SAR-studies on the importance of aromatic ring topologies in search for selective 5-HT7 receptor ligands among phenylpiperazine hydantoin derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.01.065

The current study is focused on newly developed phenylpiperazine derivatives of aromatic methylhydantoin differing in mutual positions of methyl and phenyl moieties. The new compounds were synthesized using Bucherer-Bergs reaction, two-phase alkylation, Mitsunobu reaction and/or an alkylation under microwave irradiation. The compounds developed were assessed on their affinity for serotoninergic receptors 5-HT_1A, 5-HT₆, 5-HT ₇ and α₁-ARs in radioligand binding assays. Selected compounds were tested on their inhibitory effect at human 5-HT_3A expressed in Xenopus Oocytes as well as on their activity at α₁-adrenoceptor subtypes in functional and electrophysiological bioassays, respectively. Most of investigated compounds exhibited affinities for α₁-ARs, 5-HT_1A, 5-HT₇ (K_i ～ 0.8-353 nM) significantly higher than those for 5-HT₆ receptors. Very weak inhibitory effect at 5-HT_3A accompanied with high activity at α_1D-AR subtypes were observed for selected representative compounds. Among the current series, particularly 5-(4-fluorophenyl)-3-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl) -5-methylimidazolidine-2,4-dione hydrochloride (25a) displayed the highest 5-HT₇ affinity with K_i = 3 nM and selectivity with 40-3600 fold towards 5-HT_1A, 5-HT₆, and α₁-ARs.

Full text of DOI:10.1016/j.ejmech.2014.01.065

European Journal of Medicinal Chemistry 78 (2014) 324e339
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
SAR-studies on the importance of aromatic ring topologies in search
for selective 5-HT7 receptor ligands among phenylpiperazine
hydantoin derivatives
,
b
b
c
Jadwiga Handzlik a , Andrzej J. Bojarski , Grzegorz Sata1a , Monika Kubacka ,
*
Bassem Sadek d, Abrar Ashoor d, Agata Siwek e, Ma1gorzata Wie˛cek a, Katarzyna Kucwaj a,
Barbara Filipek , Katarzyna Kiec-Kononowicz
c
a
ꢀ
a Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Medyczna 9, PL 30-688 Kraków, Poland
b Department of Medicinal Chemistry Institute of Pharmacology, Polish Academy of Sciences, Smetna 12, PL 31-343 Kraków, Poland
c Department of Pharmacodynamics, Jagiellonian University Medical College, Medyczna 9, PL 30-688 Kraków, Poland
d Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, P.O. Box 17666,
United Arab Emirates
e Department of Pharmacobiology, Jagiellonian University Medical College, Medyczna 9, PL 30-688 Kraków, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
The current study is focused on newly developed phenylpiperazine derivatives of aromatic methyl-
hydantoin differing in mutual positions of methyl and phenyl moieties. The new compounds were
synthesized using BucherereBergs reaction, two-phase alkylation, Mitsunobu reaction and/or an alkyl-
ation under microwave irradiation. The compounds developed were assessed on their affinity for
serotoninergic receptors 5-HT1A, 5-HT6, 5-HT7 and a1-ARs in radioligand binding assays. Selected com-
pounds were tested on their inhibitory effect at human 5-HT3A expressed in Xenopus Oocytes as well as
on their activity at a1-adrenoceptor subtypes in functional and electrophysiological bioassays, respec-
tively. Most of investigated compounds exhibited affinities for a1-ARs, 5-HT1A, 5-HT7 (Ki w 0.8e353 nM)
significantly higher than those for 5-HT6 receptors. Very weak inhibitory effect at 5-HT3A accompanied
with high activity at a1D-AR subtypes were observed for selected representative compounds. Among the
current series, particularly 5-(4-fluorophenyl)-3-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)
propyl)-5-methylimidazolidine-2,4-dione hydrochloride (25a) displayed the highest 5-HT7 affinity with
Ki ¼ 3 nM and selectivity with 40e3600 fold towards 5-HT1A, 5-HT6, and a1-ARs.
Received 28 August 2013
Received in revised form
5 January 2014
Accepted 7 January 2014
Available online 18 March 2014
Keywords:
Phenylpiperazines
Hydantoins
Mitsunobu reaction
5-HT1A
5-HT3A
5-HT6
5-HT7
Alpha1-adrenoceptor
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
action [3]. Their ability to combat cancer or/and bacterial multidrug
resistance [4e7] as well as their action on G-protein coupled re-
Arylpiperazine partial structure is a very popular chemical class
present in many biologically active compounds including drugs of
therapeutic implication and compounds under countless stages of
pharmacological screening. The aromatic area in combination with
positive ionizable nitrogen of piperazine meets the structural re-
quirements of binding pockets found in various protein targets that
play important physiological roles in mammal tissues. The latest
lines of evidence indicated their anticancer properties [1], anti-
tuberculosis efficacy [2], antiarrhythmic and/or antihypertensive
ceptors (GPCRs) including adenosine [8], dopaminergic [9], seroto-
nin [10,11] and adrenergic [12] receptors have been demonstrated.
Although arylpiperazine derived ligands are particularly wide-
spread for serotonin receptor 5-HT1A (1), 5-HT7 (2) [11,13e15] and all
a1-adrenoceptors subtypes (a1A) (3a), (a1B) (3b) and (a1D) (3c) [16e
19], they similarly occur in the case of 5-HT6 (4) [20] and the iono-
tropic serotonin receptors 5-HT3 [21] (5, Fig.1). Especially, the role of
arylpiperazine moiety in modulating the interactions with receptors
like 5-HT1A, 5-HT7 [11,23,24] or a1-adrenergic receptors [17,18,22] is
underlined by several pharmacophore models (Fig. 2) that were
established on the basis of large number of compounds evaluated in
radioligand binding assays. The latter models have been and are still
useful in the search for potent and selective ligands for variety of
* Corresponding author.
E-mail addresses: jhandzli@cm-uj.krakow.pl, j.handzlik@uj.edu.pl (J. Handzlik).
http://dx.doi.org/10.1016/j.ejmech.2014.01.065
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

J. Handzlik et al. / European Journal of Medicinal Chemistry 78 (2014) 324e339
325
O
CH3
O
H
N
N
O
N
S
N
N
O
O
O
N
H3C
CH3
O
1
2
O
N
O
O
F
H3C
H3C
N
CH3
N
N
N
N
N
O
O
O
F
N
OH
O
N
HN
O
F
3a
3c
3b
O
Cl
N
O
O
S
N
N CH3
N
N
F
C2H5
N
H3C
5
4
Fig. 1. Selective arylpiperazine ligands for serotonin receptors 5-HT1A (1) [13], 5-HT7 (2) [15], a1-adrenoceptor subtypes a1A (3a) [18], a1B (3b) [19] a1D (3c) [16,17], 5-HT6 (4) [20]
and the ionotropic serotonin receptors 5-HT3 (5) [21].
receptors, particularly, because any experimental 3D-structures of
these GPCRs have not yet been identified. Among these protein
targets, a special attention is given to 5-HT7 ones as they are the
most recently identified serotonin receptor subtypes and their role
in controlling various CNS functions is still increasing based on
pharmacological studies that have been performed for the last de-
cades [11]. Recent pharmacological results indicated the important
modulating role of 5-HT7 receptors in anxiety, circadian rhythm,
depression, epilepsy, learning, memory, locomotion, migraine, pain,
schizophrenia, sleep, substance abuse, and thermoregulation pro-
cesses [11]. Considering the aforementioned roles, the search for
effective and selective 5-HT7 receptor agents provides a new hope
for future therapy of a variety of CNS diseases. Arylpiperazine class is
one of the most promising groups in the search of highly potent li-
gands for 5-HT7, while their selectivity remains rather problematic
due to high probability of interactions with other protein targets [1e
12]. Thus, studies on chemical modifications of arylpiperazines
targeting an improved selectivity profile towards 5-HT7 receptors
are an important and interesting field of current medicinal chem-
istry research.
Our previous studies were focused on phenylpiperazine de-
rivatives of phenytoin, which displayed various affinities at both,
a1-drenergic and 5-HT1A receptors [3,19,25,26] Among those
compounds, the 3-methylhydantoin derivative 6a (Table 1) dis-
played comparable and moderate affinities for both of the consid-
ered GPCRs [25] as well as for 5-HT7 receptor, identified within later
assays, almost identical as that for 5-HT1A. Pharmacophore models
(Fig. 2) indicated that number and relative position of aromatic/
hydrophobic moieties, hydrogen bond acceptors, and positive
ionizable center are responsible for interactions with the consid-
ered GPCRs and they also seem to be accountable for the receptors
discrimination. Our previous studies for hydantoin derivatives have
shown that, in addition to these pharmacophoric features, a role of
substituent at 3-position of hydantoin was important for selective
interactions with the GPCRs.
enhanced selectivity profile towards 5-HT7 receptors in respect to
other competitive GPCRs, including 5-HT1A, 5-HT3A, 5-HT6 or a1
-
adrenoceptors. Our current investigations focus on four steps of
modifications of compound 6a (A-D, Table 1), which were designed
to gradually increase the structural divergence in the field of rela-
tive locations of important structural fragments within the lead
compound. Synthesis of new compounds, radioligand binding as-
says and functional bioassays were carried out, as well as struc-
tureeactivity relationship analyses were established.
2. Results and discussion
2.1. Synthesis
Syntheses of the final products 6ae9a and certain intermediates
(27, 30e32 and 41) were described elsewhere [3,4,23e25]. Phe-
nylpiperazine derivatives of hydantoin 10ae25a were obtained
within four parallel synthesis routes according to Schemes 1 and 2.
The phenylpiperazinepentyl derivative of 5,5-diphenylhydantoin 10
was synthesized within three-steps of alkylation [24] starting from
the methylation process at 3-position of 5,5-diphenylhydantoin 26
(Scheme 1a). An introduction of bromopentyl substituent at 1-
position (compound 28) was performed by two-phase alkylation
in acetone with K2CO3 and TEBA using long-term stirring at room
temperature. Contrary to its 3- or 4-carbons analogues [3], 28 did
not precipitate during simple crystallization procedure with alco-
hols. Pure precipitate of 28 was achievable by double column
chromatography separation and crystallization with ethanol sup-
ported by diethyl ether and n-hexane. The pure alkylating agent 28
was used for N-alkylation of commercial phenylpiperazine to afford
compound 10. The process was performed by reflux in microwave
reactor “Plazmatronika” in two-phase basic conditions. A special 60-
min irradiation program was elaborated on the basis of TLC control
of the reaction progress. The established program was used for
syntheses of compounds 11e13, as well.
Based on previous observations, compound 6a was selected as a
lead structure for further chemical modifications to design new
hydantoin-phenylpiperazine derivatives with higher affinity and
Compounds 11e13 were obtained within four-step syntheses
(Scheme 1b), starting from BucherereBergs condensation [25] that
allowed the synthesis of racemic 5-methyl-5-phenylhydantoin,

326
J. Handzlik et al. / European Journal of Medicinal Chemistry 78 (2014) 324e339
Fig. 2. Pharmacophore features elaborated for a1-adrenrgic- or serotonin receptors agents: (aec) pharmacophore features for a1-AR antagonists postulated by Bremner’s group in
respect to subtype-selectivity [16]; (d) the Barbaro’s model of phenylpiperazine derived a1-AR antagonists [22]; (e) the Lepailleur’s model for 5-HT1A receptor antagonists [24]; (f)
the pharmacophore features for 5-HT6 receptor antagonists elaborated by Lopèz-Rodriguez et al. [23]; (gei) the 5-HT7 agents pharmacophore features for (g) agonists and (h)
inverse agonists, described by Vermeulen et al., (i) the model of antagonists elaborated by Bojarski’s research group [11]. A- an aromatic fragment, HY or HYD- hydrophobic areas,
ꢁ
HBA-hydrogen bond acceptors, HBD-hydrogen bond donors, PI-positive ionizable nitrogen. All distances are expressed in [A].
followed by alkylation at 1- and 3-position according to the pre-
viously described methods [4]. The bromopentyl derivative of 3,5-
dimethyl-5-phenylhydantoin (32) was used as an alkylating agent
for the microwave-aided two-phase alkylation of suitable phenyl-
piperazines to give compounds 11e13. Compounds 10-13 were
obtained as free bases by column chromatography separations.
The synthetic route of phenylpiperazine 5,5-dimethylhydantoin
derivatives 14e24 was carried out according to Scheme 2a. Starting
from commercially available 5,5-dimethylhydantoin (33), alkyl-
ation at 3-position was achieved by the use of suitable (un)
substituted benzyl bromide or chlorides to afford compounds 34e
36. In the following step, the 3-benzyl derivatives 34e36 were
converted into corresponding alkylating agents (37e39) by two-
phase bromoalkylation with 1,5-dibromopentane under similar
conditions to those of used to achieve compounds 28 and 32. As no
precipitate of compounds 37e39 ensued, the compounds were
purified through column chromatography, and the resulted pure
glass-residues (37e39) were used for reactions with suitable
commercially available phenylpiperazines to give compounds 14e
24. Compounds 20e24 were obtained as free bases. However,
compounds 14e19 that did not freely precipitate from ethanol so-
lutions, were achieved through direct saturations of the solutions
with gaseous HCl to give precipitates of their hydrochlorides 14ae
19a. The phenylpiperazine derivatives 10e13, 20, 21, 23, and 24
were converted into their corresponding hydrochlorides (10ae13a,
20a, 21a, 23a and 24a) by saturation of their solutions in absolute
ethanol with gaseous HCl.
Compound 25 representing reversal position of the phenyl-
piperazinealkyl substituent (at 3-position of hydantoin) was syn-
thesized within three steps (Scheme 2b). Starting from Bucherere
Bergs cyclic condensation of 1-(4-fluorophenyl)ethanone (40), fol-
lowed by oxiranmethyl introduction at 3-position of 5-(4-
fluorophenyl)-5-methylhydantoin, the product 5-(4-fluorophenyl)-
5-methyl-3-(oxiran-2-ylmethyl)imidazolidine-2,4-dione (42) was
obtained. As described earlier [4,24], the introduction of oxir-
anmethyl at 3-position was much more complicated than that at 1-
position as the 3-oxiranmethyl derivative 42 was very sensitive to
water and ethanol resulting in undesirable ring openings of the

J. Handzlik et al. / European Journal of Medicinal Chemistry 78 (2014) 324e339
327
Table 1
Structure of the tested compounds (6ae25a).
Ph
O
O
Ph
O
Ph
Ph
OH
N
N
N
N
CH3
N
N
CH3
R
N
N
n
O
6
7-10
Group A
(Lead)
R1
F
CH3
O
Ph
O
H3C
H3C
N
CH3
O
N
OH
N
N
n
N
R
N
N
CH3
R
N
N
HN
N
N
n
OCH3
O
O
O
11-13
25
Group D
14-24
Group C
Group B
Comp.
Group
R
R1
n
a1-AR Ki (nM)
5-HT1AKi (nM)
5-HT3A% inhibition
at [30 mM] conc.
5-HT6 Ki (nM)
5-HT7Ki (nM)
[3H]-prazosin
[3H]-8-OH-DPAT
[3H]-LSD
[3H]-5-CT
6a
7a
8a
9a
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
20a
21a
542.3 ꢁ 19.4
412.9 ꢁ 21
4.7 ꢁ 1.5
2600 ꢁ 0.1
6.7 ꢁ 0.1
7.3 ꢁ 0.8
11.3 ꢁ 0.1
42.3 ꢁ 1.4
68.0 ꢁ 6.4
42.6 ꢁ 1.0
97.2 ꢁ 1.4
71.0 ꢁ 2.3
68.4 ꢁ 1.8
71.5 ꢁ 2.5
38.8 ꢁ 2.6
36.8 ꢁ 3.7
968.8 ꢁ 82.6
673.0 ꢁ 63.0
758.5 ꢁ 30.5
181.1 ꢁ 12.2
10.3 ꢁ 0.6
e
324 ꢁ 9
6.5 ꢁ 0.5
0.8 ꢁ 0.1
5.5 ꢁ 0.6
13.2 ꢁ 1.2
135 ꢁ 11
124 ꢁ 9
23.3 ꢁ 2.1
79 ꢁ 4
e
20 410 ꢁ 2037
29 070 ꢁ 3061
3567 ꢁ 251
4139 ꢁ 513
3071 ꢁ 152
9600 ꢁ 715
25 520 ꢁ 2824
14 650 ꢁ 1495
4368 ꢁ 522
8611 ꢁ 627
2926 ꢁ 134
2506 ꢁ 283
1945 ꢁ 191
7469 ꢁ 820
8104 ꢁ 367
1762 ꢁ 153
395 ꢁ 29
353 ꢁ 19
210 ꢁ 11
16 ꢁ 1
95 ꢁ 7
77 ꢁ 6
e
A
A
A
A
B
B
B
C
C
C
C
C
C
C
C
C
C
C
D
e
e
e
e
2-OCH3
2-OCH3
2-OC2H5
H
H
2-F
2- OCH3
H
2-OCH3
3-OCH3
2-F
e
3
4
3
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
e
e
e
e
e
e
36.86 ꢁ 6.65
e
e
e
e
157 ꢁ 15
34 ꢁ 2
70 ꢁ 5
72 ꢁ 4
122 ꢁ 7
135 ꢁ 9
45 ꢁ 2
172 ꢁ 8
123 ꢁ 9
46 ꢁ 3
31 ꢁ 2
78 ꢁ 4
456 ꢁ 23
3 ꢁ 0.2
e
e
19.75 ꢁ 1.76
H
H
H
H
H
H
14.28 ꢁ 1.22
26 ꢁ 2
e
45 ꢁ 3
e
76 ꢁ 3
e
4-F
201 ꢁ 16
235 ꢁ 20
299 ꢁ 17
305 ꢁ 13
88 ꢁ 7
e
2,4-diF
2,4-diF
4-F
2,3-diCl
3,4-diCl
4-Cl
e
4-F
4-F
2,4-diCl
2,4-diCl
2,4-diCl
e
e
e
e
e
e
17.22 ꢁ 0.98
22a
23a
24a
25a
13.75 ꢁ 1.29
301 ꢁ 23
2178 ꢁ 194
121 ꢁ 7
e
e
314 ꢁ 17
e
2879 ꢁ 182
10 790 ꢁ 847
e
e
16.56 ꢁ 1.29
Phentolamine
Buspirone
Olanzapine
Clozapine
LY278584
e
e
e
e
e
e
e
22 ꢁ 2
e
e
e
e
e
e
e
7.0 ꢁ 0.5
e
e
e
e
e
e
25 ꢁ 3
96.85 ꢁ 2.13
oxiran moiety. After several probes, the compound 42 was obtained
by the application of Mitsunobu reaction in dry THF.
Most of the tested compounds 6ae25a displayed moderate to
high affinities for a1-adrenoceptors, 5-HT1A and 5-HT7, whereas
their affinities for 5-HT6 were significantly lower (Table 1) with
In the last step of the synthesis route, 5-(4-fluorophenyl)-5-
methyl-3-(oxiran-2-ylmethyl)imidazolidine-2,4-dione (42) was
used as an alkylating agent for N-alkylation of 2-
methoxyphenylpiperazine in solvent-free conditions under mi-
crowave irradiation [4,24]. The pure compound 25 was obtained as
hydrochloride salt (25a) by column chromatography purification
and saturation of the pure fractions in ethanol solution using
gaseous HCl.
Kið5ꢀHT Þ values in micromolar range. The highest activity and
6
selectivity at a1-AR was observed for compounds 10ae12a with
Ki < 15 nM. Certain selectivity towards a1-AR was comprehended
for compounds 19ae21a, however their activities were lower
(30 nM < Ki < 80 nM). Furthermore, compound 8a was the most
active one when considering a1-adrenoceptors and 5-HT1A receptor
with distinct selectivity profile towards serotonin receptor
(Ki ¼ 0.8 nM). In general, the most potent 5-HT1A agents (7ae10a)
were found in group A (Table 1) with noticeable selectivity in the
case of compounds 7ae9a. Moreover, a slight selectivity profile
towards 5-HT1A was also observed for compounds 15a and 16a,
whereas tested population displayed significant submicromolar
affinity for 5-HT7 receptors (3 nM < Ki < 456 nM). In particular,
compound 25a showed the highest affinity and selectivity towards
2.2. Pharmacology
2.2.1. Radioligand binding studies
Compounds 6ae25a were examined in vitro for their affinity on
a1-adrenoceptors and serotonin receptors 5-HT1A, 5-HT6, 5-HT7 in
radioligand binding assays. The results expressed by Ki values (nM)
are shown in Table 1. The affinity for a1-adrenoceptors was tested
on rat cerebral cortex by the using of [3H]-prazosin as a specific
radioligand. In the case of serotonin receptors, the studies were
performed on human recombinant receptors expressed in
HEK293 cells using [3H]-8-OH-DPAT, [3H]-LSD and [3H]-5-CT for 5-
HT1A, 5-HT6 and 5-HT7 assays, respectively.
5-HT7, in respect to the rest of considered GPCRs (sel 5-HT7/a1
-
AR ¼ 60; sel 5-HT7/5-HT1A ¼ 40; sel 5-HT7/5-HT6 ¼ 3597). Some
selectivity towards 5-HT7 was also observed (Table 1) for the potent
agents 17a, 22a and 23a (Ki5ꢀHT < 80 nM) as well as for a moderate
7
one, 24a (Ki5ꢀHT ¼ 456 nM). Radioligand binding assays at 5-HT6
7
receptors resulted only in selecting two compounds with

328
J. Handzlik et al. / European Journal of Medicinal Chemistry 78 (2014) 324e339
a) group A:
Ph
Ph
Br
Br
Ph
Ph
O
O
Ph
Ph
Ph
O
Ph
N
Ph
NH
O
HCl
N
Ph
ii
iii
,
CH3J, i
5
,
10a
Br
N
5
N
N
N
5
N
HN
N
CH3
CH3
HN
NH
CH3
O
O
O
O
28
27
10
26 (DPH)
b) group B:
CH3
O
CH3
Ph
O
N
KCN,
(NH4)2CO3, iv
Ph
HN
HCl
R
N
N
N
11a-13a
CH3
NH
5
O
O
H3C
O
11-13
30
29
R
11 R= H
12 R= 2-F
13 R= 2-MeO
N
CH3I, i
N
H
,v
Br
Br
CH3
CH3
ii
5
,
O
O
Ph
Ph
HN
Br
N
5
N
N
CH3
CH3
O
O
31
32
Scheme 1. The synthesis route for compounds of group A and B (10e13a). i e EtONa, reflux; ii e acetone, TEBA, K2CO3, rt; iii e acetone, TEBA, K2CO3, reflux; iv e BucherereBergs
reaction, EtOH 50%, 55 ꢂC; v e acetone, TEBA, K2CO3, mv irradiation.
submicromolar affinities (22a and 23a). Both compounds, however,
displayed higher potency at 5-HT1A and 5-HT7 than that at 5-HT6
receptors (Table 1).
to better characterize their biochemical and molecular effects.
Expression of specific human 5-hydroxytryptamine type 5-HT3A
receptors (h5-HT3A) receptor subunit was constructed in Xenopus
laevis oocytes, and electrophysiological method was utilized to
evaluate the efficacy of the positive modulation of 5-HT3A-evoked
chloride currents by derivatives 10a, 13a, 14a, 21a, 22a, and 25a in
2.2.2. Functional bioassays
2.2.2.1. The a1-adrenoceptor subtypes activity. The two most se-
lective a1-adrenoceptor compounds (11a and 12a) were chosen for
further investigation, and their selectivity at different a1-adreno-
ceptor subtypes was assessed in functional experiments. In the
functional bioassays, tail artery for a1A [27,28], mouse spleen for a1B
[29] and rat aorta for subtype a1D [29] were used. On the tissues
applied, the antagonism exerted by 11a and 12a was competitive
thus enabled the calculation of functional affinities (pA2 values).
The results are shown in Fig. 3 (11a), Fig 4 (12a) and Table 2. Our
results indicated that 11a and 12a are potent antagonists at all three
receptor subtypes with pA2 values ranging from 7.312 to 8.521.
Compound 11a showed high antagonistic properties for a1D-adre-
noceptors (pA2 ¼ 8.136) and its ability to block a1A- and a1B-adre-
noceptors was approximately six fold lower. Importantly,
compound 12a displayed similar antagonistic affinity for a1B- and
a1D-adrenoceptors (pA2 ¼ 8.142 and 8.521 respectively), however,
lower antagonistic properties for a1A-subtype (pA2 ¼ 7.316).
Moreover, both compounds (11a, 12a) were not found to be selec-
tive, since the differences in potency were not high enough to
consider them as selective antagonists for a1-adrenoceptor
subtypes.
comparison with that of 0.1
5-HT3A receptors (Fig. 5, Table 1). To this end, the agonist 5-HT
(10 M) activated fast inward currents only in oocytes injected
with cRNA transcribed from cloned cDNA encoding human 5-HT3A
receptors (data not shown, n ¼ 12). Currents activated by 1 M 5-HT
were completely inhibited by 0.1 M LY278584, a specific antago-
mM LY278584, a specific antagonist of
m
m
m
nist of 5-HT3 receptor, further indicating that the 5-HT induced
current responses were mediated by the 5-HT3 receptor-ion
channel complex (n ¼ 7). 5-HT (1
mM)-evoked currents recorded
by two-electrode voltage clamp technique, were reversibly inhibi-
ted by tested compounds 10a, 13a, 14a, 21a, 22a, and 25a at a dose
of 30 mM with percent values in the range of 13.75e36.86% (n ¼ 5)
(Fig. 5, Table 1).
2.3. Structure activity relationship
The lead structure 6a is
a member of phenylpiperazine
phenytoin family incorporating derivatives with 2-hydroxypropyl
linker at 1-position of hydantoin skeleton. This family was well
defined in the field of structural properties which have been
postulated based on our previously performed studies focusing on
crystallographic analyses and molecular modeling [19,25,26].
Therefore, moderate as well as almost equal activity of compound
6a at all three investigated receptors, a1-AR, 5-HT1A and 5-HT7, can
2.2.2.2. Studies on inhibitory effect at human 5-HT3A. In this study,
the profile of compounds 10a, 13a, 14a, 21a, 22a, and 25a repre-
senting the chemical groups A-D was further investigated in order

J. Handzlik et al. / European Journal of Medicinal Chemistry 78 (2014) 324e339
329
a) group C:
R
R1
N
CH3
CH3
Br
Br
CH3
CH3
O
O
H3C
H3C
HN
O
O
H3C
HN
N
H
H3C
ii
, i
5
,
, i
N
X
R
Br
N
5
N
N
NH
N
N
5
N
O
R1
O
O
R1
R1
O
14-24
34-36
37-39
33
HCl
14
15
16
17
R= H
34, 37, 14-19 R1= H
35, 38, 20, 21 R1= 4-F
R= 2-MeO
R= 3-MeO
R= 2-F
14a-24a
36, 39, 22-24 R1= 2,4-diCl
18, 21 R= 4-F
19, 20 R= 2,4-diF
34
X = Br
22
23
24
R= 2,3-diCl
R= 3,4-diCl
R= 4-Cl
35, 36 X= Cl
b) group D:
F
F
F
KCN,
(NH4)2CO3,iii
CH3
O
CH3
OH
O
, iv
O
HN
NH
HN
N
H3C
O
O
O
H3CO
N
O
25a
41
42
40
l
C
N
H
H
,v
H3CO
N
F
CH3
O
OH
HN
N
N
O
25
Scheme 2. The synthesis route for compounds of group C and D (14ae25a). i e acetone, TEBA, K2CO3, reflux; ii e acetone, TEBA, K2CO3, rt; iii e BucherereBergs reaction, EtOH 50%,
55 ꢂC; iv e Mitsunobu reaction, DEAD, TPP, dry THF, 0 ꢂC; v e microwave irradiation, solvent-free condition.
be explained on the basis of pharmacophore models suitable for
these receptors (Fig 2). According to our previous studies [25], the
phenylpiperazine phenytoin derivatives include all structural fea-
tures required by pharmacophore models of both a1-AR and 5-HT1A
antagonists, however, some differences from spatial colocations of
ideal antagonists moieties were observed in both cases. When
considering previous results [25] compound 6a can be suggested as
a perfect agreement with pharmacophore model of a1-adreno-
ceptor antagonist in the case of PI-HY1 distance (Fig. 2d), some
agreement in PI-HBA distances (5.11e5.15 A in the case of phenyl-
piperazine phenytoin derivatives, and 5.62 in the pharmacophore
model of Barbaro) and too short distance between piperazine
protonable nitrogen (PI) and the third hydrophobic area (HY3)
matched by phenyl ring(s) at hydantoin’s 5-position [25,26]. In
particular, the presence of two hydrophobic rings at 5-position of
hydantoin, which can represent pharmacophoric feature HY3, was
in the radioligand binding assay (Ki ¼ 542.3 nM, Table 1), too. When
considering pharmacophore model of 5-HT1A-antagonist (Fig. 2e),
the previous studies on phenylpiperazine phenytoin derivatives
[25] indicated an agreement in the distance PI-A but the rest of
distances (A-HYD, A-HBA, PI-HYD and PI-HBA) were slightly too
short as compared to those of ideal antagonists. These small spatial
disagreements are highly probable to be a reason behind moderate
affinity observed for 6a at 5-HT1A. The current studies demon-
strated that affinity of the lead structure (6a) for 5-HT7 receptors is
also in submicromolar range. Although the radioligand binding
assay did not explore the way of interaction with receptor, some
differences can be observed between compound 6a and each 5-HT7
pharmacophore model, including model of agonist, inverse agonist
and antagonist (Fig. 2gei). As the highest distance-tolerance is
described for pharmacophore features of the 5-HT7 antagonist, it is
suggested that compound 6a can fit in this model with much better
agreement than those for agonists and inverse agonists. Never-
theless, its affinity for 5-HT7 is not very high (Ki ¼ 353 nM) and
almost identical as that for 5-HT1A (Table 1). The analysis of
structural properties of compound 6a based on pharmacophore
models suitable for the considered GPCRs gave some suggestions in
the field of activity and selectivity modulation. However, the
expected as a factor that limits antagonistic interaction with a1
-
adrenoceptor, resulting only in average submicromolar affinities for
a1-AR of those phenylpiperazine compounds [25,26]. In the case of
compound 6a, lack of hydrophobic substituents at o- or m-position
of phenylpiperazine phenyl ring, which fit in the HY2 pharmaco-
phoric feature, can be responsible for only mean affinity observed

330
J. Handzlik et al. / European Journal of Medicinal Chemistry 78 (2014) 324e339
Fig. 3. Effect of compound 11a on a1-adrenoceptors. Concentration-response curves to
noradrenaline (NA) in the absence (,) or presence of increasing concentrations of 11a
(filled symbols). a) rat tail artery (a1A-adrenoceptors); b) mouse spleen (a1B-adreno-
ceptors); c) rat aorta (a1D-adrenoceptors). Results are expressed as percentage of the
maximal response to NA in the first concentrationeresponse curve. Each point rep-
resents the mean ꢁ SEM (n ¼ 4e7).
Fig. 4. Effect of compound 12a on a1-adrenoceptors. Concentration-response curves to
noradrenaline (NA) in the absence (,) or presence of increasing concentrations of 12a
(filled symbols). a) rat tail artery (a1A-adrenoceptors); b) mouse spleen (a1B-adreno-
ceptors); c) rat aorta (a1D-adrenoceptors). Results are expressed as percentage of the
maximal response to NA in the first concentrationeresponse curve. Each point rep-
resents the mean ꢁ SEM (n ¼ 4).
HYD). In compounds 7ae9a, alkoxyl substituents at o-position of
phenylpiperazine were introduced to facilitate further interactions
with the receptors. Our results indicated that such modifications
presented models show mutual commonalities and therefore are
not sufficient to be followed in search to design agents of selective
action towards one of the receptor subtypes.
In this context, four modifications (AeD, Table 1) of the lead
were designed and performed to gradually expand structural
divergence from the starting compound (6a), and on this way to
improve the compounds discrimination between the considered
GPCRs with special accent on 5-HT7 receptors.
were particularly enhancing affinity and selectivity at 5-HT1A
.
Table 2
Functional affinities of test compounds 11a and 12a at a1A-AR in rat tail artery, at
a1B-AR in mouse spleen and at a1D-AR in rat aorta. Antagonist potency of test
compounds expressed as pA2 ꢁ SEM.
In consensus with suggestions resulting from pharmacophore
models as well as from our previous SAR-analyses [19,25,26],
certain prolongation of distances between pharmacophoric frag-
ments seemed to be profitable to gain biological activity at target.
Thus, the first modifications (A, Table 1) were focused on 2-
hydroxypropyl linker, which was deprived of OH-substituents to
increase flexibility and prolonged from tri- to pentamethylene
length. These intramolecular changes enabled to accommodate
distances between pharmacophoric fragments (PI-HY3, PI-HBA, PI-
Agonist:
NA compd
a1A-AR rat tail
artery pA2 ꢁ SEM
(slope ꢁ SEM)
a1B-AR mouse
spleen pA2 ꢁ SEM
(slope ꢁ SEM)
a1D-AR rat
aorta pA2 ꢁ SEM
(slope ꢁ SEM)
11a
12a
7.413 ꢁ 0.011
(0.90 ꢁ 0.01)
7.316 ꢁ 0.116
(1.02 ꢁ 0.12)
7.312 ꢁ 0.167
(0.97 ꢁ 0.08)
8.142 ꢁ 0.050
(0.91 ꢁ 0.01)
8.136 ꢁ 0.013
(0.93 ꢁ 0.01)
8.521 ꢁ 0.200
(1.06 ꢁ 0.09)
pA2 values were obtained from the linear regression of Schild plot.
Each value was the mean ꢁ SEM of 4e7 experimental results.

J. Handzlik et al. / European Journal of Medicinal Chemistry 78 (2014) 324e339
331
distance between hydantoin and piperazine, performed by intro-
duction of C5-linker, approached compounds (11ae13a) that meet
the structural requirements proposed by Barbaro et al. (Fig. 2d) for
a1-antagonist model [22]. Compounds of modifications B contain
structural moieties that can well fit into pharmacophore features of
both, a1A and a1D, adrenoceptor subtypes antagonists (Fig. 2a,c)
[16]. In the case of a1B, the hydrogen bond donor area (HBD) is
missing in the chemical structures of 11ae13a (Fig. 2b, Table 1),
however, both tested compounds (11a and 12a) demonstrated
significant antagonistic properties at the a1B subtype during the
functional bioassays. These results clearly suggest that pharmaco-
phore models of Bremner [16] are not sufficient to characterize the
group of 5-phenylhydantoin derivatives, especially those with
antagonistic properties at a1B-ARs.
Modifications C gave a series of eleven compounds with C5-
linker (14ae24a), in which an aromatic area was shifted from 5-
position into the benzyl substituent at 3-position of hydantoin
skeleton. The activities at target GPCRs were modulated by number
and position of small hydrophobic substituents at aromatic rings on
both, N1- and N3-position of hydantoin skeleton sides. The modi-
fications C were applied to increase selectivity at 5-HT7 and to
decrease those at both a1-AR and 5-HT1A receptors. The expected
results were only obtained by highly lipophilic compounds bearing
2,4-dichlorobenzyl substituents at 3-position of hydantoin and
chloride substituent(s) at the phenylpiperazine phenyl ring (22ae
Fig. 5. Inhibition of 10a, 13a, 14a, 21a, 22a, and 25a in comparison to standard 5-HT3A
receptor antagonist LY278584. Results are expressed as percentage inhibition of 5-HT-
induced currents in Xenopus laevis oocyte expressing human 5-HT3A receptors. Each
value represents the mean ꢁ SEM (n: number of oocytes tested). LY278584: 1-Methyl-
N-(8-methyl-8-azabicyclo[3.2.1]-oct-3-yl)-1H-indazole-3-carboxamide,
a
specific
antagonist for 5-HT3 receptors.
24a). The optimal 5-HT7 activity and selectivity with respect to a1
-
AR (Ki5ꢀHT ¼ 31 nM, sel a1 ¼ w31, sel
¼ w3) was observed for
5ꢀHT1A
7
Among the derivatives designed, the obtained results clearly
showed that 2-alkoxyphenylpiperazine derivatives having un-
branched 3e5-carbon linker were more effective at 5-HT1A re-
2,3-dichlorophenylpiperazine derivative 22a, whereas a slightly
higher 5-HT7 selectivity with respect to 5-HT1A was observed by 3,4-
dichlorophenylpiperazine derivative 23a (Ki5ꢀHT ¼ 78 nM, sel
7
ceptors. Moreover, compound (8a) as
a
derivative of 2-
a1 ¼ w9, sel
¼ w4). Interestingly, compounds with benzyl- or
5ꢀHT1A
methoxyphenylpiperazine with butyl linker demonstrated sub-
nanomolar affinities for 5-HT1A, whereas compound (9a) as a de-
rivative of 2-ethoxyphenylpiperazine with propyl linker displayed
the highest selectivity towards 5-HT1A compared to a1-AR (w473)
and a significant effect (w17) at 5-HT7 receptors (Table 1).
Furthermore, most members of modifications A were significantly
more potent than lead compound 6a at both 5-HT7 and a1-adren-
ergic receptors, indicating a superiority of the unbranched linkers
over the 2-hydroxypropyl ones. However, the modifications of lead
compound 6a were not sufficient to provide selectivity for 5-HT7,
thus greater structural changes starting from the lead structure
were necessary. Therefore, further modifications (B and C, Table 1)
were focused on the number and colocation of aromatic- and
methyl groups at 3- and 5-position of hydantoin.
Within the modifications B, compounds having C5-linker were
under consideration, in which one of the 5,5-diphenyl rings was
replaced with the methyl moiety (11ae13a). Analysis of the ob-
tained results showed that these changes were distinctly favorable
for interactions with a1-adrenoceptors. Although the 2-
methoxyphenyl derivative was found to be not selective at a1/5-
HT1A/and 5-HT7 receptors-selective, the unsubstituted- (11a) and
2-fluorophenylpiperazine (12a) derivatives demonstrated the
highest a1-AR selectivity when compared to the rest of other
modifications (A, C and D, Table 1). The affinities for a1-AR of all
three compounds 11ae13a were found to be significant, with Ki
values in nanomolar ranges (Table 1). The most active and selective
4-fluorobenzyl substituents (14ae21a) displayed significant affin-
ities for a1-AR (35 nM < Ki < 100 nM) even in case of halogen sub-
stituents at p-position of phenylpiperazine, which was evaluated as
a disadvantageous factor by different previous pharmacophore-
based studies [3,12,22]. Our current results clearly indicate the
importance of 4-fluorobenzyl moiety for interactions with a1-AR as
the p-substituted phenylpiperazine hydantoin derivatives of 4-
fluorobenzyl (20a and 21a) were more potent at a1-AR than the o-
methoxyphenylpiperazine derivative with unsubstituted benzyl
ring (15a). A comparison of the 4-fluorobenzylhydantoin derivatives
with their respective benzyl ones (2,4-difluorophenylpiperazine-
20a and 19a, 4-fluorophenylpiperazine derivatives 21a and 18a)
confirmed higher activities for 4-fluorobenzyl analogues observed
in both cases of the fluoropiperazines. Moreover, the results ob-
tained in the 5-HT1A radioligand binding assays evidently indicated
that substituents at benzyl ring are not as much important as types
and positions of those substituents at phenylpiperazine phenyl ring.
Thus, o- or/and m-substituents at phenylpiperazine (15ae17a and
22a) triggered significantly higher activity than those of compounds
with substituents at p-position of phenylpiperazine phenyl ring
(18ae21a, 23a and 24a). The modifications C resulted in selection of
few compounds with stronger actions at 5-HT7 receptors than those
on 5-HT1A or a1-AR, however, their selectivity profile was not
remarkable when considering 5-HT1A and 5-HT7 receptors.
In this context, we decided to depart from further systematic
modifications and perform more drastic changes within lead (6a)
structures, including introduction of 4-fluorophenyl- and methyl
moieties at 5-position and an exchange of the location of 2-
hydroxypropylphenylpiperazine substituent from 1-position into
3-position of hydantoin with simultaneous introduction of 2-
methoxy function at phenylpiperazine phenyl ring (compound
25a, Table 1). These significant changes enabled us to improve the
expected pharmacological properties, since compound 25a was
much more potent at 5-HT7 receptors among the tested hydantoin
a1-AR agents 11a and 12a, investigated on their activities at a1
-
adrenoceptor subtypes, displayed antagonistic effect at a1-AR
subtype with the highest potency for the a1D-AR in both cases.
These results are in good agreement with conclusions evolving
from our previous pharmacophore-based studies on 5,5-
diphenylhydantoin derivatives, in which the bulky 5,5-diphenyl
aromatic area did not fit well in HY3 pharmacophoric features.
The removal of one phenyl ring with a concomitant increase of

332
J. Handzlik et al. / European Journal of Medicinal Chemistry 78 (2014) 324e339
compounds (6ae24a), displaying Ki value in the nanomolar range
(3 nM) and a significant selectivity in respect to the rest of inves-
search for potent and selective 5-HT7 receptors antagonists among
phenylpiperazine derivatives of hydantoin class.
tigated GPCRs (sel a1 w60, sel
w40, sel5ꢀHT w3597).
5ꢀHT1A
6
In general, the investigated series of hydantoin phenyl-
piperazine derivatives did not show any potent interactions with 5-
HT6 receptors. It can be explained based on pharmacophore models
(Fig. 2g) which postulate more compact 5-HT6-antagonist struc-
tures with short distances between aromatic, bulky hydrophobic,
and positive ionizable areas. The central position is postulated for
aromatic fragment, and an extended (terminated) one for a positive
ionizable fragment (PI) which can be well matched by N-methyl- (4,
Fig. 1), however, not by N-phenylpiperazine derivatives. Interest-
ingly, the most hydrophobic compounds with four chloride sub-
stituents at aromatic rings (22a and 23a) demonstrated moderate
affinities for 5-HT6 receptors with Ki values in submicromolar range
(Table 1). The rest of series was found to be weakly- or almost
inactive (1792 nM < Ki < 30 000 nM).
The representative members of each modification way (AeD)
were tested on their pharmacological properties at 5-HT3A re-
ceptors. The results observed clearly show that tested compounds
displayed low inhibitory activities in the range of 13.75e36.86%.
Among tested representative derivatives, phenylpiperazinepentyl
derivative 10a was found to be the most active one with an
inhibitory value of 36.86% at 5-HT3A receptors (Table 1), however,
even this activity was much lower than that of standard antagonist
LY278584 (Fig. 5).
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 DMSO-d6 at
ambient temperature using the solvent signal as an internal stan-
dard. IR spectra were recorded on a Jasco FT/IR-410 apparatus using
KBr pellets and are reported in cmꢀ1. 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)
CHCl2/acetone 10:1; (III) toluene/acetone 10:1. (IV) toluene/acetone/
methanol 15:5:1. Melting points were determined using Mel-Temp
II apparatus and are uncorrected. The mass for compounds 10e25a
were obtained on Waters ACQUITYÔ TQD system with the TQ De-
tector (Waters, Milford, USA). The ACQUITY UPLC BEH C18, 1.7 mm,
2.1 ꢃ 50 mm column was used (Waters, Milford, USA). Elemental
analyses (C, H, N) were measured on Elemental Vario-EL III instru-
ment and are within ꢁ0.4% of the theoretical values unless stated
otherwise. Syntheses under microwave irradiation were performed
in household microwave oven Samsung M1618 or in microwave
reactor “Plazmatronika”. Syntheses of compounds 6e9, 27, 30e32
and 41 are described elsewhere [25,26].
3. Conclusion
4.1.1. Synthesis of 1-(5-bromopentyl)-3-methyl-5,5-
diphenylimidazolidine-2,4-dione (28)
The performed studies allowed to obtain a series of active GPCRs
agents among phenylpiperazine derivatives of hydantoin with
various collocation of chemical moieties, important for interaction
with a1-adrenergic and serotonin receptors. Although the 5-HT7
receptors are the main pharmacological target of the current work,
the series of hydantoin derivatives were also evaluated on their
affinities for a1-adrenergic-, serotonin 5-HT1A, and 5-HT6 receptors
in the radio-ligand binding assays, and selected representative
structures were further investigated on their action on 5-HT3A or
A mixture of 3-methyl-5,5-diphenylhydantoin 27 (15 mmol,
4.00 g), TEBA (2 mmol, 0.45 g) and potassium carbonate (44 mmol,
6 g) in acetone (30 mL) was stirred under reflux for 30 min, then
1,5-dibromopropane (20 mmol, 4.60 g) in acetone (15 mL) was
added. The mixture was stirred at room temperature for 90 h, ac-
cording to a progress controlled by TLC (I). Then, inorganic pre-
cipitate was filtered off, the mother liquor was evaporated. The
residue was purified by double chromatography columns (II)
crystallized using ethanol with diethyl ether and n-hexane to give
white crystals of 28 (2.74 g, 6.60 mmol). Yield 44%, mp 58e59 ꢂC;
TLC: Rf (I): 0.60. Anal. Calcd for C21H23BrN2O2: C, 60.73; H, 5.58; N,
a1-adrenergic subtypes, a1A, a1B and a1D, in functional bioassays.
Results of the assays showed that hydantoin derivatives tend to
strongly interact with a1-AR, 5-HT1A and 5-HT7 receptors, whereas
their actions on 5-HT3A or 5-HT6 were found to be very weak.
The pharmacophore-based SAR-analysis clearly indicated that
compounds with phenylpiperazine-alkyl substituent at 1-position
of 3-methylhydantoin: (1) are particularly active at 5-HT1A in case
of phenytoin-like compounds with unbranched alkyl linker; (2) are
particularly active at a1-AR in case of the 5-methyl-5-
phenylhydantoin compounds with pentyl linker. Regarding 5,5-
dimethyl-3-(halogen)benzylhydantoin with pentyl linker at 1-
position: (3) arylpiperazine derivatives of 3-(4-fluorobenzyl)
hydantoin are evidently the most potent at a1-AR, and (4) 3-(2,4-
dichlorobenzyl)hydantoin derivatives are selective towards 5-HT7
when chloride substituent is placed at p-position of the phenyl-
piperazine moiety.
The optimal potency and selectivity at 5-HT7 was achieved by the
compound (25a) through the following modifications: (i) reversing
phenylpiperazine-alkyl substituent of the lead (6a) hydantoin ring
from 1- to 3-position, (ii) removal of one phenyl ring from 5-position,
and (iii) introducing of lipophilic substituents at both aromatic rings.
Since the most promising 5-HT7 receptoragent (25a) was found as an
orphan’s member of its group (modifications D), it is difficult to
recognize which one of the three modifications (ieiii) was crucial for
the increasing activity of the lead compound 6a. Thus, the current
question needs further SAR-studies, in which compound 25a seems
to be a good lead structure for further chemical modifications in
6.74. Found: C, 60.77; H, 5.56; N, 6.69. 1H NMR
d (ppm): 0.70 (qu,
J ¼ 7.40 Hz, 2H, N1eCH2eCH2eCH2), 0.96 (qu, J ¼ 7.40 Hz, 2H, N1e
CH2eCH2), 1.41 (qu, J ¼ 7.40 Hz, 2H, BreCH2eCH2), 2.96 (s, 3H, Ne
CH3), 3.23e3.29 (tdef., 4H, N1eCH2, BreCH2), 7.19e7.24 (m, 4H, 2ꢃ
Ph-3,5-H), 7.40e7.44 (m, 6H, 2ꢃ Ph-2,4,6-H).
4.1.2. General procedure for synthesis of 3-benzyl derivatives of 5,5-
dimethylhydantoin (34e36)
5,5-Dimethylhydantoin 33 (50e100 mmol) was stirred under
reflux with TEBA (1.5e3 g), K2CO3 (20e40 g) in acetone (200e
500 mL) for 30 min. An appropriate benzyl chloride or bromide
(50e90 mmol) in acetone (40e100 mL) was added. Then, the
mixture was boiled under reflux for 5e6 h and left at room tem-
perature for 16 h. The precipitate was filtrated off. A pure product
was obtained from the filtrate by two methods A or B.
Method A: the pure product was precipitated from the filtrate
with water.
Method B: the filtrate was evaporated. The obtained residue
was dissolved by boiling in ethanol. The pure product was
precipitated from the ethanol solution with water.
4.1.2.1. 3-Benzyl-5,5-dimethylimidazolidine-2,4-dione
(34).
5,5-Dimethylhydantoin 33 (12.80 g, 100 mmol), K2CO3 (40.0 g),

J. Handzlik et al. / European Journal of Medicinal Chemistry 78 (2014) 324e339
333
TEBA (3.00 g) in acetone (500 mL) and benzyl bromide (17.00 g,
99 mmol) in acetone (100 mL) were refluxed for 5 h, purified by
method A to give white crystals of product 34 (16.0 g, 73 mmol).
Yield 73%, mp 86e87 ꢂC; TLC: Rf (III): 0.19. Anal. Calcd for
(qudef., 2H, CH2eCH2eN1),1.76e1.82 (qudef., 2H, CH2eCH2eBr), 3.20
(t, J ¼ 7.44 Hz, 2H, CH2eN1), 3.49 (t, J ¼ 6.67 Hz, 2H, CH2eBr), 4.52
(s, 2H, CH2-Ph), 7.11e7.18 (ddef., 2H, Ph-2,6-H), 7.23e7.28 (ddef., 2H,
Ph-3,5-H).
C
12H14N2O2: C, 66.04; H, 6.47; N, 12.84. Found: C, 66.12; H, 6.49; N,
12.88. 1H NMR
d
(ppm): 1.28 (s, 6H, 2 ꢃ CH3), 4.50 (s, 2H, CH2-Ph),
4.1.3.3. 1-(5-Bromopentyl)-3-(2,4-dichlorobenzyl)-5,5-
dimethylimidazolidine-2,4-dione (39). 3-(2,4-Dichlorobenzyl)-5,5-
dimethylimidazolidine-2,4-dione 36 (8.61 g, 30 mmol), K2CO3
(12.00 g), TEBA (0.90 g) in acetone (120 ml) and 1,5-dibromo-
pentane (17.94 g, 78 mmol) in acetone (60 ml) were stirred for 72 h
to give product 39 in form of yellow glass-oil (4.98 g, 13,6 mmol).
Yield 45%; TLC: Rf (III): 0.58. Anal. Calcd for C17H21BrCl2N2O2: C,
46.81; H, 4.85; N, 6.42. Found: C, 46.50; H, 4.54; N, 6.11. 1H NMR
7.18e7.35 (m, 5H, Ph), 8.37 (br. s, 1H, NH).
4.1.2.2. 3-(4-Fluorobenzyl)-5,5-dimethylimidazolidine-2,4-dione
(35). 5,5-Dimethylhydantoin 33 (6.40 g, 50 mmol), K2CO3 (20.00 g),
TEBA (1.50 g) in acetone (200 mL) and 4-fluorobenzyl chloride
(7.23 g, 50 mmol) in acetone (40 mL) were refluxed for 3 h, purified
by method B to give white crystals of product 35 (8.70 g, 37 mmol).
Yield 74%, mp 74e75 ꢂC; TLC: Rf (III): 0.20. Anal. Calcd for
d
(ppm): 1.35 (s, 6H, 2 ꢃ CH3), 1.35e1.43 (m, 2H, CH2eCH2eCH2e
C
12H13FN2O2: C, 61.01; H, 5.55; N, 11.86. Found: C, 60.98; H, 5.62; N,
N1), 1.52 (qudef., 2H, CH2eCH2eN1), 1.77 (qudef., 2H, CH2eCH2eBr),
3.22 (t, J ¼ 7.60 Hz, 2H, CH2eN1), 3.49 (t, J ¼ 6.70 Hz, 2H, CH2eBr),
11.80. 1H NMR
d
(ppm): 1.27 (s, 6H, 2 ꢃ CH3), 4.48 (s, 2H, CH2-Ph),
7.12e7.16 (ddef., 2H, Ph-2,6-H), 7.23e7.24 (ddef., 2H, Ph-3,5-H), 8.37
4.60 (s, 2H, CH2-Ph), 7.14 (d, J ¼ 8.20 Hz, Ph-6-H), 7.38e7.44 (ddef.
1H, Ph-5-H), 7.62 (d, J ¼ 2.30 Hz, 1H, Ph-3-H).
,
(br. s, 1H, NH).
4.1.2.3. 3-(2,4-Dichlorobenzyl)-5,5-dimethylimidazolidine-2,4-dione
(36). 5,5-Dimethylhydantoin 33 (6.40 g, 50 mmol), K2CO3 (20.00 g),
TEBA (1.50 g) in acetone (200 mL) and 2,4-dichlorobenzyl chloride
(79.77 g, 50 mmol) in acetone (40 mL) were refluxed for 3.5 h,
purified by method A to give white crystals of product 36 (12.46 g,
43 mmol). Yield 87%, mp 134e135 ꢂC; TLC: Rf (III): 0.23. Anal. Calcd
for C12H12Cl2N2O2: C, 50.19; H, 4.21; N, 9.76. Found: C, 50.38; H,
4.1.4. General procedure for synthesis of phenylpiperazine
hydantoin derivatives (10ae13a)
Commercially available phenylpiperazine (5 mmol), K2CO3
(2.00 g) and acetone (16 mL) were stirred and refluxed for 15 min in
special round flask “Plazmatronika”. Then, correspondingly
substituted bromopentyl hydantoin derivative 28 or 32
(5.50 mmol) in acetone (16 mL) was added. The mixture was placed
in microwave reactor and stirred under reflux using the following
irradiation program: time heating 10 min, maximal power 20%,
high temperature 58 ꢂC, and low temperature 45 ꢂC. The program
was repeated 6 times under TLC control (IV). Then, the solution was
stirred overnight. The inorganic precipitate was separated by
filtration. The filtrate was evaporated and purified by the chroma-
tography column (II) giving pure compound 10e13 in basic forms.
Compound 10e13 (0.50 g) was dissolved in ethanol (10 mL). To the
solution 3-5 drops of concentrated HCl was added to give precipi-
tate of desirable compound (10). As no precipitate was generated
(11e13), the solution was evaporated, the residue was dried under
vacuum at the presence of CaCl2, and crystallized with dry EtOH
and 3-6 drops of acetone to give the desirable product (11ae13a) in
the hydrochloride form.
4.32; N, 9.69. 1H NMR
d
(ppm): 1.31 (s, 6H, 2 ꢃ CH3), 4.55 (s, 2H,
CH2-Ph), 7.12 (d, J ¼ 8.20 Hz, 1H, Ph-6-H), 7.38e7.40 (ddef., 1H, Ph-5-
H), 7.63 (s, 1H, Ph-3-H), 8.46 (s, 1H, NH).
4.1.3. General procedure for synthesis of 1-bromopentyl derivatives
of 3-benzyl-5,5-dimethylhydantoins (37e39)
A
suitable 3-benzyl-5,5-dimethylohydantoin 34e36 (20e
60 mmol), TEBA (0.60e1.80 g), K2CO3 (8.00e24.00 g) was stirred in
acetone (40e120 mL) for 30 min. A solution of 1,5-dibromopentane
(26e95 mmol) in acetone (20e60 ml) was added. The mixture was
stirred at room temperature for 72e90 h. The precipitate was fil-
trated off. The filtrate was evaporated. The residue was dissolved in
methylene chloride (100 mL), washed with NaOH 2% (2 ꢃ 100 mL)
and water (2 ꢃ 100 mL) and dried with anhydrous Na2SO4 for 18 h.
After separation from the drying agent, the solution was concen-
trated and purified by column chromatography (II).
4.1.4.1. 3-Methyl-5,5-diphenyl-1-(5-(4-phenylpiperazin-1-yl)pentyl)
imidazolidine-2,4-dione hydrochloride (10a). 1-Phenylpiperazine
(0.81 g) and 1-(5-bromopentyl)-3-methyl-5,5-diphenylimi-
dazolidine-2,4-dione 28 (2.28 g) were used to give white crystals
compound 10 (0.79 g, 1.59 mmol). Yield 32%; mp 148e149 ꢂC; TLC:
Rf (IV): 0.69. MW 496.64. Monoisotopic Mass 496.28, [M þ H]þ
497.41. Anal. Calcd for C31H36N4O2: C, 74.97; H, 7.31; N, 11.28.
4.1.3.1. 3-Benzyl-1-(5-bromopentyl)-5,5-dimethylimidazolidine-2,4-
dione (37). 3-Benzyl-5,5-dimethylimidazolidine-2,4-dione 34
(13.10 g, 60 mmol), K2CO3 (24.00 g), TEBA (1.80 g) in acetone
(120 ml) and 1,5-dibromopentane (21.94 g, 95 mmol) in acetone
(60 ml) were stirred for 90 h to give product 37 in form of bright
glass-oil (13.0 g, 35 mmol). Yield 59%; TLC: Rf (III): 0.53. Anal. Calcd
for C17H23BrN2O2: C, 55.59; H, 6.31; N, 7.63. Found: C, 55.20; H,
Found: C, 75.04; H, 7.44; N, 11.19. 1H NMR for 10
d (ppm): 0.64e0.79
(qudef., 2H, PpeC2H4eCH2eC2H4-hyd), 0.90e0.98 (qudef., PpeCH2e
CH2eC3H6-hyd), 1.12e1.15 (m, 4H, PpeC3H6eCH2eCH2-hyd), 2.07e
2.10 (m, 2H, Pp-CH2), 2.46 (br.s, 4H, Pp-2,6-H), 2.97 (s, 3H, 3-CH3),
3.06 (br.s, 4H, Pp-3,5-H), 3.25 (t, J ¼ 7.70 Hz, 2H, CH2eN1-hyd), 6.73
(t, J ¼ 7.18 Hz, 1H, PpPh-4-H), 6.88 (d, J ¼ 7.94 Hz, 2H, PpPh-2,6-H),
7.16e7.23 (m, 6H, Ph-2,4,6-H), 7.42e7.44 (m, 6H, PpPh-3,5-H, Ph-
6.02; N, 7.39. 1H NMR
d
(ppm): 1.32 (s, 6H, 2 ꢃ CH3), 1.35e1.43 (m,
2H, CH2eCH2eCH2eN1), 1.45e1.59 (qudef., 2H, CH2eCH2eN1),
1.76e1.85 (qudef., 2H, CH2eCH2eBr), 3.21 (t, J ¼ 7.57 Hz, 2H, CH2e
N1), 3.49e3.55 (m, 2H, CH2eBr), 4.54 (s, 2H, CH2-Ph), 7.18e7.35 (m,
5H, Ph).
3,5-H). 13C NMR (75 MHz, DMSO-d6)
d [ppm]: 24.3, 25.8, 26.1, 27.8,
4 .1. 3 . 2 . 1 - ( 5 - B r o m o p e n t y l ) - 3 - ( 4 - fl u o r o b e n z y l ) - 5 , 5 -
41.7, 48.8, 58.1, 75.2, 115.7, 119.9, 128.6, 129.2, 129.3, 137.6, 151.8,
155.9, 173.1.
dimethylimidazolidine-2,4-dione
(38). 3-(4-Fluorobenzyl)-5,5-
dimethylimidazolidine-2,4-dione 35 (4.72 g, 20 mmol), K2CO3
(8.00 g), TEBA (0.60 g) in acetone (40 ml) and 1,5-dibromopentane
(5.98 g, 26 mmol) in acetone (20 ml) were stirred for 72 h to give
product 38 in form of white glass-oil (4.98 g, 13 mmol). Yield 65%;
TLC: Rf (III): 0.38. Anal. Calcd for C17H22BrFN2O2: C, 53.00; H, 5.76;
White powder of compound 10a. Yield 90%; mp 236e237 ꢂC;
TLC: Rf (IV): 0.69. Anal. Calcd for C31H37ClN4O2: C, 69.84; H, 7.00; N,
10.51. Found: C, 69.93; H, 7.11; N, 10.39. 1H NMR for 10
d
(ppm):
0.75e0.79 (qudef., 2H, PpeC2H4eCH2eC2H4-hyd), 0.92e0.97 (qudef.
,
PpeCH2eCH2-C3H6-hyd), 1.39 (br. s, 4H, PpeC3H6eCH2eCH2-hyd),
2.85 (br. s, 2H, Pp-CH2), 2.98 (s, 3H, 3-CH3), 3.06e3.09 (br.s, 4H, Pp-
3,5-H), 3.25 (m, 2H, CH2eN1-hyd), 3.39e3.45 (br.s, 2H, Pp-2,6-Ha),
N, 7.27. Found: C, 52.77; H, 5.49; N, 7.08. 1H NMR
d
(ppm): 1.31 (s,
6H, 2 ꢃ CH3), 1.31e1.40 (m, 2H, CH2eCH2eCH2eN1), 1.50e1.56

334
J. Handzlik et al. / European Journal of Medicinal Chemistry 78 (2014) 324e339
24). As method C*, the method C was performed for basic form
only (22).
3.74e3.78 (br.s, 2H, Pp-2,6-Hb), 6.82 (t, J ¼ 7.18 Hz, 1H, PpPh-4-H),
6.95 (d, J ¼ 7.94 Hz, 2H, PpPh-2,6-H), 7.21e7.27 (m, 6H, Ph-2,4,6-H),
7.42e7.64 (m, 6H, PpPh-3,5-H, Ph-3,5-H), 10.28 (br. s, 1H, NHþ).
Method D. The residue after evaporation was dissolved in EtOH
and stored at 4 ꢂC for 1e2 days. As no crystal appeared, the
solution was saturated with gaseous HCl, left at 4 ꢂC for 24 h to
give crystals of desirable products in hydrochloride forms (14ae
19a, 22a).
4.1.4.2. 3,5-Dimethyl-5-phenyl-1-(5-(4-phenylpiperazin-1-yl)pentyl)
imidazolidine-2,4-dione (11). 1-Phenylpiperazine (0.81 g) and 1-(5-
bromopentyl)-3,5-dimethyl-5-phenylimidazolidine-2,4-dione 32
(1.94 g) were used to give white crystals of compound 11 (0.15 g,
0.35 mmol). Yield 7%; mp 164e165 ꢂC; TLC: Rf (IV): 0.72. MW
434.57. Monoisotopic Mass 434.27, [M þ H]þ 435.39. Anal. Calcd for
4.1.5.1. 3-Benzyl-5,5-dimethyl-1-(5-(4-phenylpiperazin-1-yl)pentyl)
imidazolidine-2,4-dione hydrochloride (14a). Method D. 1-
Phenylpiperazine (2.50 mmol, 0.41 g) K2CO3 (1.0 g) in acetone
(10 ml) and 1-benzyl-1-(5-bromopentyl)-5,5-dimethylimida-
zolidine-2,4-dione 37 (2.70 mmol, 1.00 g) in acetone (10 mL)
were used to give white crystals of compound 14a (0.50 g,
1.00 mmol). Yield 38%; mp 206e207 ꢂC; TLC: Rf (IV): 0.31. MW
448.60. Monoisotopic Mass 448.28, [M þ H]þ 449.42. Anal. Calcd
for C27H37ClN4O2: C, 66.86; H, 7.69; N, 11.55. Found: C, 66.64; H,
C
26H34N4O2: C, 71.86; H, 7.89; N, 12.89. Found: C, 71.64; H, 7.80; N,
12.78. 1H NMR
d
(ppm): 1.13e1.17 (qudef., 2H, PpeC2H4eCH2eC2H4-
hyd),1.20e1.42 (qudef., 4H, CH2eCH2eCH2eCH2eCH2),1.77 (s,3H, 5-
CH3), 2.34 (br.s, 2H, Pp-CH2), 2.58 (br.s, 4H, Pp-2,6-H), 2.86e2.96
(m, 2H, CH2eN1-hyd), 2.91 (s, 3H, 3-CH3), 3.13 (br.s, 4H, Pp-3,5-H),
6.74 (t, J ¼ 7.18 Hz,1H, PpPh-4-H), 6.89 (d, J ¼ 7.95 Hz, 2H, PpPh-2,6-
H), 7.16 (t, J ¼ 7.18 Hz, 2H, PpPh-3,5-H), 7.27e7.43 (m, 5H, 5-Ph).
7.71; N, 11.50. 1H NMR
d (ppm): 1.22 (s, 2H, CH2eCH2eCH2eN1),
4.1.4.3. 1-(5-(4-(2-Fluorophenyl)piperazin-1-yl)pentyl)-3,5-
dimethyl-5-phenylimidazolidine-2,4-dione (12). 1-(2-Fluorophenyl)
piperazine (0.90 g) and 1-(5-bromopentyl)-3,5-dimethyl-5-
phenylimidazolidine-2,4-dione 32 (1.94 g) were used to give
white crystals of compound 12 (0.50 g, 1.11 mmol). Yield 22%; mp
178e179 ꢂC; TLC: Rf (IV): 0.56. MW 452.56. Monoisotopic Mass
452.26, [M þ H]þ 453.40. Anal. Calcd for C26H33FN4O2: C, 69.00; H,
1.34 (s, 6H, 2 ꢃ CH3), 1.56 (qudef., 2H, CH2eCH2eBr), 1.74 (qudef., 2H,
CH2eCH2eN1), 3.06 (d, J ¼ 7.69 Hz, 4H, Pp-3,5-H), 3.23 (t,
J ¼ 7.44 Hz, 2H, CH2eN1), 3.52 (d, J ¼ 5.39 Hz, 2H, CH2eBr), 3.79 (s,
4H, Pp-2,6-H), 4.54 (s, 2H, CH2-Ph), 6.82 (t, J ¼ 7.36 Hz, 1H, PpPh-4-
H), 6.97 (d, J ¼ 8.72 Hz, 2H, PpPh-2,6-H), 7.19e7.40(m, 7H, Ph, PpPh-
3,5-H),10.40 (br. s, 1H, NHþ). 13C NMR (75 MHz, DMSO-d6)
d [ppm]:
7.35; N,12.38. Found: C, 68.94.64; H, 7.42; N,12.17. 1H NMR
d (ppm):
23.1, 23.9, 29.0, 40.7, 41.7, 45.8, 50.8, 55.5, 62.0, 116.5, 118.4, 127.5,
127.8, 129.1, 129.6, 132.1, 137.2, 149.9, 155.1, 176.6. IR (cmꢀ1): 3067,
3036 (CeH(Ar)), 2981, 2933 (CeH(Aliph)), 2414 (NHþ), 1765 (C2]
O), 1703 (C4]O), 1598 (CeC(Ar)).
1.12e1.17 (qudef., 2H, PpeC2H4eCH2eC2H4-hyd), 1.20e1.55 (m, 4H,
CH2eCH2eCH2eCH2eCH2), 1.77 (s, 3H, 5-CH3), 2.36 (br.s, 2H, Pp-
CH2), 2.62 (br.s, 4H, Pp-2,6-H), 2.86e3.15 (m, 2H, CH2eN1-hyd),
2.91 (s, 3H, 3-CH3), 3.02 (br.s, 4H, Pp-3,5-H), 6.91e7.13 (m, 4H,
PpPh), 7.27e7.43 (m, 5H, 5-Ph). 13C NMR (75 MHz, DMSO-d6)
4.1.5.2. 3-Benzyl-1-(5-(4-(2-methoxyphenyl)piperazin-1-
yl)pentyl)-5,5-dimethylimidazolidine-2,4-dione hydrochloride (15a).
Method D. 1-(2-Methoxyphenyl)piperazine (3.50 mmol, 0.67 g)
K2CO3 (1.4 g) in acetone (12 mL) and 1-benzyl-1-(5-bromopentyl)-
5,5-dimethylimidazolidine-2,4-dione 37 (5.00 mmol, 1.84 g) in
acetone (10 mL) were used to give white crystals of compound 15a
(0.89 g, 1.70 mmol). Yield 49%; mp 225e227 ꢂC; TLC: Rf (IV): 0.21.
Initial LC/MS purity 93%, tR ¼ 4.77. MW 478.63. Monoisotopic Mass
478.29, [M þ H]þ 479.45. Anal. Calcd for C28H39ClN4O3: C, 65.29; H,
d
[ppm]: 20.4, 22.9, 23.9, 25.2, 28.6, 47.3, 51.1, 55.7, 67.2, 119.9,
125.5, 126.7, 129.0, 129.3, 137.9, 156.1, 175.1.
4.1.4.4. 1-(5-(4-(2-Methoxyphenyl)piperazin-1-yl)pentyl)-3,5-
dimethyl-5-phenylimidazolidine-2,4-dione (13). 1-(2-Methoxyphenyl)
piperazine (0.96 g) and 1-(5-bromopentyl)-3,5-dimethyl-5-pheny-
limidazolidine-2,4-dione 32 (1.94 g) were used to give white crys-
tals of compound 13 (0.90 g, 1.94 mmol). Yield 39%; mp 136e137 ꢂC;
TLC: Rf (IV): 0.62. MW 464.60. Monoisotopic Mass 464.28, [M þ H]þ
465.44. Anal. Calcd for C27H36N4O3: C, 69.80; H, 7.81; N, 12.06. Found:
7.63; N, 10.88. Found: C, 65.27; H, 7.77; N, 10.72. 1H NMR
d
(ppm):
1.28 (s, 2H, CH2eCH2eCH2eN1), 1.34 (s, 6H, 2 ꢃ CH3), 1.57 (qudef.
,
C, 69.75.64; H, 7.79; N, 11.95. 1H NMR
d (ppm): 1.12e1.18 (qudef., 2H,
2H, CH2eCH2eBr), 1.71 (qudef., 2H, CH2eCH2eN1), 2.96e3.14 (m,
4H, Pp-3,5-H), 3.23 (t, J ¼ 7.44 Hz, 2H, CH2eN1), 3.35 (s, 2H, CH2e
Br), 3.43 (s, 4H, Pp-2,6-H), 3.77 (s, 3H, CH3O), 4.54 (s, 2H, CH2-Ph),
6.88e7.01 (m, 4H, Ph-3,5-H, PpPh-2,6-H), 7.19e7.35 (m, 5H, Ph-
2,4,6-H, PpPh-3,5-H), 10.24 (br. s, 1H, NHþ). IR (cmꢀ1): 3034, 3016
(CeH(Ar)), 2985, 2964, 2938 (CeH(Aliph)), 2383 (NHþ), 1763 (C2]
O), 1698 (C4]O), 1608 (CeC(Ar)).
PpeC2H4eCH2eC2H4-hyd), 1.20e1.39 (qu def, 4H, CH2eCH2eCH2e
CH2eCH2), 1.77 (s, 3H, 5-CH3), 2.39 (br.s, 2H, Pp-CH2), 2.64 (br.s, 4H,
Pp-2,6-H), 2.83e3.15 (m, 6H, CH2eN1-hyd, Pp-3,5-H), 2.91 (s, 3H, 3-
CH3), 3.75 (s, 3H, OCH3), 6.84e6.97(m, 4H, PpPh), 7.28e7.44 (m, 5H,
5-Ph).
4.1.5. General procedure for synthesis of hydrochlorides of
phenylpiperazine hydantoin derivatives (14ae24a)
A suitable commercial phenylpiperazine (2.00e5.10 mmol),
K2CO3 (0.9e2.00 g) and acetone (7e16 mL) were stirred and
refluxed for 30 min. Then, correspondingly substituted bromo-
pentyl 3-benzyl-5,5-hydantoin derivatives 37e39 (2.20e
5.70 mmol) in acetone (9e20 mL) were added and the mixture was
refluxed for 6 h, left at room temperature overnight and separated
from the inorganic precipitate by filtration. The solvent was evap-
orated from the filtrate. The pure product (14ae24a) was obtained
from the residue using method C or D.
4.1.5.3. 3-Benzyl-1-(5-(4-(3-methoxyphenyl)piperazin-1-
yl)pentyl)-5,5-dimethylimidazolidine-2,4-dione hydrochloride (16a).
Method D. 1-(3-Methoxyphenyl)piperazine (3.50 mmol, 0.67 g)
K2CO3 (1.4 g) in acetone (12 mL) and 1-benzyl-1-(5-bromopentyl)-
5,5-dimethylimidazolidine-2,4-dione 37 (5.00 mmol, 1.84 g) in
acetone (18 mL) were used to give white crystals of compound 16a
(1.44 g, 2.8 mmol). Yield 80%; mp 198e199 ꢂC; TLC: Rf (IV): 0.31.
MW 478.63. Monoisotopic Mass 478.29, [M þ H]þ 479.53. Anal.
Calcd for C28H39ClN4O3: C, 65.29; H, 7.63; N, 10.88. Found: C, 65.19;
H, 7.65; N, 10.69. 1H NMR
CH2eN1), 1.34 (s, 6H, 2 ꢃ CH3), 1.54 (qudef., 2H, CH2eCH2eBr), 1.73
(qudef. 2H, CH2eCH2eN1), 3.04 (m, 4H,Pp-3,5-H), 3.23 (t,
d (ppm): 1.22e1.34 (m, 2H, CH2eCH2e
Method C. The residue after evaporation was crystallized with
ethanol to give precipitate of products in the basic form (20e
24). A compound in the basic form (500e900 mg) was dis-
solved in EtOH (10e15 mL) and saturated with gaseous HCl to
give hydrochlorides of the desirable product (20, 21, 23 and
,
J ¼ 7.57 Hz, 2H, CH2eN1), 3.48 (d, J ¼ 9.75 Hz, 2H, CH2eBr), 3.71 (s,
4H, Pp-2,6-H), 3.82 (s, 3H, CH3eO), 4.54 (s, CH2-Ph), 6.41e6.57 (m,
3H, PpPh-2,4,6-H), 7.11e7.37 (m, 6H, Ph, PpPh-5-H), 10.70 (b.s, 1H,

J. Handzlik et al. / European Journal of Medicinal Chemistry 78 (2014) 324e339
335
NHþ). IR (cmꢀ1): 3055, 3033 (CeH(Ar)), 2981, 2933 (CeH(Aliph)),
2413 (NHþ), 1763 (C2]O), 1704 (C4]O), 1614 (CeC(Ar)).
[M þ H]þ 503.40. Anal. Calcd for C27H33F3N4O2: C, 64.53; H, 6.62; N,
11.15. Found: C, 64.44; H, 6.83; N, 11.07. 1H NMR for 20
d (ppm): 1.27
(s, 2H, CH2eCH2eCH2eN1), 1,31 (s, 6H, 2 ꢃ CH3), 1.40 (qudef., 2H,
4.1.5.4. 3-Benzyl-1-(5-(4-(2-fluorophenyl)piperazin-1-
yl)pentyl)-5,5-dimethylimidazolidine-2,4-dione hydrochloride (17a).
Method D. 1-(2-Fluorophenyl)piperazine (3.50 mmol, 0.63 g)
K2CO3 (1.4 g) in acetone (12 mL) and 1-benzyl-1-(5-bromopentyl)-
5,5-dimethylimidazolidine-2,4-dione 37 (5.00 mmol, 1.84 g) in
acetone (18 mL) were used to give white crystals of compound 17a
(0.78 g, 1.60 mmol). Yield 44%; mp 140e141 ꢂC; TLC: Rf (IV): 0.32.
MW 466.59. Monoisotopic Mass 466.27, [M þ H]þ 467.43. Anal.
Calcd for C27H36ClFN4O3: C, 64.46; H, 7.21; N, 11.14. Found: C, 64.40;
CH2eCH2-Pp), 1.51e1.61 (qudef. 2H, CH2eCH2eN1), 2.26 (t,
,
J ¼ 7.18 Hz, 2H, CH2-Pp), 2.47 (s, 4H, Pp-2,6-H), 2.90 (t, J ¼ 4.75 Hz,
4H, Pp-3,5-H), 3.20e3.30 (m, 2H, CH2eN1), 4.51 (s, 2H, CH2-Ph),
6.95e7.07 (m, 2H, Ph-2,6-H), 7.11e7.20 (m, 3H, PpPh-3,5-H, PpPh-6-
H), 7.23e7.28 (ddef., 2H, Ph-3,5-H).
White crystals of compound 20a. Yield 37%; mp 163e164 ꢂC;
TLC: Rf (IV): 0.43. Anal. Calcd for C27H34ClF3N4O2: C, 60.16; H, 6.36;
N, 10.39. Found: C, 59.88; H, 6.40; N, 10.17. 1H NMR for 20a
d (ppm):
1.28e1.31 (m, 2H, CH2eCH2eCH2eN1), 1,32 (s, 6H, 2 ꢃ CH3), 1.53e
1.61 (qudef., 2H, CH2eCH2-Pp), 1.62e1.75 (qudef., 2H, CH2eCH2eN1),
3.07e3.27 (m, 6H, CH2-Pp, Pp-3,5-H, CH2eN1), 3.36e3.38 (ddef., 2H,
Pp-2,6-Ha), 3.49e3.53 (ddef., 2H, Pp-2,6-Hb), 4.52 (s, 2H, CH2-Ph),
6.99e7.28 (m, 7H, Ph), 10.91 (br. s, 1H, NHþ). IR (cmꢀ1): 3050 (Ce
H(Ar)), 2988, 2978, 2957, 2934 (CeH(Aliph)), 2382, 2359 (NHþ),
1765 (C2]O), 1704 (C4]O), 1620, 1608 (CeC(Ar)).
H, 7.31; N, 10.96. 1H NMR
d (ppm): 1.29 (s, 2H, CH2eCH2eCH2eN1),
1.34 (s, 6H, 2 ꢃ CH3), 1.57 (qudef., 2H, CH2eCH2eBr), 1.71 (qudef., 2H,
CH2eCH2eN1), 3.08 (d, J ¼ 7.98 Hz, 4H, Pp-3,5-H), 3.23 (t,
J ¼ 7.57 Hz, 2H, CH2eN1), 3.45 (d, J ¼ 9.49 Hz, 2H, CH2eBr), 3.54 (s,
4H, Pp-2,6-H), 4.54 (s, 2H, CH2-Ph), 7.02e7.36 (m, 9H, Ar),10.45 (b.s,
1H, NHþ). IR (cmꢀ1): 3036 (CeH(Ar)), 2988, 2977, 2934 (Ce
H(Aliph)), 2357 (NHþ), 1765 (C2]O), 1702 (C4]O), 1602 (CeC(Ar)).
4.1.5.8. 3-(4-Fluorobenzyl)-1-(5-(4-(4-fluorophenyl)piperazin-1-yl)
pentyl)-5,5-dimethylimidazolidine-2,4-dione hydrochloride (21a).
Method C. 1-(4-Fluorophenyl)piperazine (4.5 mmol, 0.81 g) K2CO3
(1.6g)in acetone (14 mL) and3-(4-fluorobenzyl)-1-(5-bromopentyl)-
5,5-dimethylimidazolidine-2,4-dione 38 (4.90 mmol, 1.90 g) in
acetone (18 mL) were used to give white crystals of compound 21
(1.55 g, 2.1 mmol). Yield 46%; mp 113e114 ꢂC; TLC: Rf (IV): 0.44. MW
484.58. Monoisotopic Mass 484.26, [M þ H]þ 485.45. Anal. Calcd for
4.1.5.5. 3-Benzyl-1-(5-(4-(4-fluorophenyl)piperazin-1-yl)pentyl)-
5,5-dimethylimidazolidine-2,4-dione
hydrochloride
(18a).
Method D. 1-(4-Fluorophenyl)piperazine (3.50 mmol, 0.63 g) K2CO3
(1.4 g) in acetone (12 mL) and 1-benzyl-1-(5-bromopentyl)-5,5-
dimethylimidazolidine-2,4-dione 37 (5.00 mmol, 1.84 g) in acetone
(18 mL) were used to give white crystals of compound 18a (0.50 g,
1.00 mmol). Yield 28%; mp 174e175 ꢂC; TLC: Rf (IV): 0.29. MW 466.59.
Monoisotopic Mass 466.27, [M þ H]þ 467.43. Anal. Calcd for
C
27H34F2N4O2: C, 66.92; H, 7.07; N, 11.56. Found: C, 66.68; H, 6.93; N,
C
27H36ClFN4O3: C, 64.46; H, 7.21; N, 11.14. Found: C, 64.34; H, 7.17; N,
11.48.1H NMR for 21
d
(ppm): 1.27 (tdef., 2H, CH2eCH2eCH2eN1),1.31
11.05. 1H NMR
d
(ppm): 1.28 (s, 2H, CH2eCH2eCH2eN1), 1.34 (s, 6H,
(s, 6H, 2 ꢃ CH3), 1.41 (qudef., 2H, CH2eCH2-Pp), 1.51 (qudef., 2H, CH2e
CH2eN1), 2.25 (t, J ¼ 7.18 Hz, 2H, CH2-Pp), 2.43 (tdef., 4H, Pp-2,6-H),
3.00 (t, J ¼ 4.87 Hz, 4H, Pp-3,5-H), 3.20 (t, J ¼ 7.70 Hz, 2H, CH2eN1),
4.51 (s, 2H, CH2-Ph), 6.88 (ddef., 2H, PpPh-3,5-H), 6.91 (ddef., 2H, PpPh-
2,6-H), 7.11 (ddef., 2H, Ph-3,5-H), 7.22 (ddef., 2H, Ph-2,6-H).
2 ꢃ CH3), 1.57 (qudef., 2H, CH2eCH2eBr), 1.72 (qudef., 2H, CH2eCH2e
N1), 3.05 (d, J ¼ 8.72 Hz, 4H, Pp-3,5-H), 3.23 (t, J ¼ 7.57 Hz, 2H, CH2e
N1), 3.51 (d, J ¼ 6.41 Hz, 2H, CH2eBr), 3.68 (d, J ¼ 8.46 Hz, 3H, Pp-2,6-
Hb), 3.86 (s,1H, Pp-2.6-Ha), 4.54 (s, 2H, CH2-Ph), 6.98e7.12 (m, 4H, Ph-
3,5-H, PpPh-2,6-H), 7.19e7.36 (m, 5H, Ph-2,4,6-H, PpPh-3,5-H), 10.62
(br. s, 1H, NHþ). IR (cmꢀ1): 3064, 3037 (CeH(Ar)), 2981, 2933 (Ce
H(Aliph)), 2420 (NHþ), 1767 (C2]O), 1703 (C4]O), 1607 (CeC(Ar)).
White crystals of compound 21a. Yield 47%; mp 175 ꢂC; TLC: Rf
(IV): 0.44. Anal. Calcd for C27H35ClF2N4O2 ꢃ H2O: C, 60.16; H, 6.92;
N, 10.39. Found: C, 59.96; H, 7.04; N, 10.32. 1H NMR for 21a
d (ppm):
1.28e1.33 (m, 2H, CH2eCH2eCH2eN1), 1,32 (s, 6H, 2 ꢃ CH3), 1.53e
1.61 (qudef., 2H, CH2eCH2-Pp), 1.73e1.78 (qudef., 2H, CH2eCH2eN1),
3.07e3.15 (qudef., 6H, CH2-Pp, Pp-3,5-H), 3.22 (t, J ¼ 7.40 Hz, 2H,
CH2eN1), 3.50e3.53 (ddef., 2H, Pp-2,6-Ha), 3.67e3.70 (ddef., 2H, Pp-
2,6-Hb), 4.52 (s, 2H, CH2-Ph), 6.98e7.28 (m, 8H, Ph), 10.91 (br. s, 1H,
NHþ). IR (cmꢀ1): 3057 (CeH(Ar)), 2982, 2948, 2933 (CeH(Aliph)),
2440 (NHþ), 1765 (C2]O), 1704 (C4]O), 1608 (CeC(Ar)). 13C NMR
4.1.5.6. 3-Benzyl-1-(5-(4-(2,4-difluorophenyl)piperazin-1-yl)pentyl)-
5,5-dimethylimidazolidine-2,4-dione
hydrochloride
(19a).
Method D. 1-(2,4-Difluorophenyl)piperazine (3.50 mmol, 0.69 g)
K2CO3 (1.4 g) in acetone (12 mL) and 1-benzyl-1-(5-bromopentyl)-
5,5-dimethylimidazolidine-2,4-dione 37 (5.00 mmol, 1.84 g) in
acetone (18 mL) were used to give white crystals of compound 19a
(0.43 g, 0.80 mmol). Yield 24%; mp 147e148 ꢂC; TLC: Rf (IV): 0.33.
MW 484.58. Monoisotopic Mass 484.26, [M þ H]þ 485.45. Anal.
Calcd for C27H36ClF2N4O3: C, 62.24; H, 6.77; N, 10.75. Found: C,
(75 MHz, DMSO-d6) d [ppm]: 23.0, 24.0, 29.0, 46.5, 50.8, 55.5, 62.0,
115.7, 115.8, 116.0, 118.3, 118.4, 129.7, 129.8, 133.4, 146.8, 155.0, 176.6.
62.17; H, 6.81; N, 10.59. 1H NMR
d
(ppm): 1.34 (s, 2H, CH2eCH2e
4 . 1 . 5 . 9 . 3 - ( 2 , 4 - D i c h l o r o b e n z y l ) - 1 - ( 5 - ( 4 - ( 2 , 3 -
dichlorophenyl)piperazin-1-yl)pentyl)-5,5-dimethylimidazolidine-
2,4-dione (22). Method C.* 1-(2,3-dichlorophenyl)piperazine hydro-
chloride (4.0 mmol,1.07 g) K2CO3 (2.2 g) in acetone (19 mL) and 3-(2,4-
dichlorobenzyl)-1-(5-bromopentyl)-5,5-dimethylimidazolidine-2,4-
dione 39 (4.90 mmol, 1.90 g) in acetone (18 mL) were used to give
white crystals of compound 22 (1.30 g, 2.1 mmol). Yield 52%; mp 91e
92 ꢂC; TLC: Rf (IV): 0.53. MW 586.38. Monoisotopic Mass 586.13,
[M þ H]þ 587.29. Anal. Calcd for C27H32Cl4N4O2: C, 55.30; H, 5.50; N,
CH2eN1), 1.34 (s, 6H, 2 ꢃ CH3), 1.57 (qudef., 2H, CH2eCH2eBr), 1.72
(qudef., 2H, CH2eCH2eN1), 3.08 (d, J ¼ 7.18 Hz, 4H, Pp-3,5-H), 3.23
(t, J ¼ 7.56 Hz, 2H, CH2eN1), 3.36 (d, J ¼ 8.46 Hz, 2H, CH2eBr), 3.51
(d, J ¼ 5.64 Hz, 3H, Pp-2,6-Hb), 3.64 (s, 1H, Pp-2,6-Ha), 4.54 (s, 2H,
CH2-Ph), 7.01e7.19 (m, 1H, PpPh-6-H), 7.20e7.36 (m, 7H, PpPh-3,5-
H, Ph), 10.64 (s, 1H, NHþ). IR (cmꢀ1): 3047 (CeH(Ar)), 2988, 2978,
2959, 2934 (CeH(Aliph)), 2377, 2356 (NHþ), 1766 (C2]O), 1702
(C4]O), 1620 (CeC(Ar)).
9.55. Found: C, 55.49; H, 5.60; N, 9.44. 1H NMR for 22
d (ppm): 1.01
4.1.5.7. 3-(4-Fluorobenzyl)-1-(5-(4-(2,4-difluorophenyl)piperazin-1-
yl)pentyl)-5,5-dimethylimidazolidine-2,4-dione hydrochloride (20a).
Method C. 1-(2,4-Difluorophenyl)piperazine (5.10 mmol, 1.00 g)
K2CO3 (2.0 g) in acetone (16 mL) and 3-(4-fluorobenzyl)-1-(5-
(qudef., 2H, CH2eCH2eCH2eN1), 1.35 (s, 6H, 2 ꢃ CH3), 1.41 (qudef., 2H,
CH2eCH2-Pp), 1.52 (qudef., 2H, CH2eCH2eN1), 2.28 (t, J ¼ 7.18 Hz, 2H,
CH2-Pp), 2.47 (s, 4H, Pp-2,6-H), 2.95 (s, 4H, Pp-3,5-H), 3.22 (t,
J ¼ 7.70 Hz, 2H, CH2eN1), 4.59 (s, 2H, CH2-Ph), 7.01e7.16 (m, 2H, PpPh-
4,6-H), 7.26e7.29 (m, 2H, Ph-6-H, PpPh-5-H), 7.35 (ddef., 1H, Ph-5-H),
bromopentyl)-5,5-dimethylimidazolidine-2,4-dione
38
(5.70 mmol, 2.20 g) in acetone (20 mL) were used to give white
crystals of compound 20 (0.76 g, 1.50 mmol). Yield 30%; mp 155e
156 ꢂC; TLC: Rf (IV): 0.43. MW 502.57. Monoisotopic Mass 502.27,
7.62 (s, 1H, Ph-3-H). 13C NMR (75 MHz, DMSO-d6)
d [ppm]: 23.1, 24.7,
26.3, 29.5, 51.4, 53.3, 58.1, 62.1, 119.9, 124.7, 126.4, 128.1, 128.9, 129.4,
130.3, 133.1, 133.1, 133.3, 151.7, 154.7, 176.5. IR (cmꢀ1): 3075, 3045 (Ce

336
J. Handzlik et al. / European Journal of Medicinal Chemistry 78 (2014) 324e339
H(Ar)), 2960, 2936 (CeH(Aliph)),1758 (C2]O),1705 (C4]O),1578 (Ce
C(Ar)).
bottom on ice-bathroom at 0 ꢂC. When the ingredients were totally
dissolved, DEAD (30 mmol, 5.22 g) in THF (10 mL) were added
dropwise for 45 min. The mixture was stirred at room temperature
for 90 h under TLC control. The solvent was evaporated. The residue
was treated with diethyl ether (200 mL). The precipitate was filtrated
of, and the filtrate was condensed to give compound 42 (7.39 g,
28 mmol) in glue-mass form. Yield 93%; TLC: Rf (IV): 0.54. Anal. Calcd
for C13H13FN2O3: C, 59.09; H, 4.96; N,10.60. Found: C, 59.45; H, 5.05;
4.1.5.10. 3-(2,4-Dichlorobenzyl)-1-(5-(4-(3,4-dichlorophenyl)piper-
azin-1-yl)pentyl)-5,5-dimethylimidazolidine-2,4-dione hydrochloride
(23a). Method C. 1-(3,4-dichlorophenyl)piperazine (2.0 mmol,
0.54 g) K2CO3 (1.0 g) in acetone (8 mL) and 3-(2,4-dichlorobenzyl)-
1-(5-bromopentyl)-5,5-dimethylimidazolidine-2,4-dione
39
(2.2 mmol, 0.96 g) in acetone (9 mL) were used to give white
crystals of compound 23 (0.61 g, 1.0 mmol). Yield 52%; mp 140 ꢂC;
TLC: Rf (IV): 0.60. MW 586.38. Monoisotopic Mass 586.13, [M þ H]þ
587.29. Anal. Calcd for C27H32Cl4N4O2: C, 55.30; H, 5.50; N, 9.55.
N,10.31. 1H NMR for 42
d (ppm): 1.66 (s, 3H, -CH3), 2.38e2.46 (m,1H,
-CH2-O-3-Ha), 2.65e2.69 (m,1H, -CH2-O-3-Hb), 3.06e3.11 (qudef., 1H,
CHeO), 3.53e3.55 (qdef., 2H, N3eCH2), 7.19e7.26 (m, 2H, Ph-2,6-H),
7.47e7.52 (m, 2H, Ph-3,5-H), 8.99 (br. s, 1H, NH).
Found: C, 55.36; H, 5.57; N, 9.37. 1H NMR for 23
d (ppm): 1.01 (qudef.,
2H, CH2eCH2eCH2eN1), 1.35 (s, 6H, 2 ꢃ CH3), 1.41 (qudef., 2H, CH2e
CH2-Pp), 1.52 (qudef., 2H, CH2eCH2eN1), 2.24 (t, J ¼ 7.18 Hz, 2H,
CH2-Pp), 2.41 (t, J ¼ 4.75 Hz, 4H, Pp-2,6-H), 3.11 (t, J ¼ 4.75 Hz, 4H,
4.1.7. Synthesis of 5-(4-fluorophenyl)-3-(2-hydroxy-3-(4-(2-
methoxyphenyl)piperazin-1-yl)propyl)-5-methylimidazolidine-2,4-
dione hydrochloride (25a)
Pp-3,5-H), 3.21 (tdef., 2H, CH2eN1), 4.59 (s, 2H, CH2-Ph), 6.88 (ddef.
,
5-(4-Fluorophenyl)-5-methyl-3-(oxiran-2-ylmethyl)imidazoli-
dine-2,4-dione 42 (4.0 mmol,1.1 g) and 2-methoxyphenylpiperazine
(3.00 mmol, 0.58 g) were dissolved in methylene chloride (5 mL). The
solvent was evaporated. The residue was irradiated in household-
microwave oven using the following program of irradiation: 300 W
(3 min), 450 W (2 ꢃ 3 min), 600 W (2 ꢃ 1 min). The obtained glue-
residue was purified with chromatography column (CH2Cl2/aceton/
MeOH). The fractions containing pure product 25 were collected and
evaporated. The residue was dissolved in 99.8% EtOH (15 mL) and
saturated with gaseous HCl to give white precipitate of 25a (0.40 g,
0.81 mmol). Yield 27%; mp 235 ꢂC; TLC: Rf (IV): 0.31. MW 456.51.
Monoisotopic Mass 456.22, [M þ H]þ 457.39. Anal. Calcd for
1H, PpPh-2-H), 7.08 (ddef., 2H, PpPh-6-H, PpPh-5-H), 7.35 (ddef., 2H,
Ph-5,6-H), 7.62 (s, 1H, Ph-3-H).
White crystals of compound 23a. Yield 59%; mp 117 ꢂC; TLC: Rf
(IV): 0.60. Anal. Calcd for C27H33Cl5N4O2 ꢃ 1/3C2H5OH: C, 52.07; H,
5.53; N, 8.78. Found: C, 51.91; H, 5.27; N, 8.89. 1H NMR for 23a
d
(ppm): 1.29e1.35 (m, 2H, CH2eCH2eCH2eN1), 1.36 (s, 6H,
2 ꢃ CH3), 1.57e1.59 (qudef., 2H, CH2eCH2-Pp), 1.71 (br. s, 2H, CH2e
CH2eN1), 3.08e3.15 (qudef., 6H, CH2-Pp, Pp-3,5-H), 3.20e3.25 (m,
2H, CH2eN1), 3.41e3.62 (m, 2H, Pp-2,6-Ha), 3.86 (br. s, 2H, Pp-2,6-
Hb), 4.60 (s, 2H, CH2-Ph), 6.97e7.01 (m, 1H, PpPh-2-H), 7.13e7.17
(m, 1H, PpPh-6-H), 7.20e7.23 (m, 1H, PpPh-5-H), 7.37e7.45 (m, 2H,
Ph-5,6-H), 6.62e7.64 (m, 1H, Ph-3-H), 10.20 (br. s, 1H, NHþ). IR
(cmꢀ1): 3074 (CeH(Ar)), 2959, 2936 (CeH(Aliph)), 2595 (NHþ),
1769 (C2]O), 1704 (C4]O), 1593 (CeC(Ar)).
C24H30ClFN4O4: C, 58.47; H, 6.13; N,11.37. Found: C, 58.54; H, 5.99; N,
11.29. 1H NMR for 25a
d
(ppm): 1.7 (br. s, 3H, CH3), 2.93e3.09 (m, 4H,
Pp- CH2, Pp-2,6-Ha), 3.20e3.24 (d, 4H, Pp-2,6-Hb, 2H, N3eCH2), 3.40
(d, J ¼ 7.18 Hz, 4H, Pp-3,5-H), 3.49 (s, 1H, CHeOH), 3.77 (s, 3H, Oe
CH3), 4.20 (br. s, 1H, OH), 6.87e7.03 (m, 4H, PpPh-3,4,5,6-H), 7.20e
7.25 (m, 2H, Ph-2,6-H), 7.50e7.55 (m, 2H, Ph-3,5-H), 8.99 (br. s, 1H,
N1H), 9.79 (s, 1H, NHþ). IR (cmꢀ1): 1608.34 (C]C; Ar), 1715.37 (C]O
(4)),1772.26 (C]O (2)), 2400.94 (NHþ), 2982.37 (CH; Aliph), 3007.44
(CH; Ar), 3314.07 (OH).
4.1.5.11. 3-(2,4-Dichlorobenzyl)-1-(5-(4-(4-chlorophenyl)piperazin-
1-yl)pentyl)-5,5-dimethylimidazolidine-2,4-dione
hydrochloride
(24a). Method C. 1-(4-chlorophenyl)piperazine (2.0 mmol, 0.40 g)
K2CO3 (0.90 g) in acetone (7 mL) and 3-(2,4-dichlorobenzyl)-1-(5-
bromopentyl)-5,5-dimethylimidazolidine-2,4-dione 39 (2.2 mmol,
0.96 g) in acetone (9 mL) were used to give white crystals of basic
compound 24 (0.78 g, 1.4 mmol). Yield 71%; mp 156 ꢂC; TLC: Rf (IV):
0.53. MW 551.94. Monoisotopic Mass 551.17, [M þ H]þ 553.32. Anal.
Calcd for C27H33Cl3N4O2: C, 58.75; H, 6.03; N,10.15. Found: C, 58.56;
4.2. Pharmacology
H, 5.97; N,10.04. 1H NMR for 24
d (ppm): 1.28 (qudef., 2H, CH2eCH2e
4.2.1. Radioligand binding assays
CH2eN1), 1.35 (s, 6H, 2 ꢃ CH3), 1.41 (qudef., 2H, CH2eCH2-Pp), 1.46
(qudef., 2H, CH2eCH2eN1), 2.25 (t, J ¼ 6.82 Hz, 2H, CH2-Pp), 2.43
(tdef., 4H, Pp-2,6-H), 3.06 (t, J ¼ 4.87 Hz, 4H, Pp-3,5-H), 3.21 (t,
J ¼ 7.44 Hz, 2H, CH2eN1), 4.59 (s, 2H, CH2-Ph), 6.89 (ddef., 2H, PpPh-
2,6-H), 7.13 (ddef., 3H, PpPh-3,5-H, Ph-6-H), 7.37 (ddef., 1H, Ph-5-H),
7.62 (ddef., 1H, Ph-3-H).
4.2.1.1. The a1-adrenoceptor binding assay. The compounds were
evaluated on their affinity for a1-adrenergic receptors by deter-
mining for each compound its ability to displace [3H]-prazosin from
specific binding sites on rat cerebral cortex. [3H]-Prazosin (19.5 Ci/
mmol) was used. The tissue was homogenized in 20 vol. of ice-cold
50 mM TriseHCl buffer (pH 7.6 at 25 ꢂC) and centrifuged at
20000 ꢃ g for 20 min. The cell pellet was resuspended in TriseHCl
buffer and centrifuged again. The final pellet was resuspended in
White crystals of compound 24a. Yield 53%; mp 71 ꢂC; TLC: Rf
(IV): 0.60. Anal. Calcd for C27H34Cl4N4O3: C, 55.11; H, 5.82; N, 9.52.
Found: C, 55.01; H, 6.11; N, 9.67. 1H NMR for 21a
d
(ppm): 1.29e1.31
TriseHCl buffer (10 mg of wet weight/ml). 240
suspension, 30 l of analyzed compound
l of [3H]-prazosin and 30
were incubated at 25 ꢂC for 30 min. To determine unspecific
binding 10 M phentolamine was used. Transfer of solutions and
ml of the tissue
(m, 2H, CH2eCH2eCH2eN1), 1.34 (s, 6H, 2 ꢃ CH3), 1.56e1.61 (qudef.
,
m
m
2H, CH2eCH2-Pp), 2.47e2.50 (m, 2H, CH2eCH2eN1), 3.07e3.27 (m,
6H, CH2-Pp, Pp-3,5-H, CH2eN1), 3.31e3.35 (m, 4H, Pp-2,6-H), 4.59
(s, 2H, CH2-Ph), 6.97e7.00 (ddef., 1H, Ph-6-H) 7.13e7.16 (ddef., 2H,
PpPh-2,6-H), 7.25e7.28 (ddef., 1H, Ph-5-H) 7.38e7.40 (ddef., 2H,
PpPh-3,5-H), 7.62 (s, 1H, Ph-3-H), 8.92 (br. s, 1H, NHþ). IR (cmꢀ1):
3073, 3055 (CeH(Ar)), 2958, 2934 (CeH(Alif)), 2489 (NHþ), 1773
(C2]O), 1703 (C4]O), 1588 (CeC(Ar)).
m
adding of reagents were performed on automated pipetting system
epMotion 5070 (Eppendorf, Germany).
After incubation reaction mix was filtered immediately onto GF/
B glass fiber filter mate presoaked using 96-well FilterMate
Harvester (PerkinElmer, USA).
The radioactivity retained on the filter was counted in MicroBeta
TriLux 1450 scintillation counter (PerkinElmer, USA). Non-linear
regression of the normalized (percent radioligand binding
compared to that observed in the absence of test or reference
compound e total binding) raw data representing radioligand
binding was performed in GraphPad Prism 3.0 (GraphPad Software)
4.1.6. Synthesis of 5-(4-fluorophenyl)-5-methyl-3-(oxiran-2-
ylmethyl)imidazolidine-2,4-dione (42)
5-(4-Fluorophenyl)-5-methylhydantoin 41 (30 mmol, 6.24 g),
oxiran-2-ylmethanol (30 mmol, 2 ml) and TPP (30 mmol, 7.86 g) in
anhydrous THF (30 mL) were stirred in the hermetic closed flask-

J. Handzlik et al. / European Journal of Medicinal Chemistry 78 (2014) 324e339
337
using the built-in three parameter logistic model describing ligand
competition binding to radioligand-labeled sites.
chamber and the other to an isometric FDT10-A force displacement
transducer (BIOPAC Systems, Inc., COMMAT Ltd., Turkey), coupled
to a MP100 analyser (BIOPAC Systems, Inc., COMMAT Ltd., Turkey)
and processed by AcqKnowledge software (BIOPAC). The arterial
rings were incubated in 30 ml chambers filled with a KrebseHen-
seleit solution (NaCl 119 mM, KCl 4.7 mM, CaCl2 1.9 mM, MgSO4
1.2 mM, KH2PO4 1.2 mM, NaHCO3 25 mM, glucose 11 mM, EDTA
0.05 mM) at 37 ꢂC and pH 7.4 with constant oxygenation (O2/CO2,
19:1) and subjected to a 0.75 g initial optimal tension. Caudal
arterial rings were incubated with preferentially a1B-adrenoceptor
4.2.1.2. The serotonin receptors binding assays
4.2.1.2.1. Cell culture and preparation of cell membranes.
HEK293 cells with stable expression of human serotonin 5-HT1AR,
5-HT6R, 5-HT7R were maintained at 37 ꢂC in a humidified atmo-
sphere with 5% CO2 and were grown in Dulbeco’s Modifier Eagle
Medium containing 10% dialyzed fetal bovine serum and 500 mg/ml
G418 sulfate. For membranes preparations, cells were subcultured
in 10 cm diameter dishes, grown to 90% confluence, washed twice
with prewarmed to 37 ꢂC phosphate buffered saline (PBS), and
were pelleted by centrifugation (200 g) in PBS containing 0.1 mM
EDTA and 1 mM dithiothreitol. Prior to membrane preparations
pellets were stored at ꢀ80 ꢂC.
alkylating agent chloroethylclonidine (CEC; 3
mM), then 30 min
later chloroethylclonidine was thoroughly washed off. During an
equilibration period of 100 min tissues were stimulated four times
with noradrenaline (NA 1
mM) followed by washout until the
contractile response had become constant. Two cumulative con-
centrationeresponse curves to noradrenaline were determined on
each arterial ring at an interval of 60 min in the absence and
presence of antagonist. Tissues were incubated with antagonists for
30 min. The experiments were conducted in the continuous pres-
4.2.1.2.2. Radioligand binding assays. Cell pellets are thawed and
homogenized in 10 volumes of assay buffer using an UltraTurrax
tissue homogenizer and centrifuged twice at 35 000 g for 20 min at
4
ꢂC, with incubation for 15 min at 37 ꢂC in between. The compo-
sition of the assay buffers is as follows: for 5-HT1AR: 50 mM Trise
HCl, 0.1 mM EDTA, 4 mM MgCl2, 10 M pargyline and 0.1% ascor-
bate; for 5-HT6R: 50 mMTriseHCl, 0.5 mM EDTA and 4 mM MgCl2,
for 5-HT7R: 50 mMTriseHCl, 4 mM MgCl2,10 M pargyline and 0.1%
ascorbate. All assays were incubated in a total volume of 200 l in
ence of yohimbine (0.1
b-adrenoceptors respectively.
mM) and propranolol (1 mM) to block a2- and
m
m
4.2.2.2. The activity at a1B-adrenoceptors in mouse spleen.
Male mice were killed by cervical dislocation. The spleen was
removed and cut longitudinally into two strips, which were set up
in 30 ml organ bath under a resting tension of 0.8 g for the
recording of isometric contractile responses in a KrebseHenseleit
solution of the above composition at 37 ꢂC and pH 7.4 with constant
oxygenation (O2/CO2, 19:1). During an equilibration period of
m
96-well microtitre plates for 1 h at 37 ꢂC except for 5-HT1AR which
were incubated at room temperature for 1 h. The process of
equilibration is terminated by rapid filtration through Unifilter
plates with a 96-well cell harvester and radioactivity retained on
the filters was quantified on
a MicroBeta plate reader. For
displacement studies the assay samples contained as radioligands:
1.5 nM [3H]-8-OH-DPAT (187 Ci/mmol) for 5-HT1AR; 2 nM [3H]-LSD
(85.2 Ci/mmol) for 5-HT6R; 0.6 nM [3H]-5-CT (39.2 Ci/mmol) for 5-
100 min tissues were stimulated with noradrenaline (10 mM) fol-
lowed by washout until the contractile response had become
constant. Two cumulative concentrationeresponse curves to
noradrenaline were determined on each tissue at an interval of
60 min in the absence and presence of antagonist. Tissues were
incubated with antagonists for 30 min. The experiments were
HT7R. Non-specific binding is defined with 10
and 5-HT7 binding experiments, whereas 10
m
M of 5-HT in 5-HT1A
mM methiothepin was
used in 5-HT6 assay. Each compound was tested in triplicate at 7
concentrations (10ꢀ10e10ꢀ4 M). The inhibition constants (Ki) were
calculated from the ChengePrusoff equation [30]. Results were
expressed as means of at least two separate experiments.
conducted in the continuous presence of propranolol (1
block -adrenoceptors.
mM) to
b
4.2.2.3. The activity at a1D-adrenoceptors in rat thoracic aorta.
The male Wistar rats were anaesthetized with thiopental sodium
(75 mg/kg ip) and the aorta was isolated, denuded of endothelium,
cut, mounted and incubated as described in a method described in
4.2.2.1. The aorta rings were stretched and maintained at optimal
tension of 2 g and allowed to equilibrate for 3 h. During an equil-
ibration period the preparations were stimulated three times with
4.2.2. Functional bioassays
The experiments were carried out on male Wistar rats (Krf:(WI),
(WU), 180e250 g), and male Albino Swiss mice (CD-1, 18e25 g).
Animals were housed in plastic cages in room at a constant tem-
perature of 20 ꢁ 2 ꢂC with natural lightedark cycles. They had free
access to standard pellet diet and water. Treatment of laboratory
animals in the present study was in full accordance with the
respective Polish regulations. All procedures were conducted ac-
cording to guidelines of ICLAS (International Council on Laboratory
Animal Science) and approved by the Local Ethics Committee on
Animal Experimentation.
Source of compounds: (ꢁ)-noradrenaline hydrochloride
(SigmaeAldrich, Germany), (ꢁ)-propranolol hydrochloride
(SigmaeAldrich, Germany), acetylcholine hydrochloride (Sigmae
Aldrich, Germany), chloroethylclonidine (CEC, SigmaeAldrich,
Germany), yohimbine hydrochloride (SigmaeAldrich, Germany),
Thiopental sodium (Biochemie Gmbh, Vienna). Other reagents
were of analytical grade from local sources.
NA (0.3
mM). Two cumulative concentrationeresponse curves to
noradrenaline were determined on each arterial ring at interval of
60 min in the absence and presence of antagonist. Tissues were
incubated with antagonists for 30 min. The experiments were
conducted in the continuous presence of yohimbine (0.1
propranolol (1 M) to block a2- and -adrenoceptors.
mM) and
m
b
Concentration-response curves were analyzed using GraphPad
Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA).
Contractile responses to vasoconstrictor (in the presence or
absence of tested compounds) are expressed as a percentage of the
maximal noradrenaline effect (Emax ¼ 100%), reached in the con-
centrationeresponse curves obtained before incubation with the
tested compounds. Data are the means ꢁ SEM of at least 4 separate
experiments. Schild analysis was performed, and the pA2 value was
determined.
4.2.2.1. The activity at a1A-adrenoceptors in rat tail artery. The male
Wistar rats were anaesthetized with thiopental sodium (75 mg/kg
ip), and the middle part of the ventral caudal artery was removed,
cleaned of surrounding tissue, denuded of endothelium by gentle
rubbing and cut into approximately 4 mm long rings. Arterial rings
were horizontally suspended between two stainless steel hooks
(diameter 0.15 mm). One hook was attached to the bottom of the
4.2.3. Inhibitory effect at human 5-HT3A expressed in Xenopus
Oocytes
Mature female X. laevis frogs were purchased from Xenopus I
(Ann Arbor, MI). They were housed in dechlorinated tap water at

338
J. Handzlik et al. / European Journal of Medicinal Chemistry 78 (2014) 324e339
18 ꢂC under a 12:12-h light/dark cycle and fed beef liver at least
twice a week. Clusters of oocytes were removed surgically under
tricaine (SigmaeAldrich, St. Louis, MO) anesthesia (0.15%), and
individual oocytes were manually dissected away in a solution
containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.8 mM
MgSO4, and 10 mM HEPES, pH 7.5. Dissected oocytes were stored
2e7 days in modified Barth’s solution containing 88 mM NaCl,
1 mM KCl, 2.4 mM NaHCO3, 0.3 mM Ca(NO3)2, 0.9 mM CaCl2,
0.8 mM MgSO4, and 10 mM HEPES, pH 7.5, supplemented with
2 mM sodium pyruvate, 10 000 IU/l penicillin, 10 mg/l strepto-
mycin, 50 mg/l gentamicin, and 0.5 mM theophylline. Oocytes
were placed in a 0.2-ml recording chamber and superfused at a
constant rate of 3e5 ml/min. The bathing solution consisted of
95 mM NaCl, 2 mM KCl, 2 mM CaCl2, and 5 mM HEPES, pH 7.5. The
amount of 5-HT3A receptor cRNA injected into oocytes varied from
1 to 30 ng, as indicated. However, the injection volume of
diethylpyrocarbonate-treated distilled water was kept at 30 nl
throughout the experiments. The cells were impaled with two
standard glass microelectrodes filled with 3 M KCl (1e3 MU). The
oocytes were routinely voltage-clamped at a holding potential of
ꢀ70 mV using a GeneClamp-500B amplifier (Molecular Devices,
Sunnyvale, CA). Current responses were digitized by A/D con-
verter and analyzed using pClamp 8 (Molecular Devices) run on
an IBM PC or directly recorded on a 2400 rectilinear pen recorder
(Gould Instrument Systems Inc., Cleveland, OH). Current-voltage
curves were generated by holding each membrane potential in a
series for 50e60 s, followed by a return to ꢀ70 mV for 5 min.
Oocyte capacitance was measured by a paired ramp method
described previously [31]. In brief, voltage ramps were used to
elicit constant capacitive current, Icap, and the charge associated
with this current was calculated by the integration of Icap. Ramps
had slopes of 2 V/s and durations of 20 ms and started at a
holding potential of 90 mV. A series of 10 paired ramps was
delivered at 1-s intervals and averaged traces were used for
charge calculations. In each oocyte, the averages of five to six
measurements were used to obtain values for membrane capac-
itance (Cm). Currents for Icap recordings were filtered at 20 kHz
and sampled at 50 kHz. Current density was calculated by
normalizing the average of three consecutive control currents to
the oocyte capacitance. Compounds were applied by addition to
the superfusate. All chemicals used in preparing the solutions
were from SigmaeAldrich. Pertussis toxin (PTX), BAPTA, actino-
4.2.3.2. Data analysis. For each test compound, six inhibitory
values were obtained in each oocyte, and three oocytes of diverse
batches were used. The average inhibitory values were calculated
as mean ꢁ S.E.M. Statistical significance was analyzed using ANOVA
or Student’s t test.
Acknowledgments
Authors thank students: Sara Borromeo, Paula Idzik and
Ma1gorzata Fra˛czek for their participation in the synthesis works.
The work was partly supported by K/ZDS/003323 and K/ZDS/
001915. Authors: J. Handzlik, A. J. Bojarski, G. Sata1a, K. Kucwaj, K.
ꢀ
Kiec-Kononowicz participate in the European COST Action CM1207
(GLISTEN).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.01.065.
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