Amine-alkyl derivatives of hydantoin: New tool to combat resistant bacteria

Article abstract of DOI:10.1016/j.ejmech.2011.09.032

A series of new 5,5-diphenylhydantoin derivatives with various amine-alkyl terminal fragments at N1-position were synthesized. Then a series of twenty-eight compounds with the same hydantoin scaffold were evaluated for their potency to combat bacterial MultiDrug Resistance (MDR). Intrinsic antibacterial activities were first evaluated. As these compounds showed no direct activity on bacteria, their influence on minimal inhibitory concentration (MIC) of nalidixic acid was tested in two strains of Enterobacter aerogenes: the reference-strain ATCC-13048 and the CM-64 strain which over-produces AcrAB-TolC efflux pump. The compounds showed moderate- or low- anti-MDR properties. According to SAR-studies, hit compounds containing 2-methoxyphenylpiperazine at N1-terminal fragment and methylcarboxyl acid one at N3-position of hydantoin have been identified for further microbiological studies and pharmacomodulations to develop efflux pump inhibitors.

Full text of DOI:10.1016/j.ejmech.2011.09.032

European Journal of Medicinal Chemistry 46 (2011) 5807e5816
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Amineealkyl derivatives of hydantoin: New tool to combat resistant bacteria
,
c
a
b
a
Jadwiga Handzlik a , Ewa Szymanska , Jacqueline Chevalier , Ewa Otre˛bska ,
ꢀ
Katarzyna Kiec-Kononowicz c, Jean-Marie Pagès b c, Sandrine Alibert b
a,
,
,c,
*
ꢀ
a Department of Technology and Biotechnology of Drugs, Jagiellonian University-Medical College, Medyczna 9, PL 30-688 Kraków, Poland
b UMR-MD-1, 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:
Received 3 May 2011
Received in revised form
20 September 2011
Accepted 20 September 2011
Available online 1 October 2011
A series of new 5,5-diphenylhydantoin derivatives with various amineealkyl terminal fragments at N1-
position were synthesized. Then a series of twenty-eight compounds with the same hydantoin scaffold
were evaluated for their potency to combat bacterial MultiDrug Resistance (MDR). Intrinsic antibacterial
activities were first evaluated. As these compounds showed no direct activity on bacteria, their influence
on minimal inhibitory concentration (MIC) of nalidixic acid was tested in two strains of Enterobacter
aerogenes: the reference-strain ATCC-13048 and the CM-64 strain which over-produces AcrAB-TolC efflux
pump. The compounds showed moderate- or low- anti-MDR properties. According to SAR-studies, hit
compounds containing 2-methoxyphenylpiperazine at N1-terminal fragment and methylcarboxyl acid
one at N3-position of hydantoin have been identified for further microbiological studies and pharma-
comodulations to develop efflux pump inhibitors.
Keywords:
Hydantoin derivatives
Chemosensitizer
Antibiotic resistance
Efflux pumps
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
strategies to circumvent the bacterial resistance mechanism are
described to restore an efficient intracellular concentration of anti-
Multidrug resistant (MDR) bacteria are a serious health problem
due to the associated failure of the therapy of infectious diseases [1,2].
OneofthemostimportantandwidespreadMDRmechanismbaseson
bacterial efflux proteins that expel all antibiotic classes out of the cell
[3,4]. Gram-negative bacteria provide efficient active expulsion of
antibiotic molecules across the membranes and periplasm using
energy-potential of membrane [3e6]. Theycontribute in both natural
insensitivity to antibiotics and high levels of antibiotics resistance by
acquiring additional mechanisms. Efflux pumps have been proposed
as a potential target in order to restore the activity of antibiotics that
are expelled by efflux pumps [7]. In the group of MDR Gram-negative
bacteria, Enterobacter aerogenes is one of the frequently described
pathogens, responsible for various hospital-acquired infections
[4,8,9]. In the case of E. aerogenes such as many Enterobacteriaceae
involved in infectious diseases, tripartite efflux pump AcrAB-TolC is
the main efflux system which can extrude a large variety of cytotoxic
substances from the cell membrane directly into the medium
[4,10,11]. According to the recent lines of evidence, three main
biotics: (1) By-passingeffluxactivity: improvingthemoleculardesign
of old antibiotics to reduce their efflux; (2) direct action on the
membrane barrier: increasing the permeability of the bacterial cell
envelope and drug uptake; (3) Blocking the efflux ability of bacterial
cell: altering pump function [12]. Concerning the third strategy,
a search for new chemical compounds with efflux pump inhibitor
(EPI) properties is a current topic in medicinal chemistry. Within the
search for new EPIs, some chemical groups of active compounds were
identifiedthatcontainring-orchainamineealkylfragmentslinked to
various aromatic moieties. Structure analysis of some potent EPIs
indicated that piperazine- (NMP, NBP, Fig. 1), primary amine-, or
guanidine fragment (PA
bN) is profitable for the activity (Fig. 1).
Among others, compound PAbN, a derivative of arginine, is a well-
known efflux pump inhibitor used in various microbiological tests
as a reference compound [7].
Our previous studies were focused on various chemical modi-
fications of hydantoin to give new GPCR-agents [13e15],
compounds with antiarrhythmic- and/or anticonvulsant activity
[16e19] as well as antibacterial agents with some syndrome to
inhibit drug resistance in mycobacterial cells [20,21]. Based on
interesting structures and pharmacological properties of the
5-aromatic-hydantoin derivatives, we decided to test this chemical
group for their potency to combat MDR. This work describes the
* Corresponding author. TMCD2-UMR-MD1, Laboratoire de Chimie Thérapeu-
tique, Faculté de Pharmacie, 27 Bd Jean Moulin, 13385 Marseille cedex 05, France.
Tel.: þ33 491 835 538; fax: þ33 491 324 606.
E-mail address: sandrine.alibert@univmed.fr (S. Alibert).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.032

5808
J. Handzlik et al. / European Journal of Medicinal Chemistry 46 (2011) 5807e5816
H2N
H
N
O
NH2
NH
HN
H
NH
N
N
NH
N
O
NBP
NMP
PAβN
Fig. 1. Structure of some potent efflux pump inhibitors.
first step of the studies focused on 5,5-diphenylhydantoin deriva-
tives. The research was divided into two stages. In the first stage
synthesis and microbiological tests were carried out for the first
generation of hydantoin derivatives 1e11 including compounds
with N3-alkyl-, ester-, acid- or free hydrogen as well as tertiary
amine: phenylpiperazine terminal fragment at N1-position
(Table 1). According to the first results obtained, further chemical
modifications of the 5,5-diphenylhydantoin derivatives 1e11 were
performed by an exchange of N1-phenylpiperazine terminal frag-
ment into primary, secondary- or tertiary amines, including
heterocyclic- or non-aromatic piperazines as well as tri-
azabicyclodecene moiety (Table 2). Syntheses of new compounds
12e28, microbiological tests using strains of E. aerogenes as well as
structureeactivity relationship studies were investigated.
The latter alkylation was performed in acetone with potassium
carbonate as a base and benzyltriethylammonium chloride (TEBA)
acting as a phase transfer catalyst. An excess (30e50%) of the
adequate dibromoalkane allows avoiding dimerization, a result of
two-bromide substitutions. Long-term stirring (90e120 h) at room
temperature gave satisfactory results. These mild conditions seem
to minimize side nucleophilic substitutions that were noted during
the pilot-reactions with boiling temperature. Bromides 33e40
were the starting materials for a number of nucleophilic substitu-
tion reactions that allowed preparing the target compounds
(Schemes 2, 3).
A series of phenylpiperazine derivatives (1e3, 5, 6, 9) were
prepared using synthetic pathway described in Scheme 2. Phenyl-
piperazine derivative 1 was synthesized within two-step process.
In the first step bromoalkyletrityl derivative of hydantoin 40
reacted with phenylpiperazine to give 41, which was next cleaved
from trityl group in acidic conditions using of 90% trifluoroacetic
acid (TFA) in methylene chloride to give 1. Phenyl-
piperazineehydantoin derivative with N3-carboxylic terminal
fragment 9 was obtained by basic hydrolysis of ester 42, performed
in aquaemethanol conditions.
Synthesis of ethoxycarbonylpiperazine derivatives 16, 18e20
was performed as two-phase alkylation of commercial ethyl
piperazine-1-carboxylate with N1-bromoalkylhydantoin deriva-
tives 36e39 (Scheme 3). Compound 17, a 3-hydantoinacetic acid
derivative, was synthesized from corresponding ester 16 by basic
hydrolysis carried out in similar conditions to those used to obtain
9. In case of free-piperazine derivative 21, synthesis came up
against difficulties due to presence of two amine groups in piper-
azine with equal sensitivity for alkylating agents. At first, a number
of pilot-syntheses was performed to obtain free-piperazine deriv-
atives by cleavage of ethoxycarbonyl group of compounds 16,
18e20. Deprotections in both, basic and acidic conditions, did not
give the target compounds. In basic conditions, the protecting
group was very stable even in case of long term heating with
concentrated KOH (6 M). Similar observation was noted in case of
acidic hydrolysis with various concentrations of HCl. Acidic
deprotection with TFA damaged hydantoin ring giving undesirable
products. The best result gave two-phase alkylation of “free”
piperazine by use of bromoalkyl derivative 39. The reaction
(Scheme 3) was performed in excess of piperazine (3 eq) allowed to
obtain pure compound 21.
Substitution of bromides 35e39 with the secondary amine 1,5,7-
triazabicyclo[4.4.0]dec-5-ene as well as potassium phthalimide was
performed in acetone at 50e60 ꢀC, in the presence of K2CO3 and
TBAB (Scheme 3). In the case of compounds 22e25, refluxconditions
led after several hours to expected products of substitution with
simultaneous hydrolysis of the ester group. In the case of 26e28,
Gabriel method [22] for primary amines synthesis was applied.
Intermediate phthalimides were treated with hydrazine mono-
hydrate in anhydrous ethanol to give 26e28 obtained as free bases.
2. Chemistry
Synthetic pathways to obtain the new hydantoin derivatives,
belonging to first (1e11) or second generation (12e28) are illus-
trated on the Schemes 1e4. Synthesis of starting hydantoins 30, 31
and 33e40 from 5,5-diphenylhydantoin (29) (Scheme 1) has been
performed according to described literature procedures [13,15,16].
Alkylation of the imidazole ring at N3-position gave methyl (30),
ester (31) or trityl (40) derivatives (Scheme 1), subsequently alky-
lated further, this time at N1-position, which allowed to obtain the
intermediate bromides (33e40) with different number of methy-
lene groups n ¼ 3, 4, 5, 6 and 8 as linkers.
Table 1
Generation I: phenylpiperazine derivatives of 5,5-diphenylhydantoin 1e11.
Cpd
R1
R2
R3
m
1
2
3
4
5
6
7
8
H
CH3
CH3
CH3
CH2COOCH3
CH2COOCH3
CH2COOCH3
CH2COOH
CH2COOH
CH3
H
H
H
OH
H
H
OH
OH
H
H
2
3
1
1
1
2
1
1
2
1
1
2-OCH3
H
H
H
H
2-OCH3
2-OCH3
H
9
10
11
H
OCOCH3
OCOCH3
CH2COOCH3
H

J. Handzlik et al. / European Journal of Medicinal Chemistry 46 (2011) 5807e5816
5809
Table 2
Generation II: 1-aminealkyl derivatives of 5,5-diphenylhydantoin 12e28.
Cpd
R1
R2
amine
n
1
Cpd
R1
R2
amine
m
6
O
O
C2H5O
12
CH3
OH
20
CH2COOCH3
N
N
O
N
N
HN
N
13
14
CH3
OH
OH
1
1
21
22
CH2COOCH3
CH2COOH
6
1
N
N
N
N
N
N
CH2COOCH3
H
N
N
N
CH3
NH
N
N
N
N
15
16
CH2COOCH3
CH2COOCH3
OH
H
1
2
23
24
CH2COOH
CH2COOH
H
H
2
3
H3C
N
N
O
C2H5O
N
N
O
C2H5O
N
N
17
CH2COOH
H
2
25
26
27
CH2COOH
CH2COOH
CH2COOH
H
H
H
4
1
2
N
N
N
eNH2
O
C2H5O
C2H5O
18
19
CH2COOCH3
CH2COOCH3
H
H
3
4
eNH2
N
N
N
O
28
CH2COOH
H
eNH2
4
N
Synthesis of hydantoin derivatives with branched propyl linker
(4, 7, 10e15) is shown in Scheme 4. 5,5-diphenylhydantoin alky-
lated at 3 position with methyl- (30) or methyl acetate (31) reacted
first with 2,3-epichlorhydrine [16] to obtain the corresponding
epoxide intermediates, which were cleaved by secondary amines
(Scheme 4) giving 2-hydroxypropylamine derivatives of hydantoin
(4, 7, 12e15, 43). Compound 8 was obtained by basic hydrolysis of
43 [15]. Acylation of the hydroxyl group of compounds 4 and 7 with
acetic anhydride (Scheme 4) yielded products 10b and 11b that
were converted into corresponding hydrochlorides 10 and 11.
Ph
Ph
Ph
O
O
O
Ph
Ph
Ph
R1X, i
ii
HN
NH
HN
N
N
N
Br
R1
R1
( )n
O
O
O
33, 34: R1 = CH3, n= 3, 5
29 (DPH)
30: R1 = CH3
35-39: R1 = CH2CO2Me, n= 3, 4, 5, 6, 8
40:
31: R1 = CH2CO2Me
32: R1 = Trityl
R1 = Trityl, n= 4
Scheme 1. Synthesis of intermediates 30e40: (i) conditions described earlier [13,15,16]; (ii) dibromoalkane, TEBA, K2CO3, acetone, rt.

5810
J. Handzlik et al. / European Journal of Medicinal Chemistry 46 (2011) 5807e5816
R3
i
O
Ph
Ph
N
N
N
N
( )n
CH3
O
O
2: R3 = H, n= 5
3: R3 = 2-OCH3, n= 3
3
Ph
R
O
R3
OCH3
N
N
NH
Ph
O
Ph
i
O
Ph
ii
Ph
Ph
N
N
N
R3 = H, 2-OCH3
Br
R1
( )n
N
N
N
N
N
N
( )4
CH2CO2H
( )n
CH2CO2Me
O
5: R3=H, n= 3
6: R3=H, n= 4
O
9
33-34: R1 = CH3, n= 3, 5
35-39: R1 = CH2CO2Me, n= 3, 4, 5, 6, 8
40:
R1 = Trityl, n= 4
42: R3=OCH3, n=4
O
Ph
O
Ph
iii
i
Ph
Ph
N
N
N
N
NH
N
N
N
( )4
( )4
Trityl
O
O
41
1
Scheme 2. (i) TEBA, K2CO3, acetone, 40e50 ꢀC; (ii) KOH, MeOH/H2O; (iii) 90% TFA, CH2Cl2, rt.
3. Results and discussion
with resistant Gram-negative bacteria E. aerogenes. Two following
strains of E. aerogenes were used: reference-strain, ATCC 13048 and
the derivative strain CM-64, which over-produces the AcrAB-TolC
efflux pump. Results are shown in Table 3. Most of the tested
compounds exhibited low intrinsic antibacterial activity except
compound 9 (0.0625 mM). However, these results show an increase
3.1. Intrinsic anti-bacterial activity
Compounds 1e28 were evaluated in vitro for their antibacterial
activity by determining the minimum inhibitory concentration (MIC)
Ph
O
Ph
HN
N
N
N
CH2CO2Me
( )8
Ph
O
O
Ph
N
O
21
N
N
( )n
O
CH2CO2Me
iii
HN
NH
O
O
H2NNH2
NH
ii
44-46
O
Ph
O
N
Ph
O
Ph
i
O
Ph
Ph
N
N
H
Ph
N
N
N
N
( )n
N
N
CH2CO2H
H2N
N
N
Br
CH2CO2H
i
( )n
CH2CO2Me
( )n
N
O
O
O
22: n= 3
23: n= 4
24: n= 5
25: n= 6
35-39: n= 3, 4, 5, 6, 8
44, 26: n= 3
26-28
45, 27: n= 4
46, 28: n= 6
iv
HN
N
CO2Et
Ph
Ph
O
Ph
O
EtO2C
Ph
EtO2C
v
N
N
N
N
N
N
N
CH2CO2H
( )4
N
CH2CO2Me
( )n
O
O
16: n=4
17
18: n= 5
19: n= 6
20: n= 8
Scheme 3. (i) TBAB, K2CO3, acetone, 60 ꢀC; (ii) ethanol, reflux; (iii) TEBA, K2CO3, acetone, 40e50 ꢀC; (iv) K2CO3, acetone, 40e50 ꢀC; (v) KOH, MeOH/H2O.

J. Handzlik et al. / European Journal of Medicinal Chemistry 46 (2011) 5807e5816
5811
Cl
1.
Cl
O
1.
2.
O
O
ii
Ph
Ph
O
O
O
O
Ph
O
N
2.
Ph
OH
Ph
NH
Ph
OH
2
O
NH
N
H
N
N
N
HN
N
N
N
N
CH2CO2Me
R1
i
CH3
O
O
O
15
12
30: R1 = CH3
31: R1 = CH2CO2Me
Ph
O
Ac
Cl
Ph
1.
O
N
O
x HCl
N
N
N
3
II
R
R1
2.
N
NH
X
10, 11
O
R3 = H, 2-OCH3
X= C, N
HCl
OCH3
N
Ph
Ph
Ph
O
O
R3
O
iv
Ph
OH
Ac
Ph
Ph
iii
X
N
O
N
OH
N
N
N
N
N
N
N
N
N
R1
R1
CH2COOH
O
10b, 11b
O
O
8
10: R1 = CH3
11: R1 = CH2CO2Me
4: R1 = CH3, R3 = H, X= C
7: R1 = CH2CO2Me, R3 = H, X= C
13: R1 = CH3, R3 = H, X= N
14: R1 = CH2CO2Me, R3 = H, X= N
43: R1 = CH2CO2Me, R3 = 2-OCH3, X= C
Scheme 4. (i) 1. TEBA, K2CO3, acetone, 60 ꢀC; 2. EtOH, reflux [17]; (ii)1. TEBA, K2CO3, acetone, rt; 2. mv-irradiation, solvent-free [13,16,17]; (iii) (CH3CO)2O, reflux; (iv) OHꢁ/H2O [15].
MIC for compounds 8 and 9 in CM-64 strain. This increase is probably
due to the over-expressed efflux pump and therefore suggests that
these compounds can be recognized and transported by AcrAB-TolC
efflux pump. But the difference in activity between the two strains
is less important than that of nalidixic acid. This suggests a lower
affinity of hydantoin derivatives for the targeted efflux pumps.
A similar range of susceptibility values was obtained for the two
strains. Most of the hydantoins had a mean value of 1.25 mM. The low
intrinsic antibacterial activity of these molecules allowed us to analyze
theireffecton the nalidixicacid susceptibilityof the MDRstrain CM-64.
as efflux pump substrate [3,6,7]. PA
and CM-64 strains) was used as a reference EPI according to
previous studies [23] at the concentration of 50 M. The first
generation of compounds 1e11 was tested in the concentration
range 62.5e333 M (Fig. 2) according to the intrinsic antibacterial
activity of each compound. Compounds of second generation
12e28 were tested in the concentration corresponding to the best
first generation chemosensitizers (62.5 mM) to facilitate compar-
ison. Anti-MDR activities of the compounds were expressed by
activity gain A on nalidixic acid susceptibility, calculated as MIC of
nalidixic acid, evaluated in absence of compound, divided by the
MIC of the antibiotic in presence of the tested compound (Fig. 2).
Generally, thecompounds showed moderateorweakpotentiating
properties, significantly lower than those obtained for strong refer-
ence effluxpump inhibitor, PAbN. Themostactivecompounds8 and9
showed significant 4-fold decrease of MIC of nalidixic acid in MDR
strain CM-64 and 2-fold MIC-decrease in reference-strain (ATCC
b
N (MIC ¼ 5 mM on ATCC 13048
m
m
3.2. Influence of hydantoins on nalidixic acid MIC
Efflux pump inhibitory properties were also evaluated in the
two strains of E. aerogenes ATCC 13048 and CM-64. To study the
possible synergistic activity, the potential inhibitors were added
during incubation with nalidixic acid that has been well described
13048)atthelowestdoseof62.5
mM(Fig. 2a). Allcompoundstestedat
Table 3
100 M (1e6 and 10) had no effect on MIC of nalidixic acid in the
m
Intrinsic antibacterial activities for alkylamine derivatives of hydantoin 1e28, tested
in two strains of E. aerogenes, ATCC 13048 (reference) and the derivative strain CM-
64 (over-producing AcrAB-TolC).
reference-strain. In this group, compounds 2 and 6 were the most
active against MDR causing 4-fold-decrease of MIC of the antibiotic in
the strain, which over-expresses AcrAB-TolC (Fig. 2b). In case of
compound 7, a decrease of MIC of nalidixic acid (4-fold, CM-64) was
Cpd
MIC [mM] EA ATCC 13048
MIC [mM] EA CM-64
1e6
7
8
>0.5
5
>0.5
5
observed at higher concentration of 333
mM. Compounds 12e28,
investigated at 62.5 M, did not significantly influence MIC of nali-
m
0.125
0.0625
>0.5
0.25
>1
>5
0.125
1.25
0.25
0.25
>0.5
0.25
>1
>5
0.125
1.25
9
dixic acid neither in ATCC 13048 reference-strain nor in CM-64 one.
10
11
12
14
15
16e28
3.3. Structureeactivity relationships
Although the tested hydantoin derivatives did not show high
chemosensitizing potency, results allowed identify some structural

5812
J. Handzlik et al. / European Journal of Medicinal Chemistry 46 (2011) 5807e5816
AcrAB-TolC but did not influence the MIC in reference-strains.
These derivatives possess N3-carboxylic terminal fragment and
2-methoxyphenylpiperazine functional group in N1 position. They
differ only within length and properties of alkyl linker between
hydantoin and piperazine. As these differences are minor (n ¼ 1 or
2, presence or not of hydroxyl group), it does not seem to affect
activity. Other compounds with 2-methoxyphenylpiperazine
fragment did not potentiate antibiotic activity in CM-64 or
decrease nalidixic acid MIC at higher concentrations (Fig. 2).
Comparing structures of the generation 1 compounds, the main
difference is shown in case of N3-substituent. Compounds with
N3eH (1) and N3-ester fragment (5e7 and 11) showed various
activities. The compounds displayed chemosensitizing activity
lower than that of compounds possessing carboxylic group (8, 9).
The best of them decreased MIC of nalidixic acid at higher doses:
100
mM (6) and 333 mM (7). Basing on this observation, the
substituent CH2COOH at N3 seems to be a structural factor
improving potentiating-properties. The influence of N3-
substituents on decrease of MIC of nalidixic acid in CM-64 is as
follows: CH2COOH > CH2COOCH3 ¼ CH3 > H.
Results obtained for first generation 1e11 revealed two most
promising chemosensitizers (8 and 9), which contain methyl-
carboxylic acid N3-substituent, and 2-methoxyphenylpiperazine.
The role of these two fragments was investigated in the next
studies of the second generation of hydantoin derivatives
(12e28). In this context, phenyl ring of phenylpiperazine moiety
was replaced by 2-furoyl (12), 2-pyridil- (13, 14), ethoxycarbonyl
(16e20) or free hydrogen (21) as well as phenylpiperazine was
exchanged into primary amine (26e28), isopropylamine (15) or
triazabicyclodecene fragment (22e25) in order to understand the
impact of the change of pharmacophore features. This new
generation of compounds possesses either N3-carboxylic
terminal fragment (17, 22e28) or the corresponding methyl
ester (14e16, 18e21) (Table 2). Results demonstrated that no
compound of the second generation (12e28) had better
chemosensitizing-properties than that of compounds 8 and 9 but
some structureeactivity relationship conclusions can be done.
This second part of the study confirms the importance of the 2-
methoxyphenyl substituent of piperazine. Its replacement by
non-aromatic functional group or an aromatic ring with a weaker
electronic density as pyridyl one led to decrease of activity gain.
Moreover, the nature of the amine group seems also to be
important. Indeed, the replacement of piperazine by primary
amine or triazabicyclodecene groups which have stronger basic
properties decrease activity gain of the chemosensitizers. Higher
basic properties lead to higher ionization of molecules, which
could affect either the interaction with the putative target
of chemosensitizers and/or the crossing through bacterial
membranes.
Fig. 2. The influence of the compounds 1e28 on MIC of nalidixic acid tested in two
strains of E. aerogenes, ATCC 13048 (reference) and CM-64 (with over-production of
AcrAB-TolC). NAL- nalidixic acid; MIC-minimal inhibitory concentration; MIC of NAL in
4. Conclusion
ATCC 13048 ¼ 8
m
g/mL; MIC of NAL in CM-64 ¼ 128/64
m
g/mL Activity gains (A) for
reference strong EPI, PA
(CM-64).
bN, at dose of 50 mg/mL, were as follows: 64 (ATCC 13048), 128
The chemosensitizing activity of 28 5,5-diphenylhydantoin
derivatives were evaluated in MDR bacterial cells which over-
produce AcrAB-TolC efflux pump in order to investigate the possi-
bility to use these compounds as EPIs. This study shows that this
series of compounds potentiates nalidixic acid activity at rather low
concentrations. Two hits and four pharmacophoric groups have
been identified as substrates of efflux pumps. However, the affinity
for the efflux target is not sufficiently higher to reverse totally the
resistance to nalidixic acid. New chemical modifications are under
consideration to improve the affinity to the target: a change in the
position of pharmacophoric groups on the hydantoin scaffold is
particularly investigated.
properties profitable for the EPI activity targeted. In the first
generation of hydantoin derivatives 1e11, 5,5-diphenyhydantoin
as well as N1-phenylpiperazine terminated fragments are fixed
structural properties for all investigated compounds. Chemical
modifications were performed for substituents in N3-position and
for alkyl linker between N1 and piperazine. Furthermore, modifi-
cations including electro-donor substituents at phenylpiperazine
ring were investigated. The most promising compounds 8 and 9
decreased MIC of nalidixic acid in strains with over-production of

J. Handzlik et al. / European Journal of Medicinal Chemistry 46 (2011) 5807e5816
5813
5. Experimental
5.1.1.4. Methyl 2-(3-(5-bromopentyl)-2,5-dioxo-4,4-diphenylimida-
zolidin-1-yl)acetate (37). Ester 31 (9.72 g), 1,5-dibromopentane
(39 mmol, 8.95 g) were used. Reactants were stirred for 134 h.
White crystals of 37 (8.5 g, 18 mmol, 59%) m.p. 114e115 ꢀC, Rf
(I):0.58. Anal. Calcd. for C23H25BrN2O4 %: C, 58.36; H,. 5.32; N, 5.92.
5.1. Chemistry
NMR spectra were recorded on following apparatus: a Varian
Mercury VX 300 MHz PFG instrument (Varian Inc., Palo Alto, CA,
USA) in [d6]-DMSO at ambient temperature using the solvent signal
as an internal standard (300 MHz for 1H and 75 MHz for 13C) and on
a Brucker ARX 200 MHz spectrometer with tetramethylsilane
(TMS) as internal reference; chemical shifts are given on the
Found: C, 58.62; H, 5.39; N, 5.88. 1H NMR (DMSO-d6)
d [ppm]:
0.71e0.76 (qu def., 2H, BreC2H4eCH2), 0.97e1.07 (qu def., 2H,
N1eCH2eCH2), 1.42e1.51 (qu def., 2H, BreCH2eCH2), 3.24e3.28 (t,
J ¼ 6.67 Hz, 2H, N1eCH2), 3.31 (t def., 2H, BreCH2), 3.70 (s, 3H,
OCH3), 4.33 (s, 2H, N3eCH2), 7.27e7.30 (m, 4H, 2ꢃ Ph-3,5-H),
7.44e7.46 (m, 6H, 2ꢃ Ph-2,4,6-H). 13C NMR (75 MHz, DMSO-d6)
d
(ppm) scale with J values in Hertz. Thin-layer chromatography
was performed on pre-coated Merck silica gel 60 F254 aluminum
sheets, the used solvent systems were: (I) toluene/acetone 40:3; (II)
toluene/acetone/methanol 15:5:1; (III) toluene/acetone/methanol
5:5:1; (IV) methylene chloride/acetone 10:2, (V) methylene chlo-
ride/methanol 1:1. Melting points were determined using Mel-
Temp II apparatus and are uncorrected. Elemental analyses were
within ꢂ0.4% of the theoretical values unless stated otherwise.
Syntheses and chemical characteristics for compounds 2e8, 12e15,
30e34, 42 and 43 were described earlier [13,15e17].
d [ppm]: 24.9, 26.9, 31.9, 34.9, 41.5, 41.6, 53.0, 74.9, 128.6, 129.3,
129.4, 137.2, 154.6, 168.4, 173.3.
5.1.1.5. Methyl 2-(3-(6-bromohexyl)-2,5-dioxo-4,4-diphenylimidazo-
lidin-1-yl)acetate (38). Ester 31 (9.72 g), 1,6-dibromohexane
(39 mmol, 9.51 g) were used. Reactants were stirred for 48 h.
White crystals of 38 (8.15 g, 16.72 mmol, 56%) m.p. 119e120 ꢀC, Rf
(I):0.66. Anal. Calcd. for C24H27BrN2O4 %: C, 59.14; H,. 5.58; N, 5.75.
Found: C, 59.25; H, 5.61; N, 5.70. 1H NMR (DMSO-d6)
d [ppm]:
0.69e0.77 (qu def., 2H, N1eC2H4eCH2), 0.85e0.95 (qu def., 2H,
BreC2H4eCH2), 1.00e1.07 (qu def., 2H, N1eCH2eCH2), 1.46e1.55
(qu def., 2H, BreCH2eCH2), 3.23e3.31 (m, 2H, N1eCH2), 3.35e3.39
(t def., 2H, BreCH2), 3.70 ( s, 3H, OCH3), 4.33 (s, 2H, N3eCH2),
7.26e7.29 (m, 4H, 2ꢃ Ph-3,5-H), 7.44e7.46 (m, 6H, 2ꢃ Ph-2,4,6-H).
5.1.1. General procedure for bromoalkyl derivatives (35e40)
A mixture of the appropriate N3-substituted derivative of 5,5-
diphenylhydantoin 31 or 32 (30 mmol), TEBA (3.96 mmol, 0.9 g)
and potassium carbonate (87 mmol, 12 g) in acetone (60 mL) was
stirred under reflux for 1 h, then an appropriate dibromoalkane
(36e42 mmol, 1.2e1.4 eq) in acetone (30 mL) was added. The
mixture was stirred at room temperature for 45e150 h, according
to a progress controlled by TLC (I). Then, inorganic precipitate was
filtered off, the mother liquor was evaporated. The residue was
crystallized with methanol.
5.1.1.6. Methyl 2-(3-(8-bromooctyl)-2,5-dioxo-4,4-diphenylimidaz-
olidin-1-yl)acetate (39). Ester 31 (9.72 g), 1,8-dibromooctane
(39 mmol, 10.61 g) were used. Reactants were stirred for 48 h.
White crystals of 39 (7.25 g, 14.07 mmol, 47%) m.p. 74e75 ꢀC, Rf
(I):0.66. Anal. Calcd. for C26H31BrN2O4 %: C, 60.58; H, 6.06; N,
5.43. Found: C, 59.96; H, 5.97; N, 5.50. 1H NMR (DMSO-d6)
5.1.1.1. 1-(4-bromobutyl)-5,5-diphenyl-3-tritylimidazolidine-2,4-dione
(40). 5,5-diphenyl-3-tritylhydantoin 32 (15.1 g), 1,4-dibromopropane
(36 mmol, 7.78 g) were used. Reactants were stirred for 85 h. White
crystals of 40 (12.24 g, 19.3 mmol, 64%) m.p. 134e135 ꢀC, Rf (I):0.78.
Anal. Calcd. for C38H33BrN2O2 %: C, 72.49; H, 5.28; N, 4.45. Found: C,
d
[ppm]: 0.71 (qu def, 2H, N1eC3H6eCH2), 0.88e0.99 (m, 6H,
BreC2H4eCH2eCH2eCH2eCH2), 1.16e1.26 (qu def., 2H,
N1eCH2eCH2), 1.64e1.73 (qu def., 2H, BreCH2eCH2), 3.24e3.29
(t def., 2H, N1eCH2), 3.44e3.48 (t, J ¼ 6.67 Hz, 2H, BreCH2), 3.70
( s, 3H, OCH3), 4.33 (s, 2H, N3eCH2), 7.26e7.29 (m, 4H, 2ꢃ Ph-
3,5-H), 7.43e7.46 (m, 6H, 2ꢃ Ph-2,4,6-H).
72.65.; H, 5.24; N, 4.58. 1H NMR (DMSO-d6)
d [ppm]: 0.65 (qu,
J ¼ 7.40 Hz, 2H, N1eCH2eCH2),1.27 (qu, J ¼ 7.40 Hz, 2H, BreCH2eCH2),
3.12e3.21 (m, 4H, N1eCH2, BreCH2), 6.93e6.96(m, 4H, 2ꢃ C5-Ph-3,5-
H), 7.15e7.28 (m,15H, 5ꢃ Ph-2,4,6-H), 7.35e7.42 (m, 6H, 3ꢃ trtPh-3,5-
5.1.2. General procedure for N1-substituted piperazine derivatives
(16, 18e20, 41)
H). 13C NMR (75 MHz, DMSO-d6)
d
[ppm]: 26.5, 29.8, 34.5, 41.1, 73.5,
An appropriate N1-substituted piperazine (7.5 mmol), K2CO3
(3 g), TEBA (0.2 g) were suspended in acetone (15e20 ml) and
stirred under reflux for 30 min. N-1-bromoalkyl- 5,5-
diphenylhydantoin derivatives 36e40 (7.5e8.5 mmol) dissolved
in acetone (20e23 ml) were added. The mixture was stirred under
reflux for 3.5e8.5 h and stirring was continued at room tempera-
ture overnight. The inorganic precipitate was separated by filtration
and washed with acetone (3 ꢃ 10 ml). Combined filtrates were
evaporated. Pure compounds (16, 18e20, 41) in basic form were
obtained from the residue using one of the following methods: (A)-
crystallization with methanol; (B)- the residue was dissolved in
methylene chloride (10e15 ml), washed with 1% solution of HCl
(2 ꢃ 10e15 ml) and water (2 ꢃ 10 ml), dried with Na2SO4 anhy-
drous, evaporated to give a new residue that was crystallized with
methanol; (C)- purification using chromatography column with
solvent system (IV).
126.9, 127.9, 128.3, 128.4, 129.3, 137.4, 137.5, 142.7, 154.9, 173.8.
5.1.1.2. Methyl 2-(3-(3-bromopropyl)-2,5-dioxo-4,4-diphenylimidaz-
olidin-1-yl)acetate (35). Methyl 2-(2,5-dioxo-4,4-diphenylimida-
zolidin-1-yl)acetate 31 (9.72 g), 1,3-dibromopropane (7.87 g) were
used. Reactants were stirred for 45 h. White crystals of 35 (9.2 g,
20.66 mmol, 69%) m.p. 126e128 ꢀC, Rf (I):0.79. Anal. Calcd. for
C21H21BrN2O4 %: C, 56.64; H,. 4.75; N, 6.29. Found: C, 56.48; H, 4.72;
N, 6.41. 1H NMR (DMSO-d6)
d [ppm]: 1.23e1.32 (qu def., 2H,
BreCH2CH2), 3.15e3.19 (t, J ¼ 6.67 Hz, 2H, N1eCH2), 3.41e3.46 (t
def., 2H, BreCH2), 3.70 (s, 3H, OCH3), 4.35 (s, 2H, N3eCH2),
7.26e7.30 (m, 4H, 2ꢃ Ph-3,5-H), 7.42e7.47 (m, 6H, 2ꢃ Ph-2,4,6-H).
5.1.1.3. Methyl 2-(3-(4-bromobutyl)-2,5-dioxo-4,4-diphenylimidazo-
lidin-1-yl)acetate (36). Ester 31 (9.72 g), 1,4-dibromobutane
(39 mmol, 8.42 g) were used. Reactants were stirred for 120 h.
White crystals of 36 (12.1 g, 26.34 mmol, 88%) m.p. 105e106 ꢀC, Rf
(I):0.56. Anal. Calcd. for C22H23BrN2O4 %: C, 57.53; H,. 5.05; N, 6.10.
5.1.2.1. Ethyl 4-(4-(3-(2-methoxy-2-oxoethyl)-2,4-dioxo-5,5-diphen-
ylimidazolidin-1-yl)butyl)piperazine-1-carboxylate
(16). Methyl
Found: C, 57.42; H, 5.11; N, 6.01. 1H NMR (DMSO-d6)
d
[ppm]:
2-(3-(4-bromobutyl)-2,5-dioxo-4,4-diphenylimidazolidin-1-yl)ace-
tate 36 (3.44 g) in acetone (23 ml) was used. Reactants were stirred
under reflux for 7 h. Method B gave white crystals of 16 (2.12 g,
3.95 mmol, 53%) m.p. 213 ꢀC, Rf (II):0.49. Anal. Calcd. for
C29H36N4O6: C, 64.91; H, 6.76; N, 10.44. Found: C, 65.11; H, 6.84; N,
0.83e0.93 (qu def., 2H, N1eCH2eCH2), 1.39e1.48 (qu def., 2H,
BreCH2eCH2), 3.19e3.23 (t, J ¼ 6.67 Hz, 2H, N1eCH2), 3.33e3.35 (t
def., 2H, BreCH2), 3.70 (s, 3H, OeCH3), 4.34 (s, 2H, N3eCH2),
7.27e7.30 (m, 4H, 2ꢃ Ph-3,5-H), 7.39e7.47 (m, 6H, 2ꢃ Ph-2,4,6-H).

5814
J. Handzlik et al. / European Journal of Medicinal Chemistry 46 (2011) 5807e5816
10.31. 1H NMR (DMSO-d6)
d
[ppm]: 0.74e0.79 (m, 2H, Pp-CH2CH2),
1.87 (t, J ¼ 7.18 Hz, 2H, Pp-CH2), 2.22 (t, J ¼ 4.62 Hz, 4H, Pp-2, 6-H),
2.97 (t, J ¼ 4.80 Hz, 4H, Pp-3, 5-H), 3.17 (t, J ¼ 7.18 Hz, 2H, N1eCH2),
6.72 (t, J ¼ 7.30 Hz, 1H, PpPh-4-H) 6.87e6.98 (m, 7H, PpPh-2,3,5,6-
H, trtPh-4-H), 7.16e7.35 (m, 16H, 5ꢃ Ph-3,5-H, 3ꢃ trtPh-2,6-H),
7.36e7.44 (m, 6H, 2ꢃ C5-Ph-2,4,6-H).
1.15e1.19 (t def, 3H, COeCH2eCH3), 1.32 (m, 2H, N1eCH2CH2),
2.75e2.91 (m, 6H, Pp-CH2, 2ꢃ Pp-2, 6-H), 3.15e3.28 (m, 4H,
N1eCH2, 2ꢃ Pp-3, 5-H), 3.70 (s, 3H, OeCH3), 4.01e4.08 (q,
J ¼ 7.10 Hz, COeCH2), 4.35 (s, 2H, N3eCH2), 7.27e7.31 (m, 4H, 2ꢃ
Ph-3, 5-H), 7.44e7.49 (m, 6H, 2ꢃ Ph-2,4,6-H). 13C NMR (75 MHz,
DMSO-d6)
d
[ppm]: 14.92, 24.9, 25.1, 41.1, 53.0, 61.8, 74.9, 128.5,
5.1.3. Synthesis of methyl 2-(2,4-dioxo-5,5-diphenyl-1-(8-(piperazin-
1-yl)octyl)imidazolidin-3-yl)acetate (21)
128.6, 129.3, 129.5, 137.1, 154.7, 154.8, 168.5, 173.2.
Piperazine (1.03 g), acetone (15 ml), K2CO3 (1.66 g), ester 39
(2.06 g) in acetone (10 ml) were used. Reactants were stirred under
reflux for 5 h. The inorganic precipitate was separated by filtration.
The filtrate was evaporated, the residue was dissolved in methylene
chloride (10 ml) washed with aqua (3 ꢃ 10 ml), dried with Na2SO4
anhydrous, evaporated to give a new residue that was purified
using chromatography column with combined solvent system (V
and MeOH). White crystals of 21 (0.11 g, 0.21 mmol, 5%) m.p. 79 ꢀC,
Rf (II):0.14. Anal. Calcd. for C30H40N4O4 %: C, 69.20; H, 7.74; N, 10.76;
5.1.2.2. Ethyl 4-(5-(3-(2-methoxy-2-oxoethyl)-2,4-dioxo-5,5-diphe-
nylimidazolidin-1-yl)pentyl)piperazine-1-carboxylate (18). Methyl
2-(3-(5-bromopentyl)-2,5-dioxo-4,4-diphenylimidazolidin-1-yl)
acetate 37 (3.57 g) in acetone (23 ml) was used. Reactants were
stirred under reflux for 5 h. Method A gave white crystals of 18
(3.00 g, 5.45 mmol, 73%) m.p. 125 ꢀC, Rf (II):0.42. Anal. Calcd. for
C30H38N4O6 ꢃ 0.66 acetone ꢃ 0.33 CH2Cl2 %: C, 62.73; H, 6.85; N,
9.14. Found: C, 62.72; H, 6.91; N, 9.16.1H NMR (DMSO-d6)
d
[ppm]: 0.72e0.75 (m, 2H, N1eC2H4eCH2), 0.84e0.94 (qu,
Found: C, 68.82; H, 7.80; N, 10.45. 1H NMR (DMSO-d6)
d [ppm]: 0.71
J ¼ 7.35 Hz, 2H, Pp-CH2eCH2), 1.05e1.10 (m, 2H, N1eCH2CH2),
1.12e1.17 (t def., 3H, COeCH2eCH3), 1.95e2.00 (t, J ¼ 7.31 Hz, 2H,
PpCH2), 2.15e2.18 (t, J ¼ 5 Hz, 4H, 2ꢃ Pp-2, 6-H), 3.27e3.29 (m,
6H, N1eCH2, 2ꢃ Pp-3, 5-H) 3.70 (s, 3H, OeCH3), 3.96e4.03 (q,
J ¼ 7.09 Hz, COeCH2), 4.33 (s, 2H, N3eCH2), 7.26e7.29 (m, 4H, 2ꢃ
Ph-3,5-H), 7.43e7.46 (m, 6H, 2ꢃ Ph-2,4,6-H). 13C NMR (75 MHz,
(m, 2H, Pp-C3H6eCH2), 0.90e1.08 (m, 6H, Pp-C2H4eCH2,
N1eC2H4eCH2CH2), 1.12 (m, 2H, Pp-CH2eCH2), 1.31 (m, 2H,
N1eCH2CH2), 2.01e2.39 (m, 7H, Pp-CH2, 2ꢃ Pp-2, 6-H, Pp-NeH),
3.23e3.26 (m, 6H, 2ꢃ Pp-3, 5-H, N1eCH2), 3.70 (s, 3H, OeCH3), 4.33
(s, 2H, N3eCH2), 7.26e7.29 (m, 4H, 2ꢃ Ph-3,5-H), 7.43e7.45 (m, 6H,
2ꢃ Ph-2,4,6-H). 13C NMR (75 MHz, DMSO-d6)
d [ppm]: 26.2, 27.0,
DMSO-d6)
d
[ppm]: 15.0, 24.2, 25.9, 27.6, 41.9, 43.7, 52.8, 53.0,
27.6, 28.6, 28.9, 42.0, 52.8, 53.0, 58.0, 74.9, 128.6, 129.2, 129.3, 137.2,
154.6, 168.4, 173.3.
57.8, 61.1, 74.9, 128.6, 129.2, 129.4, 137.2, 154.6, 155.0, 168.4, 173.3.
5.1.2.3. Ethyl 4-(6-(3-(2-methoxy-2-oxoethyl)-2,4-dioxo-5,5-diphe-
5.1.4. Synthesis of 5,5-diphenyl-1-(4-(4-phenylpiperazin-1-yl)
butyl)imidazolidine-2,4-dione (1)
nylimidazolidin-1-yl)hexyl)piperazine-1-carboxylate
(19). Methyl
2-(3-(6-bromohexyl)-2,5-dioxo-4,4-diphenylimidazolidin-1-yl)
acetate 38 (3.66 g) in acetone (23 ml) was used. Reactants were
stirred under reflux for 7 h. Method C gave white crystals of 19
(2.65 g, 4.69 mmol, 63%) m.p. 60 ꢀC, Rf (II):0.47. Anal. Calcd. for
C31H40N4O6 %: C, 65.94; H, 7.14; N, 9.92. Found: C, 65.73; H, 7.23; N,
Compound 34 (2.00 g, 2.76 mmol) was dissolved in methylene
chloride (20 ml). A 90%-concentrated aqueous solution of TFA
(18 ml) was added and the mixture was stirred at room tempera-
ture for 20 h. Then, the mixture was washed with water (3 ꢃ 20 ml),
dried with K2CO3 anhydrous and evaporated. A residue was puri-
fied by column chromatography using methylene chlorideeacetone
(10:1) to give compound 1 (0.20 g, 0.42 mmol, 15.2%), m.p.
187e188 ꢀC, Rf (II):0.39. Anal. Calcd. for C30H34N4O2: C, 74.66; H,
7.10; N, 11.61. Found: C, 74.81; H, 7.12; N, 11.54. 1H NMR (DMSO-d6)
9.90.1H NMR (DMSO-d6)
d [ppm]: 0.72 (m, 2H, Pp-C2H4eCH2),
0.89e0.92 (m, 4H, Pp-CH2CH2, N1eCH2CH2), 1.13e1.17 (t,
J ¼ 7.04 Hz, 3H, COeCH2eCH3), 2.07e2.12 (t def., 2H, Pp-CH2),
2.19e2.22 (t, J ¼ 4.87 Hz, 4H, 2ꢃ Pp-2, 6-H), 3.27e3.29 (m, 6H,
N1eCH2, 2ꢃ Pp-3, 5-H) 3.70 (s, 3H, OeCH3), 3.96e4.04 (q,
J ¼ 7.09 Hz, COeCH2), 4.33 (s, 2H, N3eCH2), 7.26e7.29 (m, 4H, 2ꢃ
Ph-3,5-H), 7.43e7.46 (m, 6H, 2ꢃ Ph-2,4,6-H).
d
[ppm]: 0.65e0.75(def. qu, 2H, Pp-CH2eCH2), 1.00e1.10( def.qu,
2H, CH2eCH2ehyd), 1.94(t, J ¼ 7.18 Hz, 2H, Pp-CH2eCH2), 2.26(t,
J ¼ 4.75 Hz, 4H, Pp-2,6-H), 2.98(t, J ¼ 4.87 Hz, 4H, Pp-3,5-H), 3.09(t,
J ¼ 7.18 Hz, 2H, CH2eCH2ehyd), 6.71(t, J ¼ 7.18 Hz, 1H, PpPh-4H),
6.86(d, J ¼ 7.69 Hz, 2H, PpPh-2.6H), 7.13e7.20(m, 6H, 2ꢃ Ph-
2,4,6H), 7.28e7.40(m, 6H, PpPh-3,5-H, 2ꢃ Ph-3, 5-H), 8.32 (s, 1H,
N3eH).
5.1.2.4. Ethyl 4-(8-(3-(2-methoxy-2-oxoethyl)-2,4-dioxo-5,5-diphen-
ylimidazolidin-1-yl)octyl)piperazine-1-carboxylate (20). Methyl 2-(3-
(8-bromooctyl)-2,5-dioxo-4,4-diphenylimidazolidin-1-yl)acetate 39
(3.87 g) in acetone (23 ml) was used. Reactants were stirred under
reflux for 8 h. Method C gave beige crystals of 20 (1.23 g, 5.06 mmol,
67%) m.p. 73 ꢀC, Rf (II):0.51. Anal. Calcd. for C33H44N4O6 %: C, 66.87; H,
7.48; N, 9.45. Found: C, 66.77; H, 7.73; N, 9.46. 1H NMR (DMSO-d6)
5.1.5. Synthesis of piperazine derivatives of 3-hydantoinacetic acid
(9, 17)
5.1.5.1. Synthesis of 2-(3-(4-(4-(2-methoxyphenyl)piperazin-1-yl)
butyl)-2,5-dioxo-4,4-diphenylimidazolidin-1-yl)acetic acid hydrochlo-
ride (9). A suspension of methyl 2-(3-(4-(4-(2-methoxyphenyl)
piperazin-1-yl)butyl)-2,5-dioxo-4,4-diphenylimidazolidin-1-yl)
acetate 42 (1.4 mmol, 0.8 g) in MeOH (5 mL) and H2O (5 mL) was
treated with KOH (8.9 mmol, 0.50 g), stirred at room temperature for
2 h, diluted with H2O (10 mL), acidified to pH ¼ 3 (35% HCl), extracted
with CH2Cl2 (3 ꢃ 5 ml), dried with Na2SO4 anhydrous and evapo-
rated. The residue was dissolved in absolute EtOH (20 ml) and
saturated with gaseous HCl to give a white powder of 9 (0.63 g,
1.1 mmol, 78.6%), m.p. 186e187 ꢀC, Rf (III): 0.83. Anal. Calcd. for
C32H36N4O5 ꢃ HCl: C, 64.80; H, 6.29; N, 9.45. Found: C, 64.67; H, 6.29;
d
[ppm]: 0.71 (m, 2H, Pp-C3H6eCH2), 0.88e0.89 (m, 6H, Pp-
CH2CH2CH2CH2CH2), 1.06e1.09 (m, 2H, N1eC2H4-CH2), 1.13e1.17 (t,
J ¼ 7.05 Hz, 3H, COeCH2eCH3), 1.25e1.32 (m, 2H, N1eCH2CH2),
2.15e2.20 (t def., 2H, Pp-CH2), 2.23e2.26 (t def, 4H, 2ꢃ Pp-2, 6-H),
3.23e3.25 (m, 6H, N1eCH2, 2ꢃ Pp-3, 5-H) 3.70 (s, 3H, OeCH3),
3.96e4.04 (q, J ¼ 7.09 Hz, COeCH2), 4.33 (s, 2H, N3eCH2), 7.26e7.29
(m, 4H, 2ꢃ Ph-3,5-H), 7.43e7.45 (m, 6H, 2ꢃ Ph-2,4,6-H).
5.1.2.5. 5,5-Diphenyl-1-(4-(4-phenylpiperazin-1-yl)butyl)-3-
tritylimidazolidine-2,4-dione (41). N-phenylpiperazine (1.08 g) in
acetone (20 ml) and compound 40 (5.31 g, 8.5 mmol) in acetone
(20 ml) were refluxed for 3.5 h. Method A gave white crystals of
compound 41 (3.12 g, 4.38 mmol, 59%) m.p. 198e199 ꢀC, Rf (II):0.8.
Anal. Calcd. for C48H46N4O2 %: C, 81.10; H, 6.52; N, 7.88. Found: C,
N, 9.51. 1H NMR (DMSO-d6)
d [ppm]: 0.78e0.82 (qu def., 2H,
PpCH2CH2), 1.40 (br. s, 2H, PpCH2CH2CH2), 2.78 (br. s, 2H, Pp-CH2),
2.92e3.05 (m, 4H, Pp-2, 6-H), 3.32e3.43 (m, 6H, Pp-3, 5-H, N1eCH2),
3.77 (s, 3H, OCH3), 4.20 (s, 2H, N3eCH2), 6.85e7.03 (m, 4H, PpPh),
7.15e7.48 (m, 10H, 2ꢃ Ph), 10.59 (br. s, 1H, NHþ). 13C NMR (75 MHz,
80.97; H, 6.62; N, 7.79. 1H NMR (DMSO-d6)
d
[ppm]: 0.62 (qu,
J ¼ 7.40 Hz, 2H, Pp-CH2CH2), 0.95 (qu, J ¼ 7.40 Hz, 2H, N1eCH2CH2),

J. Handzlik et al. / European Journal of Medicinal Chemistry 46 (2011) 5807e5816
5815
DMSO-d6)
d
[ppm]: 20.5, 25.2, 47.2, 51.3, 55.1, 55.8, 74.9, 112.4, 118.7,
1H NMR (DMSO-d6)
d
[ppm]: 1.72 ( s, 3H, CH3 Ac), 2.15 (d,
121.3, 124.0, 128.7, 129.3, 129.4, 137.3, 139.7, 152.3, 154.9, 169.0, 173.3.
J ¼ 6.41 Hz, 2H, Pp-CH2), 2.23e2.28 (m, 4H, Pp-2, 6-H), 2.93e2.96 (t
def., 4H, Pp-3, 5-H), 3.56e3.64 (m, 2H, N1eCH2), 3.68 (s, 3H, OCH3)
4.34 (2, 2H, N3eCH2), 4.34e4.40 (m, 1H, CH), 6.71 (t, J ¼ 7.50 Hz,1H,
PpPh-4-H), 6.85e6.88 (d, J ¼ 7.50 Hz, 2H, PpPh-2, 6-H), 7.14e7.19 (t
def., 2H, PpPh-3,5-H), 7.23e7.29 (m, 4H, 2ꢃ Ph-3,5-H), 7.44e7.48
5.1.5.2. Synthesis of 2-(1-(4-(4-(ethoxycarbonyl)piperazin-1-yl)
butyl)-2,4-dioxo-5,5-diphenylimidazolidin-3-yl)acetic acid (17). A
suspension of ethyl 4-(4-(3-(2-methoxy-2-oxoethyl)-2,4-dioxo-
5,5-diphenylimidazolidin-1-yl)butyl)piperazine-1-carboxylate 16
(1.96 mmol, 1.05 g) in MeOH (4 mL) and H2O (4 mL) was treated
with KOH (10.7 mmol, 0.6 g), stirred at room temperature for 3 h,
acidified to pH ¼ 6 (35% HCl) and stirred overnight. Then, the
suspension was diluted with H2O (10 mL) and extracted with
methylene chloride (3 ꢃ 10 ml). The combined organic fractions
were dried with anhydrous Na2SO4, separated from the drying
agent and evaporated to give bright crystals of 17 (0.57 g, 1.1 mmol,
56%), m.p.140e142 ꢀC, Rf (V): 0.68. Anal. Calcd. for C28H34N4O6 %: C,
64.35; H, 6.56; N, 10.72. Found: C, 64.57; H, 6.59; N, 10.58. 1H NMR
(m, 6H, 2ꢃ Ph-2,4,6-H). 13C NMR (75 MHz, DMSO-d6)
d [ppm]: 21.1,
41.05, 43.9, 48.7, 52.9, 53.3, 59.1, 68.9, 75.4, 115.8, 119.2, 128.8, 129.2,
129.4, 136.8, 151.5, 155.3, 169.7, 173.0.
5.1.6.4. Methyl 2-(3-(2-acetoxy-3-(4-phenylpiperazin-1-yl)propyl)-
2,5-dioxo-4,4-diphenylimidazolidin-1-yl)acetate hydrochloride (11).
Compound 11b (0.85 g, 1.5 mmol) in dry MeOH (15 ml) was satu-
rated with gaseous HCl to give white crystals of 9 (0.9 g, 1.38 mmol,
92%) m.p. 162e164 ꢀC, Rf (III):0.81. Anal. Calcd. for
C31H34N4O4 ꢃ HCl ꢃ CH3OH: C, 62.52; H, 6.33; N, 8.58. Found: C,
62.56; H, 6.32; N, 8.54.
(DMSO-d6)
d [ppm]: 0.77e0.87 (qu def., 2H, Pp-CH2CH2), 1.16e1.21
(t, J ¼ 7.08 Hz, 3H, COeCH2eCH3), 1.23e1.26 (m, 2H, N1-CH2CH2),
2.72e2.78 (m, 4H, 2ꢃ Pp-2, 6-H), 2.91 (m, 2H, Pp-CH2), 3.32e3.37
(m, 6H, N1eCH2, 2ꢃ Pp-3, 5-H), 4.02e4.09 (q, J ¼ 7.08 Hz, COeCH2),
4.21 (s, 2H, N3eCH2), 7.18e7.21 (m, 1H, Ph-4-H), 7.29e7.32 (m, 3H,
Ph-2,6-H, Ph-2-H), 7.38e7.49 (m, 6H, Ph-4,6-H, 2ꢃ Ph-3,5-H). 13C
1H NMR (DMSO-d6)
d [ppm]: 1.83 (s, 3H, CH3 Ac), 2.71 (br. s, 1H,
N1eCH2a), 3.01e3.20 (m, 7H, Pp-CH2, Pp-2, 6-H, N1eCH2e),
3.60e3.64 (m, 4H, Pp-3, 5-H), 3.66 (s, 3H, OCH3), 3.73e3.81 (m, 1H,
CH), 4.33 (s, 2H, N3eCH2), 6.82 (t, J ¼ 6.18 Hz, 1H, PpPh-4-H), 6.95
(d, J ¼ 7.95 Hz, 2H, PpPh-2, 6-H), 7.21e7.29 (m, 6H, 2ꢃ Ph-3,5-H,
PpPh-3,5-H), 7.47e7.52 (m, 6H, 2ꢃ Ph-2,4,6-H), 10.90 (br. s, 1H,
NHþ).
NMR (75 MHz, DMSO-d6) d [ppm]: 14.9, 21.2, 25.3, 41.24, 41.4, 51.1,
61.6, 74.9, 128.7, 129.3, 137.2, 137.3, 154.8, 155.0, 169.1, 173.4.
5.1.6. General procedure for O-acetylated derivatives (10 and 11)
Arylpiperazine derivative of hydantoin with free hydroxyl group
4 or 7 (1.4e3.1 mmol) was slowly refluxed with acetic anhydride
(4e10 ml) for 1e20 min. The solution was stored at low temperature
for 2e10 days to give a white precipitate of basic form of the product
(10b or 11b), which was converted into hydrochloride form (10 or 11)
by dissolving in methanol and saturation with gaseous HCl.
5.1.7. General procedure for hydantoin derivatives of 1,5,7-triazabicyclo
[4.4.0]dec-5-ene (22e25)
The mixture of 1,5,7-triazabicyclo[4.4.0]dec-5-ene (1.5 mmol,
210 mg), TBAB (0.15 mmol, 48 mg) and potassium carbonate
(400 mg) in acetone (4 mL) was stirred under reflux for 1 h, then an
appropriate bromide 35e38 (1 mmol) in acetone (1 mL) was added.
The mixture was refluxed for 6e7 h. After cooling the mixture was
filtered and the mother liquor was evaporated in vacuo affording
colorless oil. Hydrochlorides were obtained using an ether solution
of hydrogen chloride with methanol as co-solvent.
5.1.6.1. 1-(3-methyl-2,4-dioxo-5,5-diphenylimidazolidin-1-yl)-3-(4-
phenylpiperazin-1-yl)propan-2-yl acetate (10b). Compound 4 (1.5 g,
3.1 mmol) and Ac2O (10 ml) were refluxed for 15 min, cooled (0-
4
ꢀC) for 2 days to give white powder of 10b (1.15 g, 2.2 mmol,
5.1.7.1. 2-(3-(3-(2,3,4,6,7,8-hexahydro-1H-pyrimido[1,2-a]pyr-
imidin-1-yl)propyl)-2,5-dioxo-4,4-diphenylimidazolidin-1-yl)acetic
acid hydrochloride (22). Raw product was purified by column
chromatography (choroform:methanol). Colorless dense oil, yield
22%; Anal. Calcd. for C27H32ClN5O4: C, 61.65; H, 6.13; N, 13.31.
Found: C, 61.44; H, 6.15; N, 13.10. 1H NMR (200 MHz, MeOD):
70.5%), m.p. 178e180 ꢀC, Rf (III):0.87. Anal. Calcd. for C31H34N4O4: C,
70.70; H, 6.51; N, 10.64. Found: C, 70.59; H, 6.53; N, 10.61. 1H NMR
(DMSO-d6)
d
[ppm]: 1.72 ( s, 3H, CH3 Ac), 2.16e2.18 (d, J ¼ 6.41 Hz,
2H, Pp-CH2), 2.20e2.29 (m, 4H, Pp-2, 6-H), 2.94e2.96 (m, 7H, Pp-3,
5-H, N3eCH3), 3.55 (d, J ¼ 6.16 Hz, 2H, N1eCH2), 4.43e4.48 (qu,
J ¼ 6.16 Hz, 1H, CH), 6.71e6.76 (t, J ¼ 7.18 Hz, 1H, PpPh-4-H),
6.85e6.88 (d, J ¼ 7.95 Hz, 2H, PpPh-2, 6-H), 7.14e7.24 (m, 6H, PpPh-
3,5-H, 2ꢃ Ph-3,5-H), 7.41e7.46 (m, 6H, 2ꢃ Ph-2,4,6-H).
d
0.72e0.86 (m, 2H, CH2), 1.18e1.29 (m, 2H, CH2), 1.92e2.01 (m, 4H,
CH2), 3.10e3.30 (m, 8H, CH2), 3.53 (t, J ¼ 9 Hz, 2H, CH2), 4.00 (s, 2H,
CH2), 7.34e7.42 (m, 10H, Ar).
5.1.6.2. 1-(3-methyl-2,4-dioxo-5,5-diphenylimidazolidin-1-yl)-3-(4-
phenylpiperazin-1-yl)propan-2-yl acetate hydrochloride (10).
Compound10b (0.9g,1.71mmol)inMeOH(15ml)wassaturatedwith
gaseous HCl to give white crystals of 10 (1 g, 1.65 mmol, 96%) m.p.
180e184 ꢀC, Rf (III):0.87. Anal. Calcd. for C31H34N4O4 ꢃ 2HCl ꢃ 0.5
CH3OH: C, 61.78; H, 6.14; N, 9.22. Found: C, 61.78; H, 6.17; N, 9.28. 1H
5.1.7.2. 2-(3-(4-(2,3,4,6,7,8-hexahydro-1H-pyrimido[1,2-a]pyr-
imidin-1-yl)butyl)-2,5-dioxo-4,4-diphenylimidazolidin-1-yl)acetic
acid hydrochloride (23). Raw product was purified by column
chromatography (chloroform:methanol). White solid, yield 81%;
Anal. Calcd. for C28H34ClN5O4: C, 62.27; H, 6.35; N, 12.97. Found: C,
61.99; H, 6.37; N, 12.88. 1H NMR (200 MHz, MeOD):
d 0.74e0.80 (m,
NMR: for 5 (DMSO-d6)
d
[ppm]: 1.86 (s, 3H, COCH3), 2.71e2.73 (m,1H,
2H, CH2), 1.19e1.28 (m, 2H, CH2), 1.88e1.96 (m, 4H, CH2), 3.06e3.26
(m, 10H, CH2), 3.29e3.31 (m, 2H, CH2), 4.01 (s, 2H, CH2), 7.39e7.42
d [ppm]: 20.4, 20.5, 23.3,
24.9, 38.5, 39.5, 40.6, 46.0, 47.0, 47.4, 48.8, 75.2, 128.4, 128.6, 128.9,
136.9, 150.3, 156.0.169.0, 173.9.
CH), 2.88e2.92 (m, 2H, Pp-CH2), 2.97 (s, 3H, N3eCH3), 3.08e3.16 (m,
4H, Pp-2, 6-H), 3.21e3.31(m,2H, N1eCH2),3.63e3.72(m, 4H, Pp-3,5-
H,), 6.82 (t, J ¼ 7.30 Hz,1H, PpPh-4-H), 6.95 (d, J ¼ 7.95 Hz, 2H, PpPh-2,
6-H), 7.19e7.27 (m, 6H, PpPh-3,5-H, 2ꢃ Ph-3,5-H), 7.45e7.46 (m, 6H,
2ꢃ Ph-2,4,6-H), 10.94 (br. s, 1H, NHþ).
(m, 10H, Ar). 13C NMR (200 MHz, MeOD)
5.1.7.3. 2-(3-(5-(2,3,4,6,7,8-hexahydro-1H-pyrimido[1,2-a]pyr-
imidin-1-yl)pentyl)-2,5-dioxo-4,4-diphenylimidazolidin-1-yl)acetic
acid hydrochloride (24). Raw product was stirred in acetone at
room temperature for 30 min. White solid was filtrated off and
recrystallized from ethanol, yield 20%; Anal. Calcd. for
C29H36ClN5O4: C, 62.86; H, 6.55; N, 12.64. Found: C, 62.47; H, 6.56;
5.1.6.3. Methyl 2-(3-(2-acetoxy-3-(4-phenylpiperazin-1-yl)propyl)-
2,5-dioxo-4,4-diphenylimidazolidin-1-yl)acetate (11b). Compound 7
(1.3 g, 2.4 mmol) and Ac2O (10 ml) were refluxed for 20 min, cooled
(0e4 ꢀC) for 3 days to give white powder of 11b (1.25 g, 2.1 mmol,
89.1%), m.p. 196e198 ꢀC, Rf (III):0.81. Anal. Calcd. for C33H36N4O6: C,
67.79; H, 6.21; N, 9.58. Found: C, 67.80; H, 6.24; N, 9.62.
N, 12.51. 1H NMR (200 MHz, MeOD):
d 0.71e0.78 (m, 2H, CH2),

5816
J. Handzlik et al. / European Journal of Medicinal Chemistry 46 (2011) 5807e5816
0.95e1.17 (m, 2H, CH2), 1.28e1.39 (m, 2H, CH2), 1.95e2.01 (m, 4H,
CH2), 3.11 (t, J ¼ 12 Hz, 2H, CH2), 3.26e3.46 (m, 10H, CH2), 4.02 (s,
2H, CH2), 7.40e7.44 (m, 10H, Ar).
H, 5.76; N, 10.35. 1H NMR (200 MHz, DMSO):
d 0.78 (t def., 2H, CH2),
1.21 (t def., 2H, CH2), 3.30 (t def., 2H, CH2), 4.19 (s, 2H, CH2),
7.18e7.44 (m, 10H, Ar).
5.1.7.4. 2-(3-(6-(2,3,4,6,7,8-hexahydro-1H-pyrimido[1,2-a]pyr-
imidin-1-yl)hexyl)-2,5-dioxo-4,4-diphenylimidazolidin-1-yl)acetic
acid hydrochloride (25). Raw product was stirred in acetone at
room temperature for 30 min. White solid was filtrated off and
recrystallized from ethanol, yield 32%; Anal. Calcd. for
C30H38ClN5O4: C, 63.42; H, 6.74; N, 12.33. Found: C, 63.10; H, 6.72;
5.1.9.3. 2-(3-(6-Aminohexyl)-2,5-dioxo-4,4-diphenylimidazolidin-1-
yl)acetic acid hydrochloride (28). White solid, yield 41%; Anal.
Calcd. for C23H28ClN3O4: C, 61.95; H, 6.33; N, 9.42. Found: C, 61.58;
H, 6.18; N, 9.72. 1H NMR (200 MHz, DMSO):
d
0.72 (br. s, 2H, CH2),
0.91 (br. s, 4H, CH2), 1.20e1.33 (m, 2H, CH2), 2.58 (br. s, 2H, CH2),
3.26 (t def., 2H, CH2), 4.19 (s, 2H, CH2), 7.26e7.43 (m, 10H, Ar). 13
NMR (200 MHz, CDCl3) [ppm]: 24.2, 24.8, 38.7, 39.4, 40.8, 75.2,
C
N, 12.16. 1H NMR (200 MHz, MeOD):
d
0.83e0.89 (m, 2H, CH2),
d
1.00e1.06 (m, 4H, CH2), 1.35e1.41 (m, 2H, CH2), 1.93e2.05 (m, 4H,
CH2), 3.17 (t, J ¼ 10.5 Hz, 2H, CH2), 3.30e3.38 (m, 10H, CH2), 4.02 (s,
2H, CH2), 7.39e7.43 (m, 10H, Ar).
128.6, 128.3, 128.9, 136.9, 155.7, 169.0 173.8.
5.2. Microbiological assays
5.1.8. General procedure for phthalimides (44e46)
To determine minimal inhibitory concentrations (MICs),
approximately 106 cells were inoculated into 1 mL of Muel-
lereHinton broth containing two fold serial dilutions of nalidixic
acid. Results were read after 18 h at 37 ꢀC. To study synergistic
activity, different fixed sub-inhibitory concentrations of the
potential inhibitors, determined without antibiotic, were added
during incubation with nalidixic acid. In addition, the reference
The mixture of phthalimide (1 mmol, 185 mg), TBAB (0.15 mmol,
48 mg) and potassium carbonate (400 mg) in acetone (4 mL) was
stirred under reflux for 30 min, then an appropriate bromide 35, 36
or 38 (1 mmol) in acetone (1 mL) was added and the mixture was
refluxed for 2 h. After cooling the mixture was filtered and the
mother liquor was evaporated in vacuo. The residue was purified by
column chromatography (petroleum ether:ethyl acetate).
efflux pump inhibitor, PAbN, was used at 0.050 mM. Control
experiments were carried out without the different inhibitors.
5.1.8.1. Methyl 2-(3-(3-(1,3-dioxoisoindolin-2-yl)propyl)-2,5-dioxo-
4,4-diphenylimidazolidin-1-yl)acetate (44). White solid, yield 86%;
Acknowledgments
1H NMR (200 MHz, CDCl3):
d 0.82e0.91 (m, 2H, CH2), 3.37e3.46 (m,
4H, CH2), 3.76 (s, 3H, CH3), 4.30 (s, 2H, CH2), 7.17e7.37 (m, 10H, Ar),
7.68e7.79 (m, 4H, Ar).
Partly supported by grants: 501/N-COST/2009/0 (K/PMN/
000031), COST action BM0701 and Polonium 8127/2010.
5.1.8.2. Methyl 2-(3-(4-(1,3-dioxoisoindolin-2-yl)butyl)-2,5-dioxo-
References
4,4-diphenylimidazolidin-1-yl)acetate (45). White solid, yield 97%;
1H NMR (200 MHz, CDCl3):
d
0.77e0.87 (m, 4H, CH2), 3.37e3.44 (m,
4H, CH2), 3.78 (s, 3H, CH3), 4.31 (s, 2H, CH2), 7.17e7.38 (m, 10H, Ar),
7.70e7.82 (m, 4H, Ar). 13C NMR (200 MHz, CDCl3)
[ppm]: 25.3,
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4,4-diphenylimidazolidin-1-yl)acetate (46). White solid, yield 91%;
1H NMR (200 MHz, MeOD):
d 0.78e0.82 (m, 2H, CH2), 0.97e1.01 (m,
4H, CH2), 1.42e1.46 (m, 2H, CH2), 3.2e3.39 (m, 2H, CH2), 3.48e3.52
(m, 2H, CH2), 3.77 (s, 3H, CH3), 4.33 (s, 2H, CH2), 7.30e7.42 (m, 10H,
Ar), 7.79e7.90 (m, 4H, Ar).
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5.1.9. General procedure for primary amines 26e28
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J. Chevalier, E. Garnotel, Curr. Top. Med. Chem. 8 (2010) 1848e1857.
The mixture of an appropriate phthalimide 44e46 (1 mmol) and
hydrazine monohydrate (1 mmol, 50 mg) in anhydrous ethanol
(3 mL) was refluxed for 4 h. After cooling the reaction mixture was
filtrated from white solid and the mother liquor was evaporated in
vacuo affording yellow solid. Hydrochlorides were obtained using
an ether solution of hydrogen chloride with methanol as co-
solvent.
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5.1.9.1. 2-(3-(3-Aminopropyl)-2,5-dioxo-4,4-diphenylimidazolidin-1-
yl)acetic acid hydrochloride (26). White solid, yield 73%; Anal.
Calcd. for C20H22ClN3O4: C, 59.48; H, 5.49; N, 10.40. Found: C, 59.21;
d 1.14e1.27 (m, 2H,
CH2), 2.57e2.64 (m, 2H, CH2), 3.56e3.63 (m, 2H, CH2), 4.31 (s, 2H,
CH2), 7.40e7.48 (m, 10H, Ar).
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(1995) 355e360.
H, 5.47; N, 10.67. 1H NMR (200 MHz, MeOD):
ꢀ
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(2002) 39e44.
ꢀ
ꢀ
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[22] J.C. Sheehan, V.A. Bolhofer, J. Am. Chem. Soc. 72 (1950) 2786e2788.
[23] J. Chevalier, A. Mahamoud, M. Baitiche, E. Adam, M. Viveiros, A. Smarandache,
A. Militaru, M.L. Pascu, L. Amaral, J.-M. Pagès, Int. J. Antimicrob. Agents 36
(2010) 164e168.
5.1.9.2. 2-(3-(4-Aminobutyl)-2,5-dioxo-4,4-diphenylimidazolidin-1-
yl)acetic acid hydrochloride (27). White solid, yield 91%; Anal.
Calcd. for C21H24ClN3O4: C, 60.36; H, 5.79; N, 10.06. Found: C, 60.03;