Design, synthesis and pharmacological evaluation of new 1-[3-(4-arylpiperazin-1-yl)-2-hydroxy-propyl]-3,3-diphenylpyrrolidin-2-one derivatives with antiarrhythmic, antihypertensive, and α-adrenolytic activity

Article abstract of DOI:10.1016/j.ejmech.2009.04.028

A series of novel arylpiperazines bearing a 3,3-diphenylpyrrolidin-2-one fragment were synthesized and evaluated for their binding affinity for α₁- and α₂-adrenoceptors (ARs), as well as their antiarrhythmic, and antihypertensive act

Full text of DOI:10.1016/j.ejmech.2009.04.028

European Journal of Medicinal Chemistry 44 (2009) 3994–4003
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and pharmacological evaluation of new 1-[3-(4-arylpiperazin-
1-yl)-2-hydroxy-propyl]-3,3-diphenylpyrrolidin-2-one derivatives with
antiarrhythmic, antihypertensive, and
a
-adrenolytic activity
,
b
c
b
a
Katarzyna Kulig a , Jacek Sapa , Alicja Nowaczyk , Barbara Filipek , Barbara Malawska
*
a
´
Department of Physicochemical Drug Analysis, Jagiellonian University Medical College, Faculty of Pharmacy, 9 Medyczna Str., 30-688 Krakow, Poland
Department of Pharmacodynamics, Jagiellonian University Medical College, 9 Medyczna Str., 30-688 Krakow, Poland
c Department of Organic Chemistry, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University, Skłodowskiej-Curie 9, 85-094 Bydgoszcz, Poland
b
´
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel arylpiperazines bearing a 3,3-diphenylpyrrolidin-2-one fragment were synthesized and
evaluated for their binding affinity for a1- and a2-adrenoceptors (ARs), as well as their antiarrhythmic,
and antihypertensive activities. The highest affinity for the a1-AR was displayed by 1-{3-[4-(2-ethoxy-
phenyl)-piperazin-1-yl]-2-hydroxy-propyl}-3,3-diphenylpyrrolidin-2-one (7), which binds with
a pKi ¼ 7.28. The highest affinity for the a2-AR was shown by 1-{3-[4-(2-methoxy-phenyl)-piperazin-
1-yl]-2-hydroxy-propyl}-3,3-diphenylpyrrolidin-2-one (5), which binds with a pKi ¼ 6.68. Compound
7 was additionally evaluated in in vitro functional tests for its affinity for a1B- and a1D-AR, which gave pA2
a1B ¼ 6.55 and pA2 a1D ¼ 7.26. Among the compounds tested, compound 7 also had the highest
prophylactic antiarrhythmic activity in adrenaline-induced arrhythmia in anaesthetized rats. Its ED50
value was 1.1 mg/kg (i.v.). The compounds significantly decreased systolic and diastolic pressure in
normotensive anaesthetized rats at doses of 2.5–5.0 mg/kg (i.v.) and their hypotensive effects lasted for
longer than 1 h. It was found that the introduction of two phenyl ring substituents into the 3rd position
of the pyrrolidin-2-one fragment gave compounds with affinity for both a1- and a2-AR. The substitution
of the 2nd position in the phenyl piperazinyl fragment of the molecule was crucial for activity. To
determine detailed information concerning the structure–activity relationship, a preliminary molecular
modeling study was undertaken.
Received 18 December 2008
Received in revised form
16 April 2009
Accepted 17 April 2009
Available online 22 April 2009
Keywords:
a
-Adrenoceptors blocking activity
Antiarrhythmic
Hypotensive activity
3,3-Diphenylpyrrolidin-2-one derivatives
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
inappropriate (or a combination of inappropriate) antihypertensive
agents to treat patients and poor adherence to antihypertensive
Despite the significant progress made in prevention and treat-
ment, cardiovascular diseases are still the main cause of death
worldwide, and the number of deaths due to these diseases is
increasing [1]. Hypertension and atherosclerosis are central to the
pathogenesis of coronary artery disease (ischemia, angina,
myocardial infarction), heart failure, cerebral (stroke) and periph-
eral vascular disease. Since these two risk factors have been widely
investigated in the last few years, many therapeutic approaches are
now available.
Hypertension affects approximately 1 billion people worldwide
and is responsible for approximately 7.1 million deaths per year.
Additionally, an estimated 45% of treated patients in the United
States remained uncontrolled in 2004 [2,3].
therapy [4,5]. Therefore, with the above findings in mind, the
continued search for safer and more effective antihypertensive
drugs is essential.
The
a-adrenergic receptors (a-ARs) play a pivotal role in the
regulation of a variety of physiological processes, particularly
within the cardiovascular system and are divided into two main
subtypes a1- and a2-ARs [6]. The a1- and a2-ARs are located in the
vascular smooth muscle cell membrane, and upon stimulation by
an appropriate agonist mediate vasoconstriction. The simultaneous
occurrence of both receptor subtypes on vascular smooth muscles
makes it conceivable that a1- and a2-ARs can contribute to the
maintenance of peripheral arterial tone and may play an important
role in resistance seen in hypertension.
Several factors account for this failure and include low antihy-
pertensive efficacy of single-drug therapies, the use of
a1-ARs modulate intercellular biochemical processes in response
to changes in extracellular concentrations of the neurotransmitter
norepinephrine and the circulating hormone epinephrine [7–9].
Compounds acting as antagonists at various post-junctional a1-ARs
are frequently used in the therapy of high blood pressure, prazosin
* Corresponding author. Tel.: þ48 12 6205 465; fax: þ48 12 657 02 62.
E-mail address: mfkkulig@cyf.kr.edu.pl (K. Kulig).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.04.028

K. Kulig et al. / European Journal of Medicinal Chemistry 44 (2009) 3994–4003
3995
being the most common drug [6]. a1-AR antagonists are also used in
the treatment of benign prostatic hyperplasia, lower urinary tract
symptoms and cardiac arrhythmia [9,10].
O
R
B
C
N
N
N
linker
To date, a vast array of structurally unrelated a1-AR antagonists
have been identified, which makes it difficult to determine the
structural requirements which lead to receptor selectivity [11–17].
However, some general rules have been postulated by Barbaro et al.
[18] and Bremner et al. [19], which include an aromatic region,
a basic nitrogen atomwith at least one available protonation site and
a semipolar region. Being phenylpiperazine derivatives, these
compounds possess a positive ionizable nitrogen atom and a phenyl
ring, corresponding to the positive ionizable atom (A) and the
hydrophobic aromatic region (B). The second part of the molecule
pyrrolidin-2-one, which is connected to the phenylpiperazine by
a linker, is a hydrogen bond acceptor region (C) (Fig. 1).
A
Fig. 1. General structure of compounds tested.
hypotensive properties were studied. Taking into consideration the
pharmacophore model presented above for a1-AR antagonists,
which suggested [18,19] that a hydrophobic group larger than
a methoxy substituent may be accommodated by a hydrophobic
pocket, phenylpiperazine compounds with alkoxy moieties larger
than a methoxy group at the 2nd position of the phenyl ring were
prepared. The modifications in the arylpiperazinyl moiety also
included the introduction of 4-fluoro-; 2-methyl-; 2-trifluoro-
methyl-, and 2-, or 4-methoxy- into the phenyl ring (Fig. 2). The
newly synthesized compounds (as water-soluble hydrochlorides)
were tested for a1- and a2-AR affinity in addition to their antiar-
rhythmic and hypotensive activities.
The a2-ARs are located in the central nervous system. It has also
been shown that a2-ARs may be localized presynaptically, and act
as negative modulators in the release of catecholamine and other
neurotransmitters such as acetylcholine, g-aminobutyric acid and
nitric oxide [20,21]. In contrast to the a1-AR, the post-junctional
vascular a2-ARs have not yet been established as a target for anti-
hypertension therapy. The therapeutic uses of a2-AR antagonists
mainly concern the treatment of depression and diabetes. Other
potential clinical applications include cardiovascular disease,
obesity, Raynaud’s disease, male sexual dysfunction, Alzheimer’s
disease and Parkinson’s disease [22].
Non-selective a-AR antagonists such as phenoxybenzamine are
effective in reducing radial artery spasm during coronary artery
surgery [23].
We have previously reported that a series of 1-[3-(4-arylpiper-
azin-1-yl)-2-hydroxy]- or 2-acetoxy]-propyl-pyrrolidin-2-one
derivatives possess affinity for a1- and a2-ARs, and showed marked
hypotensive and antiarrhythmic activities. Among the compounds
tested, the most active was 1-[2-hydroxy-3(4-phenylpiperazin-1-
yl)-propyl]-pyrrolidin-2-one (I) and those which contained the
hydroxyl- or chloro-substituent in the 2nd position of the phenyl
ring [24–26]. In this context, the goal of our research was the
2. Chemistry
As a starting material for the synthesis of the title compounds
(4–10), 3,3-diphenyl-dihydro-furan-2-one (1) was used. At the first
stage, compound (1) was heated with allylamine giving 1-allyl-3,3-
diphenylpyrrolidin-2-one (2) in 69% yield. Then, oxidation of
compound (2) with meta-chloroperoxybenzoic acid (mCPBA) led to
synthesized 1-oxiranylmethyl-3,3-diphenylpyrrolidin-2-one (3) in
66% yield. Finally, aminolysis of compound (3) with N-substituted
arylpiperazines gave the relevant 1-[3-(4-arylpiperazin-1-yl)-2-
hydroxy]-propylpirolidin-2-ones (4–10). The yields of these reac-
tions ranged from 50 to 80%. The structures of the new compounds
were confirmed by elemental analysis and 1H NMR spectral data.
For the pharmacological assays, compounds (4–10) were converted
to their water-soluble hydrochlorides, by dissolving them in
anhydrous ethanol and saturating with gaseous HCl. Synthetic
routes leading to the new compounds are presented in Fig. 3.
development of novel a-AR antagonists, derivatives of arylpiper-
azine propyl-pyrrolidin-2-one. In this work, we report on the
synthesis and in vitro and in vivo pharmacological studies of a series
of new analogues of compound I; for these compounds, the influ-
ence of the introduction of two phenyl rings into the 3rd position of
the pyrrolidin-2-one and modifications in the arylpiperazinyl
moiety on their a1- and a2-AR affinity and their antiarrhythmic and
3. Pharmacology
Inthepresentstudy, severalpharmacologicaltestswerecarriedout
to assess a1- and a2-AR affinity, as well as antiarrhythmic and hypo-
tensive activities of the novel pyrrolidin-2-one derivatives (4–10).
O
O
N
N
OH
N
N
N
I
OH
N
R
4 - 10
R=-OMe, -Me, -OEt, -F, CF3
O
N
N
OH
N
II
Fig. 2. Structures of compounds (I), (II) and obtained compounds (4–10).

3996
K. Kulig et al. / European Journal of Medicinal Chemistry 44 (2009) 3994–4003
mCPBA, DCM,
H2N
rt
heat
O
O
O
O
N
N
O
1
2
3
R
HN
N
R=
4
5
6
7
8
9
-H
2-OMe
4-OMe
2-OEt
2-Me
2-CF3
2 HCl
n-PrOH, reflux, 12h
HClgas, EtOH
O
N
N
OH
N
10 4-F
R
4 - 10
Fig. 3. General route of synthesis 1-[3-(4-arylpiperazin-1-yl)-2-hydroxy-propyl]-3,3-diphenylpyrrolidin-2-one derivatives (4–10).
The pharmacological profile of the new compounds was evalu-
ated by radioligand binding assays (the ability to displace [3H]
prazosin or [3H] clonidine from a1- and a2-ARs, respectively) on rat
cerebral cortex [27,28]. All tested compounds displaced [3H]
prazosin from cortical binding sites (pKi ¼ 5.36–7.28) and [3H]
clonidine (pKi ¼ 5.29–6.68). The results obtained are presented in
Table 1.
The most potent a1-AR compound (7) was tested in in vitro
functional studies to determine its affinity to the a1B-AR and the
a1D-AR. The antagonist activity of compound (7) toward a1D-AR
present in rat aorta and toward a1B-AR present in guinea-pig spleen
was assessed by inhibition of noradrenaline-induced contractions.
The investigated compound, concentration-dependently, shifted
the noradrenaline response to the right. The Schild slopes did not
differ significantly from unity, indicating a competitive interaction
with the a1D- and a1B-adrenoceptors, and thus allowed determi-
nation of the pA2 value. The results obtained are shown in Table 2
and Figs. 4 and 5.
Table 3. The highest activity in this model of arrhythmia was dis-
played by compound (7), which had an ED50 value of 1.1 mg/kg.
The hypotensive activity of compounds (4–10) was determined
after i.v. administration to normotensive anaesthetized rats at
doses of 2.5–5.0 mg/kg. The results are presented in Table 4.
Compounds (4–9) significantly decreased systolic and diastolic
pressure. The observed effect lasted for more than 60 min.
Compound (10) was found to be inactive.
The influence of compounds (4–10) on pressor responses to
epinephrine, norepinephrine and methoxamine was tested. Intra-
venous epinephrine, norepinephrine and methoxamine were given
to rats at a dose of 2 mg/kg and 150 mg/kg, respectively, to induce
pressor response. Compounds (4, 5, 7 and 8) given at a dose of
5 mg/kg, significantly antagonized the pressor responses elicited by
epinephrine, norepinephrine and methoxamine (Figs. 6–9).
However, compounds (6, 9 and 10) did not have any significant
influence on systolic pressor response generated by epinephrine,
norepinephrine and methoxamine (data not shown).
Compounds (4–10) were tested for their prophylactic antiar-
rhythmic activity in an adrenaline-induced arrhythmia rat model
[29]. Intravenous (i.v.) injections of adrenaline at a dose of 20 mg/kg
4. Results and discussion
caused reflex bradycardia (100%), supraventricular and ventricular
extrasystoles (90%) bigeminy (75%) and blocks (100%), which led to
death in approximately 80% of rats within 10 ꢀ 5 min. Compounds
(4–5 and 7–9) injected 15 min before adrenaline administration,
decreased the occurrence of extrasystoles, bigeminy and reduced
mortality. The most active compounds were (7) and (8) and when
given at a dose of 5 mg/kg totally protected the animals from the
above disturbances. The ED50 values obtained are presented in
All the newly synthesized compounds (4–10) were found to
have an affinity toward a1- and a2-ARs, which was comparable to or
higher than the affinity of the earlier reported compounds [24–26].
The highest affinity for the a1-AR (pKi 7.28) was displayed by
compound 7, which had an ethoxy-substituent in the 2-position of
the phenyl ring. The highest affinity for the a2-AR (pKi 6.68) was
shown by compound 5, which had a methoxy group in the 2nd
position of the phenyl ring. The introduction of two phenyl groups
into the third position of pyrrolidin-2-one gave compounds which
in comparison to the parent compound (I) had higher or compa-
rable affinity for both a1- and a2-ARs. As expected, the introduction
at the 2nd position of the phenyl ring of an alkoxy group (methoxy-
(5) or ethoxy- (7)) resulted in enhancement of affinity to the a1-AR.
This effect which was proved in earlier observations showed that
a hydrophobic group larger than methoxy group could be better
Table 1
Affinity of 1-[3-(4-arylpiperazin-1-yl)-2-hydroxy-propyl]-3,3-diphenylpyrrolidin-
2-one derivatives (4–10) and model compounds (I and II) toward different a-AR
subtypes in rat cerebral cortex.
Compound
pKia1 [3H] prazosin (a1 rec.)
pKia2 [3H] clonidine (a2 rec.)
I [24]
II [24]
4
5
6
7
8
9
5.72
6.57
6.74
6.80
5.36
7.28
6.69
5.89
5.68
4.54
5.27
6.64
6.68
6.00
6.38
6.51
5.29
5.32
Table 2
Antagonistic activity for a1D- and a1B-receptors of compound (7) in functional in
vitro studies.
Tissues/receptor
pA2
Slope
Rat aorta a1D
Guinea pig spleen a1B
7.260 ꢀ 0.051
6.552 ꢀ 0.279
0.882 ꢀ 0.01
0.996 ꢀ 0.148
10
The means pKi values were obtained from three experiments. Inhibition constants
(Ki) were calculated according to the equation of Cheng and Prusoff [28].
pA2 value were obtained from the linear regression of Schild plot.
Each value was the mean ꢀ SEM of 5–8 experimental results.

K. Kulig et al. / European Journal of Medicinal Chemistry 44 (2009) 3994–4003
3997
Table 3
100
50
0
The prophylactic anarrhytmic activity of 1-[3-(4-arylpiperazin-1-yl)-2-hydroxy-
propyl]-3,3-diphenylpyrrolidin-2-one derivatives (4, 5, 7–9) and model compounds
(I and II) in adrenaline-induced arrhythmia in anaesthetized rats after i.v.
administration.
Compound
ED50 (mg/kg)
I [24]
II
4
5
7
8
9
7.6
n/a
1.9 (1.6–2.4)
2.05 (1.7–2.1)
1.1 (0.9–1.3)
1.4 (1.2–1.7)
3.9 (2.8–5.5)
3.4 (2.6–4.4)
1.05 (0.64–1.73)
Tolazoline
Propranolol
-7
-6
-5
-4
log NA [M]
n/a – Not active in this test.
compound 7 conc. 10-6
M
Each value was obtained from three experimental groups. Each group consisted of
six animals. The ED50 values and their confidence limits were calculated according to
the methods of Litchfield and Wilcoxon [42].
NA
compound 7 conc. 3x10-7
compound 7 conc. 3x10-6
M
M
Fig. 4. Concentration–response curves to noradrenaline in the guinea pig spleen in the
absence (,) or presence of 7 (- 0.3, :1, ;3 M). Results are expressed as percentage
of the maximal response to noradrenaline in the first concentration–response curve.
Each point represents the mean ꢀ SEM (n ¼ 5).
the pA2 value was equal to the pKB value [31]. The ratio of guinea-
pig spleen KB/rat aorta KB was 5.2, thus compound (7) displayed
modest tissue selectivity for a1D- over a1B-AR.
m
In order to better understand the mechanism of action displayed
by the tested compounds, their influence on the pressor response to
epinephrine, norepinephrine and methoxamine were tested. It is
generally accepted that a1-AR antagonists invert the pressor
response to epinephrine, only partially invert the response to
norepinephrine, diminish the pressor response to methoxamine,
and potentiate the hypertensive effect of norepinephrine. Whereas
the a2-AR antagonists antagonize the pressor effect of norepineph-
rine, and reverse the pressor response to epinephrine. The results of
these studies were in good agreement with the radioligand binding
accommodated by a hydrophobic pocket at the receptor binding
site [18,19]. It was observed that the 2nd substitution at the phenyl
ring was preferred. Compounds (5, 7–9) had higher or comparable
affinity for a1-AR to that of the unsubstituted compound (4). The
replacement of the methoxy group from the 2nd (5) into the 4th (6)
position of the phenyl ring resulted in decreased affinity for both
a1- and a2-ARs. The compounds tested displayed rather low
selectivity for a1-AR vs. a2-AR subtypes. The highest selectivity was
observed for compound 7, which was about 10 times more potent
against a1-AR than a2-AR (pKi a1 7.28 and pKi a2 6.38, respectively).
It is known that the a1B-AR subtype of a1-AR plays an important
role in the regulation of blood pressure due to antagonistic activity
and could be used in hypertension therapy. Antagonism of a1D-AR
may play a part in blood pressure control, because of the involve-
ment of a1D-ARs in the contraction of vessels [30]. Therefore, with
these findings in mind, compound (7) which displayed the highest
investigations and confirmed the a-AR antagonist activity of these
compounds. Compound (4) and compounds (5, 7, 8), with the
alkoxy- or methyl-substituent at the 2-position of the phenyl group,
significantly decreased the pressor response elicited by epinephrine,
norepinephrine and methoxamine. This effect can be explained by
their affinity for the a1- and a2-AR profile. All these compounds
possessed affinity for both a1- and a2-ARs which ranged from a pKi
value of 6.69–7.28 for a1-AR and a pKi value of 6.00–6.68 for a2-AR.
Compound (7) was the most active and possessed the highest
affinity for a1-AR (pKi 7.28) of all the compounds tested. Compounds
(6, 9 and 10) which displayed low affinity for both a1-AR and a2-AR
did not have a significant influence on the pressor response elicited
by epinephrine, norepinephrine and methoxamine.
affinity for a1-AR was tested for its ability to block a1B- and a1D
-
subtypes of the adrenoceptor in functional in vitro studies. It was
found that compound (7) had slightly higher affinity for a1D-AR
(pA2 ¼ 7.26) than for a1B-AR (pA2 ¼ 6.55). In both cases, compound
(7) showed a competitive, surmountable type of antagonism where
Compounds (4, 5, 7, 8 and 9) diminished or prevented the
appearance of epinephrine-induced arrhythmia symptoms. The
most active in this test, compound (7) diminished the occurrence of
extrasystoles in the anaesthetized rat in 37.5–100% of animals, and
significantly prevented mortality in 100%. The ED50 value for
compound (7) was 1.1 mg/kg and was three times lower than that
100
50
0
displayed by tolazoline (a non-selective a-AR antagonist), compa-
rable to that displayed by propranolol (the commonly used refer-
ence compound in adrenaline-induced models of arrhythmia) and
7 times lower than that displayed by compound I. These results are
in good agreement with previously published data on non-selective
and selective a-antagonists such as phentolamine, prazosin, and
abanoquil which prevented arrhythmia induced by adrenaline or
cocaine infusion [32,33]. In addition, the effectiveness of phentol-
amine and prazosin against ischemia-induced arrhythmia in
a variety of animal models of arrhythmia has also been observed
[34,35].
-8
-7
-6
-5
M
log NA [M]
compound 7 conc. 3x10-7
compound 7 conc. 10-6
NE
compound 7 conc. 10-7
M
M
The obtained pyrrolidin-2-one derivatives (4–10) were also
tested for their hypotensive activity in normotensive anaesthetized
rats. Compounds possessing a substituent at the 2-position of the
phenyl ring or 4-methoxy group (4–9) significantly decreased
Fig. 5. Concentration–response curves to noradrenaline in the rat aorta in the absence
(,) or presence of 7 (- 0.1, :0.3, ;1 M). Results are expressed as percentage of the
maximal response to noradrenaline in the first concentration–response curve. Each
point represents the mean ꢀ SEM (n ¼ 5–8).
m

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K. Kulig et al. / European Journal of Medicinal Chemistry 44 (2009) 3994–4003
Table 4
The hypotensive activity of 1-[3-(4-arylpiperazin-1-yl)-2-hydroxy-propyl]-3,3-diphenylpyrrolidin-2-one derivatives (4–9) in anaesthetized normotensive rats after i.v.
administration.
Compound
Dose mg/kg
Blood pressure (mm Hg ꢀ S.E.M.)
Time of observation (min)
0
5
10
20
30
60
4
5
6
7
8
9
5
Systolic
Diastolic
Systolic
Diastolic
Systolic
Diastolic
Systolic
Diastolic
Systolic
Diastolic
Systolic
Diastolic
140.5 ꢀ 1.1
125.5 ꢀ 0.6
139.0 ꢀ 0.6
120.0 ꢀ 1.7
125.6 ꢀ 7.6
107.2 ꢀ 7.3
142.5 ꢀ 3.6
127.0 ꢀ 8.1
141.3 ꢀ 6.4
126.0 ꢀ 4.6
138.0 ꢀ 0.9
119.0 ꢀ 1.2
135.5 ꢀ 0.8*
134.5 ꢀ 1.8**
118.6 ꢀ 1.5
135.5 ꢀ 0.9**
116.5 ꢀ 3.6
129.0 ꢀ 1.7***
113.5 ꢀ 4.4*
116.0 ꢀ 3.5***
102.5 ꢀ 0.6***
105.6 ꢀ 7.5
126.5 ꢀ 2.8***
109.5 ꢀ 4.6***
112.0 ꢀ 3.0***
98.0 ꢀ 0.9***
105.8 ꢀ 9.8
119.0 ꢀ 2.0
125.0 ꢀ 6.0*
109.0 ꢀ 1.5***
126.6 ꢀ 7.2
107.0 ꢀ 6.0
130.0 ꢀ 1.2
117.0 ꢀ 11.0
132.3 ꢀ 4.8
116.7 ꢀ 2.8
126.0 ꢀ 5.0*
103.0 ꢀ 1.5***
5
122.0 ꢀ 6.0**
107.5 ꢀ 1.9***
115.2 ꢀ 6.5
96.4 ꢀ 5.5
118.0 ꢀ 3.0***
104.0 ꢀ 1.9***
107.6 ꢀ 7.9
2.5
5
89.8 ꢀ 6.1
88.2 ꢀ 5.9*
88.2 ꢀ 9.5*
118.0 ꢀ 6.1***
105.0 ꢀ 6.0
133.7 ꢀ 2.2
118.0 ꢀ 2.1
111.0 ꢀ 7.0****
98.0 ꢀ 5.5*
113.5 ꢀ 3.6****
96.0 ꢀ 2.0*
114.0 ꢀ 2.0****
95.5 ꢀ 4.5*
5
136.3 ꢀ 2.9
119.7 ꢀ 1.8
133.0 ꢀ 3.6
116.0 ꢀ 10.8***
99.0 ꢀ 11.7***
111.0 ꢀ 3.1****
100.0 ꢀ 0.7***
117.7 ꢀ 0.9*
116.0 ꢀ 3.9***
103.0 ꢀ 3.0***
5
122.0 ꢀ 7.1*
106.5 ꢀ 2.9***
119.0 ꢀ 3.1***
104.0 ꢀ 1.8**
The data are the means of six experiments ꢀ S.E.M. Statistical analyses were performed using a one-way ANOVA test. *p < 0.05; **p < 0.02; ***p < 0.01; ****p < 0.001.
systolic and diastolic pressure. The observed effect persisted for
more than 60 min.
derivatives, the angle differences varied within 7ꢁ, and were about
4-fold less than that of the whole set (31.784ꢁ). This showed that
the presence of the diphenyl moiety at the 3,3-position of pyrroli-
din-2-one or modification of compound 4 by attachment of the
trifluoromethyl group at the ortho position of the phenylpiperazine
ring significantly influenced the ABC angle (compounds I, II, and 9).
As can be seen from Table 5, the set can be divided into two subsets,
one consisting of compounds which had similar pharmacophoric
features (4, 5, 6, 7, 8, 10) and the second consisting of compounds
which had a significant spread in the distances and angles between
the pharmacophoric points (I, II, 9). The values obtained for the first
subset corresponded to the characteristic values of the most active
compounds obtained in previously reported studies [26]. This
suggested that this subset may contain the compounds with better
In order to better define the structure–activity relationship of
the investigated compounds, a molecular modeling study was
undertaken. The three-dimensional structures of the pyrrolidin-
2-one derivatives at their neutral state were obtained through full
optimization based on the AM1 quantum chemical procedure. The
aim of this approach was the identification of a pharmacophore,
which is a template derived from the structure of these compounds,
and represents the geometry of the receptor site as a collection of
functional groups in 3D space. This work was based on our earlier
research describing a pharmacophore model for a1-AR, which
included three features: an aromatic region, a positively ionizable
group, and
a hydrogen bond acceptor. The structures were
compared to the earlier proposed pharmacophoretic features [24]
using measurements of the appropriate distances and angles. All
intramolecular distances and angles in the proposed pharmaco-
phore series of investigated compounds were measured and are
listed in Table 5.
In the preliminary analysis of the estimated distances and angles
between the pharmacophoric features of these compounds, it was
observed that the distances a, b and c were comparable, while the
angle values were appreciably different. This indicated that,
replacement of 3-(4-phenylpiperazin-1-yl) by the 3-(4-diphe-
nylmethylpiperazin-1-yl) moiety or attachment of the diphenyl
moiety at the 3,3-position of pyrrolidin-2-one had only limited
influence on the distance between the center of the phenyl ring, the
positively ionizable nitrogen atom and the hydrogen bond acceptor
(compounds I, II and 4). An extensive deflection in the angles
measured was also found in compounds (II) and (9). For all other
antiarrhythmic or hypotensive activities together with
lytic activity. It was found that compounds (4, 5, 7, 8) did possess
the most active antiarrhythmic, hypotensive, and -adrenolytic
a-adreno-
a
activities in this series. Compounds (7, 8) had the most active
profiles of antiarrhythmic activity. Compounds (4, 5, 7, 8) signifi-
cantly antagonized the pressor response. Furthermore, only
compounds (7, 8) were active in whole kinds of activity (hypoten-
sive activity, see Table 3 and Figs. 7 and 8). Surprisingly, compounds
(6) and (10) did not show either antiarrhythmic or hypotensive
activity. Despite the successive prediction of the activity of
compounds (4, 5, 7, 8) this pharmacophore model did not explain
the structure–activity relationship (SAR) of compounds (6, 10).
In order to check the applied pharmacophore model more
precisely, we extended our molecular modeling study to a two-part
calculation experiment. In the first part, single point energy (SPE)
quantum chemical calculations were carried out to predict the
65
75
Δ BP mmHg
methoxamine
Δ BP mmHg
methoxamine
65
55
45
35
25
15
5
55
methoxamine
*
45
methoxamine
**
norepinephrine
35
norepinephrine
norepinephrine
epinephrine
epinephrine
*
25
15
5
norepinephrine
epinephrine
***
*
comp. 5
5 mg/kg
epinephrine
***
comp. 4
5 mg/kg
-5
*p< 0.05
**p < 0.02
***p < 0.01
-5
* p < 0.05
*** p < 0.01
Fig. 6. The effect of compound (4) on blood pressor response to epinephrine,
Fig. 7. The effect of compound (5) on blood pressor response to epinephrine,
norepinephrine and methoxamine.
norepinephrine and methoxamine.

K. Kulig et al. / European Journal of Medicinal Chemistry 44 (2009) 3994–4003
3999
Table 5
85
75
65
55
45
35
25
15
5
Δ BP mmHg
The distances (Å) and angles (ꢁ) between pharmacophore features for compounds I,
II, 4–10.
methoxamine
O
c
Ar = Ph or H
Ar
Ar
N
N
b
N
OH
a
norepinephrine
epinephrine
R
methoxamine
norepinephrine
****
comp. 7
epinephrine
****
****
5 mg/kg
Compound
a (Å)
b (Å)
c (Å)
ABC (ꢁ)
I
8.719
8.600
8.725
8.748
8.725
8.748
8.814
8.477
8.721
3.946
3.825
3.946
4.046
3.945
4.053
4.164
4.298
3.946
4.957
4.857
4.967
4.969
4.967
4.970
4.970
4.966
4.967
156.501
164.086
156.278
151.891
156.327
151.472
149.486
132.302
156.057
-5
**** p < 0.001
II
4
5
6
7
8
9
10
Fig. 8. The effect of compound (7) on blood pressor response to epinephrine,
norepinephrine and methoxamine.
dipole moment values. In the second part, the electrostatic poten-
tial map (MEP) was generated by using quantum chemical calcu-
lations. The dipole moment values (m) and calculated MEP of the
studied compounds are listed in Table 6, for explicit understanding
the corresponding molecular structures were plotted on the MEPs.
For this investigation, the model derivatives, compounds I and II,
charge in the molecule by plotting the electrostatic charge distri-
bution contours in the plane of the pharmacophore triangle. In
Table 6, for explicit understanding, the corresponding molecular
structures are plotted on the MEP.
were chosen. Compound
I was a leading structure, while
compound II displayed activity in the binding assays but was
inactive in the in vivo tests.
The dipole moment values of the tested compounds varied
within the range 2.76–6.44 D. It was observed that modification
of compound I by replacing 3-(4-phenylpiperazin-1-yl) with the
3-(4-diphenylmethylpiperazin-1-yl) moiety or the introduction of
two phenyl rings into the 3-position of pyrrolidin-2-one resulted in
For each presented MEP, the green contours indicate less elec-
tronegative areas, which were generally found in the central region
concentrated along the primary core of the compound’s structure.
Generally these green contours were due to atoms like C and H. The
most important differences in the charge distribution concerned
the variation in the localization of the negative charges (red col-
oured contours) due to the presence of strongly electronegative
moieties (e.g. N, O, F, or phenyl ring) at a specific point in the
molecular structure. For compound (9) there was a negative charge
contour cluster enclosed by green contours localized in the upper
central part of the MEP. This molecule was highly non-planar
and had the highest number of strongly electronegative moieties
(i.e. 3–N, 2–O, 3–F, and phenyl ring) in the set. For this reason, it
differed significantly from the other compounds in the set which
had similar negative charge distribution in the upper part of the
MEP. In the MEP for compounds (I, II, 6, 10), the arylpiperazine
terminal moiety was surrounded by red regions due to the presence
of the electronegative substituent piperazine. This situation was
correlated with low or absent antiarrhythmic activity. In the MEP
(4, 5, 7, 8, 9) red areas interrupted by green contours in the plane of
the molecular skeleton which were divided into separate upper and
lower parts were observed. This type of charge distribution was
correlated with the antiarrhythmic activity of the compound. The
analysis of this activity based on the distribution of electrostatic
charge in this subset, leads us to conclude that the extension of the
negative charge in the upper part would be large, and the lower
part of the MEP would be concentrated in clusters. In the case of the
most active compound (7), there was a wide extension of red upper
contours separated by broad green areas in which there were small
clusters of negative charge in the lower part. When this cluster
became larger and started to overlap, as in the case of compounds
(8, 4, 5), the activity of the corresponding compounds decreased. It
is also significant that any substitution leading to positioning of the
terminal phenyl ring in a plane perpendicular to the pharmaco-
phoric feature plan, decreased the antiarrhythmic activity or this
activity was absent.
decreased
compounds by replacing the methoxy substituent from the 4- to
the 2-position of the phenyl ring resulted in decreased values of
m values of less than 1 D (II, 4). The modification of the
m
about 1.6 D (5, 6). Introduction of a methyl group into the second
position of the phenyl group or methoxy- into the 4- position did
not change the
m value significantly (6, 8). While introduction of
strongly electronegative substituents such as CF3 or F atom in the
2- or 4-positions of the phenyl moiety resulted in the maximum
value of
m in the set (9, 10). Low m values were estimated for
compounds with methoxy (5) or ethoxy (7) groups in the 2-posi-
tion of the phenyl ring. This detailed variability in the dipole
moment values demonstrated a multidirectional tendency.
For this reason, it was decided to include the electrostatic charge
distribution in the molecule into the structure–activity consider-
ations of the compounds in the current study. The MEP presented in
Table 6, shows the view obtained by integrating two data sets
together e.g. pharmacophoric features and the distribution of the
75
methoxamine
Δ BP mmHg
65
55
45
methoxamine
norepinephrine
epinephrine
***
35
25
15
5
norepinephrine
*
comp. 8
epinephrine
5 mg/kg
***
-5
* p < 0.05
*** p < 0.01
According to the results discussed in this work we suggest that
the distribution resembled clusters as presented in the MEP (7).
Fig. 9. The effect of compound (8) on blood pressor response to epinephrine,
norepinephrine and methoxamine.

4000
K. Kulig et al. / European Journal of Medicinal Chemistry 44 (2009) 3994–4003
Table 6
Calculated contour electrostatic potential maps and calculated dipole moment values for compounds (4–10) and parent molecules (I, II).
5. Conclusion
EL III (Elementar Analysensystem, Hanau, Germany), which gave
values within 0.4% of the theoretical values. 1H NMR and 13C NMR
data were recorded on a Varian Mercury VX 300 MHz PFG instru-
ment (Hansen Way, USA) in CDCl3 at ambient temperature. Thin
layer chromatography was carried out on Merck silica gel precoated
F254 plates (0.2 mm) (Darmstadt, Germany). The plates were
visualised with UV light or iodine solution (0.05 M in a 10% HCl).
In summary, the synthesis of several new 1-[3-(4-arylpiperazin-
1-yl)-propyl]-3,3-diphenylpyrrolidin-2-onederivatives is described.
The new compounds were tested for their affinity for a-ARs, as well
as their antiarrhythmic and hypotensive activities. As a result, each
compound was found to possess affinity for a1-AR. The most potent
and selective for a1-AR was compound (7) (1-{3-[4-(2-ethoxy-
phenyl)-piperazin-1-yl]-2-hydroxy-propyl}-3,3-diphenylpyrrolidin-
2-one; pKi a1 ¼7.28; pKi a2 ¼ 6.38) which was additionally tested in
in vitro studies for its affinity to a1B- and a1D-ARs (pA2 a1B ¼ 6.55
and pA2 a1D ¼ 7.26). It was found that the introduction of two
phenyl rings into the third position of pyrrolidin-2-one resulted in
the compounds having higher affinity than the unsubstituted
pyrrolidin-2-one derivatives for a1-AR. It was also shown that the
2nd position could play a crucial role in improving the a1-AR
antagonist properties in terms of affinity and selectivity. The
pharmacological results and binding studies suggested that the
antiarrhythmic and hypotensive properties of these compounds
were related to their adrenolytic properties. Theoretical investi-
gations showed that the charge distribution in the molecules
significantly influenced their antiarrhythmic activities. The pattern
of the charge distribution in the plane of the pharmacophoric
features was analyzed and a favorable charge distribution was
found. More extensive structure–activity studies are in progress
and will be reported in due course.
6.1.1. 1-Allyl-3,3-diphenylpyrrolidin-2-one (2)
20.0 g (84 mmol) 3,3-diphenyl-dihydro-furan-2-one (1) and
19.2 g (336 mmol) allylamine were heated in an autoclave at 200 ꢁC
for 96 h. The obtained product was crystallized from acetone. Yield:
69.4%, Anal. calc. for C19H19NO (277.37) C% 82.28, H% 6.90, N% 5.05,
found C% 82.32, H% 6.97, N% 5.04; m.p. 91–92 ꢁC; TLC Rf ¼ 0.67
(toluene:ethyl acetate (7:3)); 1H NMR (CDCl3):
J ¼ 6.5 Hz, 2H), 3.33 (t, CH2CH2C, J ¼ 6.5 Hz, 2H), 4.0 (dt, NCH2CH,
J ¼ 1.7 Hz and J ¼ 6.0 Hz, 2H), 5.08–5.20 (m, CHCH2, 2H), 5.65–5.81
(m, CH2CHCH2, 1H), 7.18–7.40 (m, arom, 10H); 13C NMR (CDCl3):
d
¼ 2.75 (t, NCH2CH2,
d
¼ 37.8 (CH2CH2CPh2), 43.8 (NCH2), 48.1 (CH2CH), 63.4 (CPh2), 117.4
(CH2CH), 126.2, 128.2, 129.8, 129.2 (arom), 131.8 (CHCH2), 176.1
(carbonyl).
6.1.2. 1-Oxiranylmethyl-3,3-diphenylpyrrolidin-2-one (3)
13.8 g (50 mmol) of compound (2) was dissolved in 50 mL of
methylene chloride and 11.4 g (50 mmol) of mCPBA. The reaction
mixture was stirred at room temperature for 2 h, and then 20.0 g of
sodium thiosulfate and 50 mL water were added. The layer was
separated and the organic layer was washed with saturated sodium
carbonate solution (15 mL) and brine (15 mL), dried over sodium
sulfate and evaporated in vacuo. The obtained residue was purified
by column chromatography using a mixture of toluene and ethyl
acetate (7:3). Yield: 65.7%; Anal. calc. C19H19NO2 (293.36) calc.
C% 77.79, H% 6.53, N% 4.77, found C% 77.83, H% 6.57, N% 4.79; m.p.
104–105 ꢁC; TLC Rf ¼ 0.55 (toluene:ethyl acetate (7:3)); 1H NMR
6. Experimental
6.1. Chemistry
Melting points were determined in open glass capillaries on
Bu¨chi 353 apparatus (Flawil, Switzerland) and were uncorrected.
Elemental analyses (C, H, N) were carried out on an Elementar Vario

K. Kulig et al. / European Journal of Medicinal Chemistry 44 (2009) 3994–4003
4001
(CDCl3):
d
¼ 2.48 (dd, CHCH2, 1H, J ¼ 2.6 Hz and J ¼ 4.7 Hz), 2.74–
found C% 65.14, H% 6.96, N% 7.38; m.p. 149–151 ꢁC; TLC: Rf ¼ 0.47
2.82 (m, CHCH2, NCH2CH2, 3H), 3.04–3.10 (m, CH2CHCH2, 1H), 3.20
(dd, NCH2CH, 1H, J ¼ 6.2 Hz, J ¼ 14.5 Hz), 3.38–3.58 (m, CH2CH2C,
2H), 3.94 (dd, NCH2CH, 1H, J ¼ 2.7 Hz, J ¼ 14.5 Hz), 7.20–7.38
chloroform:acetone (1:1); 1H NMR (CDCl3):
d
¼ 1.45 (t, CH3, 3H), 2.33
(t, NCH2CH2, 2H), 2.50 (t, CH2CH2C, 2H), 2.65–2.78 (m, piper, 4H),
3.01–3.16 (m, piper, 4H), 3.35 (dd, CH2CH(OH)CH2, 1H), 3.45–3.69 (m,
CH(OH)CH2N, CH2CH(OH)CH2Npiper, 4H), 4.15 (qw, OCH2, 2H), proton
of OH group is not visible, 6.82–7.00 (m, arom, 4H), 7.18–7.33 (m,
(m, arom, 10 H); 13C NMR (CDCl3):
d
¼ 37.7 (CH2CH2CPh2), 43.4
(NCH2), 45.3 (OCH2), 51.0 (NCH2CH), 52.9 (NCH2CH), 63.3 (CPh2),
126.2, 128.2, 129.8, 129.2 (arom), 175.8 (carbonyl).
arom, 10H); 13C NMR (CDCl3):
(NCH2CH2CPh2), 55.0 (NCH2CHOH), 56.0, 56.3 (CH2 piper), 63.3
(CPh2), 63.8 (CHCH2), 64.6 (OCH2), 66.5 (CH), 113.6, 121.2,121.7, 122.6,
126.2, 128.2, 129.2, 129.8, 142.2, 162.3 (arom), 175.8 (carbonyl).
d
¼ 14.8 (CH3), 37.7 (CH2CH2CPh2), 44.0
6.1.3. General procedure for the synthesis of the dihydrochloride
of 1-(2-hydroxy-3-substituted arylpiperazin-1-yl-propyl)-3,
3-diphenylpyrrolidin-2-one (4–10)
A solution of 2.9 g (10 mmol) of compound (3) and10 mmol of the
corresponding arylpiperazine in n-propanol (20 mL) was heated
under reflux for 12 h. After evaporating the solvent, the oily residue
was purified by column chromatography using a mixture of chloro-
form and acetone (1:1). The obtained oil was then dissolved in EtOH
and then EtOH saturated with HClgas was added until the mixture
becomes acidic. The obtained precipitate was crystallized from EtOH.
6.1.3.5. 1-{2-Hydroxy-3-[4-(2-methyl-phenyl)-piperazin-1-yl]-propyl}-
3,3-diphenylpyrrolidin-2-one dihydrochloride (8). Yield: 54.6%;
Anal. calc. C30H35N3O2 ꢂ 2 HCl (542.54) calc C% 66.41, H% 6.87, N%
7.75, found C% 66.57, H% 6.99, N% 7.92; m.p. 169–170 ꢁC; TLC:
Rf ¼ 0.59 chloroform:acetone (1:1); 1H NMR (CDCl3):
d
¼ 2.30 (s,
CH3, 3H), 2.38 (t, NCH2CH2, 2H), 2.45 (t, CH2CH2C, 2H), 2.65–2.76
(m, piper, 4H), 2.80–2.95 (m, piper, 4H), 3.40 (dd, CH2CH(OH)CH2,
1H), 3.52–3.63 (m, CH(OH)CH2N, CH2CH(OH)CH2Npiper, 4H), proton
of OH group is not visible, 6.85–7.00 (m, arom, 4H), 7.12–7.49 (m,
6.1.3.1. 1-[2-Hydroxy-3-(4-phenylpiperazin-1-yl)-propyl]-3,3-diphe-
nylpyrrolidin-2-one dihydrochloride (4). Yield: 86.5%; Anal. calc.
C29H33N3O2 ꢂ 2 HCl (528.51) calc. C% 65.90, H% 6.67, N% 7.95, found
C% 66.03, H% 6.79, N% 8.09; m.p. 189–190 ꢁC; TLC: Rf ¼ 0.48 chlor-
arom, 10H); 13C NMR (CDCl3):
d
¼ 17.9 (CH3), 37.7 (CH2CH2CPh2),
44.0 (NCH2CH2CPh2), 55.0 (NCH2CHOH), 56.0, 56.3 (CH2 piper), 63.3
(CPh2), 63.8 (CHCH2), 64.6 (OCH2), 66.5 (CH), 118.9, 123.2, 126.2,
126.6, 128.2, 129.2, 129.8, 131.4, 132.8, 151.0 (arom), 175.8
(carbonyl).
oform:acetone (1:1); 1H NMR (CDCl3):
d
¼ 2.28 (t, NCH2CH2, 2H),
2.39–2.50 (m, CH2CH2C, 2H), 2.65–2.78 (m, piper, 4H), 3.10–3.18
(m, piper, 4H), 3.34 (dd, CH2CH(OH)CH2, 1H), 3.46–3.62 (m,
CH(OH)CH2N, NCH2CH(OH), 3H), 3.82–3.96 (m, NCH2CH(OH), 1H),
proton of OH group is not visible, 6.80–6.92, 7.17–7.37 (m,
6.1.3.6. 1-{2-Hydroxy-3-[4-(2-triflouromethyl-phenyl)-piperazin-1-
yl]-propyl}-3,3-diphenylpyrrolidin-2-one dihydrochloride (9). Yield:
55.5%; Anal. calc. C30H32O2N3F3 ꢂ 2 HCl (596.51) calc. C% 60.40, H%
5.75, N% 7.04, found C% 60.58, H% 5.89, N% 7.19; m.p. 220–222 ꢁC;
arom, 15H); 13C NMR (CDCl3):
(NCH2CH2CPh2), 55.0 (NCH2CHOH), 56.0, 56.3 (CH2 piper), 63.3
(CPh2), 63.8 (CHCH2), 66.5 (CH), 114.3, 121.9, 126.2, 128.2, 129.2,
129.6, 129.8, 149.6 (arom), 175.8 (carbonyl).
d
¼ 37.7 (CH2CH2CPh2), 44.0
TLC: Rf ¼ 0.52 chloroform:acetone (1:1); 1H NMR (CDCl3):
¼ 2.26
d
(t, NCH2CH2, 2H), 2.45 (t, CH2CH2C, 2H), 2.65–2.79 (m, piper, 4H),
2.84–2.93 (m, piper, 4H), 3.45 (dd, CH2CH(OH)CH2, 1H), 3.57–3.67
(m, CH(OH)CH2N, CH2CH(OH)CH2Npiper, 4H), proton of OH group is
not visible, 7.15–7.40 (m, arom, 4H), 7.42–7.68 (m, arom, 10H); 13C
6.1.3.2. 1-{2-Hydroxy-3-[4-(2-methoxy-phenyl)-piperazin-1-yl]-pro-
pyl}-3,3-diphenylpyrrolidin-2-one dihydrochloride (5). Yield: 52.6%;
Anal. calc. C30H35N3O3 ꢂ 2 HCl (558.54) calc. C% 64.51, H% 6.68%, N%
7.52, found C% 64.67, H% 6.78, N% 7.69; m.p. 202–203 ꢁC; TLC:
NMR (CDCl3):
d
¼ 37.7 (CH2CH2CPh2), 44.0 (NCH2CH2CPh2), 55.0
(NCH2CHOH), 56.0, 56.3 (CH2 piper), 63.3 (CPh2), 63.8 (CHCH2), 64.6
(OCH2), 66.5 (CH), 110.4, 113.1, 118.5, 126.2, 127.4, 128.2, 129.2, 129.8,
132.9, 143.4 (arom), 125.8 (CF3), 175.8 (carbonyl).
Rf ¼ 0.50 chloroform:acetone (1:1); 1H NMR (CDCl3):
¼ 2.30 (t,
d
NCH2CH2, 2H), 2.51–2.59 (m, CH2CH2C, 2H), 2.66–2.81 (m, piper,
4H), 3.03–3.16 (m, piper, 4H), 3.37 (dd, CH2CH(OH)CH2, 1H), 3.52–
3.67 (m, CH(OH)CH2N, 2H), 3.85 (s, OCH3, 3H), 3.90–4.00 (m,
NCH2CH(OH), 2H), proton of OH group is not visible, 6.82–7.02 (m,
6.1.3.7. 1-{2-Hydroxy-3-[4-(4-flouro-phenyl)-piperazin-1-yl]-propyl}-
3,3-diphenylpyrrolidin-2-one dihydrochloride (10). Yield: 53.8%;
Anal. calc. C29H32N3O2F ꢂ 2 HCl (546.50) calc. C% 60.40, H% 5.75, N%
7.04, found C% 60.54, H% 5.92, N% 7.14; m.p. 175–177 ꢁC; TLC:
arom, 4H), 7.20–7.40 (m, arom, 10H); 13C NMR (CDCl3):
d
¼ 37.7
(CH2CH2CPh2), 44.0 (NCH2CH2CPh2), 55.0 (NCH2CHOH), 55.8
(OCH3), 56.0, 56.3 (CH2 piper), 63.3 (CPh2), 63.8 (CHCH2), 66.5 (CH),
113.5, 121.9, 122.1, 123.0, 126.2, 128.2, 129.2, 129.6, 141.1, 162.2
(arom), 175.8 (carbonyl).
Rf ¼ 0.52 chloroform:acetone (1:1); 1H NMR (CDCl3):
d
¼ 2.26 (t,
NCH2CH2, 2H), 2.45 (t, CH2CH2C, 2H), 2.65–2.79 (m, piper, 4H),
2.84–2.93 (m, piper, 4H), 3.45 (dd, CH2CH(OH)CH2, 1H), 3.57–3.67
(m, CH(OH)CH2N, CH2CH(OH)CH2Npiper, 4H), proton of OH group is
not visible, 7.15–7.40 (m, arom, 4H), 7.42–7.68 (m, arom, 10H); 13C
6.1.3.3. 1-{2-Hydroxy-3-[4-(4-methoxy-phenyl)-piperazin-1-yl]-pro-
pyl}-3,3-diphenylpyrrolidin-2-one dihydrochloride (6). Yield: 57.2%;
Anal. calc. C30H35N3O3 ꢂ 2 HCl (558.54) calc. C% 64.51, H% 6.68%, N%
7.52, found C% 64.73, H% 6.73, N% 7.65; m.p. 196–197 ꢁC; TLC:
NMR (CDCl3):
d
¼ 37.7 (CH2CH2CPh2), 44.0 (NCH2CH2CPh2), 55.0
(NCH2CHOH), 56.0, 56.3 (CH2 piper), 63.3 (CPh2), 63.8 (CHCH2), 64.6
(OCH2), 66.5 (CH), 113.2, 126.0, 127.4, 128.2, 129.2, 129.8, 152,9
(arom), 124.1 (CF3), 175.8 (carbonyl).
Rf ¼ 0.52 chloroform:acetone (1:1); 1H NMR (CDCl3):
¼ 2.30 (t,
d
NCH2CH2, 2H), 2.44–2.50 (m, CH2CH2C, 2H), 2.66–2.81 (m, piper,
4H), 3.03–3.16 (m, piper, 4H), 3.37 (dd, CH2CH(OH)CH2, 1H), 3.52–
3.67 (m, CH(OH)CH2N, 2H), 3.78 (s, OCH3, 3H), 3.90–4.00 (m,
NCH2CH(OH), 2H), proton of OH group is not visible, 6.82–7.02 (m,
6.2. Pharmacology
arom, 4H), 7.20–7.40 (m, arom, 10H); 13C NMR (CDCl3):
(CH2CH2CPh2), 44.0 (NCH2CH2CPh2), 55.0 (NCH2CHOH), 55.8 (OCH3),
56.0, 56.3 (CH2 piper), 63.3 (CPh2), 63.8 (CHCH2), 66.5 (CH), 115.2,
115.3, 126.2, 128.2,129.2,129.8, 146.3, 152.8 (arom), 175.8 (carbonyl).
d
¼ 37.7
6.2.1. Materials and methods
6.2.1.1. Compounds. [3H] clonidine (Amersham), epinephrine
(Adrenalinum hydrochloricum, Polfa), norepinephrine (Levonor,
Polfa), methoxamine (Sigma-Aldrich Chemie GmbH), [3H] pra-
zosin (Amersham), tyramine (Sigma-Aldrich Chemie GmbH),
sodium heparin (Polfa), thiopental sodium (Biochemie GmbH,
Vienna).
6.1.3.4. 1-{2-Hydroxy-3-[4-(2-ethoxy-phenyl)-piperazin-1-yl]-propyl}-
3,3-diphenylpyrrolidin-2-one dihydrochloride (7). Yield: 58.8%; Anal.
calc. C31H37N3O3 ꢂ 2 HCl (572.56) calc. C% 65.03%, H% 6.87, N% 7.34,

4002
K. Kulig et al. / European Journal of Medicinal Chemistry 44 (2009) 3994–4003
6.2.2. Animals
yohimbine (0.1
mM) and propranolol (1 mM) to block a2- and
The experiments were carried out on male Wistar rats (180–
250 g). Animals were housed in constant temperature facilities,
exposed to a 12/12 h light–dark cycle and maintained on a standard
pellet diet and tap water which was given ad libitum. Control and
experimental groups consisted of 8–10 animals in each group. All
procedures were carried out according to the Animal Care and Use
Committee Guidelines and approved by the Ethical Committee of
b
-adrenoceptors [37].
6.2.6.2. a1B-Adrenoceptors: guinea pig spleen. Male guinea pigs
(350–400 g) were killed by cervical dislocation. The spleen was
removed and cut longitudinally into strips of approximately
20 mm, which were set up in a 30 mL organ bath under a resting
tension of 1 g to record isometric contractile responses in Krebs–
Henseleit solution of the above composition at 37 ꢁC and pH 7.4
with constant oxygenation (O2/CO2, 19:1). During an equilibration
´
the Jagiellonian University, Krakow, Poland.
6.2.3. Reference compounds
period of 100 min, tissues were stimulated with NA (0.1–10 mM)
Compound I was used as a reference.
followed by washout until the contractile response had become
constant. Two cumulative concentration–response curves to NA
were determined for each tissue at an interval of 60 min in the
absence and presence of antagonist. Antagonists were incubated
for 30 min. Experiments were conducted in the presence of
6.2.4. Statistical analysis
The data were expressed as mean ꢀ S.E.M. The statistical
significance was calculated using one-way ANOVA. Differences
were considered significant when p < 0.05.
continuous propranolol (1 mM) to block b-adrenoceptors [38].
Concentration–response curves were analyzed 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) were expressed as a percentage of
the maximal noradrenaline effect (Emax ¼ 100%), reached in the
concentration–response curves obtained before incubation with
the tested compounds. Data were expressed as the mean ꢀ SEM of
at least 5 separate experiments. Curves were fitted to all the data by
non-linear regression to determine Hill slopes for the agonist
concentration–response curves, and to calculate EC50 values. The
EC50 value in the presence and absence of antagonists was used to
determine the concentration ratio (CR). Schild analysis was per-
formed, and where the slope was not significantly different from
unity, the pA2 value was determined by plotting the log (CR-1)
against the ꢃlog of antagonist concentration [38].
6.2.5.
a-Adrenoceptor radioligand binding assay
These experiments were carried out on rat cerebral cortex. [3H]
prazosin (19.5 Ci/mmol, an a1-adrenergic receptor antagonist) and
[3H] clonidine (70.5 Ci/mmol, an a2-adrenergic receptor agonist)
were used. Rat brains were homogenized in 20 volumes of ice-cold
50 mM Tris–HCl buffer (pH 7.6) and were centrifuged at 20 000 ꢂ g
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, USA). The final incu-
bation mixture (final volume 300
membrane suspension, 30
L of [3H] prazosin (0.2 nM) or [3H]
clonidine (2 nM) solution and 30 L of the buffer containing seven
to eight concentrations (10ꢃ11–10ꢃ4 M) of the tested compounds.
To measure unspecific binding, phentolamine, 10 M (in the case of
[3H] prazosin), or [3H] clonidine, 10 M (in the case of [3H] cloni-
mL) consisted of 240 mL of the
m
m
m
m
dine), was applied [27]. The incubation was terminated by rapid
filtration over glass fiber and placed in scintillation vials with
a liquid scintillation cocktail. Radioactivity was measured in
a WALLAC 1409 DSA liquid scintillation counter (PerkinElmer, USA).
All assays were carried out in duplicate. Radioligand binding was
analyzed using an iterative curve-fitting routine (GraphPAD/Prism,
Version 4.0 – San Diego, CA, USA). Ki values were calculated by the
method of Cheng and Prusoff [28].
6.2.7. Prophylactic antiarrhythmic activity in a model of
adrenaline-induced arrhythmia according to Szekeres and Papp [29]
Arrhythmia was evoked in thiopental (60 mg/kg, ip) – anaes-
thetized rats by i.v. injection of adrenaline (20 mg/kg). The tested
compounds were administered intravenously 15 min before
adrenaline. The criterion for antiarrhythmic activity was the lack of
premature beats and the inhibition of rhythm disturbances in
comparison with the control group (ventricular bradycardia,
atrioventricular block, ventricular tachycardia or ventricular
fibrillation). The cardiac rhythm disturbances were recorded for
15 min after adrenaline injection. ECGs were analyzed according to
the guidelines of the Lambeth Convention [39] on ventricular
premature beats (VBs), bigeminy, salvos (less than four successive
VBs), ventricular tachycardia (VT, four or more successive VBs) and
ventricular fibrillation (VF).
6.2.6. In vitro functional studies
6.2.6.1. a1D-Adrenoceptors: rat thoracic aorta. Male Wistar rats
weighing 200–350 g were anaesthetized with thiopental sodium
(75 mg/kg ip) and the aorta was dissected and placed in 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, and EDTA 0.05 mM) and surrounding
fat tissues were cleaned. 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 Krebs–
Henseleit solution 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 aorta rings were stretched and maintained at
an optimal tension of 2 g and allowed to equilibrate for 3 h.
During the equilibration period, the preparations were stimu-
6.2.8. The influence on blood pressure
Normotensive male Wistar rats were anaesthetized with thio-
pental (50–75 mg/kg, ip). The right carotid artery was cannulated
with a polyethylene tube filled with heparin in saline to facilitate
pressure measurement using the Datamax apparatus (Columbus
Instruments, USA). The studied compounds were injected in
a single dose of 2.5 or 20 mg/kg or into the caudal vein after a 5 min
stabilization period at a volume equivalent to 1 mL/kg.
6.3. Molecular modeling
lated three times with noradrenaline (NA) (0.3 mM). Two cumu-
lative concentration–response curves to NA were determined for
each arterial ring at an interval of 60 min in the absence and
presence of antagonist. Antagonists were incubated for 30 min.
Experiments were conducted in the presence of continuous
To identify the effect of substituents on the antiarrhythmic and
hypotensive activities of novel pyrrolidin-2-one derivatives a confor-
mational analysis was preformed. An earlier report suggested that
1-[2-hydroxy-3-(4-phenylpiperazin-1-yl)propyl]-pyrrolidin-2-one (I)

K. Kulig et al. / European Journal of Medicinal Chemistry 44 (2009) 3994–4003
4003
[10] G. Chiu, S. Li, P.J. Connolly, V. Pulito, J. Liu, S.A. Middleton, Bioorg. Med. Chem.
Lett. 18 (2008) 640–644.
[11] O.M. Becker, Y. Marantz, S. Sharam, B. Ibal, A. Heifetz, O. Kalid, S. Bar-Haim,
D. Warshaviak, M. Fichman, S. Noiman, Proc. Natl. Acad. Sci. U.S.A. 101 (2004)
11304–11309.
[12] A. Leonardi, D. Barlocco, F. Montesano, G. Cignarella, G. Motta, R. Testa, E. Poggesi,
M. Seeber, P.G. De Benedetti, F. Fanelli, J. Med. Chem. 47 (2004) 1900–1918.
[13] M.-Y. Li, H. Fang, L. Xia, Bioorg. Med. Chem. Lett. 15 (2005) 3216–3219.
[14] E. Patane, V. Pittala, F. Guerrera, L. Salerno, G. Romeo, M.A. Siracusa, F. Russo,
F. Manetti, M. Botta, I. Mereghetti, A. Cagnotto, T. Mennini, J. Med. Chem. 48
(2005) 2420–2431.
[15] M. Lopez-Rodriguez, M.J. Morcillo, E. Fernandez, B. Benhamu, I. Tejada,
D. Ayala, A. Viso, M. Campillo, L. Prado, M. Delgado, J. Manzanares, J. Fuentes,
J. Med. Chem. 48 (2005) 2548–2558.
[16] M.A.H. Ismail, M.N.Y. Aboul-Enein, K.A.M. Abouzid, R.A.T. Serya, Bioorg. Med.
Chem. 14 (2006) 898–910.
[17] L. Betti, M. Zanelli, G. Ginnaccini, F. Manetti, S. Schenone, G. Stappaghetti,
Bioorg. Med. Chem. 14 (2006) 2828–2836.
[18] R. Barbaro, L. Botti, M. Botta, F. Corelli, G. Giannaccini, L. Maccari, F. Manetti,
G. Strappaghetti, S. Corsano, J. Med. Chem. 44 (2001) 2118–2132.
[19] J.H. Bremner, B. Coban, R. Griffith, K.M. Groenewoud, B.F. Yates, Bioorg. Med.
Chem. 8 (2000) 201–214.
[20] F. Gentili, M. Pigini, A. Piergentili, M. Giannella, Curr. Top. Med. Chem. 7 (2007)
163–186.
[21] P.-A. Crassous, C. Denis, H. Paris, J.M. Senard, Curr. Top. Med. Chem. 7 (2007)
187–194.
[22] M.R. Marien, F.C. Colpaert, A.C. Rosenquist, Brain Res. Rev. 45 (2004) 38–78.
[23] A. Saina, J. Lindsay, E.-G. Ahmed, Ann. Thorac. Surg. 85 (2008) 1483–1489.
[24] B. Malawska, K. Kulig, B. Filipek, J. Sapa, D. Macia˛g, M. Zygmunt, L. Antkiewicz-
Michaluk, Eur. J. Med. Chem. 37 (2002) 183–195.
[25] B. Malawska, K. Kulig, A. Gippert, B. Filipek, J. Sapa, D. Macia˛g, Il Farmaco 60
(2005) 793–803.
[26] K. Kulig, J. Sapa, D. Macia˛g, B. Filipek, B. Malawska, Arch. Pharm. Chem. Life Sci.
340 (2007) 466–475.
[27] J. Maj, V. Klimek, G. Nowak, J. Pharmacol. 119 (1985) 113–116.
[28] Y.C. Cheng, W.H. Prusoff, Biochem. Pharmacol. 22 (1973) 3099–3108.
[29] L. Szekeres, G. Papp, in: J. Schimier, O. Eichler (Eds.), Handbook of Experi-
mental Pharmacology, Springer Verlag, Berlin, Heidelberg, New York, 1975.
[30] M. Thiyagarajan, Pharmacology 65 (2002) 119–128.
[36] and 1-[2-hydroxy-3-(4-diphenylmethylpiperazin-1-yl)propyl]-
pyrrolidin-2-one (II) [24] were model structures for the current
investigations. The core for the synthesis of new species comprised
a skeleton of 1-[2-hydroxy-3-(4-phenylpiperazin-1-yl)propyl]-3,3-
diphenylpyrrolidin-2-one (4) containing the 4-(para-substituted
phenyl)piperazin-1-yl moiety (6, 10) or the 4-(ortho-substituted
phenyl)piperazin-1-yl moiety (5, 7, 8, 9).
For the molecular 3D structure calculations, the Gaussian 03
program [40] was used. Each compound was subjected to the
following calculation procedure. Firstly, three-dimensional struc-
tures of the pyrrolidin-2-one derivatives at their neutral state were
obtained through full optimization based on the AM1 quantum
chemical procedure. Force field calculations were used to ascertain
whether the resulting structures were energy minima. All the
molecules were minimized until the root mean square (RMS)
gradient value was smaller than 10ꢃ6 a.u. Then the single point
energy (SPE) calculations were performed at the DFT/B3LYP level of
theory using the 6-31G** basis set. Later, using the surface data
generated from Gaussian checkpoint files, and the GaussView 4.1
program [41], the distribution of charge in a molecule was calcu-
lated. To obtain a 2D contour plot of the MEP, the electrostatic
potential cube file was calculated from total SCF density. The
contours were then drawn using the following parameters: the two
grid direction fields specified as ꢃ15 to 15 Å, the distance between
the grid point of 0.1 Å, and the isovalues were 0.001. The plane in
the contour was determined by the same point as was used to
define the pharmacophore model (i.e. A – the center of the phenyl
ring, B – the less negative ionizable nitrogen atom in the piperazine
ring and C – the hydrogen bond acceptor). All computations were
performed on a HP-6200 wx workstation.
[31] T. Kenakin, S. Jenkinson, C. Watson, J. Pharmacol. Exp. Ther. 319 (2006) 710–723.
[32] M. Maze, C.M. Smith, Anesthesiology 59 (1983) 322.
[33] G.E. Billman, J. Pharmacol. Exp. Ther. 269 (1994) 409–416.
[34] P.B. Corr, K.A. Heathers, K.A. Yamada, Am. J. Med. 87 (Suppl. 2A) (1989) 19S.
[35] O.E. Brode, H. Bruck, K. Leineweber, J. Pharm. Sci. 100 (2006) 323–337.
[36] B. Filipek, J. Sapa, B. Malawska, K. Kulig, L. Antkiewicz-Michaluk, Arch. Pharm.
Pharm. Med. Chem. 330 (1997) 225–231.
[37] M. Eltze, R. Boer, M.C. Michel, P. Hein, R. Testa, W.R. Ulrich, N. Kolassa,
K.H. Sanders, Naunyn Schmiedebergs Arch. Pharmacol. 363 (2001) 649–662.
[38] O. Arunlakshana, H.O. Schild, Br. J. Pharmacol. 14 (1959) 48–52.
[39] M.J. Walker, M.J. Curtis, D.J. Hearse, R.W. Campbell, Cardiovasc. Res. 22 (1988)
447–452.
Acknowledgements
The authors would like to thank Prof. Dr. hab Gabriel Nowak and
Ms. Ma1gorzata Dyba1a for implementation of radioligand studies.
The study was supported by the Polish Ministry for Science and
High Education, grant 3 P 05 F 03622.
[40] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman,
J.A. Montgomery Jr., T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar,
J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A. Petersson,
H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida,
T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J.E. Knox, H.P. Hratchian,
J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann,
O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala,
K. Morokuma, G.A. Voth, P. Salvador, J.J. Dannenberg, V.G. Zakrzewski,
S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck,
K. Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul, S. Clifford,
J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi,
R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara,
M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen, M.W. Wong, C. Gonzalez,
J.A. Pople, Gaussian 03, Revision D.01, Gaussian, Inc., Wallingford, CT, 2004.
[41] GaussView, Version 4.1, Semichem, Inc., Shawnee Mission, KS, 2003.
[42] J.T. Litchfield, F.A. Wilcoxon, J. Pharmacol. Exp. Ther. 96 (1949) 99–113.
References
[1] A.D. Lopez, C.D. Mathers, M. Ezzati, D.T. Jamison, C.L. Murray, Lancet 367
(2006) 1747–1757.
[2] W.B. White, Am. J. Med. 118 (2005) 695–705.
[3] J. Varon, Drugs 68 (2008) 283–297.
[4] A. Ferro, R. Gilbert, H. Krum, Int. J. Clin. Pract. 60 (2006) 577–581.
[5] F.H. Messerli, B. Williams, E. Ritz, Lancet 370 (2007) 591–603.
[6] K.S. Jain, J.B. Bariwal, M.K. Kathiravan, M.S. Phoujdar, R.S. Sahne, B.S. Chauhan,
A.K. Shah, M.R. Yadav, Bioorg. Med. Chem. 16 (2008) 4759–4800.
[7] T. Koshimizu, A. Tanoue, A. Hirasawa, J. Yamauchi, G. Tsujimoto, Pharmacol.
Ther. 98 (2003) 235–244.
[8] M.A. Patane, A.L. Scott, T.P. Broten, R.S.L. Chang, R.W. Ranson, J. DiSalvo,
C. Forray, M.G. Bock, J. Med. Chem. 41 (1998) 1206–1208.
[9] C.R. Chapple, BJU Int. 94 (2004) 738–744.