Pharmacophore models based studies on the affinity and selectivity toward 5-HT1A with reference to α1-adrenergic receptors among arylpiperazine derivatives of phenytoin

Article abstract of DOI:10.1016/j.bmc.2010.11.051

The study is focused on (2-alkoxy)phenylpiperazine derivatives of 1-(2-hydroxy-3-(4-arylpiperazin-1-yl)propyl)-5,5-diphenylimidazolidine-2, 4-dione with alkyl or ester substituents at N3 of hydantoin ring, as well as a new designed and synthesized series

Full text of DOI:10.1016/j.bmc.2010.11.051

Bioorganic & Medicinal Chemistry 19 (2011) 1349–1360
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Bioorganic & Medicinal Chemistry
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Pharmacophore models based studies on the affinity and selectivity toward
5-HT1A with reference to a1-adrenergic receptors among arylpiperazine
derivatives of phenytoin
a
a
b
c
d
´
Jadwiga Handzlik , Ewa Szymanska , Krystyna Ne˛dza , Monika Kubacka , Agata Siwek ,
c
e
c
a,
⇑
´
Szczepan Mogilski , Jarosław Handzlik , Barbara Filipek , Katarzyna Kiec-Kononowicz
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 Pharmacobiology, Jagiellonian University Medical College, Medyczna 9, PL 30-688 Kraków, Poland
e Faculty of Chemical Engineering and Technology, Cracow University of Technology, Warszawska 24, PL 31-155 Kraków, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
The study is focused on (2-alkoxy)phenylpiperazine derivatives of 1-(2-hydroxy-3-(4-arylpiperazin-1-
yl)propyl)-5,5-diphenylimidazolidine-2,4-dione with alkyl or ester substituents at N3 of hydantoin ring,
as well as a new designed and synthesized series of compounds with a free N3H group or N3-acetic acid
Received 20 March 2010
Revised 17 November 2010
Accepted 20 November 2010
Available online 5 December 2010
terminal fragment. The compounds were assessed on their affinity for 5-HT1A and a1-adrenoceptors and
evaluated in functional bioassays for antagonistic properties. Classical molecular mechanics (MMFFs
force field, MCMM, MacroModel) and DFT methods (B3LYP functional, Gaussian 0.3) were used to inves-
tigate 3D structure of the compounds. SAR analysis was based on two pharmacophore models, the one
Keywords:
Arylpiperazine derivatives
Phenytoin derivatives
5-HT1A receptor
described by Barbaro et al. for
a1-adenoceptor antagonist and the model of Lepailleur et al. for 5-HT1A
receptor ligands. All compounds exhibited significant to moderate affinities for 5-HT1A receptors in nano-
molar range (7–610 nM). The highest activity (7 nM) and selectivity (17.38) for 5-HT1A was observed
for 1-(3-(4-(2-ethoxyphenyl)piperazin-1-yl)-2-hydroxypropyl)-3-methyl-5,5-diphenylimidazolidine-2,4-
dione (13a). Among new synthesized compounds 1-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-
yl)propyl)-5,5-diphenylimidazolidine-2,4-dione hydrochloride (20a) displayed the highest affinity
N-Triphenylmethyl protection
(16.6 nM) and selectivity (5.72) for a1-AR.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
sedative and hypotensive properties. Some of them (7, 8) were
tested in clinical trials as effective tranquilizers (Fig. 1). Antinoci-
ceptive activity of compounds with phenylpiperazine moiety
was confirmed by studies of Cesari et al.5 performed for aryl-
piperazinylalkylpyridazinones. Especially, arylpiperazine derivatives
seem to be an interesting target in the search for antidepressant
drugs. So far several phenylpiperazine agents, including Trazodone
(4), Nefazodone (5), Aripiprazole (3) or Flesinoxan (6) (Fig. 1),
achieved pharmaceutical market as psychoactive drugs useful in
major depressive disorder.6 For N-phenylpiperazine dioxolones
hypocholesterolemic activity was found.7 Among arylpiperazines
activity against gram-positive bacteria8 or efflux pump inhibitors,9
promising in therapy of infectious diseases, has been confirmed as
well.
Arylpiperazine moiety is a popular chemical fragment that can
be found in active compounds with various biological specificities.
Many lines of evidence1–9 have indicated a potential usage of aryl-
piperazine derivatives in the treatment of circulation diseases,1–4
CNS failures4–6 and bacterial infections.8,9 Arylpiperazine deriva-
tives (Fig. 1), Urapidil (1) and Naftopidil (2), are known antihyper-
tensive agents. Recently Malawska et al.1 has described a group of
phenylpiperazine derivatives of pyrrolidin-2-one with significant
antiarrhythmic and hypotensive activity in primary screening
in vivo. Phenylpiperazine derivatives of hydantoin presented in
our previous works2,3 showed antiarrhythmic activity in the
adrenaline induced model of arrhythmia and influenced ECG of
rats in similar way to that of class Ia and III of Vaughan-Williams
classification of antiarrhythmic agents. Hayao et al.4 obtained phe-
nylpiperazine derivatives of trimethoxybenzoate that displayed
The most important therapeutic potency of arylpiperazine
derivatives is in close connection to their interactions with GPCRs
(G-protein coupled receptors) including a
-adrenergic,10,11 5-HT or
dopamine receptors.12,13 The competition in binding towards
several GPCRs-members is a serious factor that significantly limits
selectivity of pharmacological properties of active arylpiperazine
⇑
Corresponding author. Tel.: +48 012 620 55 81; fax: +48 012 620 55 96.
E-mail address: mfkonono@cyf-kr.edu.pl (K. Kiec´-Kononowicz).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.051

1350
J. Handzlik et al. / Bioorg. Med. Chem. 19 (2011) 1349–1360
O
N
H
N
Cl
N
O
N
N
O
N
OCH3
Cl
N
O
N
O
N
N
OH
H
H3C
CH3
O
2 (Naftopidil)
3 (Aripiprazole)
1 (Urapidil)
O
N
N
N
O
N
O
Cl
N
N
N
O
N
Cl
N
HO
O
N
N
H
N
O
F
4 (Trazodone)
6 (Flesinoxan)
5 (Nefazodone)
OCH3
OCH3
N
O
N
OCH3
OCH3
N
O
N
O
OCH3
8
O
OCH3
7
Figure 1. Structure of arylpiperazine derivatives with various therapeutic potency; compounds useful in therapy of hypertension (1, 2), anti-depressant drugs (3–6),
tranquilizers under clinical trials (7, 8).
derivatives and their ability for therapeutic usage. A main problem
is a weak selectivity between 5-HT1A and 1-adrenoceptors. In last
Mokrosz et al.23 and Chilmonczyk et al.25 (Fig. 2) and a modern
model of Lepailleur et al.,26 elaborated by use of CATALYST, compa-
a
years pharmacophore models created for active compounds and
describing the structural features responsible for binding proper-
rable to Barbaro’s model for
Our previous works2,27 described synthesis and affinity of
N1-arylpiperazine Phenytoin (23) derivatives for both 1- and
2-adrenoceptors.27 Results of SAR studies supported by molecular
modelling calculation confirmed a close relationship between a
range of affinity for 1-adrenoceptor and five pharmacophore fea-
tures of Barbaro’s model (Fig. 3). In fact, the 2-alkoxyphenylpiper-
azine derivatives fitting two hydrophobic areas (HY1, HY2) showed
the highest activity.27 Results indicated that a kind of a substituent
at N3 of the hydantoin ring, a feature not included in Barbaro’s
a1-AR antagonists (Fig. 3).
ties at selected GPCRs were reported. In case of
a
1-adrenoceptor
a
antagonists, the early pharmacophore model, elaborated by De
Marinis,14 proposed three following important features: aromatic
area, basic nitrogen and semi-polar or internal lipophilic area.
Further evolutions of pharmacophore study were given by
De Benedetti’s model15 (Fig. 2), models of Bremner et al. that
a
a
respected
a
1-adrenoceptor subtypes selectivity16,17 and model of
Barbaro,18 especially adequate for arylpiperazine derivatives
(Fig. 3). Pharmacophore models of serotonin receptors ligands have
been created for above thirty years.13,19 The basic model, discrim-
inating agonistic and antagonistic properties at 5-HT1A, is known
as Hilbert’s model20 and postulates two distances crucial for inter-
actions with the receptor. During the next twenty years a number
of pharmacophore models of 5-HT1A ligands have been elabo-
rated,21–26 including simple quantitative models developed by
model, influenced a1-affinity and a2/a1 selectivity among the de-
scribed arylpiperazines. As this influence was unclear, the further
investigation was needed. Basing on this conclusion, the current
study is focused on N1-(2-alkoxy)phenylpiperazine derivatives
with various substituents at N3 of hydantoin (Table 1), including
alkyl (9a, 12a–14a), ester moieties (10a, 11a, 15a–18a) as well as
a series of new designed and synthesized compounds with free
NH group (19a–21a) or acetic acid terminal fragment (22a). The
new compounds (19a–22a) were assessed on their affinity to
a1-adrenoceptors, for their antagonistic properties determination
the functional bioassays were performed. For compounds
9a–22a, their affinity to 5-HT1A was evaluated in radioligand bind-
ing assays. Selectivity studies concerned
a1/5-HT1A selectivity for
all compounds (9a–22a). The work is focused on the SAR study
to resolve two following problems: (i) search for structural param-
eters responsible for activity and selectivity a1/5-HT1A, (ii) useful-
ness of available pharmacophore models to search for selective
agents among arylpiperazine derivatives of phenytoin. In this field,
molecular modelling by use of the newest calculation methods was
performed. SAR analysis was based mainly on two pharmacophore
models, created by the comparable calculating methods (CATA-
LYST), described by Barbaro et al.18 and Lepailleur et al.26 for
a1-AR and 5-HT1A receptors, respectively (Fig. 3).
2. Results and discussion
2.1. Synthesis
Figure 2. Pharmacophore models. (a) De Benedetti’s model for
a1-adrenoceptor
antagonist: P (red), protonated nitrogen; Ar1, Ar2 (dark yellow) aromatic systems;
distances P-Ar1 and P-Ar2 should be in range of 4–7 Å.15 (b) Chilmonczyk’s model
for buspiron-like 5-HT1A receptor ligands; the three centres from a triangle with
sides defined by: d1 = 7.07 Å, d2 = 4.30 Å, d3 = 4.88 Å distances.13
The synthesis of compounds 9a–18a was described previ-
ously.2,27 Compounds 19a–21a were obtained in four-step synthesis,

J. Handzlik et al. / Bioorg. Med. Chem. 19 (2011) 1349–1360
1351
Figure 3. Pharmacophore features of selected GPCR’s ligand elaborated by the use of CATALYST. (a) The Barbaro’s model of
a1-adrenoceptor antagonist (mapped with a
phenylpiperazine antagonist, (3-(2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl)-4a,5-dihydro-1H-pyrimido[5,4-b]indole-2,4(3H,9bH)-dione); PI, positive ionisable centre;
HY1-3, three hydrophobic moieties; HBA, hydrogen bound acceptor). Selected distances are crucial for the activity.18 (b) The 5-HT1A pharmacophore model described by
Lepailleur et al. (maped with phenylpiperazine derivatives, NAN-190); PI, positive ionisable centre; HYD, hydrophobic moiety; AR, aromatic ring; HBA, hydrogen bound
acceptor. Six selected distances are important for expected activity.26
Table 1
Structure and binding properties at 5-HT1A receptors in comparison to
a
1-AR for the tested N1-arylpiperazine derivatives of hydantoin 9a–22a
R2
OR3
O
x HCl
N
N
N
N
R1
*
R,S
O
Compounds 9a-22a
Compd
R1
R2
R3
Ks (nM)
5-HT1A
Kia (nM)
Sel
1/5-HT1A
Antagonist potency
pKB SEM
a1-AR
a
9a
10a
–C2H5
–CH2COOC2H5
H
OCH3
H
H
610 25
88 12
529 9.5
160 21.3
0.86
1.82
4.52
CH3
O
CH3
11a
OCH3
H
30
4
135.7 31.3
*
R,S O
12a
13a
14a
15a
16a
–CH3
–CH3
–CH3
–CH2COOCH3
–CH2COOCH3
OCH3
H
H
22 0.1
160.7 13.6
121.6 14.9
607 74.7
197.8 25.4
251.6 3.8
7.27
17.37
15.97
2.29
OC2H5
OC2H5
OCH3
7
1
–COCH3
H
H
38 0.5
86 13
OC2H5
37
3
6.8
CH3
O
17a
18a
OCH3
H
104
2
103.9 4.2
1.00
CH3
*
R,S O
CH3
O
OC2H5
H
28
3
167.7
8
5.99
CH3
*
R,S O
19a
20a
21a
22a
Buspirone
Prazosin
H
H
H
H
H
H
H
H
573 48
731,9 33.2
16,6 1.0
67,4 17.8
4800 500
—
1.28
0.17
0.81
8.35
—
OCH3
OC2H5
OCH3
95
83
575
16
7
5
0
3
7.79 0.025
7.44 0.025
—
–CH2COOH
—
0.24 0.05
a
Results for compounds 9a–18a from earlier Ref. 27.
according to Scheme 1. In the first step, 5,5-diphenylhydantoin
(23) was protected at N3-position giving 5,5-diphenyl-3-tritylimi-
dazolidine-2,4-dione (24) by the introduction of a triphenylmethyl
(trityl) group using trityl chloride in a diluted solution of methy-
lene chloride in a presence of TEA (triethylamine). In the second
step of synthesis, compound 24 was converted into 25 by

1352
J. Handzlik et al. / Bioorg. Med. Chem. 19 (2011) 1349–1360
two-phase alkylation in a basic condition, similar to that described
previously (acetone/K2CO3/TEBA).2,27 The pure oxiran derivative 25
was used in the reaction with respective ortho-(un)substituted
N-phenylpiperazines to give 26–28. The reactions were performed
under microwave irradiation in a simple microwave oven. The
method was a next modification of microwave-aided reaction de-
scribed earlier,2,27 adopted for reactants with higher difference of
melting points. Before irradiating, compound 25 with equimolar
amount of respective arylpiperazine was carefully dissolved in
methylene chloride, then the solvent was evaporated and the
residue was irradiated under TLC-control as long as the reaction
progress stopped. In the case of trityl derivatives 26–28, the time
(7–25 min) and the power of irradiation were higher in compari-
son to those described for the products 10–18.2,27 It seems to be
connected with high melting point of the starting material (25)
and a rest of CH2Cl2 in reactants-mixture that poorly transmits
microwaves as a nonpolar solvent. In contrast to the synthesis of
compounds 9–18, compounds 26–28 easily crystallized from
methanol giving pure white solids with good yield (68–92%). In
the last step of synthesis, compounds 26–28 were deprotected
from the trityl group.
N3-position over N1-position. Furthermore, the protecting group
had to be stable in basic conditions during the second step of syn-
thesis and easily eliminated at the end of synthesis. In this context,
two protecting methods were considered: a method with ethyl
chloroformate28 and the method with triphenylmethyl chloride.
In the first one, the protecting group was easily introduced in
N3-position giving ethyl 2,5-dioxo-4,4-diphenylimidazolidine-
1-carboxylate, but the protection was partly eliminated during
the alkylation in two phase-transfer catalytic conditions in the
presence of potassium carbonate and benzyltriethylammonium
chloride (TEBA) to give a number of by-products. On the contrary,
the N-trityl protection was stable during all synthesis steps, but it
was hard to remove it to obtain the target products (19–21). Com-
pounds 26–28 were stable in the basic conditions and resistant to
deprotection by the long-term heating in the concentrated HCl
(38%) yielding hydrochlorides of 1-(2-hydroxy-3-(4-arylpipera-
zin-1-yl)propyl)-5,5-diphenyl-3-tritylimidazolidine-2,4-diones.
The successful deprotection was accomplished using long-term
stirring of compounds 26–28 dissolved in methylene chloride with
the concentrated trifluoroacetic acid (TFA). The final products were
converted into hydrochloric forms 19a–21a with gaseous HCl and
precipitated from methanol (19a) or the mixture of methanol with
diethyl ether (20a, 21a).
N-Protection was the main problem in the presented synthesis
of the target hydantoins possessing an alkyl substituent at N1-
position and the free N3-position of the ring, as the nitrogen atom
N3 is more susceptible for alkylation. Thus, it was necessary to find
a protecting group appropriate for amide nitrogen and selective for
Synthesis of N3-acetic acid derivative 22a (Scheme 1) was per-
formed as a basic hydrolysis of the corresponding ester (10), syn-
thesized within our previous work.2 The crude product of the
O
O
O
NH
O
ii
,
(Ph)3 CCl, i
Cl
N
N
HN
N
HN
O
O
O
O
25
23 (DPH)
24
R2
iii
,
NH
N
R2
R2
+, iv
OH
O
O, H
H2
OH
O
N
N
N
N
N
N
N
NH
O
O
26-28
19-21
v
26
27
28
R2=H
R2=OCH3
R2=OC2H5
R2
i - CH2Cl2, TEA
OH
O
NH
ii - acetone, TEBA, K2CO3
iii - microwave irradiation
iv - CH2Cl2, 90% TFA
N
N
N
x HCl
O
v - MeOH, gaseous HCl
19a-21a
OCH3
OCH3
N
1) KOH, H2O/EtOH
2) HCl
O
O
OH
N
OH
O
O
O
OH
N
N
N
N
N
N
x HCl
O
O
22a
10
Scheme 1. Synthesis of compounds 19a–22a.

J. Handzlik et al. / Bioorg. Med. Chem. 19 (2011) 1349–1360
1353
hydrolysis, dissolved in absolute ethanol, was converted into
100
(a)
hydrochloride 22a by the use of gaseous HCl.
Compounds 9a–22a possessing one or two chiral centres were
obtained and tested as racemates.
phenylephrine
20a 10 nM
20a 100 nM
50
2.2. Pharmacology
2.2.1. Radioligand binding study
Affinity for 5-HT1A receptors was determined using [3H] 8-OH
DPAT as a radioligand, based on the screening protocol described
earlier.29 The ligands affinities expressed as estimated Ks values
(nM) are shown in Table 1.
0
-8
-7
-6
-5
-4
log [phenylephrine] (M)
100
Affinity for
a1-adrenoceptors of compounds 9a–18a was evalu-
(b)
ated previously.27 Compounds 19a–22a were tested for their
phenylephrine
21a 30 nM
in vitro affinity for
a1-adrenoceptors on rat cerebral cortex by
the radioligand binding assays using [3H]-prazosin as a specific
21a 100 nM
50
0
radioligand. The affinities described by Ki values (nM) are shown
in Table 1. Selectivity toward 5-YN1F over
a1-AR was calculated
as Ki 1/Ks5HT1A. For 5-HT1A receptors compounds 9a–22a showed
a
affinity within the range of 7–610 nM, whereas the affinity values
for a1-AR of new compounds (19a–22a) were included in the wide
range of 16.6–4800 nM.
In the case of affinity for 5-HT1A, three groups of the activity
may be seen: (I) the group of potent ligands with affinities in range
of 7–38 nM (11a–14a, 16a, 18a), (II) the group of active ligands
with affinities in range of 83–104 nM (10a, 15a, 17a, 20a, 21a)
and (III) the compounds with moderate affinities in range of
573–610 nM (9a, 19a, 22a).
-8
-7
-6
-5
log [phenylephrine] (M)
Figure 4. Concentration–response curves to phenylephrine in the rat aorta in the
absence (h) or presence of (a) 20a (j 10 and N 100 nM); (b) 21a (j 30 and N
100 nM). Results are expressed as percentage of the maximal response to
phenylephrine in the first concentration–response curve. Each point represents
the mean SEM (n = 4).
directing mostly towards piperazine. Compound 10a has the ester
fragment directed outside the molecule (Fig. 5)—as a matter of fact,
similar conformations have been also found for other structures
with longer N3 substituents as energetic local minima; their po-
tential energy was not higher than 2 kJ/mol over global minimum.
In all presented conformations internal hydrogen bonds have been
found: between the hydroxy group and the carbonyl in the position
2 of hydantoin ring (9a–13a, 15a–22a) or between the protonated
piperazine nitrogen N4 and the acetyl oxygen (14). Results of cal-
culated spatial properties in the comparison to both Barbaro’s
and Lepailleur’s pharmacophore models are shown in Figure 7.
Properties of substituents at nitrogen N3 for selected compounds
with different pharmacological activities (10a, 13a, 20a and 22a)
are displayed in Figure 8.
Concerning Barbaro’s model, the computed results indicated a
high spatial similarity of the compounds 9a–22a in the area of
two pharmacophoric features, PI-HY1 (X1) and PI-HY2 (X2). In
case of the distance X1 between a positive ionisable centre PI
and a hydrophobic area HY1 (Fig. 7a), the values in range of
5.68–5.69 Å are almost identical with the one described for the
2.2.2. Functional bioassays results
The antagonist activity of compounds 20a and 21a toward
a1-adrenoceptors present in rat aorta from adult Wistar rats was
assessed by inhibition of phenylephrine induced contractions.
The investigated compounds shifted the phenylephrine response
to the right. The antagonist affinities were reported as pKB esti-
mates (Table 1, Fig. 4). Compounds 20a and 21a at higher concen-
trations depressed the maximum effect of phenylephrine.
Reduction of phenylephrine maxima indicates a more complex
interaction between the tested compounds, a1-adrenoceptors and
probably other receptor systems present in the rat aorta which
limits the maximal effect of phenylephrine. Other possibilities in-
clude a slow dissociation of the agent from a receptor and an allo-
steric modification of receptors.30
Compound 20a displayed an ability to block the contractions in-
duced by phenylephrine and it shifted the phenylephrine response
to the right giving a pKB estimate of 7.785 0.025. Compound 21a
also shifted the phenylephrine response to the right indicating an
interaction with a1-adrenoceptors present in rat aorta. The pKB va-
lue was 7.441 0.025. It is noticeable that the affinity from the
functional test for compounds 20a and 21a was in the same con-
centration range as determined at radioligand binding assay.
2.3. Molecular modelling study
The goal of our molecular modelling was to search for 3D-struc-
ture properties of arylpiperazine derivatives (9a–22a), responsible
for discrimination between
tors. The studies are based on two pharmacophore models: the
Barbaro’s18 model for
1-adrenoceptor antagonists and Lepaill-
a1-adrenoceptors and 5-HT1A recep-
a
eur’s26 model for ligands of 5-HT1A receptors. The calculations per-
formed previously27 were modified using newer, more precise
methods.31–38 The lowest energy conformation for each compound
was found (Fig. 6) and considered as the bioactive one. In case of
9a–11a, 15a–18a, 22a, substituted at N3 position of the hydantoin
ring with longer fragments (ethyl, ester, acetic acid), the substitu-
ent places perpendicularly to the plane of the imidazole ring,
Figure 5. Molecular modelling results. Superimposition of global energy minimum
conformations of 9a–22a after molecular mechanics conformational analysis. Non
polar hydrogens are not displayed. Conformation of 14 is coloured green.

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J. Handzlik et al. / Bioorg. Med. Chem. 19 (2011) 1349–1360
rently obtained compounds (19a–22a), showed the role of the
modified fragment for the biological activities (Table 1). In case
of activity at 5-HT1A, three classes of activity may be observed
(I–III). Within the series of 2-alkoxyphenylpiperazine derivatives
(R2 = OCH3, OC2H5) compounds substituted at N3 with methyl
group (12a–14a), 2-methyl-propionic ester (11a and 18a) and ace-
tic acid ester (16a) belong to the class of the most potent ligands
(class I), while esters (10a, 15a and 17a) as well as N3H-free deriv-
atives (20a and 21a) represent the class of active ligands (class II).
In the group of moderate activity (class III), compounds with
unsubstituted phenylpiperazine phenyl ring (R2 = H, 9a, 19a) and
with N3-acetic acid end (22a) can be found.
On the other hand, the presence of carboxylic moiety at N3
position critically decreased affinity for a1-adrenoceptors, what is
observed in case of compound 22a (Ki = 4800 nM) in comparison
to the corresponding ester 10a (Ki = 160 nM). The conserved NH
group at N3-position (20a and 21a, Table 1) improved affinity
Figure 6. Molecular modelling results. Superimposition of global energy minimum
conformations of 9a–22a after reoptimization (DFT) calculations. Non polar
hydrogens are not displayed. Conformation of 14 is coloured green.
for
a1-adrenoceptors of corresponding N3-substituted hydantoin
9a–18a (Table 1).27
pharmacophore model. Similarly, the calculated distance PI-HY2
(X2) fits almost perfectly in the pharmacophore model, placing in
the narrow range of 6.18–6.20 Å for the compounds 10a–18a and
20a–22a. The calculated distance PI-HBA (X3) is in similar range
(5.11–5.15 Å) for all 2-hydroxypropyl derivatives (9a–13a and
15a–22a), lower than that of Barbaro’s model in 8–9%. On the other
hand, in case of acetoxy derivative 14a, the X3 value is 6.8% higher
than the one given by Barbaro. The distance between the positive
ionisable centre and the centre of most peripheral aromatic ring
HY3 (X4) was computed as distinctly lower than that of the phar-
macophore model of Barbaro (9.78 Å), in range of 7.95–8.08 Å for
2-hydroxypropyl derivatives (9a–13a and 15a–22a) and in the
value of 7.60 Å for the compound 14a.
The pharmacological studies performed for arylpiperazine
hydantoin derivatives 9a–22a indicated that most of the tested
compounds (10a–16a, 18a, 19a and 22a) acted stronger on
5-HT1A than on
a1-adrenergic receptors (Table 1). The significantly
higher activity toward
a1-AR comparing to 5-HT1A was observed
only in case of 1-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-
1-yl)propyl)-5,5-diphenylimidazolidine-2,4-dione hydrochloride
(20a), a compound with NH-free at position 3 of hydantoin. In case
of compounds with unsubstituted phenylpiperazine phenyl ring
(9a, 19a) as well as 2-methoxyphenylpiperazine derivative with
methyl 2-propionate end (17a), the affinity values for both,
5-HT1A and
a1-adrenergic receptors, are comparable. According
to the binding assays results (Table 1), unsubstituted phenylpiper-
As Lepailleur’s pharmacophore features (Fig. 3), six following
distances were considered: between hydrophobic area HYD and
hydrogen bound acceptor HBA (Y1), HBA and aromatic ring AR
(Y2); HBA and positive ionisable centre PI (Y3), HYD and AR (Y4),
HYD and PI (Y5) and between PI and AR (Y6).26 Results of the
molecular modelling study (Fig. 7b) indicate a high convergence
only in case of distances Y6 for all investigated compounds (9a–
22a). The compounds display values of distance Y1 in range of
5.53–5.62 Å, significantly higher than the pharmacophoric one
(4.4 Å). In case of distances Y2–Y5, a decrease, in comparison with
an ideal Lepailleur’s pharmacophoric features, can be observed. All
2-hydroxypropyl derivatives show approximately 17% shortening
of distance Y2 (10.08–10.23 Å) in comparison with that of Lepaill-
eur’s model (12.2 Å), whereas a much better accordance is seen for
acetoxy derivative 14a (11.68 Å). Similarly, compounds 9a–13a
and 15a–19a have distance Y3 in range of 5.11–5.15 Å while this
distance for compound 14a (6.0 Å) is closer to that of pharmaco-
phore model (6.6 Å). The distances Y4 are approximately 2 Å short-
er than the pharmacophoric one (15.8 Å) for 2-hydroxypropyl
derivatives (13.65–13.69 Å) and almost 3.5 Å shorter for com-
pound 14a (12.24 Å). The molecular modelling calculations gave
distances HYD-PI (Y5) in range of 7.95–8.08 Å for 2-hydroxypropyl
derivatives and 7.6 Å for 2-acetoxypropyl derivative 14a, which
is clearly lower than the Y5-distance of 5-HT1A pharmacophore
model (10.2 Å).
azine phenyl ring (9a, 19a) is less profitable for both 5-HT1A and
a1-adrenergic receptors affinities. In case of affinity for a1-adreno-
ceptors, the decrease of activity may be explained basing on the
pharmacophore model of Barbaro,18 as the unsubstituted phenyl-
piperazine derivatives do not include pharmacophore feature
HY2, desirable for a1-AR-antagonistic properties (Fig. 3). The Lepa-
illeur model for 5-HT1A receptor ligand26 does not respect this
structural feature as necessary for the activity (Fig. 3), however,
our results demonstrated a significant influence of this feature on
the affinity for 5-HT1A receptors.
Generally, the introduction of an alkyl-5,5-diphenylhydantoin
fragment at phenylpiperazine2,27 seems to be profitable for the
affinity for 5-HT1A but this is limited by N3-substitution.
N3-methyl (12a) and N3-ethyl propionate substituents are the
most profitable (11a) as they increased the 5-HT1A affinity of cor-
responding free 2-methoxyphenylpiperazine (Ki = 68 nM).13 The
following order of activity for N3-substituents can be observed:
methyl > ethyl propionate > methyl 2-propionate > H > ethyl ace-
tate > methyl acetate > acetic acid (Table 1). The highest decrease
of affinity for 5-HT1A (approximately 10-fold) is observed when
2-methoxyphenylpiperazine is linked to DPH with N3-acetic acid
end (22a). However, the acidic end of the compound 22a seems
to be much more disadvantageous for activity at
a1-AR (Table 1).
The compound 22a displayed almost 30-fold decrease of activity
of its corresponding esters (10a, 15a). Our results also demon-
strated that the 2-ethoxyphenylpiperazine fragment is more prof-
itable for 5-HT1A affinity than the 2-methoxyphenylpiperazine one.
Comparing four pairs of corresponding 2-ethoxy- and 2-methoxy-
phenylpiperazine derivatives (13a–12a, 16a–15a, 18a–17a and
21a–20a), it can be concluded that the 2-ethoxyphenylpiperazine
derivative was more active in each pair (Table 1). An analysis of
an influence of an alkyl spacer on affinity for both 5-HT1A and
2.4. SAR-study
As a continuation of previously started study,2,27 the presented
modifications were focused on the substituent at N3-position of
hydantoin. The performed synthesis allowed to save free NH at po-
sition N3 (19a–21a) as well as deprotect previously obtained ester
giving carboxylic end (22a). Results of affinity for both
a
1-AR and
a1-AR indicated that the acetoxy substituent at the alkyl chain
5-HT1A receptors, determined for previously (9a–18a) and cur-
linking piperazine to hydantoin fragment caused fivefold decrease

J. Handzlik et al. / Bioorg. Med. Chem. 19 (2011) 1349–1360
1355
(a) Structural factors responsible for α1-AR binding
(b) Structural factors responsible for 5 -HT1A binding
b1)
a1)
O
HBA
R1
PI
HY2
HY1
PI
HY3
R2
N
N
N
N
OR3
OR3
O
O
R2
N
N
N
N
R1
AR
HYD
O
HBA
b2)
a2)
Y2
PI
X2
HY2
HY1
HBA
HY3
X4
Y3
Y6
X3
AR
X1
Y5
Y1
PI
Y4
HBA
HYD
b3)
a3)
12.2 A
HY2
HY1
6.17 Ǻ
HY3
6.6 A
HBA
9.78 Ǻ
5.62 Ǻ
5.7 A
PI
4.4 A
10.2 A
AR
PI
5.69 Ǻ
15.8 A
HBA
Cpd
9a
X1=Y6 [Å]
X2[Å] X3=Y3[Å] X4=Y5[Å]
Y1[Å]
Y2[Å]
Y4[Å]
13.71
5.68
5.68
5.68
5.68
5.68
5.69
5.68
5.69
5.69
5.69
5.69
5.69
5.69
5.69
-
5.13
5.14
5.15
5.13
5.12
6.00
5.13
5.14
5.12
5.14
5.11
5.12
5.13
5.13
8.05
8.05
8.05
7.95
8.08
7.60
8.05
8.04
8.02
8.02
8.06
8.06
8.07
8.05
5.62
10.08
10.16
10.22
10.16
10.14
11.68
10.17
10.17
10.13
10.18
10.08
10.13
10.13
10.23
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
20a
21a
22a
6.19
6.18
6.18
6.19
6.20
6.18
6.19
6.20
6.18
-
5.57
5.58
5.61
5.62
5.53
5.59
5.62
5.58
5.55
5.62
5.62
5.62
5.59
13.66
13.65
13.69
13.71
12.24
13.69
13.91
13.68
13.65
13.71
13.70
13.71
13.70
6.19
6.19
6.20
Figure 7. Structural parameters of compounds 9a–22a in the comparison with two pharmacophore models. Distances corresponding with both, Barbaro’s and Lepailleur’s
models, according to the molecular modelling calculation for compounds 9a–22a (below the pictures). (a) Barbaro’s model for 1-adrenoceptor antagonists; (a1) five
pharmacophore features mapped by phenylpiperazine derivatives of phenytoin; (a2) four distances affect binding properties at 1-adrenoceptor; X1(PI-HY1), X2 (PI-HY2), X3
a
a
(PI-HBA) and X4 (PI-HY3); (a3) distances X1-X4 for ideal a1-AR antagonist. (b) Lepailleur’s model for 5-HT1A receptor ligands; (b1) four pharmacophore features mapped by
phenylpiperazine derivatives of phenytoin; (b2) six distances affect binding properties at 5ÀYN1F receptor; Y1(HYD-HBA), Y2 (AR-HBA), Y3 (PI-HBA), Y4 (HYD-AR), Y5 (PI-HYD)
and Y6 (PI-AR); (b3) distances Y1–Y6 for ideal 5-HT1A ligand.
of the affinity for both receptors (compounds 13a and 14a,
Table 1).
14a can be seen. Similarly, all compounds 9a–22a exhibit some
deviation in the location of the third hydrophobic feature (HY3).
There are two aromatic rings present in the non-phenylpiperazine
area, but each of them is located too close to the positive ionisable
nitrogen (PI) comparing to the ideal distance described by Barbaro
(Fig. 7). Although the most distant aromatic ring was considered as
the HY3-area for each compound (9a–22a), the distances between
the HY3 centre and PI are shorter (17–19%) for 2-hydroxypropyl
derivatives (9a–13a, 15a–22a) and, particularly, for 2-acetoxypro-
pyl derivative 14a (22.3%). These differences may explain a de-
The results of the pharmacological assays exhibited that most of
modifications performed for compound AZ–99 (9a)2,27 resulted in
compounds with higher activity at 5-HT1A than that of
a1-AR.
However, the pharmacophore-based SAR analysis did not confirm
this conclusion. Compounds 9a–22a displayed a significantly high-
er accordance with Barbaro’s pharmacophore features, dedicated
to
a1-adrenoceptor antagonists, than with Lepailleur’s model for
5-HT1A ligands. The compounds 9a–22a possess all structural frag-
ments characteristic for both, Barbaro’s and Lepailleur’s models,
but some divergence in the features collocation is observed
(Fig. 7). As Barbaro’s model is concerned, all compounds (9a–
22a) display high accordance with the ideal antagonist behaviour
within the areas of HY1, PI and HY2 (10a–18a, 20a–22a). Differ-
ences in the distances PI-HBA (X3) for compounds 9a–22a are low-
er than 10% in comparison to the ideal antagonist. The shortening
of X3-distance is observed for all compounds with a hydroxypropyl
linker, while a little prolongation for 2-acetoxypropyl derivative
crease of
a1-adrenoceptors affinity for compound 14a comparing
to the corresponding 2-hydroxypropyl derivative (13a).27
The presented group of arylpiperazine derivatives 9a–22a
exhibits also a good agreement with a previously described
a
1-AR antagonist pharmacophore model of De Benedetti15 which
requires a presence of protonated nitrogen (P) and two aromatic
systems (Ar1, Ar2) located 4–7 Å from P (Fig. 2). Within the
group of compounds 9a–22a a distance from the positive ionisable
nitrogen PI to the centre of phenylpiperazine phenyl ring HY1

1356
J. Handzlik et al. / Bioorg. Med. Chem. 19 (2011) 1349–1360
Figure 8. Properties of substituents at nitrogen N3 for selected arylpiperazine derivatives of hydantoin with different pharmacological activity; HBA-hydrogen bond acceptor
site; HBD-hydrogen bond donor site. (a) N–H, the strongest 1-AR agent 20a. (b) N–CH3, the strongest 5-HT1A agent 13a. (c) N–CH2COOC2H5, a long ester N3-substituent of
a
10a, a compound active for both receptors. (d) N–CH2COOH, acid substituent of compound 22a displaying a noticeable decrease of activity for both,
a1-AR and 5-HT1A,
receptors.
(5.68–5.69 Å, Fig. 7a) fits in De Benedetti’s distance P-Ar1. De
Benedetti’s feature Ar2 can match the closer hydantoin phenyl
ring, which is placed 4.98 Å from protonated nitrogen for the
compound 14a and 5.58–6.16 Å for the rest of compounds
(9a–13a and 15a–22a). In both cases, the considered distances
are between 4 Å and 7 Å—in agreement with Benedetti’s pharma-
cophore requirements.
toward a1-AR in comparison to 5-HT1A receptors. The 5-HT1A/a1
selectivities for 2-methoxyphenylpiperazine pyridazinone deriva-
tives were clearly lower. In our group of hydantoin derivatives
9a–22a, a reversal behaviour can be observed (12a–13a, 15a–
16a, 17a–18a and 20a–21a). An influence of N3-ester end on the
selectivity did not seem to be significant, while unsubstituted
N3H fragment (20a, 21a) conduced an evident increase of selectiv-
On the other hand, it is hard to explain differences in 5-HT1A
affinity for compounds 9a–22a basing on the pharmacophore mod-
el of Lepailleur. The analysis of the Lepailleur’s pharmacophore fea-
tures collocation for an ideal 5-HT1A ligand (Fig. 7b) demonstrates
that the hydantoin derivatives 9a–22a match the model only with-
in one out of six crucial distances (Y6). In case of distance Y1,
hydrophobic centre and hydrogen bond acceptor are located 26–
28% too far in comparison to the pharmacophore model distance.
In case of distances Y2–Y5, the target pharmacophore features
are located too close, giving a shortening of the following distances
for compounds with hydroxypropyl linker: Y2 (17%), Y3 (23%), Y4
(13%) and Y5 (22%). A different behaviour can be noticed for acet-
oxy derivative 14a, which demonstrated a higher accordance with
the pharmacophore model within the distances Y2 (4.3% shorten-
ing) and Y3 (9% shortening), whereas its deviations from the dis-
tances HYD-AR (Y4) and HYD-PI (Y5) were significantly higher,
that is, 22% (Y4) and 25% (Y5). SAR-studies based on a simple phar-
macophore model for buspiron-like 5-HT1A receptor ligands,
described by Chilmonczyk et al.,13,25 gave similar inferences.
Compounds 9a–22a show significant deviations (13–43%) from
the ideal distances d1–d3 (Fig. 2).
ity toward a1-AR comparing to 5-HT1A (5-HT1A/a1).
Our results indicated that compounds 9a–22a showed high 3D
convergence within arylpiperazine-hydantoin area (Figs. 5 and 6),
covering all Barbaro’s and Lepailleur’s pharmacophore features,
nevertheless their affinity and selectivity for
a1/5-HT1A receptors
were different. This may suggest that both pharmacophore
models18,26 are not sufficient to describe the differences in affinity
and selectivity for a1/5-HT1A receptors in the group of phenylpip-
erazine hydantoin derivatives. Additionally, the kind of N3-substi-
tuent should be taken into consideration.
Analysis of N3-substituent properties (Fig. 8) revealed that a
free NH group, the smallest fragment possessing hydrogen bond
donor (HBD) properties, is especially favourable for a1-adrenocep-
tors affinity (20a, 21a). On the other hand, a presence of methyl
group, a small H-bond-neutral substituent, placed in the neigh-
bourhood of N3-moiety, is particularly profitable for affinity for
5-HT1A receptors (12a–14a). The introduction of methyl group into
N3-position deprived the compounds 20a and 21a of a HBD-site. A
replacement of NH group with longer N-ester fragment (10a, 11a,
15a–18a) also removed the hydrogen bond donor site, but it
increased the number of hydrogen bond acceptors (HBA) as the
new C@O fragment was added. This modification seems to have
The tested compounds 9a–22a showed various a1/5-HT1A selec-
tivity values (Table 1) in range of 0.17–17.37. The most selective
compounds (13a, 14a) belong to the N3-methyl derivatives of
hydantoin with 2-ethoxyphenylpiperazine fragment. On the other
hand, Betti et al.39 described a group of 2-ethoxyphenylpiperazine
derivatives of pyridazinone with explicit selectivity (1.5–119-fold)
rather moderate influence on the affinity and selectivity for a1/5-
HT1A receptors. In the case of compound 22a, the N3-substituent
with carboxylic end can play both a role of HBD- and HBA-site.
The carboxylic end seems to be a hindrance in the interaction with
both, 5-HT1A and
a1-adrenergic receptors as this modification

J. Handzlik et al. / Bioorg. Med. Chem. 19 (2011) 1349–1360
1357
decreased affinity of corresponding esters (10a, 15a) for 5-HT1A
and, particularly, for a1-AR.
4.1.2. Preparation of 1-(oxiran-2-ylmethyl)-5,5-diphenyl-3-
tritylimidazolidine-2,4-dione (25)
A suspension of 24 (35 mmol, 17.29 g), K2CO3 (14 g), TEBA
(1.05 g), in acetone (70 mL) was stirred at room temperature for
3. Conclusion
15 min, then,
a solution of freshly distiled epichlorohydrin
(36 mmol, 3.56 g) in acetone (42 mL) was added dropwise. The
suspension was stirred for the next 54 h, then, the precipitate
was removed by filtration. The solvent was evaporated. The resi-
due was heated with CH2Cl2 (150 ml) under reflux for 10 min
and separated from insoluble inorganic precipitate by filtration.
The filtrate was evaporated and the residue was purified by
crystallization from acetone/water to give bright crystals of 25
(10.6 g, 19 mmol, 53%) mp 224–226 °C, Rf (I): 0.52. 1H NMR for
25 (DMSO-d6) d (ppm): 1.97 (dd, J1 = 2.82 Hz, J2 = 4.87 Hz., 1H,
CHoxiran), 2.26–2.30 (dd def. 2H, CH2oxiran), 2.02–3.09 (dd def., 1H,
N1-CH2a), 3.24–3.28 (m, 1H, N1-CH2b), 6.90–7.46 (m, 25H,
5 Â Ph). Anal. Calcd for C37H30N2O3 Â 0.5 H2O: C, 76.93; H, 5.76;
N, 4.85. Found: C, 76.40; H, 5.49; N, 4.70.
Our study performed for the group of arylpiperazine deriva-
tives of hydantoin 9a–22a provided new information in the field
of structural properties responsible for affinity and selectivity
toward 5-HT1A receptors comparing to
a1-adrenoceptors. The
molecular modelling aided SAR-analysis demonstrated the role
of three following structural parameters: the substituent at phe-
nylpiperazine phenyl ring, the substituent at the alkyl spacer and
the substituent at N3-position. 2-Ethoxyphenylpiperazine moiety
as well as N3-methyl substituent were particularly profitable for
affinity and selectivity for 5-HT1A, while 2-methoxyphenylpiper-
azine fragment and free N3H group were preferable for
a1-AR.
Phenylpiperazine moiety with the unsubstituted phenyl ring,
the N3-acetic acid end or acetoxy substituent at the alkyl spacer
were unfavourable for affinity for both, 5-HT1A and
a1-AR.
4.1.3. General procedure for preparation of 1-(2-hydroxy-3-(4-
arylpiperazin-1-yl)propyl)-5,5-diphenyl-3-tritylimidazolidine-
2,4-diones (26–28)
Among considered pharmacophore models, the model of Bar-
baro18 seems to be the best corresponding with the group of
arylpiperazine hydantoin derivatives 9a–22a. Nevertheless, none
of the investigated pharmacophore models was sufficient to
Equimolar (5–6 mmol) amounts of appropriate arylpiperazine
and
1-(oxiran-2-ylmethyl)-5,5-diphenyl-3-tritylimidazolidine-
explain all differences in affinity and selectivity for 5-HT1A
/
2,4-dione (25) were dissolved in CH2Cl2 (20–30 ml) in a flat-
bottomed flask and mixed for 2 min. The solvent was evaporated,
then, the residue was irradiated in a standard household micro-
wave oven using various powers (450–600 W) and times (7–
25 min) of irradiation for each prepared compound, respectively.
The progress of reaction was controlled with TLC (II). After irradi-
ation, the glassy residue was heated with methanol (15–20 ml) un-
der reflux for 30 min. During that time, the glassy residue was
converted into suspension of white precipitate. The suspension
was left at 0–4 °C overnight. The precipitate was filtrated to give
ready compounds 26–28.
a1-adrenoceptors among the arylpiperazine hydantoin deriva-
tives. Our results suggest that non-pharmacophoric N3-substitu-
ent is an important factor which, according to its chemical
properties, can affect affinity and discriminate between 5-HT1A
and
a1-adrenoceptors.
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 standard. IR spectra were recorded on a Jasco FT/IR-410
apparatus using KBr pellets and are reported in cmÀ1. Thin-layer
chromatography was performed on pre-coated Merck silica gel
60 F254 aluminium sheets, the used solvent systems were: (I) tolu-
ene/acetone 40:3; (II) toluene/acetone/methanol 5:5:1; (III) meth-
anol/dichloromethane 7:1. Melting points were determined using
Mel-Temp II apparatus and are uncorrected. Elemental analyses
were within 0.4% of the theoretical values unless stated other-
wise. Syntheses under microwave irradiation were performed in
household microwave oven Samsung M1618.
4.1.3.1.
1-(2-Hydroxy-3-(4-phenylpiperazin-1-yl)propyl)-5,5-
diphenyl-3-tritylimidazolidine-2,4-dione (26). Compound 25
(5 mmol, 2.75 g), 1-phenylpiperazine (5 mmol, 0.81 g) and 20 mL
of CH2Cl2 were used. The irradiation: (450 W) 7 Â 1 min. The puri-
fication from 15 ml of methanol was performed. White crystals of
26 (2.8 g, 3.9 mmol, 79%) mp 186–190 °C, Rf (II):0.86. 1H NMR for
26 (DMSO-d6) d (ppm): 1.78–1.99 (m, 2H, Pp-CH2), 2.11–2.14 (t
def., 4H, Pp-2,6-H), 2.80–2.89 (m, 1H, CHOH), 2.89–2.94 (t def.,
4H, Pp-3,5-H), 3.08–3.11 (d def., 2H, N1-CH2), 3.25–3.60 (m, 1H,
OH), 6.72 (t, J = 7.31 Hz, 1H, PpPh-4-H), 6.86–7.45 (m, 29H, PpPh-
2,3,5,6-H, 5 Â Ph). Anal. Calcd for C47H44N4O3: C, 79.19; H, 6.22;
N, 7.86. Found: C, 79.26; H, 6.20; N, 7.82.
4.1.3.2.
1-(2-Hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)-
propyl)-5,5-diphenyl-3-tritylimidazolidine-2,4-dione (27). Com-
pound 25 (6 mmol, 3.30 g), 1-(2-methoxyphenyl)piperazine
(6 mmol, 1.15 g) and 30 mL of CH2Cl2 were used. The irradiation:
(450 W) for 15 min (3 Â 1 min and 8 Â 1.5 min). The purification
from 20 ml of methanol was performed. White crystals of 27 (4.1 g
, 5.5 mmol, 92%) mp 188–192 °C, Rf (II):0.79. 1H NMR for 27
(DMSO-d6) d (ppm): 1.83–1.98 (m, 2H, Pp-CH2), 2.14 (br s, 4H, Pp-
2,6-H), 2.77 (br s, 4H, Pp-3,5-H), 2.90–3.02 (m, 1H, CHOH), 3.06–
3.12 (m, 2H, N1-CH2), 3.76 (s, 3H, OCH3), 4.38 (d, J = 4.88 Hz, 1H,
OH), 6.82–7.43 (m, 29H, PpPh, 5 Â Ph). Anal. Calcd for C48H46N4O4:
C, 77.60; H, 6.24; N, 7.54. Found: C, 77.48; H, 6.27; N, 7.51.
4.1.1. Preparation of 5,5-diphenyl-3-tritylimidazolidine-2,4-
dione (24)
A mixture of 23 (50 mmol, 12.6 g) in CH2Cl2 (80 mL) and TEA
(100 mmol, 10 g) in CH2Cl2 (40 mL) was stirred in room tempera-
ture for 15 min. Trityl chloride (50 mmol, 13.9 g) in CH2Cl2
(300 mL) was slowly added (10 min). The reactants were mixed
in room temperature for 30 h. The solution was washed with water
(250 mL) and twice with diluted NaOH solution (1%, 250 mL). The
organic phase was dried with anhydrous Na2SO4. After evaporation
of the solvent, the residue was crystallized from 1-butanol giving
pure white crystals of 24 (14.6 g, 29 mmol, 58%) mp 252–253 °C,
Rf (I): 0.49. 1H NMR for 24 (DMSO-d6) d (ppm): 7.03–7.08 (m, 4H,
2 Â 5-Ph-3,5-H), 7.15–7.38 (m, 21H, 2 Â 5-Ph-2,4,6-H, 3 Â Phtrityl),
9.47 (s, 1H, NH). Anal. Calcd for C34H26N2O2: C, 82.57; H, 5.30; N,
5.67. Found: C, 82.78; H, 5.31; N, 5.40.
4.1.3.3. 1-(3-(4-(2-Ethoxyphenyl)piperazin-1-yl)-2-hydroxypro-
pyl)-5,5-diphenyl-3-tritylimidazolidine-2,4-dione (28). Com-
pound 25 (5 mmol, 2.75 g), 1-(2-ethoxyphenyl)piperazine
(5 mmol, 1.03 g) and 20 mL of CH2Cl2 were used. The irradiation:

1358
J. Handzlik et al. / Bioorg. Med. Chem. 19 (2011) 1349–1360
450 W for 4 min (4 Â 1 min), then (600 W) for 21 min (3 Â 1 min
and 9 Â 2 min). The purification from 15 ml of methanol was per-
formed. White crystals of 28 (2.5 g, 3.3 mmol, 66%) mp 190–194 °C,
Rf (II):0.84. 1H NMR for 28 (DMSO-d6) d (ppm): 1.33 (t, J = 7.08 Hz,
3H, OCH2CH3), 1.80–1.93 (m, 2H, Pp-CH2), 2.13 (br s, 4H, Pp-2,6-H),
2.80 (br s, 4H, Pp-3,5-H), 2.85–2.95 (m, 1H, CHOH), 3.09 (d,
J = 6.67 Hz, 2H, N1-CH2), 3.96 (q, J = 6.97 Hz, 2H, OCH2CH3), 4.40
(br s, 1H, OH), 6.78–6.95 (m, 4H, PpPh), 7.06–7.45 (m, 25H,
5 Â Ph). Anal. Calcd for C49H48N4O4: C, 77.75; H, 6.39; N, 7.40.
Found: C, 77.77; H, 6.38; N, 7.38.
(OH), 3062 (N3-H), 3004 (CH), 2484 (NH+), 1764 (C2@O), 1715
(C4@O), 1595(Ar). Anal. Calcd for
C
29H32N4O4  HCl  1.4
H2O Â CH2Cl2 (20a): C, 61.62; H, 6.33; N, 9.88. Found: C, 62.12;
H, 6.35; N, 10.00.
4.1.4.3.
1-(3-(4-(2-Ethoxyphenyl)piperazin-1-yl)-2-hydroxy-
hydrochloride
propyl)-5,5-diphenylimidazolidine-2,4-dione
(21a). 1-(3-(4-(2-Ethoxyphenyl)piperazin-1-yl)-2-hydroxypro-
pyl)-5,5-diphenyl-3-tritylimidazolidine-2,4-dione 28 (1.32 mmol,
1 g) was stirred with TFA for 20 h. The pure 21a was precipitated
from methanol/diethyl ether to give white powder (0.30 g ,
0.55 mmol, 42%) mp 234–236 °C, Rf (II): 0.63. 1H NMR for 21
(DMSO-d6) d (ppm): 1.29 (t, J = 7.05 Hz, 3H, OCH2CH3), 2.02–2.32
(d def., 6H, Pp-CH2, Pp-2,6-H), 2.71–2.96 (m, 5H, Pp-3,5-H, CHOH),
3.24–3.33 (m, 2H, N1-CH2), 3.93 (q, J = 6.92 Hz, 2H, OCH2CH3), 4.43
(d, J = 5.13 Hz, 1H, OH) 6.80–6.88 (m, 4H, PpPh-4,6-H, 2 Â Ph-4-H),
7.12–7.26 (m, 4H, 2 Â Ph-2,6-H), 7.42–7.48 (m, 6H, 2 Â Ph-3,5-H,
PpPh-3,5-H), 11.33 (br s, 1H, N3-H). Anal. Calcd for
4.1.4. General procedure for preparation of 1-(2-hydroxy-3-(4-
arylpiperazin-1-yl)propyl)-5,5-diphenylimidazolidine-2,4-dione
hydrochlorides (19a–21a)
1-(2-Hydroxy-3-(4-arylpiperazin-1-yl)propyl)-5,5-diphenyl-3-
tritylimidazolidine-2,4-dione (26–28) (1.32–1.4 mmol, 1 g) was
dissolved in CH2Cl2 (10 ml) by an intensive stirring for 5–10 min.
A water solution of TFA (90%, 10 mL) was added. The mixture was
stirred at room temperature for 18–20 h. Then, the solution was
twice washed with water (2 Â 30 mL). The organic phase was dried
with anhydrous potassium carbonate. The solution was condensed
by evaporation of CH2Cl2 and purified by column chromatography
using silica gel and dichloromethane: acetone (10:1)/MeOH as
eluents to give a compound (19–21) as a bright oil. The oil was dis-
solved in methanol and was saturated with dried gaseous hydrogen
chloride until acidic pH. The pure crystals of a desirable hydrochlo-
ride were obtained after cooling at 0–4 °C overnight (19a) or were
precipitated with diethyl ether (20a and 21a).
C
30H34N4O4 Â CH2Cl2 Â 0.5 H2O (21): C, 61.18; H, 6.13; N, 9.21.
Found: C, 60.94; H, 6.21; N, 9.11. 1H NMR for 21a (DMSO-d6) d
(ppm): 1.35 (t, J = 6.92 Hz, 3H, OCH2CH3), 2.63–2.71 (m, 1H, CHOH),
2.80–3.05 (m., 6H, Pp-CH2, Pp-2,6-H), 3.14–3.41 (m, 6H, Pp-3,5-H,
N1-CH2), 3.98 (q, J = 6.92 Hz, 2H, OCH2CH3), 6.02 (br s, 5H, CHOH,
H2O) 6.83–7.10 (m, 4H, PpPh-4,6-H, 2 Â Ph-4-H), 7.12–7.32 (m,
4H, 2 Â Ph-2,6-H), 7.43–7.51 (m, 6H, 2 Â Ph-3,5-H, PpPh-3,5-H),
10.09 (br s, 1H, NH+), 11.46 (s, 1H, N3-H). IR (KBr) (cmÀ1): 3292
(OH), 3121 (N3-H), 2971 (CH), 2430 (NH+), 1770 (C2@O), 1720
(C4@O), 1606 (Ar). Anal. Calcd for C30H34N4O4  HCl  H2O (21a):
C, 63.32; H, 6.55; N, 9.84. Found: C, 62.98; H, 6.57; N, 9.79.
4.1.4.1.
1-(2-Hydroxy-3-(4-phenylpiperazin-1-yl)propyl)-5,5-
diphenylimidazolidine-2,4-dione hydrochloride (19a). 1-(2-
Hydroxy-3-(4-phenylpiperazin-1-yl)propyl)-5,5-diphenyl-3-trity-
limidazolidine-2,4-dione 26 (1.4 mmol, 1 g) was stirred with TFA
for 18 h. The pure 19a was precipitated from methanol to give
bright-pink powder (0.14 g, 0.26 mmol, 18%) mp 250–252 °C, Rf
(II): 0.67. 1H NMR for 19a (DMSO-d6) d (ppm): 2.61–2.68 (t def.,
1H, CHOH), 2.76–2.82 (m, 2H, Pp-CH2), 2.93–3.11 (m, 4H, Pp-2,6-
H), 3.14–3.40 (m, 4H, Pp-3,5-H), 3.66–3.70 (m, 2H, N1-CH2), 5.88
(br s, 5H, CHOH, H2O), 6.81 (t, J = 7.31 Hz, 1H, PpPh-4-H), 6.94 (d,
J = 7.69 Hz, 2H, PpPh-2,6-H), 7.21–7.30 (m, 6H, 2 Â Ph-2,4,6-H),
7.41–7.49 (m, 6H, 2 Â Ph-3,5-H, PpPh-3,5-H), 10.13 (br s, 1H,
NH+), 11.46 (s, 1H, N3-H). IR (KBr) (cmÀ1): 3266 (OH), 3062 (N3-
H), 3002 (CH), 2485 (NH+), 1765 (C2@O), 1716 (C4@O), 1594
(Ar). Anal. Calcd for C28H30N4O3  HCl  1.6 H2O  0.1 CH2Cl2: C,
61.89; H, 6.42; N, 10.31. Found: C, 62.06; H, 6.47; N, 10.27.
4.1.5. Preparation of 2-(3-(2-hydroxy-3-(4-(2-methoxyphenyl)-
piperazin-1-yl)propyl)-2,5-dioxo-4,4-diphenylimidazolidin-1-
yl)acetic acid hydrochloride (22a)
A
suspension of ethyl 2-(3-(2-hydroxy-3-(4-(2-methoxy-
phenyl)piperazin-1-yl)propyl)-2,5-dioxo-4,4-diphenylimidazolidin-
1-yl)acetate 10 (1.7 mmol, 1 g) in EtOH (5 mL) and H2O (5 mL)
was treated with KOH (8.9 mmol, 0.50 g), stirred at room temper-
ature for 90 min, diluted with H2O (10 mL), acidified to pH 2 (35%
HCl) and evaporated. The residue was dissolved in absolute EtOH
(20 ml) and saturated with gaseous HCl. Ethyl ether (20 ml) was
added to precipitate a white powder of 22a (0.56 g, 0.94 mmol,
55.4%), mp 186–187 °C, Rf (III): 0.83. 1H NMR for 22a (DMSO-d6)
d (ppm): 2.76 (br s, 3H, Pp-CH2, CHOH), 2.90–2.98 (m, 4H, Pp-
2,6-CH2), 3.34–3.40 (m, 6H, Pp-3,5-CH2, N1-CH2), 3.78 (s, 3H,
OCH3), 4.23 (s, 2H, N3-CH2), 4.40–5.90 (br s, 7H, OH, H2O) 6.87–
6.94 (m, 2H, PpPh-4,6-H), 6.96–7.03 (m, 2H, PpPh-3,5-H), 7.30–
7.37 (m, 4H, 2 Â Ph-3,5-H), 7.46–7.48 (m, 6H, 2 Â Ph-2,4,6-H),
10.06 (br s, 1H, NH+). IR (KBr) (cmÀ1): 3423 (OH) 3250 (OH car-
boxyl), 3001 (CH), 2486 (NH+), 1768 (C2@O), 1738 (C@O carboxyl),
1710 (C4@O), 1611 (Ar). Anal. Calcd for C31H34N4O6 Â HCl: C,
62.57; H, 5.93; N, 9.41. Found: C, 62.44; H, 5.94; N, 9.37.
4.1.4.2.
1-(2-Hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-
yl)propyl)-5,5-diphenylimidazolidine-2,4-dione hydrochloride
(20a). 1-(2-Hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)pro-
pyl)-5,5-diphenyl-3-tritylimidazolidine-2,4-dione 27 (1.35 mmol,
1 g) was stirred with TFA for 18 h. The pure 20a was precipitated
from methanol/diethyl ether to give white powder (0.26 g,
0.46 mmol, 34%) mp 240–242 °C, Rf (II): 0.65.1H NMR for 20
(DMSO-d6) d (ppm): 1.97–1.99 (m, 2H, Pp-CH2), 2.19 (br s, 4H,
Pp-2,6-H), 2.78 (br s, 4H, Pp-3,5-H), 2.96 (br s, 1H, CHOH), 3.22–
3.28 (m, 2H, N1-CH2), 3.73 (s, 3H, OCH3), 4.39 (br s, 1H, OH),
6.81–6.91 (m, 4H, PpPh-3,4,5,6-H), 7.21–7.26 (m, 4H, 2 Â Ph-3,5-
H), 7.43–7.46 (m, 6H, 2 Â Ph-2,4,6-H, PpPh-5-H), 11.31 (br s, 1H,
N3-H). Anal. Calcd for C29H32N4O4 Â H2O Â CH2Cl2 (20): C, 59.70;
H, 6.01; N, 9.28. Found: C, 59.34; H, 5.89; N, 9.32. 1H NMR for
20a (DMSO-d6) d (ppm): 2.58–2.66 (m, 1H, CHOH), 2.79–3.09 (m,
6H, Pp-CH2, Pp-2,6-H), 3.27–3.48 (m, 6H, Pp-3,5-H, N1-CH2), 3.78
(s, 3H, OCH3), 5.15 (br s, 6H, CHOH, H2O, CH2Cl2), 6.87–6.88 (m,
2H, PpPh-4,6-H), 6.94–7.03 (m, 2H, 2 Â Ph-4-H), 7.22–7.31 (m,
4H, 2 Â Ph-2,6-H), 7.43–7.51 (m, 6H, 2 Â Ph-3,5-H, PpPh-3,5-H),
9.82 (br s, 1H, NH+), 11.44 (s, 1H, N3-H). IR (KBr) (cmÀ1): 3268
4.2. Pharmacology
4.2.1. General information
The pharmacological studies were carried out on male Wistar
rats ((KRF.(WI).WU), Animal House, Faculty of Pharmacy, Jagiello-
nian University Medical College, Cracow) weighing 170–350 g.
Treatment of laboratory animals in the present study was in full
accordance with the respective Polish regulations. All procedures
were conducted according to guidelines of ICLAS (International
Council on Laboratory Animal Science) and approved by the Local
Ethics Committee on Animal Experimentation.
Source of compounds: Phenylephrine hydrochloride, acetylcho-
line hydrochloride, ( )-noradrenaline hydrochloride (Sigma,

J. Handzlik et al. / Bioorg. Med. Chem. 19 (2011) 1349–1360
1359
Aldrich Chemie Gmbh); Thiopental sodium (Biochemie Gmbh,
Vienna); [3H]-Prazosin (Amersham). Other reagents were of ana-
lytical grade from local sources.
Cumulative concentration–response curves to phenylephrine
(0.003 to 3
M) were obtained by the method of Van Rossum.41
l
Following the first phenylephrine curve, aortae rings were incubated
with tested compound (one concentration of the antagonist was used
in each arterial ring in every experiment) for 20 min and the next
cumulative concentration curve to phenylephrine was constructed.
In order to avoid fatigue of the aortae preparation, a 60 min recovery
period was allowed between phenylephrine curves.
4.2.2. Radioligand binding tests
4.2.2.1. 5-HT1A receptor binding assay. The in vitro affinity for
native serotonin 5-HT1A receptors was determined by inhibiting
[3H]-8-OH-DPAT (170.2 Ci/mmol; PerkinElmer) binding to rat hip-
pocampal membranes. Membrane preparation and a general assay
procedure were carried out according to the previously published
protocols.29,40
Concentration–response curves were analysed using GraphPad
Prism 4.0 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 phenylephrine effect (Emax = 100%), reached in the
concentration–response curves obtained before incubation with
the tested compounds. Data are the means SEM of four separate
experiments. Schild analysis could not be performed as a result of
depression of the maximal response by higher antagonist concen-
trations and the resulting nonparallel slopes of the concentration–
response curves. The affinity was estimated with the equation
pKB = log (concentration ratio À 1) À log (molar antagonist concen-
tration), where the concentration ratio is the ratio of equieffective
agonist concentrations in the absence and in the presence of the
antagonist.
Two compound concentrations were tested: 0.1 and 1 lM, each
run in triplicate. Radioactivity was determined by liquid scintilla-
tion counting in a Beckman LS 6500 apparatus. The KS values, esti-
mated on the basis of three independent binding experiments,
were reproducible in 20%.
4.2.2.2.
a1-Adrenoceptor binding test. The compounds were
evaluated on their affinity for
a
1-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 homogenised in 20 vol. of ice-cold 50 mM
Tris–HCl buffer (pH 7.6 at 25 °C) and centrifuged at 20,000g
for 20 min. The cell pellet was resuspended in Tris–HCl buffer
and centrifuged again. The final pellet was resuspended in Tris–HCl
4.3. Molecular modelling methods
The 3D molecule structures of 9a–22a were built using Schrö-
dinger Maestro molecular modelling environment31 basing on the
crystal structure of the (S)-isomer of phenylpiperazine phenytoin
derivative JH–9a.27 Basic piperazine nitrogens N4 in all structures
were protonated and the charge of +1 was assigned. For each com-
pound a conformational search was then performed using the
Monte Carlo method (MCMM) as implemented in MacroModel
9.732 with MMFFs force field and Polak-Ribiere conjugate gradient
(PRCG) options. The conformational analysis was carried out for
aqueous solutions with continuum solvation treatment (General-
ised Born/Solvent Accessible, GB/SA). Found global minimum
energy conformations of the ligands were superimposed by a
least-squares method; all heavy atoms of imidazole ring and the
basic nitrogen atom N4 of piperazine were chosen as fitting points
(Fig. 5). All compounds adopt an extended conformation as the
global minimum energy. The geometries of the lowest energy struc-
tures obtained from the conformational analysis were finally
optimised using density functional theory (DFT) and the Berny algo-
rithm with redundant internal coordinates.33 Becke’s three-param-
eter B3LYP hybrid functional34,35 and the 6-31G(d,p) basis set were
applied. Harmonic vibrational frequencies were calculated for each
structure to confirm the potential energy minimum. The DFT calcu-
lations were carried out using the Gaussian 03 suite of programs.36
For the graphic presentation of selected structures (10a, 13a, 20a
and 22a), PyMOL37 and Materials Studio v.4.4 software38 were used.
buffer (10 mg of wet weight/ml). 240
30 l of analysed compound were incu-
l of [3H]-prazosin and 30
bated at 25 °C for 30 min. To determine unspecific binding 10
phentolamine was used. Transfer solutions and adding reagents
was performed on automated pipetting system epMotion 5070
(Eppendorf, Germany).
After incubation reaction mix was filtered immediately onto GF/
B glass fibre filter mate presoaked using 96-well FilterMate Har-
vester (Perkin–Elmer, USA).
The radioactivity retained on the filter was counted in MicroB-
eta TriLux 1450 scintillation counter (Perkin–Elmer, USA). Non-
linear regression of the normalised (percent radioligand binding
compared to that observed in the absence of test or reference com-
pound—total binding) raw data representing radioligand binding
was performed in GraphPad Prism 3.0 (GraphPad Software) using
the built-in three parameter logistic model describing ligand com-
petition binding to radioligand-labelled sites.
ll of the tissue suspension,
l
l
lM
4.2.3. Functional bioassay
Isolated rat aorta was used in order to test antagonistic activity
of investigated compounds for a1-adrenoceptors. The male Wistar
rats weighting 200–350 g were anaesthetized with thiopental
sodium (75 mg/kg ip) and the aorta was dissected and placed in
a Krebs-Henseleit solution and cleaned of surrounding fat tissues.
The thoracic aorta was denuded of endothelium and cut into
approximately 4 mm long rings. The aorta rings were incubated
in 30 ml chambers filled with a Krebs–Henseleit solution (NaCl
118 mM, KCl 4.7 mM, CaCl2 2.25 mM, MgSO4 1.64 mM, KH2PO4
1.18 mM, NaHCO3 24.88 mM, glucose 10 mM, C3H3O3Na 2.2 mM,
EDTA 0.05 mM) at 37 °C and pH 7.4 with constant oxygenation
(O2/CO2, 19:1). Two stainless steel pins were inserted through
the lumen of each arterial segment: one pin was attached to the
bottom of the chamber and the other to an isometric FDT10-A force
displacement transducer (BIOPAC Systems, Inc., COMMAT Ltd,
Turkey). The aortae rings were stretched and maintained at
optimal tension of 2 g and allowed to equilibrate for 2 h. The lack
of endothelium was confirmed by the absence of acetylocholine
Acknowledgements
Authors thank to Mrs. Maria Kaleta for her assistance in synthe-
sis. Authors also thank very much Professor Andrzej J. Bojarski for
his helpful suggestions concerning this study. The works were
partly supported by grant K/ZDS/000727. Computing resources
from Academic Computer Centre CYFRONET AGH (Grants
MNiSW/SGI3700/UJ/078/2008) are gratefully acknowledged.
Supplementary data
(1
l
M) vasorelaxant action in aortic rings precontracted by
M).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.11.051.
noradrenaline (0.1
l

1360
J. Handzlik et al. / Bioorg. Med. Chem. 19 (2011) 1349–1360
23. Mokrosz, M. J.; Duszyn´ ska, B.; Bojarski, A. J.; Mokrosz, J. L. Bioorg. Med. Chem.
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