Synthesis, α1-adrenoceptor antagonist activity, and SAR study of novel arylpiperazine derivatives of phenytoin

Article abstract of DOI:10.1016/j.bmc.2008.04.058

In the search for new antiarrhythmic agents, some active 2-methoxyphenylpiperazine derivatives of phenytoin were obtained as a chemical modification of compound AZ-99 (3-ethyl-1-[2-hydroxy-3-(4-phenylpiperazin-1-yl)-propyl]-2,4-dioxo-5,5-diphenylimidazolidine). These compounds possessed structural properties similar to those of α₁-adrenoceptor antagonists. In the present study, the affinities of the 2-methoxyphenylpiperazine derivatives (1a-3a) for α₁- and α₂-adrenoceptors were evaluated using radioligand ([³H]prazosin, [³H]clonidine) binding assays. In the next step, a new series of phenylpiperazine derivatives of phenytoin (4a-16a) containing 2-methoxyphenyl-, 2-ethoxyphenyl-, 2-pyridyl- or 2-furoylpiperazine moiety, as well as, various ester or alkyl substituents at 3-position of hydantoin ring were synthesized. The newly synthesized compounds were tested for their affinity to α₁- and α₂-adrenoceptors. They have shown affinities for α₁-adrenoceptors at nanomolar to submicromolar range. Some compounds were moderately selective ligands of α₁-adrenoceptors. Selected compounds (3a-5a, 7a, 13a, 14a) were also evaluated for their α₁-adrenoceptor antagonistic properties in functional bioassays. A SAR study indicated that the most active compounds contain 2-alkoxyphenylpiperazine moieties and methyl or 2-methylpropionate substituent at 3-N position in hydantoin. The exchange of 2-alkoxyphenyl moiety into 2-furoyl or 2-pyridyl group significantly decreased affinities for α₁-adrenoceptors. Molecular modelling results obtained using conformational analysis CONFLEX and PM5 method for geometry optimization, allowed for comparison of the spatial properties of tested compounds with pharmacophore model created by Barbaro et al. for the ideal α₁-adrenoceptor antagonist.

Full text of DOI:10.1016/j.bmc.2008.04.058

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5982–5998
Synthesis, a1-adrenoceptor antagonist activity, and SAR study
of novel arylpiperazine derivatives of phenytoin
Jadwiga Handzlik,a Dorota Macia˛g,b Monika Kubacka,c Szczepan Mogilski,c
c
d
a,
*
´
Barbara Filipek, Katarzyna Stadnicka and Katarzyna Kiec-Kononowicz
aDepartment of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Medyczna 9,
30-688 Krako´w, Poland
bDepartment of Pharmacobiology, Jagiellonian University Medical College, Medyczna 9, 30-688 Krako´w, Poland
cDepartment of Pharmacodynamics, Jagiellonian University Medical College, Medyczna 9, 30-688 Krako´w, Poland
dFaculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krako´w, Poland
Received 8 January 2008; revised 22 April 2008; accepted 23 April 2008
Available online 26 April 2008
Abstract—In the search for new antiarrhythmic agents, some active 2-methoxyphenylpiperazine derivatives of phenytoin were
obtained as a chemical modification of compound AZ-99 (3-ethyl-1-[2-hydroxy-3-(4-phenylpiperazin-1-yl)-propyl]-2,4-dioxo-5,5-
diphenylimidazolidine). These compounds possessed structural properties similar to those of a1-adrenoceptor antagonists. In the
present study, the affinities of the 2-methoxyphenylpiperazine derivatives (1a–3a) for a1- and a2-adrenoceptors were evaluated using
radioligand ([3H]prazosin, [3H]clonidine) binding assays. In the next step, a new series of phenylpiperazine derivatives of phenytoin
(4a–16a) containing 2-methoxyphenyl-, 2-ethoxyphenyl-, 2-pyridyl- or 2-furoylpiperazine moiety, as well as, various ester or alkyl
substituents at 3-position of hydantoin ring were synthesized. The newly synthesized compounds were tested for their affinity to a1-
and a2-adrenoceptors. They have shown affinities for a1-adrenoceptors at nanomolar to submicromolar range. Some compounds
were moderately selective ligands of a1-adrenoceptors. Selected compounds (3a–5a, 7a, 13a, 14a) were also evaluated for their
a1-adrenoceptor antagonistic properties in functional bioassays. A SAR study indicated that the most active compounds contain
2-alkoxyphenylpiperazine moieties and methyl or 2-methylpropionate substituent at 3-N position in hydantoin. The exchange of
2-alkoxyphenyl moiety into 2-furoyl or 2-pyridyl group significantly decreased affinities for a1-adrenoceptors. Molecular modelling
results obtained using conformational analysis CONFLEX and PM5 method for geometry optimization, allowed for comparison of
the spatial properties of tested compounds with pharmacophore model created by Barbaro et al. for the ideal a1-adrenoceptor
antagonist.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
selective a1 antagonist, it has been proposed that a1-
adrenoceptors can be divided into two types, a1H (high
sensitivity) and a1L (low sensitivity) (see Fig. 1). Three
subtypes of human a1H-adrenoceptors: a1A, a1B and
The adrenergic receptors, divided into three subclasses,
a1, a2, and b, belong to the superfamily of G-protein
coupled receptors that are targets of the catecholamines,
such as adrenaline and noradrenaline.1,2 The a1-adreno-
ceptors subclass, discovered as an independent group in
1974,3 contains multiple subtypes which were identified
based on pharmacological studies and cloning tech-
niques. According to the pharmacological classification,
and based on sensitivity to the prazosine, a potent and
a
1D, have been identified by pharmacological and clon-
ing studies. At this time, the a1L-adrenoceptors are
recognised as a separate subtype of a1-adrenoceptors
in various pharmacological assays but a discrete protein
with a1L-adrenoceptor characteristics has yet to be
cloned. Some lines of evidence suggest that a1L-adreno-
ceptors may represent a functional phenotype of a1A
-
adrenoceptors.2,4
Keywords: Arylpiperazine derivatives; Phenylpiperazine derivatives;
Phenytoin derivatives; Hydantoin derivatives; Adrenergic receptors;
a1-Adrenoceptor antagonists.
Corresponding author. Tel.: +48 012 657 04 88; fax: +48 012 657 04
88; e-mail: mfkonono@cyf-kr.edu.pl
The a1-adrenoceptors are involved in sympathetic and
central nervous system functions,1–7 and are responsible
for many physiological effects including: vascular
smooth muscle contraction, blood pressure regulation,1,2
*
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.058

J. Handzlik et al. / Bioorg. Med. Chem. 16 (2008) 5982–5998
5983
NH2
N
O
N
O
H3CO
H3CO
CH3
N
H
N
O
N
OCH3
OCH3
N
N
N
O
N
N
N
N
O
H3C
CH3
O
Prazosin
5-Methylurapidil
BMY-7378
OCH3
N
N
O
CH3
O
O
O
N
N
N
N
OCH3
N
N
N
H
N
N
OCH3
N
H
O
WAY-100635
REC-15/2739
RA36
Figure 1. Structure of potent a1-adrenoceptor antagonists.
the human prostate smooth muscle contraction,5 or reg-
ulation of cerebral microcirculation.6
of Barbaro, five structural fragments, common for tar-
get compounds, corresponding to five pharmacophore
features can be found (Fig. 2). As 2-substituted phenyl-
piperazine derivatives, all considered compounds
possess a positive ionisable nitrogen atom and an
ortho-substituted phenyl ring corresponding to PI and
both HY1 and HY2, respectively. These compounds
also contain an additional or fused aromatic moiety,
ending the heterocyclic fragments, which can corre-
spond to the HY3-feature. Additionally, they possess
an alkyl spacer between piperazine nitrogen and nitro-
gen placed close to carbonyl oxygen (HBA) at a chain
or at heterocyclic rings. The alkyl spacer may consist
of 2–7 carbons and may additionally be substituted with
alkyl or hydroxyl moieties, respectively. Some lines of
evidence indicate that acetylation of the hydroxyl moie-
ties significantly increases the affinity for a1-AR.18
Thus, a1-adrenoceptor antagonists can be useful in the
treatment of hypertension, benign prostatic hyperplasia
(BPH), lower urinary tract symptoms (LUTS), or car-
diac arrhythmia.1,2,5,6 In this context, the search for
selective a1-adrenoceptor antagonists has been, and still
is, an important topic in medicinal chemistry. In recent
decades, various new a1-adrenoceptor antagonists have
been designed and successfully synthesized.8–19 Analysis
of a number of chemical structures of selective a1-adre-
noceptor antagonists8 indicates that a large group of ac-
tive compounds contain arylpiperazine moieties. Some
known phenylpiperazine derivatives such as 5-methylu-
rapidil, BMY-7378, WAY-100635, REC-15/2739, and
RA36 (Fig. 1) can be considered as classical examples
of potent a1-adrenoceptor antagonists useful in various
pharmacological assays including binding studies, as
well as, in vivo tests.8,9 Furthermore, in recent years,
new families of phenylpiperazine derivatives with high
binding potency for a1-adrenoceptors have been synthe-
sized.9–17 The general structures of compounds and
ranges of their affinities for a1-adrenoceptors, described
by Romeo et al.,9,16 Betti et al.,10–13 Barbaro et al.,14
Strappaghetti et al.,15 and Kuo et al.,17 are presented
in Table 1. The large database of active compounds al-
lowed to evaluate pharmacophore models of the a1-
antagonist.8,14 The most recent models, elaborated using
radioligand binding data and computer aided methods
with Catalyst have been proposed by Bremner et al.8
and Barbaro et al.14 Barbaro’s model, especially useful
for phenylpiperazine derivatives, has postulated five
pharmacophore features: a positive ionisable atom
(PI), three hydrophobic regions (HY1–HY3), and a
hydrogen bond acceptor (HBA). Considering the struc-
ture of active arylpiperazine a1-antagonists (Fig. 1 and
Table 1) in comparison with the pharmacophore model
On the other hand, our previous studies were focused on
evaluating various biologically active phenytoin (DPH)
derivatives.19–21 We carried out the chemical modifica-
tion of phenytoin, a known anticonvulsant and antiar-
rhythmic agent, in order to increase its antiarrhythmic
potency and decrease its anticonvulsant action. We
introduced various amine moieties into phenytoin struc-
tures to minimize its interaction with the CNS. Among
others, we obtained 3-ethyl-1-[2-hydroxy-3-(4-phenylpi-
perazin-1-yl)-propyl]-2,4-dioxo-5,5-diphenylimidazolidine
(AZ-99, Table 2) that was further modified to give a ser-
ies of pharmacologically active compounds.19 Chemical
structures of the phenytoin derivatives display a similar-
ity to many phenylpiperazine a1-adrenoceptor antago-
nists. These compounds possess phenylpiperazine
moieties, a heterocyclic ring of hydantoin with two phe-
nyl rings (position 5) and a 2-hydroxypropyl spacer
between piperazine and 1-N-hydantoin moieties. Espe-
cially, three derivatives of AZ-99, compounds 1–3 (Ta-
ble 2), show high similarity to a1-adrenoceptor

5984
J. Handzlik et al. / Bioorg. Med. Chem. 16 (2008) 5982–5998
Table 1. Selected general structures of recently discovered a1-adrenoceptor antagonists
Structures
a1-Adrenoceptor affinity
range ꢀKia (nM)
R1
O
N
R2
N
N
N
R1=
ꢀ0.2–12010,11
,
,
N N
N
N
N
n
R2 = Cl, OCH3
n=2, 3, 4, 5, 6, 7
OCH3
O
R2
R1=
,
O
,
O
Cl
ꢀ0.05–6012–14
O
N
N
N
N
n
O
OC2H5
R1
N
N
,
O
O
R2 = OCH3, OC2H5, OCH(CH3)2
n=2, 3, 4, 5, 6, 7
R1
R3
N
O
R2
R1 = H, CH3
R2 = H, CH3
R3 = OCH3
ꢀ0.1–1516
N
X
X
N
N
n
O
n = 1,2
X = NH, NCH3, S, O
N
O
R1
ꢀ0.5–209
N
X = NH, S
R1 = 2-CH3O
R2 = H, 5-Cl
N
N
R2
O R2
OH
H
N
N
N
N
R1 = H, 4-Cl, 3,4-Cl2, 4-CH3, 4-C(CH3)2,
3-N(CH3)2, 2-CH3, 2-OCH3, 4-OCH3, 3-CF3
R2 = CH3, C2H5, CH(CH3)2
ꢀ0.5–30.017
O
X
R1
X = O, S, NH
a Results of radioligand binding assays performed for a1-AR (rat cortex) or cloned a1-adrenoceptor subtypes (a1a, a1b, a1d), respectively (see Refs. 9–
14, 16, 17).
antagonists as members of 2-methoxyphenylpiperazine
derivatives family.
1a–16a within SAR studies including molecular model-
ling calculations.
2. Results
In our present work we decided to evaluate the com-
pounds (1–3) for their a1- and a2-adrenoceptor affinity
in radioligand binding assays. Furthermore, we designed
and synthesized a series of new compounds (4–16) as
further derivatives of AZ-99 (Table 2) possessing 2-
methoxyphenyl-, 2-ethoxyphenyl-, 2-furoyl-, or 2-pyri-
dylpiperazine fragments, as well as, various substituents
at position 3-N of the hydantoin ring. In the case of
compound 8, we modified, additionally, the hydroxy-
propyl chain by acetylation of the hydroxyl group.
The new compounds were converted into hydrochloric
salts 4a–16a and initially tested for their affinities to
both a1- and a2-adrenoceptors in radioligand binding
assays. Selected compounds (3a–5a, 7a, 13a, and 14a)
were then tested for their antagonistic properties at the
vascular a1-adrenoceptor within functional bioassays.
Finally, we analysed the role of the modified chemical
fragments in a1-antagonistic properties of compounds
2.1. Synthesis
The synthesis of compounds 1a–3a was described ear-
lier.19 Compounds 4a–16a were obtained according to
Scheme 1. Compounds 4–7 and 9–16 were synthesized
by three-step alkylation using phenytoin (17) as a start-
ing product. At first, phenytoin was alkylated at 3-N po-
sition giving methyl- or methyl ester derivatives 18–21,
respectively. The synthesis of compounds 18 and 19
was performed via the alkylation with CH3I in EtONa
(18) or the reaction with methyl 2-bromoacetate in two
phase-transfer catalytic conditions in the presence of
potassium carbonate and benzyltriethylammonium
chloride (TEBA) as phase-transfer catalyst (19), accord-
ing to the methods described earlier.20,21 Two new
methyl esters, derivatives of 2-methylpropionate (20)

J. Handzlik et al. / Bioorg. Med. Chem. 16 (2008) 5982–5998
5985
mercially available methyl 2-bromopropionate or
methyl 2-bromobutyrate, respectively. The reactions
were carried out in similar conditions to those for com-
pound 19.
The next steps of synthesis were based on double-alkyl-
ating properties of epichlorohydrin. At first, epichloro-
hydrin was used, as an alkyl chloride, to introduce
oxiranylmethyl moiety into the 1-N position of com-
pounds 18–21 to give compounds 22–25. The reaction
was carried out at room temperature by stirring the
reactants in phase-transfer catalytic conditions accord-
ing to the method described earlier.20,21 In the case of
new compounds 24 and 25, the previous method was
modified by changing stirring time and methods of puri-
fication. The synthesis was performed under TLC con-
trol (toluene–acetone 40:3). Compound 24 was
obtained as a result of long-time stirring for 54.5 h.
The compound precipitated as pure crystals from con-
densed filtrate that was obtained after the separation
of the solid phase from the reaction mixture. According
to TLC control, synthesis of compound 25 needed 69 h
of stirring. The pure compound 25 was obtained by crys-
tallization from methanol.
Figure 2. Structural features (A–E) of phenylpiperazine a1-adreno-
ceptor antagonists9–17 compared to pharmacophore model of Barbaro
et al. (PI, HY1–3, HBA).14 (A) (Un)substituted phenylpiperazine
phenyl ring corresponding to hydrophobic features (HY1 and HY2);
(B) positive ionisable nitrogen of phenylpiperazine corresponding to
PI; (C) (un)substituted alkyl spacer between two nitrogens. (D)
Carbonyl moiety corresponding to HBA; (E) heterocyclic moiety with
condensed or substituted phenyl ring corresponding to HY3 in
Barbaro’s model. Draft pharmacophore model mapped with an
example phenylpiperazine antagonist (3-(2-(4-(2-methoxyphenyl)pip-
erazin-1-yl)ethyl)-4a,5-dihydro-1H-pyrimido[5,4-b]indole-2,4(3H,9bH)-
dione) made basing on Ref. 14.
and 2-methyl butyrate (21), were synthesized by heating
compound 17 in acetone with TEBA, K2CO3, and com-
Table 2. Compound AZ-99 and its chemical modifications
OR3
O
R1
OH
O
R2
N
N
N
N
N
N
N
N
C2H5
O
O
Compounds 1-16
AZ-99
Compound
R1
R2
R3
H
Compound
R1
R2
R3
H
OCH3
OCH3
OCH3
OCH3
OC2H5
OCH3
1
–C2H5
9
–CH2COOCH3
–CH2COOCH3
–CH2COOCH3
OC2H5
O
2
3
4
5
–CH2COOC2H5
H
H
H
H
10
11
12
13
H
H
H
H
CH3
O
CH3
O
O
–CH3
–CH2COOCH3
N
CH3
OCH3
OC2H5
OCH3
O
–CH3
–CH3
–CH3
CH3
O
CH3
O
O
6
7
H
H
14
15
H
H
CH3
O
O
CH3
O
CH3
N
O
CH3
O
OC2H5
OC2H5
8
–CH3
16
H
O
CH3
CH3
O

5986
J. Handzlik et al. / Bioorg. Med. Chem. 16 (2008) 5982–5998
O
O
O
R1X, i
O
i
i
Cl
NR1
HN
NR1
N
HN
NH
O
O
O
O
R1X = CH3I
18, 22 R1 = CH3
18-21
22-25
19, 23 R1 =CH2COOCH3
17
R1X =BrCH2COOCH3
R1X =BrCH(CH3)COOCH3
R1X =BrCH(C2H5)COOCH3
20, 24 R1 =CH(CH3)COOCH3
21, 25 R1 =CH(C2H5)COOCH3
R2
N
NH
, iii
OR3
HCl, v
O
OH
O
R2
N
N
N
NR1
R2
N
N
N
O
NR1
x HCl
O
4-7, 9-16
4a-16a
HCl, v
O
O
OH
O
(CH3CO)2O, iv
O
O
O
N
N
N
N
N
N
N
N
O
O
5
8
Scheme 1. Synthesis of compounds 4a–16a. Reagents and conditions: (i) EtONa or K2CO3, TEBA, EtOH; (ii) K2CO3, TEBA, acetone, 20 ꢁC; (iii)
mw-irradiation; (iv) reflux; (v) MeOH or EtOH, gaseous HCl.
Synthesis of the final products 4–7 and 9–16 was based on
N-alkylating properties of the oxiranyl rings of com-
pounds 22–25 in reactions with secondary amines. The
oxiranyl rings of compounds 22–25 were opened via reac-
tions with 2-alkoxyphenyl-, 2-furoyl-, or 2-pyridylpipera-
zine, respectively. The reactions were carried out with
equimolar amounts of dry reagents under microwave irra-
diation using a standard household microwave oven.19
solved in anhydrous alcohol for saturation with
gaseous HCl (method B). In the case of compound 8a,
a glue residue of compound 8 was dissolved in anhy-
drous methanol for the saturation with HCl. All hydro-
chlorides (4a–16a) precipitated from alcohols to give
pure bright crystals.
Additionally, syntheses of two compounds (4 and 5)
were carried out using the microwave oven ‘CEM-Dis-
cover’, qualified for organic synthesis performed in var-
ious conditions including high pressure, reactants in
solvent or in dry conditions. This professional micro-
wave oven allows to create process conditions by the
set of following parameters: temperature, starting time
to achieve a desirable temperature and continuing irra-
diation time. During the process, temperature, power
and pressure were automatically controlled by computer
monitoring. The reactants were magnetically stirred in
flat-bottom flask placed in microwave oven and covered
by special polymer protector. Special protecting system
does not allow open microwave oven until the tempera-
ture decreases to a safe value. In the case of compound
4, reactants were irradiated for three periods of time
controlling the colour of the melting mixture. After
the first period of irradiation at 120 ꢁC, about half of
the reactants was melted. Irradiation was prolonged
but the result was not satisfying as the part of reactants
was still not melted. When the temperature was raised to
140 ꢁC, in the third irradiation period, a little part of
As a result of irradiation glassy residues were obtained.
Contrary to compounds 11, 15, and 16, for compounds
4–7, 9, 10, and 12–14, pure products were obtained by
recrystallization of glass residues from ethanol or meth-
anol, respectively. Synthesis of 3-(4-(2-ethoxyphenyl)
piperazin-1-yl)-1-(3-methyl-2,4-dioxo-5,5-diphenylimi-
dazolidin-1-yl)propan-2-yl acetate (8) was performed
by acetylation of compound 5 using acetic anhydride
to give a gluey residue.
Finally, all compounds (4–16) were converted into
hydrochloric salts (4a–16a) using gaseous HCl.19 Com-
pounds 5–7, 9, 10, and 12–14, obtained as pure precipi-
tates, were dissolved in anhydrous alcohol and saturated
with HCl (method A). Compounds 4, 6, 11, 15, and 16
did not crystallize from alcohols, giving gluey residues.
These residues were dissolved in methylene chloride
and washed from the rest of starting piperazines by
the use of diluted hydrochloric acid. After solvent evap-
oration, the residues of 4, 6, 11, 15, and 16 were dis-

J. Handzlik et al. / Bioorg. Med. Chem. 16 (2008) 5982–5998
Table 3. Binding properties of compounds 1a–16a
5987
white starting product was still present in the flask and
reactant mixture was getting dark yellow. Despite mix-
ing and computer-controlled conditions, an appearance
of partial overheatings was observed. Therefore the irra-
diation process was interrupted.
Compound
Affinity Ki (nM)
a2-AR
a1-AR
a2/a1
1a
2a
292.7 1.1
160 21.3
135.7 31.3
160.7 13.6
121.6 14.9
>100 000
415.63
1.42
2.59
8.48
2.15
4.40
—
413.7 59.3
1150 170
344.2 44.3
534.7 54
28300 5300
693.2 100
1900 200
800 160
852 70
3a
4a
In case of compound 5, dry reactants were dissolved in
CH2Cl2 and the solvent was evaporated to give a homo-
geneous reactant mixture as glassy residue. A flask with
the residue was placed in microwave oven ‘CEM-dis-
cover’ and the process was performed in the same man-
ner as that of compound 4. Unfortunately, the reactant
mixture was getting dark brown after the second irradi-
ation period at 120 ꢁC giving burnt product.
5a
6a
7a
8a
691.9 16.5
607 74.7
197.8 25.4
251.6 3.8
7600 1150
1100 100
103.9 4.2
1.01
3.13
4.04
3.39
0.74
0.9
9a
10a
11a
12a
13a
14a
15a
16a
AZ-99a
Prazosin
5600 900
990 250
448.6 25.9
733.8 90
5600 400
23800 900
564.1 28.6
—
4.32
4.38
1.81
3.05
1.07
—
The experiments indicated that professional microwave
oven did not allow to create universal repeatable condi-
tions for synthesis of the presented phenylpiperazine
derivatives. Furthermore, the longer time of irradiation
seems to be dangerous for reactant mixture in profes-
sional microwave oven, too. Thus, short-irradiation-ses-
sions (1–2 min) with reactants-semblance control and
TLC monitored reaction progress are recommended to
assure stable and efficient process conditions.
167.7
8
3100 200
7800 500
529 9.5
0.24 0.05a
Inhibition constants (Ki) were calculated according to the equation of
Cheng and Prusoff. Radioligand binding assays to rats cortex mem-
brane using [3H]prazosin (a1) and [3H]clonidine (a2), respectively.
a According to Ref. 11.
Summing up performance of the reactions especially in
solvent-free conditions was not better in professional
microwave oven comparing to that in domestic micro-
wave oven. In the literature there are some refer-
compounds, concentration-dependently, shifted the
phenylephrine response to the right. For compound 4a
a Schild slope did not differ significantly from unity,
indicating a competitive interaction with the a1-adreno-
ceptors, and thus allowing for the unambiguous deter-
mination of the pA2 value. In other cases affinities
were reported as pKB estimates, since Schild slopes were
lower than unity. This means that the antagonism was
not competitive and can suggest that the responses were
mediated by more than one receptor. Other possibilities
include a slow dissociation of the ligand from the recep-
tor or an allosteric modulation of receptors.26
ences.22–24
confirming
our
observations
and
conclusions concerning solvent-free reactions performed
in domestic microwave ovens.
2.2. Pharmacology
2.2.1. Radioligand binding results. Compounds 1a–16a
were tested for their in vitro affinity to a1- and a2-
adrenoceptors in rat cerebral cortex by radioligand
binding assays using [3H]prazosin and [3H]clonidine as
specific radioligands, respectively.25 The affinities de-
scribed by Ki values (nM) are shown in Table 3. Selectiv-
ity towards a1-AR in respect of a2-AR was calculated as
Kia2/Kia1. All compounds displayed lower affinities for
a1-AR when compared to prazosin. A large group of
compounds (1a–5a, 9a, 10a, 13a, and 14a) showed high-
er affinities for a1-AR than that of AZ-99, with Ki values
100–300 nM. Compounds 7a and 8a showed affinities
slightly lower than that of the lead compound AZ-99.
The rest of the compounds (6a, 11a, 12a, 15a, and
16a) had a low affinity for both the a1- and a2-adreno-
ceptors. Especially, 2-furoyl derivative 6a did not show
any affinity for a1-AR at tested concentrations. Gener-
ally, the target compounds possessed a slightly higher
selectivity for a1-AR over a2-AR. The most selective
compound (3a) showed 8.48-fold higher affinity for a1-
AR than for a2-AR (Table 3).
The following order of activity was found for tested
compounds: 13a > 5a > 3a > 4a ꢁ 14a > 7a (Table 4,
Figs. 3 and 4). The strongest antagonistic activity was
from compounds 13a and 5a with pKB estimates of
7.084 and 7.022, respectively. For compounds 3a, 4a,
and 5a pKB values ranged from 6.860 to 6.611. Com-
pound 4a gave a pA2 value of 6.616 with a slope
Table 4. Functional bioassay results for selected compounds 3a–5a,
7a, 13a, and 14a
Compound
pKB/pA2 SEM (Slope SEM)
3a
4a
pKB = 6.860 0.07
pA2 = 6.616 0.12a (0.99 0.13)
pKB = 7.022 0.06
5a
7a
pKB = 6.133 0.05
pKB = 7.084 0.05
pKB = 6.611 0.08
13a
14a
2.3. Functional bioassays results
Antagonistic potency of selected compounds, expressed as pA2or
pKB SEM values, in isolated rat thoracic aorta (a1-AR).
The a1-adrenoceptor antagonistic activity of compounds
3a–5a, 7a, 13a, and 14a was studied in rat aorta from
adult Wistar rats and was assessed by inhibition of
phenylephrine-induced contractions. The investigated
pKB values were calculated according to relationship pKB = log(con-
centration ratio À 1) À log(molar antagonist concentration).
a pA2 value was obtained from the linear regression of Schild plot.
Each value was the mean SEM of 5–8 experimental results.

5988
J. Handzlik et al. / Bioorg. Med. Chem. 16 (2008) 5982–5998
100
100
50
0
a
a
phenylephrine
phenylephrine
3a 0.1 μM
3a 0.3 μM
3a 1 μM
7a 1 μM
7a 3 μM
7a 10μM
3a 3 μM
50
-8
-7
-6
-5
0
log [Phenylephrine] (M)
-8
-7
-6
-5
log [Phenylephrine] (M)
b100
phenylephrine
4a 0.1 μM
4a 0.3 μM
4a 1 μM
100
phenylephrine
13a 0.1 μM
13a 0.3 μM
13a 1 μM
b
50
50
0
-8
-7
-6
-5
0
log [Phenylephrine] (M)
-8
-7
-6
-5
log [Phenylephrine] (M)
100
c
phenylephrine
5a 0.1 μM
5a 0.3 μM
5a 1 μM
100
phenylephrine
14a 0.1 μM
14a 1 μM
c
50
0
14a 3 μM
50
-8
-7
-6
-5
-4
log [Phenylephrine] (M)
0
Figure 3. Concentration–response curves to phenylephrine in the rat
aorta in the absence (h) or presence of (a) 3a (j 0.1, m 0.3, .1 and Ç
3 lM); (b) 4a (j 0.1, m 0.3, .1 lM); (c) 5a (j 0.1, m 0.3, .1 lM).
Results are expressed as a percentage of the maximal response to
phenylephrine in the first concentration–response curve. Each point
represents the mean SEM (n = 5–8).
-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) 7a (j 1, m 3, .10 lM); (b)
13a (j 0.1, m 0.3, .1 lM); (c) 14a (j 0.1, m 1, .3 lM). Results are
expressed as a percentage of the maximal response to phenylephrine in
the first concentration–response curve. Each point represents the
mean SEM (n = 5–8).
0.99 0.13. Compound 7a showed the weakest antago-
nistic potency, giving a pKB estimate of 6.133. It is
noticeable that the affinity from the functional test for
compounds 3a–5a, 7a, 13a, and 14a was in the same
concentration range as determined in the radioligand
binding assay.
exchange of the appropriate substituents in JH-9a using
the computer programme HyperChem 7.5 (Hypercube).
The primary optimisation of the prepared structures was
carried out using the semi-empirical method PM5, avail-
able by the MOPAC implement in CAChe 5.0. In the
next step, a conformational analysis using the CON-
FLEX method was performed to give different
conformer populations for each calculated compound
1a–16a. Selected conformers were re-optimized using
PM5. The lowest-energy conformer for each compound
(1a–16a) was selected as representative for the real
3D-structure and was evaluated on its geometrical
parameters. Four distances were analysed, important
for a1-antagonistic properties that correspond to
Barbaro’s model (Fig. 6). In this context, the following
distances were assessed between positive ionisable
2.4. SAR studies
2.4.1. Molecular modelling. The goal of the present
molecular modelling was to determine the 3D-structural
properties of the phenylpiperazine derivatives 1a–16a in
order to compare them with ideal a1-antagonist proper-
ties according to the pharmacophore model of
Barbaro.14
3D-structures of compounds 1a–16a were built based on
the crystal structure of phenylpiperazine phenytoin
derivative JH-9a (Fig. 5). The structures were built by

J. Handzlik et al. / Bioorg. Med. Chem. 16 (2008) 5982–5998
5989
Figure 5. The starting point for molecular modelling calculations.
Crystal structure of phenylpiperazine derivative of phenytoin JH-9a
(1-(3-(4-(2-chlorophenyl)piperazin-1-yl)-2-hydroxypropyl)-3-methyl-
5,5-diphenylimidazolidine-2,4-dione hydrochloride) determined by X-
ray analysis. Element colours: C, cyan; H, white; N, blue; O, red; F,
black.
nitrogen of piperazine and a centre of arylpiperazine
aryl (furoyl in the case of compounds 6a and 11a) ring
(X1) corresponding to PI–HY1, between positive ionisa-
ble nitrogen of piperazine and alkoxyl substituent at
phenylpiperazine phenyl ring (X2) corresponding to
PI–HY2; between positive ionisable nitrogen of pipera-
zine and carbonyl oxygen in position 2 of hydantoin
(X3) corresponding to PI–HBA and between positive
ionisable nitrogen of piperazine and a centre of the most
distant phenyl ring at hydantoin fragment (X4) as corre-
sponding to PI–HY3 in Barbaro’s model. Most of the
considered compounds showed values of PI–HY1 (X1)
˚
distance in the range of 6.5–6.6 A, similar to that of
ideal a1-antagonist. Only two furoyl derivatives (6a
and 11a) showed slightly lower X1 distances in the range
˚
of 6.0 A. In case of PI–HY2 (X2), most of the com-
pounds displayed lower values than that of the ideal
˚
antagonist, in the range of 5.08–5.78 A. The best agree-
Figure 6. Structural parameters of compounds 1a–16a in the compar-
ison with pharmacophore model of Barbaro. (a) Spatial properties
required for the ideal a1-antagonist described by Barbaro et al.14
Distances between proton ionisable nitrogen (PI) and following
regions: HY1, HY2, HY3, and HBA. (b) The corresponding distances
for compounds 1a–16a according to the molecular modelling calcu-
lation (CONFLEX conformational analysis, PM5-geometry optimiza-
tion): protonated nitrogen—centre of aromatic (acyl-aromatic)
piperazine substituent (X1); protonated nitrogen—substituent at
aromatic ring (X2); protonated nitrogen—carbonyl oxygen (X3);
protonated nitrogen—centre of most distant phenyl ring (X4).
ment between the ideal a1-antagonist and the com-
pounds was observed for the ethoxyphenyl derivatives
5a and 8a. Compounds 10a, 13a, and 15a showed the
˚
best values of X3 distances (5.51–5.59 A), similar to that
˚
of the ideal antagonist (5.63 A). The rest of compounds
˚
possessed lower X3-values (5.03–5.42 A). All of the ob-
tained compounds 1a–16a possess the most distant
hydantoin phenyl rings placed significantly closer to
˚
the positive ionisable centre (7.85–8.09 A) when com-
˚
pared to the ideal PI–HY3 value (9.78 A) described by
Barbaro et al.14
2.4.2. SAR analysis. In order to obtain the considered
phenylpiperazine derivatives of phenytoin 1a–16a, three
structural fragments of the lead compound AZ-99 were
modified: the phenylpiperazine phenyl ring, the 3-N-
substituent at the hydantoin moiety, and the 2-hydroxy-
propyl chain. In this context, we analysed the influence
of each of these three structural fragments on affinities
for a1-adrenoceptors (Tables 2 and 3). At first, we ana-
lysed two example groups of compounds differing only
in the area of the phenylpiperazine aryl moiety (4a–7a
and 9a–12a). The first group contained 3-N-methyl
derivatives (4a–7a) with 2-methoxyphenyl-, 2-ethoxy-
phenyl-, 2-pyridyl and 2-furoylpiperazine moieties,
respectively. Similarly, the second group included 3-N-
methyl acetate derivatives (9a–12a). The influence of
the substituent at the 3-N-hydantoin ring on a1-adreno-
ceptors affinities can be seen in the case of 2-methoxy-
phenylpiperazine derivatives (1a–4a, 9a, 13a, and 15a).
In the next step, the study of the relationship between
the affinity of the compounds for a1-adrenoceptors
and the a1-adrenoceptor antagonistic potency was car-
ried out. Figure 7 demonstrates a comparison of func-
tional bioassay results (pKB, pA2) to radioligand
binding results (pKi) for selected compounds (3a–5a,
7a, 13a, and 14a). For these compounds a linear quanti-
tative pKi–pKB relationship was found (Fig. 7). Correla-
tion of the affinities for a1-AR from binding studies with
those obtained from functional studies in rat aorta was
statistically significant (R2 = 0.89, p < 0.005).

5990
J. Handzlik et al. / Bioorg. Med. Chem. 16 (2008) 5982–5998
binding properties
antagonistic properties
phenyl rings at hydantoin situated too close to positive
ionisable nitrogen compared to that required for the
ideal a1-adrenoceptor antagonist. In the case of the most
distant phenyl rings the distance PI–HY4 (X4) is ap-
prox. 20% shorter. Furthermore, the second phenyl
rings at hydantoin are situated much closer to PI (ꢀ5–
7.2
6.8
6.4
˚
6 A). The rest of the distances (X1–X3) do not show
too much difference when compared to the ideal value
and in the case of the selected compounds high confor-
mity is observed (X2 for 5a; X3 for 13a, see Fig. 6).
Thus, we assume that the presence of two phenyl rings
in close proximity to the PI-centre may cause some de-
crease in the a1-antagonistic properties in all of the
investigated phenylpiperazine derivatives 1a–16a.
6
13a
pK
5a
3a
4a*
14a
7a
7.2
B
= 1.10 pK
i
- 0.70
R2 = 0.89
6.8
6.4
6
In our study, the most active compounds (2a–5a, 9a,
13a, and 14a) contained 2-alkoxyphenylpiperazine moi-
eties, 2-methoxyphenyl or 2-ethoxyphenyl. Although,
most of 2-alkoxyphenylpiperazine derivatives (1a–5a,
9a, 10a, 13a, and 14a) showed a1-affinities in the range
of 100–300 nM, it is difficult to recognise which type
of alkoxyl substituent is superior for target activity. In
the group of 3-N-methyl derivatives, the 2-methoxy-
phenyl derivative (4a) showed a slightly lower affinity
for a1-AR than the 2-ethoxyphenyl derivative (5a). In
the case of the three pairs of 3-N-methyl ester derivatives
(9a and 10a, 13a and 14a, and 15a and 16a), the opposite
situation was observed.
6
6.2
6.4
6.6
pKi
6.8
7
7.2
Figure 7. The relationship between a1-adrenoceptor affinities and
antagonistic properties for compounds: 3a–5a, 7a, 13a, and 14a,
expressed as pKi and pKB (*4a: pKB = pA2) values. Quantitative
relationship pKi–pKB is expressed by a linear equation. The correlation
was calculated using Pearson worksheet function (R2 = 0.89;
p < 0.005).
Comparing two groups of compounds containing the
same substituents at 3-N-hydantoin rings and different
at 4-N-piperazine fragments, similar effects on affinities
for a1-AR can be observed for 2-pyridyl- and 2-furoyl
moieties. In both groups, the pKi values are decreasing
in the following order: 2-alkoxyphenyl- > 2-pyridyl-
> 2-furoyl- and the difference between the affinities of
2-pyridyl- (7a) and analogical 2-furoyl derivative (6a)
is much higher in the group of 3-N-methyl derivatives
than in the group of 3-N-methyl acetate derivatives.
The affinity of compound 7a can be considered as a
moderate value (Kiꢁ 0.7 lM) while compound 6a did
not display any receptor binding. In the case of methyl
2-(3-(2-hydroxy-3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)-2,
5-dioxo-4,4-diphenylimidazolidin-1-yl)acetate dihydro-
chloride (12a) and methyl 2-(3-(3-(4-(furan-2-car-
bonyl)piperazin-1-yl)-2-hydroxypropyl)-2,5-dioxo-4,4-
diphenylimidazolidin-1-yl)acetate hydrochloride (11a),
both compounds were weakly active with affinities for
a1-AR in the micromolar range. Generally, the replace-
ment of 2-alkoxyphenyl moiety with 2-pyridyl- or,
particularly, 2-furoyl moieties is not beneficial for affin-
ities to a1-AR in the presented group of phenytoin–aryl-
piperazine derivatives. This observation is comparable
to results of Barbaro et al. in the group of 1,4-benzodi-
oxan-arylpiperazine derivatives.15 In the case of com-
pounds with a propyl spacer, according to Barbaro et
al.,15 affinities for a1-adrenoceptors were decreasing in
the following order: 2-methoxyphenyl- > phenyl- > 2-
furoyl- > 2-pyridylpiperazine derivatives. In contrast to
our results, the 1,4-benzodioxan derivatives showed
higher activity with the 2-furoyl- than with the 2-pyridyl
substituent. The decrease of activity for furoyl- and pyr-
idyl derivatives can be explained based on our molecular
3. Discussion
As a result of the chemical modification of compound
AZ-99, the new group of compounds with a wide range
of affinities to a1-adrenoceptors (103.9–100,000 nM) and
a relatively weak selectivity for a1-adrenoceptors over
a2-adrenoceptors has been synthesized and character-
ised. Based on radioligand binding results (Table 3), it
can be concluded that the type of substituent at 4-N
of piperazine significantly influences the affinity for a1-
adrenergic receptors. The introduction of 2-methoxyl
moiety into the phenylpiperazine phenyl ring (1a) en-
hanced the a1-binding potency of the lead compound
AZ-99. These results are in agreement with many other
reports, which have also confirmed the importance of 2-
methoxyphenylpiperazine moiety for the a1-adrenocep-
tor antagonistic properties.9–17 It is important to note
that the work of Romeo et al.,16 carried out on a group
of pyrimido-2,4-dione phenylpiperazine derivatives,
indicated an increase of the a1-AR affinity following
the introduction of methoxyl moiety at position ortho
in the phenylpiperazine phenyl ring. Although the com-
pounds described by Romeo et al. were more active than
our compounds 1a–16a, each phenylpiperazine deriva-
tive showed lower affinity for the a1-adrenoceptor than
the corresponding 2-methoxyphenyl derivative.
The lower activity of presented phenylpiperazine deriva-
tives 1a–16a, compared to reference a1-antagonists9–17
with corresponding arylpiperazine moieties, can be in
part explained based on molecular modelling results.
The results indicated that all compounds 1a–16a possess

J. Handzlik et al. / Bioorg. Med. Chem. 16 (2008) 5982–5998
5991
modelling results (Fig. 6). In the case of furoyl- and pyr-
idyl derivatives (6a, 7a, 11a, 12a), the structural param-
eters significantly differ from the ideal a1-antagonist
parameters. These compounds do not possess any frag-
ment that could map HY2 of Barbaro’s model. Further-
more, furoyl derivatives 6a and 11a, contrary to the rest
of the obtained compounds (1a–5a, 7a–10a, and 12a–
16a), possess distinctly shorter PI–HY1 distances than
that described for the ideal antagonist. Finally, the fur-
oyl- and pyridyl derivatives (6a, 7a, 11a, and 12a), sim-
ilarly to all the tested group of compounds (1a–16a),
showed significantly shorter PI–HY3 distances in com-
parison to the ideal a1-antagonist described by Barbaro
et al.14
corresponding 2-ethoxyphenyl derivatives 5a and 8a,
differing in the area of the 2-hydroxypropyl spacer,
an almost 5-fold decrease of affinity for a1-adrenocep-
tors was observed in the case of acetylated compound
8a (Table 3).
In terms of selectivity, compounds 1a–16a were weakly
selective (0.74- to 8.48-fold) towards a1-AR over a2-
AR (Table 3). The most active compounds (2a–5a,
9a, 13a, and 14a) displayed a slightly higher selectivity
than the lead compound AZ-99. The highest selectivity
of compound 3a may suggest that ethyl 2-propionate
moiety at position 3 in hydantoin is responsible for
the selectivity. This cannot be fully confirmed in the
present work because compound 3a is the only mem-
ber of ethyl 2-propionate derivatives. In the case of
similar compounds possessing methyl 2-propionate
moieties (13a and 14a), the a2/a1 selectivity is almost
2-fold lower. The 3-methyl derivatives group, including
five compounds (4a–8a) shows heterogeneous a2/a1
selectivity. Similar trends can be observed in the group
of methyl 2-acetate derivatives (9a–12a). The active 2-
alkoxyphenylpiperazine derivatives (9a and 10a) were
3- to 4-fold more potent at a1-ARs while the weakly
active compounds (11a and 12a) displayed higher
affinity for a2- than for a1-ARs. Thus, a substituent
at 3-N-hydantoin does not seem to be crucial for tar-
get selectivity. Likewise, the presence of 2-alkoxyphe-
nyl does not affect selectivity. On the other hand, the
introduction of 2-pyridyl- or 2-furoyl moiety reduces
the selectivity towards a1- in relation to a2-adrenocep-
tors dramatically.
Although a favourable influence of 2-alkoxyphenylpiper-
azine moieties on a1-adrenoceptor antagonistic properties
is obvious, we also obtained some 2-alkoxyphenylpiper-
azine derivatives (8a, 15a, and 16a) which were clearly
less active than the lead compound AZ-99. This can sug-
gest that other structural fragments can strongly limit
affinities for a1-AR in the considered chemical group
(1a–16a). Considering an importance of 3-N-substitu-
ents, the a1-adrenoceptor affinities for 2-methoxyphen-
ylpiperazine derivatives (1a–4a, 9a, 13a, bf 15a) were
analysed. The results showed that the methyl 2-propio-
nate substituent (13a) was the most favourable. The
other ester derivative, ethyl 2-(3-(2-hydroxy-3-(4-(2-
methoxyphenyl)piperazin-1-yl)propyl)-2,5-dioxo-4,4-
diphenylimidazolidin-1-yl)propionate
hydrochloride
(3a), showed slightly lower affinity for a1-adrenoceptors
but the highest selectivity to a2-AR. Interestingly, this
compound (3a) was the most active antiarrhythmic
agent in the adrenaline-induced model of arrhythmia.19
The a1-adrenoceptor affinities for 2-methoxyphenyl
derivatives were decreasing in the following order:
methyl 2-propionate- (13a) > ethyl 2-propionate-
(3a) > ethyl acetate- (2a) > methyl- (4a) > methyl ace-
tate- (9a) > ethyl- (1a) > methyl 2-butyrate derivative
(15a). Generally, compounds 1a–4a, 9a, and 13a showed
affinities in the range of 10À7 M. Only compound 15a
was significantly less active, displaying an affinity for
a1-AR in the micromolar range (3.1 lM). This may sug-
gest an unprofitable effect of large methyl 2-butyrate
moiety at position 3-N in hydantoin. A similar situation
can be observed in the case of 2-ethoxyphenylpiperazine
derivatives (5a, 10a, 14a, and 16a). Compound 16a, pos-
sessing a methyl 2-butyrate fragment, was much less ac-
tive than the rest of the compounds (5a, 10a, and 14a).
Based on the radioligand binding results, we selected a
smaller group of compounds, including active 2-alk-
oxyphenylpiperazine derivatives (3a–5a, 13a, and 14a)
and the moderately active compound 7a, for functional
bioassay to evaluate the nature of their interaction with
a1-adrenoceptors. The obtained results showed a1-adre-
noceptor antagonistic properties for all tested com-
pounds,
but
only
1-(2-hydroxy-3-(4-(2-
methoxyphenyl)piperazin-1-yl)propyl)-3-methyl-5,5-diph-
enylimidazolidine-2,4-dione hydrochloride (4a) showed
competitive interaction with a1-adrenoceptors in rat
aorta. Furthermore, the results of antagonistic potency
for the tested compounds were in agreement with radi-
oligand binding results. The most similar results were
obtained for the ethyl 2-propionate derivative 3a,
which showed almost identical pKi- and pKB values
(Fig. 7). In addition, radioligand binding test and func-
tional bioassay results were also consistent for the less
active 2-pyridyl derivative 7a. In the case of most ac-
tive compounds (13a and 5a) pKB values were slightly
higher than corresponding pKi values. In contrast, for
compounds 4a and 14a both the pA2- and pKB values
were slightly lower than the corresponding pKi values.
The similarity of binding results to functional bioassays
results, obtained for antagonists 3a, 4a, 5a, 7a, 13a and
14a, indicated an almost linear correlation for the pKi–
pKB relationship (Fig. 7) with a R2-value of 0.89
(p < 0.005). Although the results of both functional
and radioligand binding assays are not fully compara-
ble because of different experimental conditions,9 the
Malawska et al.20 indicated that acetylation of the 2-
hydroxypropyl spacer may improve the affinity for
a1-adrenoceptors. They described two pairs of arylpip-
erazine derivatives of pirolidyn-2-one possessing 2-
hydroxypropyl- or 2-acetoxypropyl moiety. In the case
of 2-methoxyphenylpiperazine derivatives, the com-
pound possessing a 2-acethoxypropyl group had a
1.68-fold higher affinity for a1-AR than its hydroxy-
propyl analogue. In the pair of 2-chlorophenylpiper-
azine derivatives, the 2-acethoxypropyl derivative was
2.23-fold more potent than its 2-hydroxypropyl ana-
logue. In our present work, we obtained the opposite
results. Considering the binding properties of the two

5992
J. Handzlik et al. / Bioorg. Med. Chem. 16 (2008) 5982–5998
obtained consistency seems to confirm the accuracy of
the performed tests.
5. Experimental
5.1. Chemistry
4. Conclusion
1H NMR spectra were recorded on Varian Mercury VX
300 MHz PFG instrument in DMSO-d6 at ambient tem-
perature. Chemical shifts are given in parts per million
relative to tetramethylsilane, coupling constants given
in Hz. IR spectra were recorded on a Jasco FT/IR-410
The results of pharmacological tests confirmed our
hypothesis that previously obtained derivatives (1a–
3a) with antiarrhythmic properties possess antagonistic
activity towards a1-adrenoceptors. Furthermore,
through modifications of compound AZ-99, a series
of new a1-adrenoceptor antagonists were synthesized
(4a, 5a, 9a, 10a, 13a, and 14a). The SAR study has dis-
tinctly indicated a favourable effect of 2-alkoxyphenyl-
piperazine moieties for a1-adrenoceptor antagonistic
properties in the considered group. The results of both
radioligand binding assays and functional bioassays
demonstrate that methyl 2-(3-(2-hydroxy-3-(4-(2-
methoxyphenyl)piperazin-1-yl)propyl)-2,5-dioxo-4,4-
diphenylimidazolidin-1-yl)propionate hydrochloride (13a)
and 1-(3-(4-(2-ethoxyphenyl)piperazin-1-yl)-2-hydroxypro-
pyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione hydro-
chloride (5a) are the most active a1-adrenoceptor
antagonists among tested arylpiperazine derivatives of
phenytoin (1a–16a). As all 2-alkoxyphenylpiperazine
derivatives with higher potency (3a, 4a, 5a, 13a, and
14a) displayed the similar magnitude of a1-adrenocep-
tor blocking activity, the SAR study was not able to
estimate which of the two 2-alkoxyl groups, as well
as, which of the corresponding four types of 3-N-sub-
stituents is distinctly preferable for the observed a1-
adrenolytic properties. In contrast, the SAR study
clearly indicated some structural disadvantages for
decreasing the affinity for a1-AR: 2-furoyl- or 2-pyri-
dylpiperazine group, methyl 2-butyrate moiety at posi-
tion 3-N as well as the acetylation of 2-hydroxylpropyl
chain. The molecular modelling results helped to ex-
plain the lowest activities of the 2-pyridyl and 2-furoyl
derivatives. These results indicated that compounds 6a,
7a, 11a, and 12a poorly map the pharmacophore mod-
el of a1-adrenoceptor antagonist because the distances
between PI-nitrogen and hydrophobic fragments
(HY1 and HY3) are significantly shorter than those
for the ideal a1-antagonist and the compounds do
not possess any substituent corresponding to HY2 re-
gion. Furthermore, results of the molecular modelling
showed that all of the investigated compounds (1a–
16a) hardly map pharmacophore hydrophobic region
HY3 as they possess appropriate aromatic rings situ-
ated too close to positive ionisable region comparing
to the ideal a1-antagonist. This may be the main disad-
vantage limiting a1-adrenoceptor antagonistic proper-
ties in this group of phenylpiperazine derivatives of
phenytoin.
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) toluene/acetone 40:3;
(II) toluene/acetone/methanol 5:5:1. Melting points were
determined using Mel-Temp II apparatus and are
uncorrected. Analyses indicated by the symbols of the
elements or functions were within 0.4% of the theoret-
ical values unless stated otherwise (Table 5). Syntheses
under microwave irradiation were performed in house-
hold microwave oven Samsung M1618. Syntheses of
compounds 18, 19, 22, and 23 were described earlier.20,21
5.1.1. General method for preparation of esters 20 and 21.
Compounds 20 and 21 were obtained based on the pro-
cedure described for similar esters.19 A mixture of DPH
17 (100 mmol, 25.2 g), TEBA (13 mmol, 3 g), and
K2CO3 (290 mmol, 40 g) in acetone (500 mL) was
heated under reflux for 30 min. Appropriate methyl
bromoester (100 mmol) in acetone (100 mL) was added.
The mixture was heated under reflux for 3–5 h and stir-
red at room temperature overnight. The inorganic pre-
cipitate was separated by filtration. The filtrate was
evaporated and the residue was crystallised from meth-
anol giving pure products 20 and 21, respectively.
5.1.1.1. Methyl 2-(2,5-dioxo-4,4-diphenylimidazolidin-
1-yl)propionate (20). One-hundred millimoles of educt
was used. Bright crystals (19.8 g, 58.6 mmol, 59%) mp
106–108 ꢁC, Rf(I): 0.26.
5.1.1.2. Methyl 2-(2,5-dioxo-4,4-diphenylimidazolidin-
1-yl)butyrate (21). Eighty millimoles (20.16 g) of educt
was used. White crystals (20.1 g, 56.8 mmol, 71%) mp
152–153 ꢁC, Rf(I): 0.24.
5.1.2. Preparation of methyl 2-(3-(oxiran-2-ylmethyl)-2,5-
dioxo-4,4-diphenylimidazolidin-1-yl)esters 24 and 25.
Compounds 24 and 25 were obtained based on the pro-
cedures described earlier for similar esters.19
5.1.2.1. Methyl 2-(3-(oxiran-2-ylmethyl)-2,5-dioxo-
4,4-diphenylimidazolidin-1-yl)propionate (24). A suspen-
sion of 20 (50 mmol, 16.19 g), K2CO3 (20 g), and TEBA
(1.5 g) in acetone (100 mL) was stirred at room temper-
ature for 30 min, then, a solution of freshly distilled epi-
chlorohydrin (55 mmol, 5.06 g) in acetone (60 mL) was
added dropwise. The suspension was stirred for the next
54 h, then, the precipitate was removed by filtration. The
filtrate was condensed to 1/3 of the starting volume and
kept at 0–4 ꢁC overnight. The obtained solid was
collected by filtration and dried to give bright crystals
of 24 (13.2 g, 34 mmol, 67%) mp 121–122 ꢁC, Rf(I):
0.30.
As a continuation of this study, further modifications
of compound AZ-99 are necessary to improve its a1-
adrenoceptor antagonistic activity. Furthermore, the
antiarrhythmic properties of phenylpiperazine deriva-
tives of phenytoin (4a–16a) presented here need to
be evaluated. Also, the relationship between a1-antag-
onistic- and antiarrhythmic activity needs to be
established.

J. Handzlik et al. / Bioorg. Med. Chem. 16 (2008) 5982–5998
Table 5. Elemental analysis of compounds 4a–16a and corresponding basic forms 4–16
5993
Compound
Empirical formula
% C
% H
% N
Calcda
Found
Calcd
Found
Calcd
Found
4
C
C
30H34N4O4
30H34N4O4 · HCl
70.02
65.38
70.43
65.89
62.39
69.26
58.67
64.43
66.59
62.18
66.87
61.82
60.35
65.85
56.00
67.56
63.15
66.98
61.48
61.76
64.55
67.44
68.17
66.99
67.63
70.12
65.64
70.29
65.41
62.21
69.24
58.80
64.39
66.55
62.14
66.87
61.79
60.29
65.66
56.19
67.37
63.35
66.95
61.64
61.79
64.65
67.4
6.66
6.40
6.86
6.60
5.80
6.43
6.35
6.39
6.37
6.20
6.58
6.45
5.57
6.15
5.95
6.53
6.34
6.78
6.68
6.66
6.66
5.36
5.72
5.62
5.92
6.65
6.40
6.85
6.53
5.78
6.44
6.41
6.44
6.26
6.17
6.53
6.46
5.56
6.06
5.91
6.56
6.28
6.77
6.64
6.58
6.57
5.51
5.65
5.67
5.89
10.89
10.17
10.60
9.91
10.39
14.42
12.00
9.11
9.71
9.06
9.46
8.74
9.38
12.80
10.88
9.55
8.93
9.19
8.44
8.47
8.60
8.28
7.95
7.10
6.86
10.88
10.08
10.57
9.66
10.21
14.15
12.14
9.14
9.36
8.97
9.37
8.65
9.12
12.70
10.91
9.43.
8.88
9.10
8.52
8.44
8.63
8.26
8.06
7.09
6.83
4a
5
C31H36N4O4
C31H36N4O4 · HCl
C28H34N4O5 · HCl
5a
6a
7
C
28H31N5O3
7a
8a
9
C28H31N5O3 · 1.75 HCl · H2O · 0.33 CH3OH
C33H37N4O5 · HCl · 0.5 H2O
C32H36N4O6
9a
10
10a
11a
12
12a
13
13a
14
14a
15a
16a
20
21
24
25
C32H36N4O6 · HCl · 0.5 H2O
C33H38N4O6
C33H38N4O6 · HCl · H2O
C
30H32N4O7 · HCl
30H33N5O5
C
C30H33N5O5 · 2 HCl · 1.5 H2O
C33H38N4O6
C
C
33H38N4O6 · HCl · 0.25 H2O
34H40N4O6
C34H40N4O6 · HCl · 1.5 H2O
C34H40N4O6 · HCl · 1.33 H2O
C35H42N4O6 · HCl
C
19H18N2O4
C20H20N2O4
C22H22N2O5
C23H24N2O5
68.47
66.98
67.78
a Theoretical results of elementary analysis were calculated for the given empirical formulas.
5.1.2.2. Methyl 2-(3-(oxiran-2-ylmethyl)-2,5-dioxo-
4,4-diphenylimidazolidin-1-yl)butyrate (25). A suspension
of 21 (20 mmol, 7.04 g), K2CO3 (8 g), and TEBA (0.6 g),
in acetone (40 mL) was stirred at room temperature for
60 min, then, a solution of freshly distilled epichlorohy-
drin (22 mmol, 5.06 g) in acetone (24 mL) was added
dropwise. The suspension was stirred for the next 68 h,
then, the precipitate was removed by filtration. The fil-
trate was condensed by evaporation. The residue was
purified by crystallisation from methanol to give white
powder of 25 (5.8 g, 14 mmol, 71%) mp 110–112 ꢁC,
Rf(I): 0.29.
irradiation was continued at mean power (300–450 W)
for the next 7–12 min under TLC control. A product
was precipitated by methanol from the glassy residue
giving pure crystals of desired compound in basic form
(4–7, 9, 10, and 12–14). The obtained basic form (4–7,
9, 10, and 12–14) was dissolved in anhydrous methanol
and was saturated with dried gaseous hydrogen chloride
until acidic pH. The mixture was left at 0–4 ꢁC overnight
to give a precipitate of a desirable hydrochloride (4a–7a,
9a, 10a, and 12a–14a).
5.1.3.1.1. Hydrochloride of 1-(2-hydroxy-3-(4-(2-
methoxyphenyl)piperazin-1-yl)propyl)-3-methyl-5,5-diph-
enylimidazolidine-2,4-dione (4a). Compound 4: Com-
pound 22 (1.56 g,) and 1-(2-methoxyphenyl)piperazine
(0.96 g) were melted (450 W) for 3 min, then for 2 min
(450 W), 4 min (300 W). White precipitate (1.2 g,
5.1.3. Synthesis of arylpiperazine phenytoin derivatives
(4a–16a). Preparation of compounds 4, 6, 7, 9, 11–13,
and 15 was performed using commercially available free
bases of 2-methoxyphenyl-, 2-furoyl-, and 2-pyridylpi-
perazine, respectively. In the case of synthesis of com-
pounds 5, 8, 10, 14, and 16, commercially available
monohydrochloride salt of 2-ethoxyphenylpiperazine
was previously converted into free base according to
the method described earlier.27
1
2.3 mmol, 46%) mp 124–125 ꢁC, Rf(II): 0.66. H NMR
for 4 (DMSO-d6) d [ppm]: 1.96–2.01 (m, 2H, Pp-CH2),
2.19 (br s, 4H, Pp-2,6-H), 2.78 (br s, 4H, Pp-3,5-H),
2.97–3.03 (m, 4H, N3-CH3, CHOH), 3.26–3.34 (m,
2H, N1-CH2), 3.72 (s, 3H, OCH3), 4.40 (d,
J = 4.95 Hz, 1H, OH) 6.81–6.83 (m, 2H, PpPh-4,6-H),
6.86–6.92 (m, 2H, 2· Ph-4-H), 7.22–7.27 (m, 4H, 2·
Ph-2,6-H), 7.40–7.45 (m, 6H, 2· Ph-3,5-H, PpPh-3,5-H).
5.1.3.1. General procedure for preparation of arylpip-
erazine phenytoin derivatives (4a–7a, 9a, 10a, and 12a–
14a ). Method A. Equimolar (5–10 mmol) amounts of
appropriate arylpiperazine and 2-(3-(oxiran-2-yl-
methyl)-2,5-dioxo-4,4-diphenylimidazolidin-1-yl deriva-
tive (22–25) were placed in a flat-bottomed flask and
irradiated in a standard household microwave oven,
using various times of irradiation for each prepared
compound, respectively. The reactants were melted at
higher irradiation-power (450 W) for 3–5 min, then the
Compound 4a: Using compound 4 (1 g, 1.94 mmol) in
15 mL of methanol. White crystals of 4a from ethanol
(1.05 g, 1.91 mmol, 98%) mp 260–262 ꢁC, Rf(II): 0.66.
1H NMR for 4a (DMSO-d6) d [ppm]: 2.61–2.68 (m,
1H, CHOH), 2.80–2.95 (m, 6H, Pp-CH2, Pp-2,6-H),
2.98 (s, 3H, N3-CH3), 3.23–3.46 (m, 6H, Pp-3,5-H,
N1-CH2), 3.78 (s, 3H, OCH3), 4.05 (br s, 1H, OH),
6.86–6.87 (m, 2H, PpPh-4,6-H), 6.94–7.03 (m, 2H, 2·

5994
J. Handzlik et al. / Bioorg. Med. Chem. 16 (2008) 5982–5998
Ph-4-H), 7.21–7.32 (m, 4H, 2· Ph-2,6-H), 7.43–7.49 (m,
6H, 2· Ph-3,5-H, PpPh-3,5-H), 9.96 (br s, 1H, NH+). IR
(KBr) [cmÀ1]: 3292 (OH), 2953 (CH), 2549 (NH+), 1770
(C2@O), 1716 (C4@O), 1593 (Ar).
5.1.3.1.4. Hydrochloride of methyl 2-(3-(2-hydroxy-3-
(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-2,5-dioxo-
4,4-diphenylimidazolidin-1-yl)acetate (9a).Compound 9:
Compound 23 (2.15 g, 6 mmol) and N-2-metoxyphenyl-
piperazine (1.1 g, 6 mmol)) were melted (450 W) for
5 min, then for 9 min (300 W). White precipitate (1.7 g,
5.1.3.1.2. Hydrochloride of 1-(3-(4-(2-ethoxyphenyl)-
piperazin-1-yl)-2-hydroxypropyl)-3-methyl-5,5-diphenylim-
idazolidine-2,4-dione (5a). Compound 5: Compound 22
(1.55 g, 5 mmol) and 1-(2-ethoxyphenyl)piperazine
(1.03 g, 5 mmol) were melted (450 W) for 3 min, then
for 3 min (450 W), 4 min (300 W). White crystals
1
3 mmol, 50%) mp 100–103 ꢁC, Rf(II): 0.67. H NMR
for 9 (DMSO-d6) d [ppm]: 1.99 (d, J = 6.05 Hz, 2H,
Pp-CH2), 2.21 (br s, 4H, Pp-2,6-H), 2.79 (br s, 4H, Pp-
3,5-H), 2.96–3.07 (m, 1H, CHOH), 3.21–3.36 (m, 2H,
N1-CH2), 3.68 (s, 3H, OCH3), 3.73 (s, 3H, COOCH3),
4.34 (s, 2H, N3-CH2), 4.38–4.42 (m, 1H, OH), 6.81–
7.05 (m, 4H, PpPh-4,6-H, 2· Ph-4-H), 7.29–7.34 (m,
4H, 2· Ph-2,6-H), 7.44–7.49 (m, 6H, 2· Ph-3,5-H,
PpPh-3,5-H).
1
(1.4 g, 2.7 mmol, 54%) mp 120–122 ꢁC, Rf(II): 0.69. H
NMR for 5 (DMSO-d6) d [ppm]: 1.28 (t, J = 7.05 Hz,
3H, OCH2CH3), 2.0 (d, J = 5.8 Hz, 2H, Pp-CH2), 2.19
(br s, 4H, Pp-2,6-H), 2.80 (br s, 4H, Pp-3,5-H), 2.90–
3.02 (s, 1H, CHOH), 2.97 (s, 3H, N3-CH3), 3.22–3.36
(m, 2H, N1-CH2), 3.92 (q, J = 6.98 Hz, 2H, OCH2CH3),
4.40 (d, J = 4.95 Hz, 1H, OH) 6.80–6.87 (m, 4H, PpPh-
4,6-H, 2· Ph-4-H), 7.22–7.27 (m, 4H, 2· Ph-2,6-H),
7.40–7.47 (m, 6H, 2· Ph-3,5-H, PpPh-3,5-H).
Compound 9a: Using compound 9 (1.3 g, 2.25 mmol)
and 15 mL of methanol. White crystals (1.1 g,
1.78 mmol, 80%) mp 215–217 ꢁC, Rf(II): 0.67. 1H
NMR for 9a (DMSO-d6) d [ppm]: 2.75–2.81 (m, 3H,
CHOH, Pp-CH2), 2.92–3.08 (m, 4H, Pp-2,6-CH2),
3.14–3.36 (m, 6H, N1-CH2, Pp-3,5-H), 3.67 (s, 3H,
COOCH3), 3.77 (s, 3H, OCH3), 4.37 (s, 2H, N3-CH2),
5.23 (br s, 1H, OH), 6.87–6.90 (m, 2H, PpPh-4,6-H),
6.94–7.03 (m, 2H, 2· Ph-4-H), 7.28–7.44 (m, 4H, 2·
Ph-2,6-H), 7.47–7.53 (m, 6H, PpPh-3,5-H, 2· Ph-3,5-
H), 10.13 (br s, 1H, NH+). IR (KBr) [cmÀ1]: 3458
(OH), 2950 (CH), 2432 (NH+), 1780 (C2@O), 1744
(C@O ester), 1728 (C4@O), 1603 (Ar).
Compound 5a: Using compound 5 (1 g, 1.9 mmol) in
15 mL of methanol. Pure white crystals (1.05 g,
1.86 mmol, 98%) mp 245–246 ꢁC, Rf(II): 0.69. 1H
NMR for 5a (DMSO-d6) d [ppm]: 1.36 (t, J = 7.02 Hz,
3H, OCH2CH3), 2.66–2.90 (m, 7H, Pp-CH2, Pp-2,6-H,
CHOH), 2.98 (s, 3H, N3-CH3), 3.14–3.49 (m, 6H, Pp-
3,5-H, N1-CH2), 3.98 (q, J = 6.88 Hz, 2H, OCH2CH3),
5.64 (br s, 1H, OH), 6.85–6.89 (m, 2H, PpPh-4,6-H),
6.91–6.99 (m, 2H, 2· Ph-4-H), 7.22–7.34 (m, 4H, 2·
Ph-2,6-H), 7.44–7.49 (m, 6H, 2· Ph-3,5-H, PpPh-3,5-
H), 9.70 (br s, 1H, NH+). IR (KBr) [cmÀ1]: 3274
(OH), 2973 (CH), 2452 (NH+), 1770 (C2@O), 1714
(C4@O), 1589 (Ar).
5.1.3.1.5. Hydrochloride of methyl 2-(3-(3-(4-(2-
ethoxyphenyl)piperazin-1-yl)-2-hydroxypropyl)-2,5-dioxo-
4,4-diphenylimidazolidin-1-yl)acetate (10a). Compound
10: Compound 23 (1.99 g, 5 mmol) and 1-(2-ethoxy-
phenyl)piperazine (1.03 g, 5 mmol)) were melted
(450 W) for 3 min, then for 3 min (450 W), for 6 min
(300 W). White precipitate (1.35 g, 2.28 mmol, 46%)
5.1.3.1.3. Dihydrochloride of 1-(2-hydroxy-3-(4-(pyri-
din-2-yl)piperazin-1-yl)propyl)-3-methyl-5,5-diphenylim-
idazolidine-2,4-dione (7a). Compound 7: Compound 22
(3.1 g, 10 mmol) and 1-(2-pyridiny)lpiperazine (1.66 g,
10 mmol) were melted (450 W) for 3 min, then for
4 min (300 W). White crystals (1.29 g, 2.66 mmol, 27%)
1
mp 126–128 ꢁC, Rf(II): 0.78. H NMR for 10 (DMSO-
d6) d [ppm]: 1.28–1.33 (t, J = 7.05 Hz, 3H, OCH2CH3),
1.99 (d, J = 6.1 Hz, 2H, Pp-CH2), 2.20 (br s, 4H, Pp-
2,6-H), 2.81 (br s, 4H, Pp-3,5-H), 3.03–3.09 (m, 1H,
CHOH), 3.22–3.35 (m, 2H, N1-CH2), 3.68 (s, 3H,
OCH3), 3.93 (q, J = 6.9 Hz, 2H, OCH2CH3), 4.34 (s,
2H, N3-CH2), 4.41 (d, J = 4.95 Hz, 1H, OH), 6.78–6.87
(m, 4H, PpPh-4,6-H, 2· Ph-4-H), 7.30–7.34 (m, 4H, 2·
Ph-2,6-H), 7.41–7.49 (m, 6H, 2· Ph-3,5-H, PpPh-3,5-H).
1
mp 95–96 ꢁC, Rf (II): 0.65. H NMR for 7 (DMSO-d6)
d
[ppm]: 1.92–2.04 (m, 2H, Pp-CH2), 2.12 (t,
J = 4.81 Hz, 4H, Pp-2,6-H), 2.97 (s, 3H, N3-CH3),
3.00–3.16 (m, 1H, CHOH), 3.12–3.38 (m, 6H, Pp-3,5-
H, N1-CH2); 4.41 (d, J = 4.95 Hz, 1H, OH), 6.56 (dd,
J1 = 6.5 Hz, J2 = 4.95 Hz, 1H, Pd-5-H), 6.72 (d,
J = 8.8 Hz, 1H, Pd-3-H), 7.21–7.26 (m, 4H, 2· Ph-2,6-
H), 7.32–7.50 (m, 7H, 2· Ph-3,4,5-H, Pd-4-H), 8.04
(dd, J1 = 4.8 Hz, J2 = 1.8 Hz, 1H, Pd-6-H).
Compound 10a: Using compound 10 (1.0 g, 1.7 mmol)
and 15 mL of methanol. White crystals (1.07 g,
1.67 mmol, 98%) mp 130–131 ꢁC, Rf(II): 0.78. 1H
NMR for 10a (DMSO-d6)
d
[ppm]: 1.34 (t,
Compound 7a: Using compound 7 (1 g, 2.1 mmol) and
15 mL of methanol. Bright crystals (1.1 g, 1.9 mmol,
91%) mp 108–110 ꢁC, Rf(II): 0.65. 1H NMR for 7a
J = 6.88 Hz, 3H, OCH2CH3), 2.05–2.07 (m, 2H, Pp-
CH2), 2.77–3.06 (m, 5H, Pp-2,6-H, CHOH), 3.31–3.58
(m, 6H, Pp-3,5-H, N1-CH2), 3.66 (s, 3H, OCH3), 3.98
(q, J = 6.97 Hz, 2H, OCH2CH3), 4.37 (s, 2H, N3-
CH2), 5.63 (br s, 1H, OH), 6.85–6.91 (m, 2H, PpPh-
4,6-H), 6.94–6.99 (m, 2H, 2· Ph-4-H), 7.29–7.37 (m,
4H, 2· Ph-2,6-H), 7.44–7.53 (m, 6H, 2· Ph-3,5-H,
PpPh-3,5-H), 9.88 (br s, 1H, NH+); IR (KBr) [cmÀ1]:
3357 (OH), 2980, 2940 (CH), 2427 (NH+), 1777
(C2@O), 1751 (C@O ester), 1722 (C4@O), 1592 (Ar).
5.1.3.1.6. Dihydrochloride of methyl 2-(3-(2-hydroxy-
3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)-2,5-dioxo-4,4-
diphenylimidazolidin-1-yl)acetate (12a). Compound 12:
(DMSO-d6)
d [ppm]: 2.65–2.89 (m, 3H, CHOH,
Pp-CH2), 2.97 (s, 3H, N3-CH3), 2.97–3.05 (m, 2H, Pp-
2,6-Ha), 3.21–3.49 (m, 6H, Pp-2,6-He, Pp-3,5-Ha, N1-
CH2), 4.34–4.43 (m, 3H, Pp-3,5-He, OH), 6.91 (t,
J = 6.3 Hz, 1H, Pd-5-H), 7.14–7.28 (m, 5H, Pd-3-H, 2·
Ph-2,6-H), 7.44–7.48 (m, 7H, 2· Ph-3,4,5-H, Pd-1-
NH+-H), 7.88 (t, J = 7.7 Hz, 1H, Pd-4-H), 8.09 (dd,
J1 = 5.8 Hz, J2 = 1.7 Hz, 1H, Pd-6-H), 10.24 (br s, 1H,
NH+). IR (KBr) [cmÀ1]: 3404 (OH), 2946 (CH), 2458
(NH+), 1771 (C2@O), 1707 (C4@O), 1541 (Ar).

J. Handzlik et al. / Bioorg. Med. Chem. 16 (2008) 5982–5998
5995
Compound 23 (1.9 g, 5 mmol) and 1-(2-pyridinyl)pipera-
zine (0.83 g, 5 mmol)) were melted (450 W) for 4 min,
then for 9 min (300 W). White precipitate (1.4 g,
2.56 mmol, 51%) mp 134–135 ꢁC, Rf(II): 0.63. 1H NMR
for 12 (DMSO-d6) d [ppm]: 1.97 (d, J = 6.05 Hz, 2H,
Pp-CH2), 2.15 (br s, 4H, Pp-2,6-H), 3.04–3.16 (m, 1H,
CHOH), 3.23–3.36 (m, 6H, Pp-3,5-H, N1-CH2), 3.66 (s,
3H, OCH3), 4.34 (s, 2H, N3-CH2), 4.45 (br s, 1H, OH),
6.57 (dd, J1 = 6.6 Hz, J2 = 4.95 Hz, 1H, Pd-5-H), 6.73
(d, J = 8.8 Hz, 1H, Pd-3-H), 7.29–7.34 (m, 6H, 2· Ph-
2,4,6-H), 7.44–7.50 (m, 5H, 2· Ph-3,5-H, Pd-4-H), 8.04
(dd, J1 = 4.95 Hz, J2 = 1.4 Hz, 1H, Pd-6-H).
(450 W) for 3 min, then for 1 min (450 W), for 3 min
(300 W). White precipitate (2.0 g, 3.28 mmol, 66%) mp
134–135 ꢁC, Rf(II): 0.71. H NMR for 14 (DMSO-d6)d
1
(ppm): 1.32 (t, J = 6.88 Hz, 3H, OCH2CH3), 1.49 (d,
J = 7.15 Hz, 3H, N3-CHCH3), 2.03–2.10 (m, 2H, Pp-
CH2), 2.23 (br s, 4H, Pp-2,6-H), 2.85 (br s, 4H, Pp-
3,5-H), 3.14–3.21 (m, 1H, CHOH), 3.29–3.35 (m, 2H,
N1-CH2,), 3.64 (s, 3H, OCH3), 3.96 (q, J = 7.0 Hz,
2H, OCH2CH3), 4.41 (br s, 1H, OH), 4.88 (q,
J = 7.24 Hz, 1H, N3-CHCH3) 6.84–6.91 (m, 4H,
PpPh-4,6-H, 2· Ph-4-H), 7.29–7.36 (m, 4H, 2· Ph-2,6-
H), 7.46–7.54 (m, 6H, 2· Ph-3,5-H, PpPh-3,5-H).
Compound 12a: Using compound 12 (1.1 g, 2.0 mmol)
and 15 mL of methanol. White crystals (1.1 g,
1.71 mmol, 85%) mp 94–95 ꢁC, Rf(II): 0.63. H NMR
Compound 14a: Using compound 14 (1.0 g, 1.7 mmol) in
15 mL of methanol. White crystals of 14a (1.1 g,
1.64 mmol, 97%) mp 148–150 ꢁC, Rf(II): 0.71. 1H
1
for 12a (DMSO-d6) d [ppm]: 2.77–2.91 (m, 3H, Pp-
CH2, CHOH), 3.04–3.14 (m, 4H, Pp-2,6-H), 3.30–3.56
(m, 6H, Pp-3,5-H, N1-CH2), 3.66 (s, 3H, OCH3), 4.36
(s, 2H, N3-CH2), 4.39–4.51 (m, 1H, OH), 6.94 (t,
J = 6.33 Hz, 1H, Pd-5-H), 7.27–7.35 (m, 5H, 2· Ph-
2,6-H, Pd-3-H), 7.44–7.53 (m, 7H, 2· Ph-3,4,5-H, Pd-
1-NH+), 7.92 (t, J = 7.7 Hz, 1H, Pd-4-H), 8.08 (dd,
J1 = 5.78 Hz, J2 = 1.38 Hz, 1H, Pd-6-H), 10.39 (br s,
1H, NH+); IR (KBr) [cmÀ1]: 3441 (OH), 2949, 2936
(CH), 2464 (NH+), 1779 (C2@O), 1754 (C@O ester),
1704 (C4@O), 1590 (Ar).
NMR for 14a (DMSO-d6)
d
[ppm]: 1.35 (t,
J = 6.80 Hz, 3H, OCH2CH3), 1.51 (d, J = 7.44 Hz, 3H,
N3-CHCH3), 2.71–2.88 (m, 4H, Pp-CH2, Pp-2,6-Ha),
3.00–3.03 (m, 3H, CHOH, Pp-2,6-He), 3.35–3.43 (m,
6H, Pp-3,5-H, N1-CH2), 3.61 (s, 3H, COOCH3), 3.98–
4.05 (m, 3H, OCH2CH3, OH), 4.88 (q, J = 7.18 Hz,
1H, N3-CHCH3), 6.86–6.98 (m, 4H, PpPh-4,6-H, 2·
Ph-4-H), 7.25–7.36 (m, 4H, 2· Ph-2,6-H), 7.47–7.52
(m, 6H, 2· Ph-3,5-H, PpPh-3,5-H), 9.82 (br s, 1H,
NH+). IR (KBr) [cmÀ1]: 3417 (OH), 2956 (CH), 2347
(NH+); 1772 (C2@O), 1750 (C@O ester), 1716
(C4@O), 1611 (Ar).
5.1.3.1.7. Hydrochloride of methyl 2-(3-(2-hydroxy-3-
(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-2,5-dioxo-
4,4-diphenylimidazolidin-1-yl)propionate (13a). Com-
pound 13: Compound 24 (1.97 g, 5 mmol) and 1-(2-
methoxyphenyl)piperazine (0.91 g, 5 mmol)) were
melted (450 W) for 4 min, then for 2 min (450 W), for
4 min (300 W). White precipitate (1.6 g, 2.73 mmol,
55%) mp 115–118 ꢁC, Rf(II): 0.75. 1H NMR for 13
(DMSO-d6) d (ppm): 1.46 (d, J = 7.15 Hz, 3H, N3-
CHCH3), 2.01–2.09 (m, 2H, Pp-CH2), 2.21 (br s, 4H,
Pp-2,6-H), 2.80 (br s, 4H, Pp-3,5-H), 3.10–3.18 (m,
1H, CHOH), 3.27–3.34 (m, 2H, N1-CH2), 3.68 (s, 3H,
OCH3), 3.76 (s, 3H, COOCH3), 4.42 (d, J = 4.95 Hz,
1H, OH), 4.86 (q, J = 7.20 Hz, 1H, N3-CHCH3) 6.78–
6.89 (m, 4H, PpPh-4,6-H, 2· Ph-4-H), 7.20–7.34 (m,
4H, 2· Ph-2,6-H), 7.40–7.52 (m, 6H, 2· Ph-3,5-H,
PpPh-3,5-H).
5.1.3.2. General procedure for preparation of arylpip-
erazine phenytoin derivatives (6a, 11a, 15a, and 16a).
Method B. Equimolar (5–10 mmol) amounts of appro-
priate arylpiperazine and 2-(3-(oxiran-2-ylmethyl)-2,5-
dioxo-4,4-diphenylimidazolidin-1-yl derivative (22–25)
were placed in a flat-bottomed flask and irradiated in a
standard household microwave oven at similar condi-
tions to those of method A. After irradiation, the glassy
residue was dissolved by heating with alcohol. The solu-
tion was left at 0–4 ꢁC for 7–14 days. Since no precipitate
appeared, the solvent was removed by evaporation. A
residue was dissolved in CH2Cl2 (20–25 mL) and washed
with hydrochloric acid (1%, 3 · 15 mL). The organic
phase was evaporated. A residue was dissolved in anhy-
drous alcohol and was saturated with dried gaseous
hydrogen chloride until acidic pH. The mixture was left
at 0–4 ꢁC overnight to give a desired precipitate of hydro-
chloride (6a, 11a, 15a, and 16a).
Compound 13a: Using compound 13 (1.0 g, 1.7 mmol) in
15 mL of methanol. White crystals (1.0 g, 1.6 mmol,
94%) mp 168–169 ꢁC, Rf(II): 0.75. 1H NMR for 13a
(DMSO-d6) d [ppm]: 1.54 (d, J = 7.15 Hz, 3H, N3-CH
CH3), 2.74–3.07 (m, 7H, CHOH, Pp-CH2, Pp-2,6-H),
3.38–3.47 (m, 6H, Pp-3,5-H, N1-CH2), 3.63 (s, 3H,
COOCH3), 3.81 (s, 3H, OCH3), 4.01 (br s, 1H, OH),
4.89 (q, J = 7.15 Hz, 1H, N3-CHCH3), 6.91–6.98 (m,
2H, PpPh-4,6-H), 7.00–7.07 (m, 2H, 2· Ph-4-H), 7.28–
7.39 (m, 4H, 2· Ph-2,6-H), 7.51–7.56 (m, 6H, 2· Ph-
3,5-H, PpPh-3,5-H), 9.97 (br s, 1H, NH+). IR (KBr)
[cmÀ1]: 3415 (OH), 2950 (CH), 2418 (NH+), 1773
(C2@O), 1752 (C@O ester), 1716 (C4@O), 1609 (Ar).
5.1.3.1.8. Hydrochloride of methyl 2-(3-(3-(4-(2-
ethoxyphenyl)piperazin-1-yl)-2-hydroxypropyl)-2,5-dioxo-
4,4-diphenylimidazolidin-1-yl)propanoate (14a). Com-
pound 14: 24 (1.97 g, 5 mmol) and 1-(2-ethoxy-
phenyl)piperazine (1.03 g, 5 mmol)) were melted
5.1.3.2.1. Hydrochloride. of 1-(3-(4-(furan-2-car-
bonyl)piperazin-1-yl)-2-hydroxypropyl)-3-methyl-5,5-
diphenylimidazolidine-2,4-dione (6a).Compound 22
(1.55 g, 5 mmol) and 1-(2-furoyl)piperazine (0.9 g,
5 mmol) were melted (450 W) for 4 min, then irradiation
was continued for 2 min (300 W), 5 min (450 W). White
crystals of 6a from methanol (1.80 g, 3.24 mmol, 65%)
1
mp 280–281 ꢁC, Rf(II): 0.57. H NMR for 6a (DMSO-
d6) d [ppm]: 2.47–2.48 (m, 1H, CHOH), 2.57–2.64 (m,
2H, Pp-CH2), 2.73–2.89 (m, 2H, Pp-3,5-Ha), 2.97 (s,
3H, N3-CH3), 2.97–3.10 (m, 2H, Pp-3,5-He), 3.30–3.42
(m, 4H, Pp-4,6-H), 4.31–4.41 (m, 2H, N1-CH2), 5.68
(br s, 1H, OH), 6.64 (dd, J1 = 3.6, J2 = 1.7 Hz, 1H,
fur-4-H), 7.07 (d, J = 3.0 Hz, 1H, fur-3-H), 7.21–7.28
(m, 4H, 2· Ph-2,6-H), 7.43–7.45 (m, 6H, 2· Ph-3,4,5-
H), 7.88 (d, J = 0.8 Hz, 1H, fur-5-H), 10.29 (br s, 1H,

5996
J. Handzlik et al. / Bioorg. Med. Chem. 16 (2008) 5982–5998
NH+); IR (KBr) (cmÀ1): 3421(OH), 2944 (CH), 2433
(NH+); 1767 (C2@O), 1741 (C@O furoilo), 1717
(C4@O), 1581 (Ar).
1-yl)propan-2-yl acetate (8a). To compound
5
(1.9 mmol, 1 g) in a flat-bottomed flask acetic anhydride
(8 mL) was added. The mixture was refluxed for 2 min
and was left at 0–4 ꢁC for 7 days. As no precipitate ap-
peared, a glue residue was dissolved in anhydrous meth-
anol and was saturated with dried hydrogen chloride.
The mixture was left at 0–4 ꢁC overnight to afford white
crystals of 8a (0.65 g, 1.1 mmol, 56%) mp 156–157 ꢁC,
5.1.3.2.2. Hydrochloride of methyl 2-(3-(3-(4-(furan-
2-carbonyl)piperazin-1-yl)-2-hydroxypropyl)-2,5-dioxo-
4,4-diphenylimidazolidin-1-yl)acetate (11a). Compound
23 (1.9 g, 5 mmol) and 1-(2-furoyl)piperazine (0.9 g,
5 mmol) were melted (450 W) for 2 min, then for 2 min
(300 W), for 2 min (450 W). White crystals of 11a from
methanol (0.83 g, 1.39 mmol, 28%) mp 230–231 ꢁC,
Rf(II): 0.59. 1H NMR for 11a (DMSO-d6) d [ppm]:
2.49–2.71 (m, 3H, CHOH, Pp-CH2), 2.80–3.14 (m, 2H,
Pp-3,5-Ha), 3.24–3.63 (m, 6H, Pp-3,5-He, Pp-2,6-H),
3.66 (s, 3H, OCH3), 4.30 (m, 2H, N1-CH2), 4.36 (s,
2H, N3-CH2), 5.71 (br s, 1H, OH), 6.65 (dd,
J1 = 3.34 Hz, J2 = 1.7 Hz, 1H, fur-4-H), 7.07 (d,
J = 3.6 Hz, 1H, fur-3-H), 7.27–7.36 (m, 4H, 2· Ph-2,6-
H), 7.42–7.52 (m, 6H, 2· Ph-3,4,5-H), 7.88 (d,
J = 1.7 Hz, 1H, fur-5-H), 10.20 (br s, 1H, NH+). IR
(KBr) [cmÀ1]: 3211 (OH), 2962 (CH), 2427 (NH+),
1776 (C2@O), 1748 (C@O ester), 1718 (C4@O), 1571
(Ar).
1
Rf(II): 0.89. H NMR for 8a (DMSO-d6) d [ppm]: 1.36
(t, J = 6.92 Hz, 3H, OCH2CH3), 1.88 (s, 3H, CH3CO),
2.67–2.79 (m, 1H, CH), 2.83–2.98 (m, 2H, Pp-CH2),
2.98 (s, 3H, N3-CH3), 3.11–3.14 (m, 4H, Pp-2,6-H),
3.17–3.37 (m, 4H, Pp-3,5-H), 3.57–3.73 (m, 2H, N1-
CH2), 3.98 (q, J = 6.92 Hz, 2H, OCH2CH3), 6.85–6.86
(m, 2H, PpPh-4,6-H), 6.89–6.99 (m, 2H, 2· Ph-4-H),
7.19–7.32 (m, 4H, 2· Ph-2,6-H), 7.44–7.46 (m, 6H, 2·
Ph-3,5-H, PpPh-3,5-H), 10.74 (br s, 1H, NH+); IR
(KBr) [cmÀ1]: 2998 (CH), 2409 (NH+), 1768 (C2@O),
1736 (CH3C@O), 1714 (C4@O), 1598 (Ar).
5.2. Pharmacology
5.1.3.2.3. Hydrochloride of methyl 2-(3-(2-hydroxy-3-
(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-2,5-dioxo-
4,4-diphenylimidazolidin-1-yl)butyrate (15a). Compound
25 (2.05 g, 5 mmol) and 1-(2-methoxyphenyl)piperazine
(0.9 g, 5 mmol) were melted (450 W) for 4 min, for
4 min (300 W), for 2 min (450 W). White crystals of
15a from methanol (1.22 g, 1.85 mmol, 37%) mp 182–
184 ꢁC, Rf(II): 0.76. 1H NMR for 15a (DMSO-d6) d
[ppm]: 0.72 (t, J = 7.30 Hz, 3H, N3-CHCH2CH3),
1.95–2.08 (m, 2H, N3-CH-CH2-CH3), 2.70–3.04 (m,
7H, CHOH, Pp-CH2, Pp-2,6-H), 3.28–3.46 (m, 6H,
Pp-3,5-H, N1-CH2), 3.64 (s, 3H, COOCH3), 3.77 (s,
3H, OCH3), 4.41 (br s, 1H, OH), 4.65 (t, J = 5.30 Hz,
1H, N3-CHCH2CH3), 6.87–6.88 (m, 2H, PpPh-4,6-H),
6.94–7.03 (m, 2H, 2· Ph-4-H), 7.25–7.36 (m, 4H, 2·
Ph-2,6-H), 7.45–7.53 (m, 6H, 2· Ph-3,5-H, PpPh-3,5-
H), 9.99 (br s, 1H, NH+). IR (KBr) [cmÀ1]: 3401
(OH), 2969 (CH), 2426 (NH+), 1770 (C2@O), 1745
(C@O ester), 1711 (C4@O), 1608 (Ar).
5.1.3.2.4. Hydrochloride of methyl 2-(3-(2-hydroxy-3-
(4-(2-ethoxyphenyl)piperazin-1-yl)propyl)-2,5-dioxo-
4,4-diphenylimidazolidin-1-yl)butyrate (16a). Compound
25 (2.87 g, 7 mmol) and 1-(2-methoxyphenyl)piperazine
(1.44 g, 7 mmol) were melted (450 W) for 3 min, then
for 2 min (450 W). White crystals of 16a from methanol
(1.20 g, 1.84 mmol, 26%) mp 203–205 ꢁC, Rf(II): 0.70.
1H NMR for 16a (DMSO-d6) d [ppm]: 0.72–0.75 (m,
3H, CHCH2CH3), 1.37 (t, J = 6.92 Hz, 3H, OCH2CH3),
1.98–2.07 (m, 2H, CHCH2CH3), 2.60–3.03 (m, 7H,
CHOH, Pp-CH2, Pp-2,6-H), 3.16–3.39 (m, 6H, N1-
CH2, Pp-3,5-H), 3.64 (s, 3H, OCH3), 3.98 (q,
J = 6.92 Hz, 2H, OCH2CH3), 4.68–4.72 (m, 1H,
CHCH2CH3), 5.68 (br s, 1H, OH), 6.85–6.86 (m, 2H,
PpPh-4,6-H), 6.91–6.98 (m, 2H, 2· Ph-4-H), 7.25–7.37
(m, 4H, 2· Ph-2,6-H), 7.44–7.53 (m, 6H, 2· Ph-3,5-H,
PpPh-3,5-H), 9.92 (br s, 1H, NH+). IR (KBr) [cmÀ1]:
3255 (OH), 2977 (CH), 2486 (NH+), 1776 (C2@O),
1754 (C@O ester), 1726 (C4@O), 1591 (Ar).
5.2.1. General information. The pharmacological studies
were carried out on male Wistar rats ((KRF.(WI).WU),
Animal House, Faculty of Pharmacy, Jagiellonian Uni-
versity Medical College, Cracow) weighing 170–350 g.
Treatment of laboratory animals in the present study
was in 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,
acetylcholine hydrochloride, ( )-noradrenaline hydro-
chloride (Sigma, Aldrich Chemie Gmbh); thiopental so-
dium (Biochemie Gmbh, Vienna); [3H]prazosin,
[3H]clonidine (Amersham). Other reagents were of ana-
lytical grade from local sources.
5.2.2. Radioligand binding test. The compounds were
evaluated for their affinity to a1- and a2-adrenoceptors
by determining for each of them its ability to displace
[3H]prazosin or [3H]clonidine from specific binding sites
in rat cerebral cortex. [3H]prazosin (19.5 Ci/mmol, a1-
adrenergic receptor) and [3H]clonidine (70.5 Ci/mmol,
a2-adrenergic receptor) were used. Rat brains were
homogenised in 20 volumes of ice-cold 50 mM Tris–
HCl buffer (pH 7.6), and centrifuged at 20,000g for
20 min (0–4 ꢁC). The cell pellet was resuspended in
Tris–HCl buffer and centrifuged again. Radioligand
binding assays were performed in plates (MultiScreen/
Millipore). The final incubation mixture (final volume
300 lL) consisted of 240 lL membrane suspension,
30 lL of [3H]prazosin (0.2 nM) or [3H]clonidine
(2 nM) solution, and 30 lL of buffer containing from se-
ven to eight concentrations (10À11–10À4 M) of tested
compounds. For measuring unspecific binding, phentol-
amine À10 lM (in the case of [3H]prazosin) and cloni-
dine À10 lM ( in the case of [3H]clonidine) were
applied. The incubation was terminated by rapid filtra-
tion over glass fibre filters (Whatman GF/C) using a vac-
uum manifold (Millipore). The filters were then washed
5.1.3.3. Hydrochloride of 3-(4-(2-ethoxyphenyl)pipera-
zin-1-yl)-1-(3-methyl-2,4-dioxo-5,5-diphenylimidazolidin-

J. Handzlik et al. / Bioorg. Med. Chem. 16 (2008) 5982–5998
5997
twice with the assay buffer and placed into scintillation
vials with liquid scintillation cocktail. Radioactivity
was measured using WALLAC 1409 DSA—liquid scin-
tillation counter. All assays were done in duplicate.
the ratio of equieffective agonist concentrations in the
absence and in the presence of the antagonist.
Acknowledgments
Radioligand binding data were analysed25 using itera-
tive curve fitting routines (GraphPAD/Prism, Version
3.0—San Diego, CA, USA) Ki values were calculated
from the Cheng and Prusoff equation.28
Authors thank Prof. Barbara Malawska and Mr.
Krzysztof Wie˛ckowski for help in the performance of
two microwave reactions and Mrs. Małgorzata Dybała
for her assistance in radioligand binding tests. The work
was partly supported by Polish State Committee for Sci-
entific Research, Grant No. 3P05F 03125 and pro-
gramme K/ZDS/000727.
5.2.3. Functional bioassay. Isolated rat aorta was used in
order to test antagonistic activity of investigated com-
pounds for a1-adrenoceptors. The male Wistar rats
weighing 200–350 g were anaesthetised with thiopental
sodium (75 mg/kg ip) and the aorta was dissected and
placed into a Krebs–Henseleit solution and cleaned of
surrounding fat tissue. The thoracic aorta was denuded
of endothelium and cut into approximately 4-mm long
rings. The aorta rings were incubated in 30-mL cham-
bers 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, glu-
cose 10 mM, C3H3O3Na 2.2 mM, and EDTA
0.05 mM) at 37 ꢁC and pH 7.4 with constant oxygena-
tion (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 displace-
ment transducer (BIOPAC Systems, Inc., COMMAT
Ltd, Turkey). The aorta rings were stretched and main-
tained at optimal tension of 2 g and allowed to equili-
brate for 2 h. The lack of endothelium was confirmed
by the absence of acetylocholine (1 lM) vasorelaxant ac-
tion in aortic rings precontracted by noradrenaline
(0.1 lM).
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