM). The compounds were
tested at 1/4 of the MIC to ensure a lack of a direct antibacterial ef-
fect. As a role of drug-hydantoin combinations in the chemosensi-
tizer action is concerned, the combination with nalidixic acid
seems to be superior. Eight members of the tested population (3,
4, 12, 14–16, 18, 19) generated statistically different values of
activity (A) comparing the over-producing MDR efflux pump strain
(CM-64) to the wild one (ATCC 13048), significantly higher in the
case of CM-64. The weakest hydantoin-drug collaboration was ob-
served during the assays with sparfloxacin in the strain CM-64.
Only one compound, the diethylaminobenzylidene derivative 14,
generated statistically different higher A value for the strain
over-producing MDR efflux pump AcrAB-TolC. In contrary, four
compounds (12, 15 > 11 > 6) caused statistically different stronger
increase of sparfloxacin action in the reference strain ATCC 13048.
The results obtained within the assays using strain CM-64 allow
to divide the series of hydantoin derivatives (3–21) into following
classes of potential efflux pump inhibitors: (I) the promising
compounds which caused fourfold decrease of MIC of at least
one antibiotic (3, 4, 11, 12, 14–16) at the lower dose (63 lM);
(II) compounds with slight efflux-modulator properties (5–8, 10,
2.4. Structure–activity relationship
17–21) causing the twofold decrease at lower concentration or
fourfold one at the higher concentrations (100–200 lM); and (III)
The arylidenehydantoin derivatives (3–21) differ within three
structural fragments, what gives an opportunity to evaluate an

J. Handzlik et al. / Bioorg. Med. Chem. 21 (2013) 135–145
141
hydroxyethylpiperazine (3, 4, 12, 14 and 16) and isopropylamine
(11 and 15). The hydroxyethylpiperazine fragment is particularly
favorable to improve the antibacterial effect of nalidixic acid,
whereas a role of this fragment is shared with isopropyl moiety
in the case of assays with sparfloxacin and chloramphenicol. The
compounds 14 and 15 may be identified as the most active chemo-
sensitizers of the resistant E. aerogenes because of their fourfold
MIC reduction for all the tested antibiotics (Table 3). The com-
pounds contain an aromatic arylidene fragment which is signifi-
cantly activated by electron donating substituents as follows: a
strong activating group of diethylamine at position para (14) or
two moderate activating groups of 2,4-dimethoxyl substituents
(15). Considering the influence of substitution at the arylidene aro-
matic rings on the tested biological activities, it can be noted that
4-diethylamine- (14), 2,4-dimethoxy- (15 and 16) and 4-chloro-
(11 and 12) substituents are the most favorable. Furthermore,
the substituent-free benzylidene fragment (3) seems to be more
profitable than the 4-methoxybenzylidene (6–10). The role of 4-
bromide substituent is hard to evaluate since the compounds pos-
sessing this fragment (4, 13) caused an aggregation allowing to test
them only with nalidixic acid. The 4-chlorobenzylidene fragment,
present in compounds 11 and 12, improved efficacy of sparfloxacin
similarly- (11) or higher (12) than PAbN in the reference strain of
E. aerogenes. This suggests that the p-Cl substituent may promote
some unknown mechanism of anti-MDR action which disappears
when tripartite efflux pump AcrAB-TolC is the predominant resis-
tance mechanism, as in strain CM-64. A role of linkers between
amine and hydantoin is difficult to explain on the basis of the ob-
tained results. Although the compounds possessing acyl linker (3
and 4) were relatively less active than compounds with the
hydroxypropyl one (11, 14–16), the structural differences of the
both groups involves other fragments (e.g., substitutuents at ary-
lidene moieties).
Figure 3. Molecular modeling results. The superimposition of the most active
chemosensitizer 14 (purple) with the rest of the compounds in respect to the three
structural groups: (a) compounds of the group A (yellow); (b) compounds of the
group B (blue); (c) compounds of the group C (black). All conformations represent
the global energy minimum point. The hydrogen atoms are not shown.
Chemical properties of the most active compounds (14, 15), as-
sessed basing on their 3D-structure, allow to propose pharmaco-
phore features which may be responsible for chemosensitizing
action on the MDR pump AcrAB-TolC, observed among the tested
hydantoins (Fig. 4). It could be seen that an arylidene moiety at
position 5 (AR) as well as a basic positive ionizable area linked to
position 3 of the hydantoin (PIs) are crucial for the activity. A
non-aromatic hydrophilic substituent at the positive ionizable area
(HYL) seems to improve reversal-MDR activity of compounds, par-
ticularly in the case of nalidixic acid, whereas electron donating
substituents in para- and ortho-position of the aromatic ring (ED)
enable to develop the activity for all tested antibiotics. An aromatic
influence of the fragments on the compound properties to restore a
susceptibility to investigated antibiotics. Considering an influence
of the amine terminated fragments, non-aromatic amines (group
A and B, Table 1) seem to be more profitable than phenylpiperazine
(group C). Among phenylpiperazine derivatives (17–21), only two
compounds (18 and 19) caused fourfold decrease of one antibiotic
(NAL) but their action was identified at higher doses (100 lM) than
that of corresponding non-aromatic amine derivatives (Table 3,
Fig. 2). Two non-aromatic amine moieties seem to be beneficial,
(a)
(b)
Compound
X1 (PI-HC) [Å]
X2 (HYL-HC) [Å]
X3 (AR-HC) [Å]
X4 (ED-HC) [Å]
3
7.68
5.94
5.99
6.00
5.94
6.00
-
10.31
4.85
4.85
4.85
4.87
4.84
4.84
-
11
12
14
15
16
-
-
7.80
7.81
7.56
7.46
7.46
8.76
8.76
-
11.40
11.40
-
8.76
11.40
Figure 4. Pharmacophore features proposed based on properties of a most promising compound (14); (a) HC-hydantoin core, AR-aromatic ring, ED-electron donating
substituent at aromatic ring, PI(s)-area with positive ionizable center(s), HYL-hydrophilic non-aromatic substituent; (b) distances from the center of hydantoin core to PI (X1),
HYL (X2), AR (X3) and ED (X4) estimated for the most promising compounds 3, 11, 12, 14–16.

142
J. Handzlik et al. / Bioorg. Med. Chem. 21 (2013) 135–145
substituent at the piperazine area PI can be considered as an unde-
sirable feature decreasing anti-MDR properties of the compounds.
This can underline a role of both, HYL and PIs, since the hydropho-
bic aromatic substituent at piperazine nitrogen drastically changes
its basic properties and causes a huge increase of lipophilicity of
the whole substituent at position 3 of the hydantoin.
activity. Results of the comprehensive studies performed here will
be a base for rational design of new MDR efflux pump inhibitors
possessing hydantoin scaffold. The most active compounds, de-
tected here, will be investigated using other bacterial species and
antibiotic classes to understand their exact effect on various bacte-
rial mechanisms of resistance and their precise target, a special
attention will be paid for tripartite AcrAB-TolC efflux pump de-
scribed in the MDR Enterobacterial isolates.
Molecular modeling calculations allowed to estimate topology
of four pharmacophoric areas (PI, HYL, AR, ED) of the promising
compounds (3, 11, 12, 14–16) in respect to the centre (HC) of the
hydantoin scaffold (Fig. 4). Analysis of the calculated distances
(X1-X4) indicated that the rotation of the arylidene phenyl ring
(AR) by 57° or À57° with reference to the imidazol ring (HC) did
not affect the distance X3 between both fragments. The distance
was in range of 4.84–4.87 Å (Fig. 4) for all the tested compounds
(3–21, Fig. 4). Only small differences can be noticed also in the case
of the X1 value, expressed as a distance between the hydantoin
ring and the closest basic positive ionizable centre (PI). Superimpo-
sition of 14 with the low-energy poses of compounds from coming
B or C groups shows overlapping of the basic nitrogen atoms in
piperidine/morpholine/piperazine as well as isopropylamine frag-
ments. In the first case (10, 12, 14, 16, 17–21) the X1 distance
was 6.00 Å (8.72–8.76 Å for the second basic nitrogen atom, if pos-
sible), while this distance was around 5.94 Å for isopropyl deriva-
tives 11, 13 and 15. In the case of piperazinamides 3 and 4 from the
group A, the distance X1 was extended to 7.68 Å (Fig. 3b).
4. Experimental
4.1. Chemistry
1H NMR spectra were recorded on a Varian Mercury VX
300 MHz PFG instrument (Varian Inc., Palo Alto, CA, USA) in CDCl3
or DMSO-d6 at ambient temperature using the solvent signal as an
internal standard. IR spectra were recorded on a Jasco FT/IR-410
apparatus using KBr pellets and are reported in cmÀ1. Thin-layer
chromatography was performed on pre-coated Merck silica gel
60 F254 aluminium sheets, the used solvent systems were (I)
CH2Cl2/acetone 17:3; CH2Cl2, (II) CH2Cl2/acetone 19:1; (III) CHCl3/
i-PrOH/NH3 9:11:3; (IV) MeOH/acetone/benzene 1:1:1; (V) tolu-
ene/acetone/methanol 15:5:1. Melting points were determined
using Mel-Temp II apparatus and are uncorrected. Elemental anal-
yses were within 0.4% of the theoretical values unless stated
otherwise. Syntheses under microwave irradiation were performed
in household microwave oven Samsung M1618. Synthesis of com-
pounds 3, 4, 17–21, 23–29, 31, and 34–38 has been described
elsewhere.19,24
3. Conclusions
In summary, the work contributes to the search of new tools to
combat multidrug resistant bacteria among chemical compounds
possessing hydantoin scaffold. From our previous studies,9 a new
series of N3-aminealkyl arylidenehydantoin derivatives was
designed, synthesized and evaluated for their ability to improve
antibiotic efficacy in Gram-negative E. aerogenes bacteria over-
producing AcrAB-TolC efflux pump. The SAR analysis was per-
formed on basis of the microbiological assays and the molecular
modeling calculation. In comparison to the previous series of N1-
aminealkyl derivatives of 5,5-diphenylhydantoin,9 the line of
chemical modifications of hydantoin described here (3–21) seems
to be more favorable as it gave a higher number of new compounds
displaying chemosensitizing action on the MDR bacteria. Seven of
the tested compounds (3, 4, 11, 12, 14–16), used in the concentra-
tion closed to the active dose of PAbN, displayed significant four-
fold increase of antibacterial activity of nalidixic acid (3, 4, 12,
14–16), chloramphenicol (11, 14–16) and/or sparfloxacin (11, 14,
15). The general SAR derived from the studies indicates that an
amphiphilic character of the compounds is beneficial for the ex-
pected chemosensitizing action on AcrAB-TolC, what can be ex-
pressed by ‘antipodal’ positions of both, the hydrophilic basic
amine fragment and the hydrophobic aromatic one, in the case of
the most active compounds. The SAR study also demonstrates that
strong electron-donor properties of substituent(s) at the aryliden-
ene aromatic ring, that is seen in the case of (Z)-5-(4-(diethyl-
amino)benzylidene)-3-(2-hydroxy-3-(4-(2-hydroxyethyl)piperazin-
1-yl)propyl)imidazolidine-2,4-dione (14) and (Z)-5-(2,4-dimeth-
oxybenzylidene)-3-(2-hydroxy-3-(isopropylamino)propyl)imidaz-
olidine-2,4-dione (15), could be responsible for similar
chemosensitizing properties observed in the assays with different
antibiotics (NAL, CHL, SFX) in the strain over-producing MDR efflux
pump AcrAB-TolC.
4.1.1. General procedure for preparation of 5-arylidene-3-
(oxiran-2-ylmethyl)imidazolidine-2,4-diones (30, 32 and 33)
5-Arylideneimidazolidine-2,4-dione
(10 mmol),
oxiran-2-
ylmethanol (15 mmol, 1.11 g) and triphenylphosphine (10 mmol,
2.62 g) were added to 50 mL of dry DMF at 0 °C. The mixture was
stirred on the ice-bath until the reactants were totally dissolved
(ꢀ20 min). A solution of diethyl azodicarboxylate DEAD (10 mmol,
1.74 g) in dry DMF (10 mL) was added to the mixture dropwise for
45 min and stirring was continued at room temperature for further
3 h. Then, the mixture was poured into 100 mL of cold water to
precipitate and left at 0–4 °C overnight. After filtration, pure prod-
uct was obtained from precipitate by column chromatography
with CH2Cl2/acetone and additional crystallization with EtOH or
MeOH.
4.1.1.1. (Z)-5-benzylidene-3-(oxiran-2-ylmethyl)imidazolidine-
2,4-dione (30).
(Z)-5-benzylideneimidazolidine-2,4-dione 23
(1.88 g) was used. Crystallization with isopropanol gave white
crystals of 30 (200 mg, 0.82 mmol, 8.2%), mp: 170–172 °C, Rf (I):
0.74. Anal. Calcd for C13H12N2O3: C, 63.93; H, 4.95; N, 11.47. Found:
C, 63.85; H, 5.10; N, 11.04. 1H NMR (CDCl3) d (ppm): 2.69 (dd,
J1 = 4.87 Hz, J2 = 2.56 Hz, 1H, N3-CH2CHCHa(H)), 2.81–2.84 (m,
1H, N3-CH2CHCHb(H)), 3.22–3.28 (m, 1H, N3-CH2CHCH2), 3.75
(dd, J1 = 14.36 Hz, J2 = 4.87 Hz, 1H, N3-CHc(H)), 3.90 (dd,
J1 = 14.36 Hz, J2 = 5.13 Hz, 1H, N3-Hd(H)), 6.78 (s, 1H, CH@C),
7.35–7.48 (m, 5H, Ar-2,3,4,5,6-H), 8.37 (s, 1H, N1H). IR KBr
(cmÀ1): 3276 (N1H), 1764 (C2@O), 1711 (C4@O), 1656 (CH@C),
1573 (Ar).
4.1.1.2. (Z)-5-(4-chlorobenzylidene)-3-(oxiran-2-ylmethyl)imi-
Although the studies gave a promising group of potential MDR
efflux pump inhibitors, the most active compounds (14 and 15)
displayed only moderate chemosensitizing properties, lower than
that of peptidomimetics (PAbN). Thus, further modifications within
the group of hydantoin derivatives are necessary to improve their
dazolidine-2,4-dione (32).
(Z)-5-(4-chlorobenzylideneimi-
dazolidine-2,4-dione 25 (2.23 g) was used. Crystallization with
isopropanol gave white crystals of 33 (750 mg, 2.6 mmol; 26.9%),
mp: 207–212 °C, Rf (I): 0.67. Anal. Calcd for C13H11ClN2O3: C,
56.03; H, 3.98; N, 10.05. Found: C, 56.20.85; H, 4.03; N, 10.01. 1H

J. Handzlik et al. / Bioorg. Med. Chem. 21 (2013) 135–145
143
NMR (CDCl3) d (ppm): 2.69 (dd, J1 = 4.87 Hz, J2 = 2.57 Hz, 1H, N3–
CH2CHCHa(H)), 2.83–2.86 (m, 1H, N3–CH2CHCHb(H)), 3.23–3.29
(m, 1H, N3–CH2CHCH2), 3.76–3.89 (m, 2H, N3–CH2), 6.72 (s, 1H,
CH@C), 7.38–7.44 (m, 4H, Ar-2,3,5,6-H), 8.86 (s, 1H, N1H).
4.1.2.3. (Z)-5-(4-methoxybenzylidene)-3-(2-hydroxy-3-(piperi-
din-1-yl)propyl)imidazolidine-2,4-dione hydrochloride
(7). (Z)-5-(4-methoxybenzylidene)-3-(oxiran-2-ylmethyl)imi-
dazolidine-2,4-dione 33 (0.77 g) and 1-methylpiperazine (0,24 g)
were irradiated at 600 W during 6 min and next at 750 W in
18 min (4 Â 2 min, 2 Â 3 min, 2 min, 2 Â 1 min, 2 min). Methanol
was used to crystallization. After conversion into the hydrochloride
form crystallization with dry ethanol and next ethanol gave
white crystals of 7 (0.04 g, 0.11 mmol, 4%), mp: 171–176 °C, Rf
(III): 0.26. Anal. Calcd for C19H26N3O4Cl: C, 57.64; H, 6.62;
N, 10.61; Found: C, 57.81; H, 6.34; N, 10.21. 1H NMR (DMSO-d6)
4.1.1.3. (Z)-5-(4-methoxybenzylidene)-3-(oxiran-2-ylmethyl)-
imidazolidine-2,4-dione (33).
(Z)-5-(4-methoxybenzylidene -
imidazolidine-2,4-dione 26 (2.18 g) was used. Purification by
column chromatography gave white crystals of 33 (1.34 g,
4.8 mmol, 49%), mp: 176–179 °C, Rf (II): 0.59. Anal. Calcd for
C
14H14N2O4: C, 61.31; H, 5.14; N, 10.21. Found: C, 61.44; H, 5.25;
N, 10.28. 1H NMR (DMSO-d6) d (ppm): 2.52–2.55 (m, 2H, CH2O),
3.15–3.17 (m, 1H, CHO), 3.63–3.65 (d, J = 4.87 Hz, 2H, N3-CH2),
3.79 (s, 3H, OCH3), 6.52 (s, 1H, CH@C), 6.95–6.98 (m, 2H,
Ar-3,5-H), 7.60–7.63 (m, 2H, Ar-2,6-H), 10.72 (s, 1H, N1H).
d (ppm): 1.28–1.42 (m, 6H, Pip-3,4,5-H), 2.22–2.32 (m, 4H,
Pip-2,6-H), 3.40–3.52 (m, 4H, N3-CH2, Pip-CH2), 3.78 (s, 3H,
OCH3), 3.91 (m, 1H, CHOH), 4.87–4.88 (m, 1H, OH), 6.43 (s, 1H,
CH@C), 6.93–6.96 (m, 2H, Ar-3,5-H), 7.59–7.62 (m, 2H, Ar-2,6-H),
10.59 (s, 1H, N1H).
4.1.2. General procedure for preparation of hydrochloride form
of amine derivatives of 5-arylideneimidazolidine-2,4-diones
(5–7)
4.1.3. General procedure for preparation of amine derivatives of
5-arylideneimidazolidine-2,4-diones (8, 9)
(Z)-5-(4-methoxybenzylidene)-3-(oxiran-2-ylmethyl)imidaz-
olidine-2,4-dione (33) (1.6–3.0 mmol) and appropriate amine (2.8–
6.0 mmol) were irradiated in a standard household microwave
oven at various power (600–750 W) and time intervals (16–
24 min) of irradiation for each prepared compound (5–7), respec-
tively. The reaction progress was controlled with TLC (toluene
(15):acetone (5):methanol (1)). After irradiation, the glassy residue
was crystallized and saturated with gaseous HCl to give a pure
product in hydrochloride form. The precipitate of pure product
(5–7) was separated by filtration.
(Z)-5-(4-methoxybenzylidene)-3-(oxiran-2-ylmethyl)imidaz-
olidine-2,4-dione 33 (2.2–5 mmol) and appropriate amine (2.2–
5 mmol) were irradiated in a standard household microwave oven
at various power (450–750 W) and time intervals (16–28 min) of
irradiation for each prepared compound (8, 9), respectively. The
reaction progress was controlled with TLC (V). After irradiation,
the glassy residue was crystallized to give a pure product in basic
form. The precipitate of pure product (8, 9) was separated by
filtration.
4.1.3.1. (Z)-5-(4-methoxybenzylidene)-3-(3-(4-acetylpiperazin-
4.1.2.1. (Z)-5-(4-methoxybenzylidene)-3-(2-hydroxy-3-(4-meth-
1-yl)-2-hydroxypropyl)imidazolidine-2,4-dione (8).
(Z)-5-(4-
ylpiperazin-1-yl)propyl) imidazolidine-2,4-dione hydrochloride
methoxybenzylidene)-3-(oxiran-2-ylmethyl)imidazolidine-2,4-
dione 33 (1.37 g) and 1-acetylpiperazine (0,64 g) were irradiated at
450 W during 5 min (2 Â 1 min, 3 min) next at 600 W during 4 min
and next at 750 W during 7 min (2 min, 5 min). Crystallization
with methanol with activated coal gave white crystals of 8
(1.21 g, 0.003 mmol, 60%), mp: 178–181 °C, Rf (III): 0.81. Anal.
Calcd for C20H26N4O5: C, 59.69; H, 6.51; N, 13.92. Found: C,
59.63; H, 6.42; N, 13.90.1H NMR (DMSO-d6) d (ppm): 1.93 (s, 3H,
CH3), 2.25–2.40 (m, 10H, Pip-2,3,4,5,6-H, Pp-CH2), 3.46–3.49 (m,
2H, N3-CH2), 3.78 (s, 3H, OCH3), 3.95–3.96 (m, 1H, CHOH), 4.95–
4.97 (d, J = 5.13 Hz, 1H, OH), 6.47 (s, 1H, CH@C), 6.94–6.97 (d,
J = 8.97 Hz, 2H, Ar-3,5-H), 7.58–7.61 (d, J = 8.72 Hz 2H, Ar-2,6-H),
10.63 (s, 1H, N1H).
(5).
(Z)-5-(4-methoxybenzylidene)-3-(oxiran-2-ylmethyl)imi-
dazolidine-2,4-dione 33 (0.82 g) and 1-methylpiperazine (0,60 g)
were irradiated at 600 W during 6 min and next at 750 W in
18 min (4 Â 2 min, 2 Â 3 min, 2 min, 2 Â 1 min, 2 min). Methanol
was used to crystallization. After conversion into the hydrochloride
form crystallization with dry ethanol and next ethanol gave white
crystals of 5 (0.18 g, 0.5 mmol, 17%), mp: 193–199 °C, Rf (III):
0.13. Anal. Calcd for C19H28N4O4Cl2 Â 2.5 H2O: C, 48.28; H, 6.16;
N, 11.81. Found: C, 48.11; H, 6.59; N, 11.81.1H NMR (DMSO-d6) d
(ppm): 2.20–2.40 (m, 3H, N-CH3), 2.72–3.12 (m, 8H, 2 Â Pp-
2,3,5,6-H), 3.39–3.46 (m, 2H, N3-CH2), 3.78 (s, 3H, OCH3), 3.90–
3.98 (br s, 1H, CHOH), 4.95–5.05 (br s, 1H, OH), 6.49 (s, 1H, CH@C),
6.95–9.98 (m, 2H, Ar-3,5-H), 7.59–7.62 (m, 2H, Ar-2,6-H), 10.65 (m,
1H, N1H).
4.1.3.2. (Z)-5-(4-methoxybenzylidene)-3-(2-hydroxy-3-morpho-
linopropyl)imidazolidine-2,4-dione (9).
benzylidene)-3-(oxiran-2-ylmethyl)imidazolidine-2,4-dione
(Z)-5-(4-methoxy-
33
4.1.2.2.
methylpiperazin-1-yl)propylamino)propyl)imidazolidine-2,4-
dione hydrochloride (6). (Z)-5-(4-methoxybenzylidene)-3-
(Z)-5-(4-methoxybenzylidene)-3-(2-hydroxy-3-(3-(4-
(0.60 g) and morpholine (0,19 g) were irradiated at 450 W during
3 min, next at 600 W during 6 min (4 min, 2 min) and next
19 min (3 Â 5 min,4 min). Crystallization with methanol gave
white crystals of 9 (0.18 g, 0.5 mmol, 23%), mp: 170–173 °C, Rf
(III): 0.23. Anal. Calcd for C18H23N3O5: C, 59.82; H, 6.41; N, 11.63.
Found: C, 59.85; H, 6.45; N, 11.49. 1H NMR (DMSO-d6) d (ppm):
2.27–2.32 (m, 4H, Mor-2,6-H), 2.34–2.39(m, 2H, Mor-CH2), 3.46–
3.50 (m, 6H, CH2OCH2, N3-CH2), 3.78 (s, 3H, OCH3), 3.92–3.98 (q,
J = 6.07 Hz, 1H, CHOH), 4.94–4.96 (d, J = 5.39 Hz, 1H, OH), 6.47 (s,
1H, CH@C), 6.94–6.97 (d, J = 8.72 Hz, 2H, Ar-3,5-H), 7.58–7.61 (d,
J = 8.72 Hz 2H, Ar-2,6-H), 10.62 (s, 1H, N1H).
(oxiran-2-ylmethyl)imidazolidine-2,4-dione 33 (0.44 g) and 3-(4-
methylpiperazin-1-yl)propan-1-amine (0,50 g) were irradiated at
600 W during 2 min, next at 750 W in 6 min (3 Â 2 min) and next
at 600 W in 8 min (4 Â 2 min). Methanol was used to crystalliza-
tion. After converting into the hydrochloride form, crystallizing
with ethanol with water and precipitating with dry ethanol gave
white crystals of 6 (0.15 g, 0.35 mmol, 22%), mp: 213–219 °C, Rf
(III): 0.33. Anal. Calcd for C22H35N5O4Cl2 Â 3H2O: C, 47.10; H,
7.32; N, 12.52. Found: C, 47.31; H, 7.40; N, 12.54. 1H NMR
(DMSO-d6) d (ppm): 1.95–2.05 (m, 2H, Pp-CH2-CH2), 2.47–2.50 (s,
3H, CH3), 2.78–2.95 (m, 6H, Pp-CH2, Pp-2,6-H), 2.98–3.15 (m, 4H,
Pp-3,5-H), 3.20–3.30 (m, 4H, CH2NHCH2), 3.52–3.59 (m, 2H, N3-
CH2), 3.79 (s, 3H, OCH3), 4.11 (m, 1H, CHOH), 5.95 (br s, 1H, OH),
6.51 (s, 1H, CH@C), 6.95–9.98 (d, J = 8.98 Hz, 2H, Ar-3,5-H), 7.60–
7.63 (d, J = 8.98 Hz, 2H, Ar-2,6-H), 8.65–8.90 (m, 2H, NH2+), 10.71
(s, 1H, N1H).
4.1.3.3. (Z)-5-(4-methoxybenzylidene)-3-(2-hydroxy-3-(pipera-
zin-1-yl)propyl)imidazolidine-2,4-dione
(10). (Z)-5-(4-methoxybenzylidene)-3-(3-(4-acetylpiperazin-
1-yl)-2-hydroxypropyl)imidazolidine-2,4-dione (0.40 g) was
dihydrochloride
8
added to 15% hydrochloride acid (2 mL). 10% NH3(aq) was been add-
ing to hot solution until obtain pH 7. White crystals of 10 were

144
J. Handzlik et al. / Bioorg. Med. Chem. 21 (2013) 135–145
obtained (0.32 g,8.9 mmol, 89%), mp: 269–272 °C, Rf (III): 0.23.
Anal. Calcd for C18H26N4O4Cl2: C, 59.99; H, 6.71; N, 15.55. Found:
C, 59.61; H, 6.90; N, 15.19. 1H NMR (DMSO-d6) d (ppm): 1.21 (s,
1H, Pp-NH), 2.35–2.40 (t.def, 2H, Pp-CH2), 2.57–2.61 (m, 4H, Pp-
2,6-H), 2.99 (br s, 4H, Pp-3,5-H), 3.45–3.48 (d, J = 6.41 Hz, 2H,
N3-CH2), 3.78 (s, 3H, OCH3), 3.92–3.94 (m, 1H, CHOH), 5.00–5.02
(d, J = 5.13 Hz, 1H, OH), 6.48 (s, 1H, CH@C), 6.94–6.97 (d,
J = 8.72 Hz, 2H, Ar-3,5-H), 7.59–7.62 (d, J = 8.72 Hz 2H, Ar-2,6-H),
7.68–9.25 (br s, 2H, Pp-NH+, Pp-NH2), 9.5–11.2 (m, 1H, N1H).
C, 62.24; H, 7.72; N, 15.40. 1H NMR (DMSO) d (ppm): 1.20 (t,
J = 6.80 Hz, 6H, 2 Â CH3), 2.20–2.50 (m, 12H, Pp-H, C2H4–OH),
3.30–3.60 (m, 8H, CH2–CHOH–CH2, CH2NCH2), 3.90–4.00 (m, 1H,
CHOH), 4.30 (s, 1H,OH), 4.90 (1H, OH) 6.50 (s, 1H, CH@C), 6.60
(d, J = 8.80 Hz, 2H, Ar-3,5-H), 7.40 (d, J = 8.80 Hz, 2H, Ar-2,6-H),
10.30 (s, 1H, N1H). IR KBr (cmÀ1): 3430 (OH), 3400 (NH), 1750
(C4@O), 1710 (C2@O), 1580 (Ar).
4.1.4.5. (Z)-5-(2,4-dimethoxybenzylidene)-3-(2-hydroxy-3-(iso-
propylamino)propyl) imidazolidine-2,4-dione (15).
(Z)-5-(2,4-
4.1.4. General procedure for preparation of amine derivatives of
5-arylideneimidazolidine-2,4-diones (11–16)
dimethoxybenzylidene)-3-(oxiran-2-ylmethyl)imidazolidine-2,4-dione
35 and isopropylamine were used to give white crystals of 15.
50%, mp: 148–150 °C, Rf (IV): 0.25. Anal. Calcd for C18H25N3O5: C,
59.50; H, 6.70; N, 10.94. Found: C, 59.32; H, 7.19; N, 11.53.
1H NMR for hydrochloride of 15 (DMSO) d (ppm): 1.19 (d,
J = 3.40 Hz, 3H, CH3), 1.22 (d, J = 3.40 Hz, 3H, C–CH3), 2.84 (qu
def., 1H, CHNH), 3.00–3.15 (m, 1H, CHNH) 3.22–3.38 (m, 1H,
CHNH) 3.40–3.58 (m, 2H, NCH2), 3.80 (s, 3H, OCH3), 3.85 (s, 3H,
OCH3), 4.10 (s, 1H, CH–OH), 5.80 (d, J = 6.46 Hz, 1H, OH), 6.50–
6.69 (m, 2H, Ar-3,5-H), 6.71 (s, 1H, CH@C), 7.75 (d, 1H,
J = 9.50 Hz, Ar-6-H), 8.35 (s, 1H, NH), 8.55 (s, 1H, NH+), 10.56 (s,
1H, N1H). IR KBr (cmÀ1): 3420 (OH), 3380 (NH), 1750 (C4@O),
1720 (C2@O), 1580 (Ar).
5-Arylidene-3-(oxiran-2-ylmethyl)imidazolidine-2,4-diones 30,
31, 34 or 35 (10 mmol) and primary or secondary amine (40 mmol)
and MeOH (30 mL) were mixed in flask. The mixture was refluxed
for 3 h. The solvent was evaporated until dry residue was obtained.
The residue was purified by crystallization with methanol.
4.1.4.1.
(Z)-5-(4-chlorobenzylidene)-3-(2-hydroxy-3-(isopro-
(Z)-5-(4-
pylamino)propyl) imidazolidine -2,4-dione (11).
chlorobenzylidene)-3-(oxiran-2-ylmethyl)imidazolidine-2,4-dione
32 and isopropylamine were used to give white crystals of 11. 48%;
mp: 184–186 °C, Rf (IV): 0.20. Anal. Calcd for C16H20N3O3Cl: C,
56.88; H, 5.97; N, 12.44. Found: C, 56.81; H, 5.68; N, 12.30. 1H
NMR (DMSO) d (ppm): 0.90 (d, J = 6.20 Hz, 6H, C-(CH3)2), 2.45–
2.65 (m, 2H, CH2NH) 2.72 (qu def., 1H, CH–(CH3)2), 3.40–3.55 (m,
2H, N3-CH2), 3.75–4.00 (m, 1H, CHOH), 5.35 (s, 1H, OH), 6.50 (s,
1H, CH@C), 7.45 (d, J = 8.20 Hz, 2H, Ar-3,5-H), 7.70 (d, J = 8.00 Hz,
2H, Ar-2,6-H), 10.80 (s, 1H, N1H). IR KBr (cmÀ1): 3435 (NH),
1770 (C4@O), 1725 (C2@O), 1580 (Ar).
4.1.4.6. (Z)-5-(2,4-dimethoxybenzylidene)-3-(2-hydroxy-3-(4-
(2-hydroxyethyl)piperazin-1-yl)propyl)imidazolidine-2,4-dione
(16).
(Z)-5-(2,4-dimethoxybenzylidene)-3-(oxiran-2-ylmethyl)-
imidazolidine-2,4-dione 35 and 2-(piperazin-1-yl)ethanol were
used to give white crystal of 16. 57%, mp: 162–163 °C, Rf (IV):
0.36. Anal. Calcd for C21H30N4O6: C, 58.05; H, 6.96; N, 12.89. Found:
C, 58.10; H, 6.73; N, 12.84. 1H NMR (DMSO) d (ppm): 2.20–2.40 (m,
12H, Pp-H, C2H4–OH), 3.40–3.50 (m, 4H, CH2–CHOH–CH2), 3.80 (s,
3H, OCH3), 3.85 (s, 3H, OCH3), 3.93 (m, 1H, CHOH), 4.95 (s, 1H, OH)
6.60 (d, J = 9.00 Hz, 2H, Ar-3,5-H), 6.70 (s, 1H, CH@C), 7.60 (d,
J = 8.40 Hz, 1H, Ar-6-H), 9.60 (s, 1H, N1H). IR KBr (cmÀ1): 3415
(OH), 3415 (NH), 1770 (C4@O), 1720 (C2@O), 1595 (Ar).
4.1.4.2.
hydroxyethyl)piperazin-1-yl)propyl)imidazolidine-2,4-dione
(12). (Z)-5-(4-chlorobenzylidene)-3-(oxiran-2-ylmethyl)imi-
(Z)-5-(4-chlorobenzylidene)-3-(2-hydroxy-3-(4-(2-
dazolidine-2,4-dione 32 and 2-(piperazin-1-yl)ethanol were used
to give white crystals of 12. 82%, mp: 181–182 °C, Rf (IV): 0.26.
Anal. Calcd for C15H25N4O4Cl: C, 55.80; H, 6.16; N, 13.16. Found:
C, 55.52; H, 5.73; N, 13.16. 1H NMR (DMSO) d (ppm): 2.20–2.40
(m, 10H, Pp-H, CH2–CH2–OH), 3.40–3.50 (m, 4H, CH2–CHOH–
CH2), 3.78 (qu, J = 6.80 Hz, 1H, CHOH), 3.97 (t, J = 6.00 Hz, 2H,
CH2OH), 5.50 (s, 1H, OH), 6.50 (s, 1H, CH@C), 7.45 (d, J = 8.60 Hz,
2H, Ar-3,5-H), 7.65 (d, J = 8.50 Hz, 2H, Ar-2,6-H), 10.80 (s,1H,
N1H). IR KBr (cmÀ1): 3410 (OH), 3310 (NH), 1760 (C4@O), 1720
(C2@O), 1590 (Ar).
4.2. Molecular modeling methods
The 3D models of compounds 3–21 were built using Schrödinger
Maestro30 molecular modeling environment basing on the solved
crystal structure of 19, crystallized in an unprotonated form and
described previously.24 Only (S)-isomers of alcohols 5–21 were ta-
ken into account. Compounds were submitted to further analysis
as free bases. For each compound, a conformational search was
then performed using the mixed torsional method as implemented
in MacroModel 9.8 with MMFFs force field and TNCG (Truncated
Newton Conjugate Gradient) method of energy minimization.
The conformational analysis was carried out for aqueous solutions
with continuum solvation treatment (Generalised Born/Solvent
Accessible, GB/SA) and terminated when the root means square
4.1.4.3.
(Z)-5-(4-bromobenzylidene)-3-(2-hydroxy-3-(isopro-
(Z)-5-(4-
pylamino)propyl) imidazolidine-2,4-dione (13).
bromobenzylidene)-3-(oxiran-2-ylmethyl)imidazolidine-2,4-dione
31 and isopropylamine were used to give white crystals of 13. 52%,
mp: 180–182 °C, Rf (IV): 0.26. Anal. Calcd for C16H20N3O3Br: C,
50.26; H, 5.27; N, 10.92. Found: C, 49.86; H, 5.41; N, 10.43. 1H
NMR for hydrochloride of 13 (DMSO)
d
(ppm): 1.19 (d,
(RMS) of conjugate gradient was below 0.05 kJ molÀ1 ÅÀ1
.
J = 3.90 Hz, 3H, CH3), 1.22 (d, J = 3.85 Hz, 3H, C–CH3), 2.48 (qu,
1H, CH-(CH3)2), 3.04–3.10 (m, 1H, CHNH), 3.20–3.35 (m, 1H,
CHNH), 3.40–3.55 (m, 2H, NCH2), 4.09 (s, 1H, CHOH), 5.82 (d,
J = 4.88 Hz, 1H, OH) 6.25 (s, 1H, CH@C), 7.43–7.45 (d, J = 6.70 Hz,
2H, Ar-2,6-H), 7.46–7.48 (d, 2H, J = 6.90 Hz, Ar-4,5-H), 8.40 (s, 1H,
NH), 8.64 (s, 1H, NH+), 10.87 (s, 1H, N1H). IR KBr (cmÀ1): 3440
(OH), 3440 (NH), 1765 (C4@O), 1715 (C2@O), 1595 (Ar).
Found global minimum energy poses of the ligands were super-
imposed by a least-squares method; heavy atoms of hydantoin
fragment (carbon, nitrogen and oxygen atoms) were chosen as fit-
ting points (Fig. 3). The distances between structural fragments of
molecules were measured in Maestro using centroids defined for a
phenyl (AR) and a hydantoin ring (HC). For the graphic presenta-
tion of superimposed lowest energy conformations PyMOL soft-
ware31 was used.
4.1.4.4. (Z)-5-(4-(diethylamino)benzylidene)-3-(2-hydroxy-3-(4-
(2-hydroxyethyl) piperazin-1-yl)propyl)imidazolidine-2,4-dione
4.3. Microbiological assays
(14).
(Z)-5-(4-(diethylamino)benzylidene)-3-(oxiran-2-ylmethyl)-
imidazolidine-2,4-dione 34 and 2-(piperazin-1-yl)ethanol were
used to give white crystals of 14. 37%, mp: 108–110 °C, Rf (IV):
0.23. Anal. Calcd for C23H35N5O4: C, 61.90; H, 7.92; N, 15.72. Found:
To determine minimal inhibitory concentrations (MICs),
approximately 106 cells were inoculated into 1 mL of Mueller-
Hinton broth containing twofold serial dilutions of nalidixic acid

J. Handzlik et al. / Bioorg. Med. Chem. 21 (2013) 135–145
145
Walravens, K.; Langton, K.; Herbert, R. B.; Skurray, R. A.; Paulsen, I. T.; O’Reilly,
J.; Rutherford, N. G.; Brown, M. H.; Bill, R. M.; Henderson, P. J. F. Curr. Drugs
Targets 2006, 7, 793.
or chloramphenicol or sparfloxacin according to the Clinical and
Laboratory Standards Institute (CLSI, http://www.clsi.org/) guide-
lines. Triplicate results were read after 18 h at 37 °C. To study syn-
ergistic activity, different fixed sub-inhibitory concentrations of
the potential inhibitors, determined without antibiotic, were
added during incubation with nalidixic acid or chloramphenicol
or sparfloxacin. In addition, the reference efflux pump modulator,
PAbN, was used at 0.050 mM. Control experiments were carried
out without the different inhibitors. MIC values are means of three
independent determinations.
9. Handzlik, J.; Szyman´ ska, E.; Chevalier, J.; Otre˛bska, E.; Kiec´-Kononowicz, K.;
Pagès, J.-M.; Alibert, S. Eur. J. Med. Chem. 2011, 46, 5807.
10. Gulshan, K.; Moye-Rowley, S. Eucariot. Cell 1933, 2007, 6.
11. Lomovskaya, O.; Zgurskaya, H. I.; Totrov, M.; Watkins, W. J. Nat. Rev. Drug Disc.
2007, 6, 56.
12. Nobili, S.; Landini, I.; Giglioni, B.; Mini, E. Curr. Drugs Targets 2006, 7, 861.
13. Teodori, E.; Dei, S.; Scapecchi, S.; Gualtieri, F. Farmaco 2002, 57, 385.
14. Handzlik, J.; Macia˛g, D.; Kubacka, M.; Mogilski, S.; Filipek, B.; Stadnicka, K.;
´
Kiec-Kononowicz, K. Bioorg. Med. Chem. 2008, 16, 5982.
15. Handzlik, J.; Szyman´ ska, E.; Ne˛dza, K.; Kubacka, M.; Siwek, A.; Mogilski, S.;
´
Handzlik, J.; Filipek, B.; Kiec-Kononowicz, K. Bioorg. Med. Chem. 2011, 19,
1349.
Acknowledgements
16. Handzlik, J.; Pertz, H. H.; Gornemann, T.; Jahnichen, S.; Kiec´-Kononowicz, K.
Bioorg. Med. Chem. Lett. 2010, 20, 6152.
17. Kiec´-Kononowicz, K.; Stadnicka, K.; Mitka, A.; Pe˛kala, E.; Filipek, B.; Sapa, J.;
Zygmunt, M. Eur. J. Med. Chem. 2003, 38, 555.
18. Handzlik, J.; Bajda, M.; Zygmunt, M.; Macia˛g, D.; Dybała, M.; Bednarski, M.;
Partly supported by grants: 501/N-COST/2009/0 (K/PMN/
000031), COST action BM0701, Polonium 8127/2010 and ANR-
11-BS07–019–02 ‘IBEF’.
´
Filipek, B.; Malawska, B.; Kiec-Kononowicz, K. Bioorg. Med. Chem. 2012, 20,
2290.
19. Pe˛kala, E.; Stadnicka, K.; Broda, A.; Zygmunt, M.; Filipek, B.; Kiec´-Kononowicz,
Supplementary data
K. Eur. J. Med. Chem. 2005, 40, 259.
20. Kiec´-Kononowicz, K.; Szyman´ ska, E.; Motyl, M.; Holzer, W.; Białecka, A.;
Kasprowicz, A. Pharmazie 1998, 53, 680.
21. Szyman´ ska, E.; Kiec´-Kononowicz, K.; Białecka, A.; Kasprowicz, A. K. Farmaco
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.10.053.
2002, 57, 39.
´
´
22. Szymanska, E.; Kiec-Kononowicz, K. Farmaco 2002, 57, 355.
23. Subtelna, I.; Atamanyuk, D.; Szyman´ ska, E.; Kiec´-Kononowicz, K.;
Zimenkovsky, B.; Vasylenko, O.; Gzella, A.; Lesyk, R. Bioorg. Med. Chem. 2010,
18, 5090.
24. Handzlik, J.; Szyman´ ska, E.; Wójcik, R.; Dela, A.; Jastrze˛bska-Wie˛sek, M.;
Karolak-Wojciechowska, J.; Fruzin´ ski, A.; Siwek, A.; Filipek, B.; Kiec´-
Kononowicz, K. Bioorg. Med. Chem. 2012, 20, 4245.
25. Marton, J.; Enisz, J.; Hosztafi, S.; Timar, T. J. Agric. Food Chem. 1993, 41, 148.
26. Sau-Fun, T. J. Phys. Org. Chem. 1990, 3, 559.
27. Tan, S.-A.; Ang, K.-P.; Fong, Y.-F. J. Chem. Soc., Perkin Trans. 2 1986, 1941.
28. Wojciechowska-Karolak, J.; Szyman´ ska, E.; Mrozek, A.; Kiec´-Kononowicz, K. J.
Mol. Struct. 2009, 930, 126.
References and notes
1. Chopra, I.; Schofield, C.; Everett, M.; O’Neill, A.; Miller, K.; Wilcox, M.; Frère, J.
M.; Dawson, J. M.; Czaplewski, L.; Urleb, U.; Courvalin, P. Lancet Infect. Dis.
2008, 8, 133.
2. Mahamoud, A.; Chevalier, J.; Davin-Régli, A.; Barbe, J.; Pagès, J.-M. Curr. Drugs
Targets 2006, 7, 843.
3. Pagès, J.-M.; Amaral, L. Biochim. Biophys. Acta 2009, 1794, 826.
4. Pagès, J. M.; Alibert-Franco, S.; Mahamoud, A.; Bolla, J. M.; Davin-Régli, A.;
Chevalier, J.; Garnotel, E. Curr. Top. Med. Chem. 1848, 2010, 8.
5. Bolla, J.-M.; Alibert-Franco, S.; Handzlik, J.; Chevalier, J.; Mahamoud, A.; Boyer,
´
´
´
29. Wojciechowska-Karolak, J.; Szymanska, E.; Fruzinski, A.; Kiec-Kononowicz, K. J.
Mol. Struct. 2010, 966, 14.
´
G.; Kiec-Kononowicz, K.; Pagès, J.-M. FEBS Lett. 2011, 585, 1682.
6. Pagès, J.-M.; Amaral, L.; Fanning, S. Curr. Med. Chem. 2011, 18, 2969.
7. Nikaido, H.; Takatsuka, Y. Biochim. Biophys. Acta 2009, 1794, 769.
8. Saidijam, M.; Benedetti, G.; Ren, Q.; Xu, Z.; Hoyle, C. J.; Palmer, S. L.; Ward, A.;
Bettaney, K. E.; Szakonyi, G.; Meuller, J.; Morrison, S.; Pos, M. K.; Butaye, P.;
30. Maestro v. 9.0, MacroModel v.9.7, Glide v.5.5; Schrödinger, LLC., New York, NY,
2009.
31. PyMOL Molecular Graphics System v. 0.99, DeLano Scientific.