Department of Bioorganic Chemistry, Chair of Organic Chemistry1; Department of Pharmacodynamics2; Department
of Cytobiology and Histochemistry, Laboratory of Pharmacobiology3, Faculty of Pharmacy Medical College; Faculty of
Chemistry4, Jagiellonian University Krakow, Poland
Synthesis, ?-adrenoceptors affinity and ?1-adrenoceptor antagonistic
properties of some 1,4-substituted piperazine derivatives
H. Marona1, M. Kubacka2, B. Filipek2, A. Siwek3, M. Dybała3, E. Szneler4, T. Pociecha1, A. Gunia1,
A. M. Waszkielewicz1
Received March 24, 2011, accepted April 25, 2011
Dr. Anna M. Waszkielewicz, Department of Bioorganic Chemistry, Chair of Organic Chemistry, Faculty of Pharmacy,
Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Krakow, Poland
Pharmazie 66: 733–739 (2011)doi: 10.1691/ph.2011.1543
A series of different 1,4-substituted piperazine derivatives (1–11) was synthesized. It comprised 1-
(substituted-phenoxyalkyl)-4-(2-methoxyphenyl)piperazine derivatives (1–5); 1,4-bis(substituted-phenoxy-
ethyl)piperazine derivatives (6–8) and 1-(substituted-phenoxy)-3-(substituted-phenoxyalkylpiperazin-1-
yl)propan-2-ol derivatives (9–11). All compounds were evaluated for affinity toward ?1- and ?2-receptors by
radioligand binding assays on rat cerebral cortex using [3H]prazosin and [3H]clonidine as specific radioli-
gand, respectively. Furthermore ?1-antagonistic properties were checked for most promising compounds
(1–5 and 10) by means of inhibition of phenylephrine induced contraction in isolated rat aorta. Antago-
nistic potency stayed in agreement with radioligand binding results. The most active compounds (1–5)
displaced [3H]prazosin from cortical binding sites in low nanomolar range (Ki=2.1−13.1nM). Compound
antagonistic activity with pA2 values ranging from 8.441 to 8.807. Compound 1 gave a pA2 value of
7.868, while compound 10 showed the weakest antagonistic potency, giving a pA2 value of 6.374. 1-
[3-(2-Chloro-6-methylphenoxy)propyl]-4-(2-methoxyphenyl)piperazine hydrochloride (5) showed the best
?1- affinity properties with a Ki(?1) value of 2.1nM and it was 61.05 fold more selective toward ?1than ?2-
receptors. The best properties showed 1-[3-(2,6-dimethylphenoxy)propyl]-4-(2-methoxyphenyl)piperazine
hydrochloride (4) with a Ki(?1) value of 2.4nM, a 142.13 fold better selectivity to ?1- over ?2-adrenoceptors
and the best antagonistic potency (pA2=8.807). It is worth to emphasized that all most promising com-
pounds possessed an 1-(o-methoxyphenyl)piperazine moiety which probably plays an important role in
the affinity to ?-adrenoceptors.
coupled seven-transmembrane spanning receptors. They are
divided into three subclasses: ?1, ?2and ?, and further into sev-
eral subtypes (i.e. ?1A,?1B,?1D,?2A, ?2B, ?2C, ?1, ?2,?3). The
classification is based on structural similarity, localization and
pharmacology as well as on cloning techniques (Bishop 2007;
Docherty 1998; Gauthier et al. 2007). The ?-adrenoceptors are
involved in central and peripheral nervous system functions
(Gyires et al. 2009) while ?-adrenoceptors are mainly responsi-
ble for the physiological function of the cardiovascular system,
respiratory tract and of fat tissue (Johnson 2006). More detailed
?1receptors roles are connected with vascular smooth muscle
contraction, increasing blood pressure, dilation of the pupil, the
human prostate smooth muscle contraction (Jain et al. 2008)
as well as regulation of cerebral microcirculation (Yokoo et al.
intestinal secretion, aggregation of the platelets and inhibition
of neurotransmitters release (Jain et al. 2008; Giovannoni et al.
2009; Gyires et al. 2009).
Considering the physiological function of ?-adrenoceptors lig-
ands both agonist and antagonists of those receptors were intro-
tions. ?1-Antagonists have been used with considerable success
in the treatment of hypertension over the past two decades. It
as overactivity of the symphatetic nervous system constitutes a
common phenomenon in resistant forms of hypertension. Addi-
tively ?1-antagonists are currently first-line therapy for lower
hyperplasia (BPH). They improve both symptom score and uri-
nary flow in that condition. It is worth to mention that selective
urinary ?1-blockers (?1A/?1D) constitute important treatment
tools for hypertensive patients with BPH since such patients
are best treated by the separate management of each condition
this role is currently not important. However new fields of their
application have occurred, particularly in the management of
pain and anesthesia (Crassous et al. 2007; Gyires et al. 2009).
In this context, the search for ?-adrenoceptor antagonists con-
stitutes an important area in medicinal chemistry. Furthermore,
Pharmazie 66 (2011)733
Scheme 1: Synthesis of compounds 1–8
et al. 2007).
In recent years various new adrenergic receptor ligands have
been synthesized. A large group of them contains an arylpiper-
azine moiety and more detailed – a phenylpiperazine moiety
(Betti et al. 2004; Bremner et al. 2000; Ismail et al. 2006;
Pittalá et al. 2006). The encouraging fact is that potent
?-receptors antagonists – prazosin, terazosin and doxazosin,
which have a well established position in pharmacotherapy,
posses a structural fragment of piperazine (Heran et al. 2009).
It is also worth to mention other ?-receptors blockers urapidil
moiety (Cherney and Straus 2002; Muramatsu et al. 1991).
activity of compounds in the group of 1,4-substituted piper-
azine derivatives that proved ?1- and ?2-adrenoceptors binding
properties (Maci˛ ag et al. 2003, 2008; Marona et al. 2008).
The best results showed aroxyethylpiperazine derivatives e.i.
dihydrochlorides with Kivalues related to inhibiting in vitro
[3H]prazosin binding to cortical ?1-receptors of 26.0±6.9,
87.5±19.0, 84.6±16.1nM and Kivalues related to inhibit-
ing in vitro [3H]clonidine binding to cortical ?2-receptor of
197.9±40.8, 252.3±49.7, 282.1±39.0nM, respectively. At
the same time all substances proved ?1antagonistic properties
by showing hypotensive activity in normotensive rats. The sub-
stances showed also ?1-receptors binding properties as well as
antiarrhythmic activity (Maci˛ ag et al. 2003).
Taking into consideration those facts and our former experience
we synthesized a series of 1,4-substituted piperazine deriva-
tives. Among them there were both aroxyethyl and aroxypropyl
derivatives of piperazine, in addition compounds 9, 10 and
11 contained a structural moiety of propranolol while com-
pounds 1–5 and 10 possessed a 1-(o-methoxyphenyl)piperazine
fragment in their molecule. All compounds were subjected
to preliminary pharmacological screening. The present paper
reports on the results of their in vitro affinity to ?1 and
?2-adrenoceptors as well as their antagonistic properties at ?1-
adrenoceptors on isolated rat aorta.
2. Investigations and results
2.1. Chemical synthesis
Synthesis of compounds 1–8 is shown in Scheme 1.
Compounds 1–5 were obtained in reaction between 1-
(2-methoxyphenyl)piperazine and appropriate phenoxyalkyl
bromides carried out in toluene in the presence of anhydrous
734Pharmazie 66 (2011)
Scheme 2: Synthesis of compounds 9–11
potassium carbonate according to well known procedures
(Maci˛ ag et al. 2003; Marona et al. 2004). Compounds 6–8
were synthesized in multistep reaction according to earlier
published methodology (Marona et al. 2004, 2009). At first
appropriate phenoxyethyl bromides were used in the reaction
potassium carbonate, intermediate products were not separated.
Hydrolysis in 85% potassium hydroxide of derivatives gave the
corresponding 1-[2-(phenoxyethyl)piperazine]. The raw prod-
ucts were used in further reaction with another appropriate
phenoxyethyl bromides. Bases received as oils were converted
into hydrochlorides using an excess of ethanol saturated with
gas hydrochloride. Raw salts were recrystallized from ethanol.
Compounds 9–11 were obtained according to well known
procedures via epoxy derivatives (Hlaváˇ cová et al. 1995;
Marona et al. 1997, 1998; Mlynárova et al. 1996). The
way of synthesis is presented in Scheme 2. At first
2,6-dimethylphenol was used in the reaction with (+/-)-
epichlorohydrin in alkaline medium. Then the crude oil product
i.e. (+/-)-1,2-epoxy-3-(2,6-dimethylphenoxy)propane was used
in the reaction of aminolyzis by means of an appropriate 2-
(phenoxyethyl)piperazine carried out in n-propanol. Received
crude oil bases were converted into hydrochlorides using an
excess of ethanol saturated with HCl. Raw salts were recrystal-
lized from ethanol/acetone mixture (1:1).
2.2.1. Radioligand binding results
Compounds 1–12 were tested for their in vitro affinity toward
?1- and ?2-adrenoceptors on rat cerebral cortex by radioligand
binding assays using [3H]prazosin and [3H]clonidine as spe-
cific radioligands (Maj et al. 1985). The affinities described by
Kivalues [nM] are shown in Table 1. Selectivity toward ?1-AR
with respect to ?2-AR was calculated as Ki?2/Ki?1. Compounds
1–5 displaced [3H]prazosin from cortical binding sites in low
nanomolar range (Ki=2.1−13.1nM). Compound 10 showed a
Pharmazie 66 (2011)
slightly lower affinity for the ?1-adrenoceptor (Ki=781nM),
whereas other compounds (6–9, 11, 12) had a low affinity for
both ?1- and ?2-adrenoceptors, moderately inhibiting radioli-
gands binding in the ?M-range. The most selective compounds
(4 and 5) showed a 142.1- and 61-fold higher affinity for ?1-
than for ?2-adrenoceptors.
2.2.2. Functional bioassays results
The antagonist activity of compounds 1–5 and 10 toward
?1-adrenoceptors present in rat aorta from adult Wistar rats
was assessed by inhibition of phenylephrine induced contrac-
tions. The investigated compounds, concentration-dependently,
shifted the phenylephrine response to the right. For all test com-
pounds Schild slopes did not differ significantly from unity,
Table 1: Binding properties of compounds 1–11
Inhibition constants (Ki) were calculated according to the equation of Cheng and Prusoff. Radioli-
gands binding assays to rats cortex membrane using [3H]-prazosin (?1) and [3H]-clonidine (?2),
Table 2: Functional bioassays results for selected compounds
Antagonist potency of selected compounds, expressed as pA2±SEM values, in isolated rat thoracic
aorta (?1-AR) pA2values were obtained from the linear regression of Schild plot
Each value was the mean±SEM of 5–8 experimental results
and thus allowing for the determination of the pA2values. The
obtained results are shown in Table 2 as well as Figs. 1 and 2.
The strongest antagonistic activity occurred with compounds
2–5 with pA2values ranging from 8.441 to 8.807. Compound
1 gave a pA2 value of 7.868 which was comparable with
the pA2value obtained for the reference compound, urapidil
(pA2=7.334). Compound 10 showed the weakest antagonistic
potency, giving a pA2value of 6.374. It is worth to note that
the affinity from the functional test for all test compounds and
urapidil was in the same concentration range as determined in
radioligand binding assay.
The aim of this study was to evaluate ?1and ?2-adrenoceptors
affinity of 11 different 1,4-substituted piperazine derivatives.
We focused on piperazine derivatives bearing in mind literature
data (Betti et al. 2004; Bremner et al. 2000; Ismail et al. 2006;
Pittalá et al. 2006) and our former research (Maci˛ ag et al.
2003, 2008; Marona et al. 2008). Compounds which are pre-
sented in this work can be divided into three chemically
different groups namely: I-1-(substituted-phenoxyalkyl)-4-
(2-methoxyphenyl)piperazine derivatives (compounds 1–5); II-
1,4-bis(substituted-phenoxyethyl)piperazine derivatives (com-
pounds 6–8) and III-1-(substituted-phenoxy)-3-(substitutedph-
enoxyalkylpiperazin-1-yl)propan-2-ol derivatives (compounds
Among the tested compounds the values of inhibition constants
(Ki) to ?1-adrenergic receptors ranged from 2.1 to 9600nM.
The best affinity was characteristic for the group I, where all
compounds possessed Kivalues at the level of a few nM. All of
them had an o-methoxyphenyl piperazine moiety. Our research
is consistent with the notion that this structural fragment exerts
Taking into consideration the structure of the compounds 1 – 5
it could be stated that the substitution in phenyl ring with either
2 methyl groups or with 1 methyl group and the chlorine atom
did not play an important role in the activity. On the other hand
there was a slight difference in the affinity of the compounds
possessing either a 2 or a 3 carbon atoms linker wherein the
latter seemed to be favorable.
Compound 10 is the only one in group III which had an o-
methoxyphenyl moiety but it was not directly connected with
piperazine. Its Ki(?1) value was in the middle between those
for most and least active substances. Considering results for the
structures 9 – 11 it was once again proved that the substitution
in a phenyl ring in ortho position is beneficial as compared to
meta as well as no substitution.
towards ?1-AR compared to ?2-AR (0.02 to 142.13-fold). The
1 30 nM
1 100 nM
1 300 nM
log [Phenylephrine] (M)
2 30 nM
2 100 nM
2 300 nM
log [Phenylephrine] [M]
3 10 nM
3 30 nM
3 100 nM
log [Phenylephrine] (M)
Fig. 1: Concentration-response curves to phenylephrine in the rat aorta in the
absence (?) or presence of a) 1 (? 30, ? 100 and ? 300nM); b) 2 (? 30,
?100 and ?300nM); c) 3 (? 10, ? 30 and ? 100nM). Results are expressed
as percentage of the maximal response to phenylephrine in the first
concentration-respose curve. Each point represents the mean±SEM
most interesting results are characteristic for group I, where all
substances possessed very good affinity to ?1-adrenoceptors.
Only compound 4 displayed noticeable selectivity. Consider-
ing the chemical structure it was the only one from the group
which possessed a 2,6-dimethyl substituted phenyl ring. The
results could suggest that this kind of substitution is favourable
in case of ?1as compared to ?2selectivity. Comparing com-
pounds 2 and 5, the first showed almost the same affinity to
both kind of adrenergic receptors while the second was more
selective towards ?1-AR. The only difference in their structures
was a linker consisting of 2 or 3 carbon atoms, respectively. The
affinities of compounds 1 and 3 did not confirm the relationship
between the number of carbon atoms in the linker and selec-
736Pharmazie 66 (2011)
-8-7 -6-5 -4
4 10 nM
4 30 nM
4 100 nM
log [Phenylephrine] (M)
-8 -7-6-5 -4
5 10 nM
5 30 nM
5 100 nM
log [Phenylephrine] (M)
10 1 µM
10 3 µM
10 10 µM
log [Phenylephrine] (M)
???????? ???? ??
???????? ?? ??
Fig. 2: Concentration-response curves to phenylephrine in the rat aorta in the
absence (?) or presence of a) 4 (? 10, ? 30 and ? 100nM); b) 5 (? 10, ? 30
and ? 100nM); c) 10 (? 1, ? 3 and ? 10?M); d) urapidil (? 0.1, ? 0.3 and
? 1?M). Results are expressed as percentage of the maximal response to
phenylephrine in the first concentration-respose curve. Each point represents
the mean±SEM (n=4–8).
tivity. In group III all compounds were highly more selective
towards ?2-adrenoceptors. In group II surprisingly compound
6 displayed significant selectivity toward ?2compared to ?1
but there was no noticeable relationship between structure and
Based on the radioligand binding results, the 6 most promis-
ing compounds were evaluated for their interaction with
?1-adrenoceptors in functional bioassays. The results showed
?1-AR antagonistic activity for all the tested compounds.
All substances displayed competitive interaction with ?1-
characteristic for compound 4 which at the same time showed
the highest ?1/?2selectivity.
The outcome of pharmacological tests confirmed that among
1-(o-methoxyphenyl)piperazine derivatives the affinity to ?-
adrenoceptors could be expected, however, the proposed
was left unchanged (compounds 1–5), the values of affinity to
?1-adrenergic receptors (Ki) were at the level of a few nanomol
(2.1 – 5.1nM). The affinity was even better than that of the ref-
erence compound – urapidil. It was also proved that compounds
2–5 possessed good antagonistic activity to the receptors. The
ther research regarding pharmacological properties of the most
were uncorrected. Analyses of percentage content of carbon, hydrogen and
nitrogen were within 0.4% of the theoretical values. Thin-layer chromatog-
raphy was performed on precoated aluminium sheets (silica gel 60 F254,
Merck) using as a mobile phase a mixture of methanol and ethyl acetate in
volume ratio 1:1. The results were visualized by means of ultraviolet light.
The proton magnetic resonance spectra were recorded on Bruker Avance II
500, Bruker Avance II 300 or Bruker AMX 500 spectrometer using TMS
as an internal standard. Compounds were dissolved in DMSO-d699.8% in
300K. The results were presented in the following format: chemical shift
? (ppm), multiplicity, coupling constants (J) values in Hertz (Hz), number
of protons, proton’s position (where “pip” indicates piperazin, “a”-axial,
“e”-equatorial). Multiplicities were shown as the abbreviations: s (singlet),
bs (broad singlet), d (doublet), ddq (double doublet of quintets), m (multi-
plet). The IR spectra were recorded on a Jasco FT/IR 410 apparatus using
KBr pellets and are reported in cm−1. The theoretical values of the parti-
tion coefficient (LogP) of the tested compounds were calculated by means
of ACDLABS 12.0 program. Reagents were purchased from Alfa Aesar
commercially available materials of reagent grade.
piperazine hydrochloride (1)
3.90 (s, 3H, Ar-O-CH3), 4.45 (t, J=5.0Hz, 2H, Ar-O-CH2-), 6.78−7.11
(m, 7H, Ar), 11.71 (bs, 1H, NH+); IR 3434.60, 2976.59, 2327.66, 1460.81,
1262.18. 756.92; LogP=4.55+/− 0.44; Rf=0.84
piperazine dihydrochloride (2)
(m, 4H, pip(e)), 3.49−3.77 (m, 6H, pip(a)+-CH2-NH+), 3.81 (s, 3H, Ar-O-
CH3), 4.38 (t, J=5.2Hz, 2H, Ar-O-CH2-), 6.87−7.38 (m, 7H, Ar), 11.73
(bs, 1H, NH+); IR 3433.64, 2980.45, 2335.37, 1458.89, 1262.18, 1024.98,
767.53; LogP=4.64+/− 0.51; Rf=0.84
Pharmazie 66 (2011)737
piperazine hydrochloride (3)
Yield: 59%. M.p.=222–224◦C;1H NMR 2.10 (s, 3H, Ar-CH3), 2.22 (s,
3H, Ar-CH3), 2.23−2.32 (m, 2H, -CH2-CH2-CH2-), 3.02−3.33 (m, 6H,
pip(e))+-CH2-NH+), 3.44−3.69 (m, 4H, pip(a)), 3.80 (s, 3H, Ar-O-CH3),
4.03 (t, J=5.9Hz, 2H, Ar-O-CH2-), 6.75−7.08 (m, 7H, Ar), 11.03 (bs,
piperazine hydrochloride (4)
Yield: 65%. M.p.=216–218◦C;
CH3), 2.25−2.33 (m, 2H, -CH2-CH2-CH2-), 3.14−3.30 (m, 4H, pip(e)),
3.33−3.42 (m, 2H, -CH2-NH+), 3.47−3.65 (m, 4H, pip(a)), 3.81 (s, 3H,
Ar-O-CH3), 3.82 (t, J=6.0Hz, 2H, Ar-O-CH2-), 6.89−7.08 (m, 7H, Ar),
11.48 (bs, 1H, NH+); IR 3409.53, 2959.23, 2349.84, 1461.78, 1263.15,
1200.47, 778.14; LogP=4.99+/− 0.44; Rf=0.84
1H NMR 2.24 (s, 6H, Ar-CH3,Ar-
piperazine hydrochloride (5)
Yield: 58%. M.p.=199–201◦C;1H NMR 2.30 (dd, J=0.8Hz, J=0.6Hz,
3H, Ar-CH3), 2.24−2.39 (m, 2H, -CH2-CH2-CH2-), 3.19−3.67 (m, 10H,
pip+-CH2-NH+), 3.81 (s, 3H, Ar-O-CH3), 3.96 (t, J=5.9Hz, 2H, Ar-O-
CH2), 6.89−7.05 (m, 4H, Ar), 7.06 (dd, J=7.8Hz, J=7.7Hz, 1H, Ar(5),
1463.71, 1264.11, 772.35; LogP=5.07+/− 0.51; Rf=0.77
piperazine dihydrochloride (6)
Yield: 60%. M.p.=218–220◦C;1H NMR 2.31 (s, 3H, Ar-CH3), 3.14−4.21
(m, 12H, N-CH2), 4.43 (t, J=4.9Hz, 2H, Ar-O-CH2-), 4.52 (t, J=4.5Hz,
2H, Ar-O-CH2-), 6.77−7.40 (m, 8H, Ar), 12.34 (bs, 1H, NH+); IR 3433.64,
2974.66, 2244.74, 1490.70, 1459.85, 1246.75, 759.82; LogP=4.03+/−
piperazine dihydrochloride (7)
Yield: 63%. M.p.=226–228◦C;1H NMR 2.31 (s, 3H, Ar-CH3), 3.30−3.97
(m, 12H, N-CH2), 4.40 (t, J=5.0Hz, 2H, Ar-O-CH2-), 4.41 (t, J=5.0Hz,
2H, Ar-O-CH2-), 6.83−7.40 (m, 8H, Ar), 12.17 (bs, 1H, NH+); IR
3433.64, 2380.48, 1242.90, 750.17; LogP=4.17+/− 0.40; Rf=0.61;
C21H29N2O2Cl3, M = (447.86).
methylphenoxy)ethyl]piperazine dihydrochloride (8)
4.42 (t, J=4.7Hz, 2H, Ar-O-CH2-), 7.39−6.82 (m, 6H, Ar), 12.32 (bs, 1H,
-NH+); IR 3432.67, 2360.44, 1479.13, 1174.44, 762.71; LogP=5.18+/−
piperazin-1-yl]propan-2-ol dihydrochloride (9)
Yield: 64%. M.p.=214–216◦C;
3.23−4.10 (m, 14H, -O-CH2+ =N-CH2,=N-CH2, pip), 4.44 (bs, 3H, -O-
IR 3374.82, 2928.28, 2375.87, 1476.24, 1200.47. 752.10; LogP=3.89+/−
1H NMR 2.25 (s, 6H, Ar-(CH3)2),
ethyl]-piperazin-1-yl}propan-2-ol dihydrochloride (10)
Yield: 61%. M.p.=215–217◦C;
3.27−4.11 (m, 14H, (-CH2-NH+)+CH2-O-), 3.79 (s, 3H, Ar-O-CH3),
4.38−4.50 (m, 3H, Ar-O-CH2-, CH-OH), 6.03 (bs, 1H, OH), 7.09−6.89
1H NMR 2.25 (s, 6H, Ar-(CH3)2,
(m, 7H, Ar), 11.64 (bs, 1H, NH+), 12.37 (bs, 1H, NH+); IR 3239.82,
2957.30, 2308.37, 1508.06, 1254.47, 1023.05, 764.64; LogP=3.72+/−
0.45; Rf=0.69; C24H36N2O4Cl2, M=487.48.
phenoxy)-ethyl]piperazin-1-yl}-propan-2-ol dihydrochloride (11)
Yield: 54%.; M.p.=216–218◦C;
3.10−4.00 (m, 12H, NH+-CH2), 3.71 (s, 3H, Ar-O-CH3), 3.70−3.77 (m,
2H, Ar-O-CH2-), 4.36 (t, J=4.7Hz, 2H, CH2-O-Ar), 4.40−4.48 (m, 1H,
CH2-CH(OH)-CH2), 5.90 (bs, 1H, OH), 6.87−7.05 (m, 7H, Ar), 11.43 (bs,
1H, NH+), 12.81 (bs, 1H, NH+); IR 3261.04, 2406.73, 1509.03, 1224.58,
1035.59, 832.13; LogP=3.86+/− 0.47; Rf=0.73
1H NMR 2.23 (s, 6H, Ar-(CH3)2,
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 −350g. Treatment of
laboratory animals in the present study was in full accordance with the
respective Polish regulations. All procedures were conducted according to
Animal Care and Use Committee guidelines, and approved by the Ethical
Committee of Jagiellonian University, Krakow.
Source of compounds: phenylephrine hydrochloride, acetylcholine
hydrochloride, (±)-noradrenaline hydrochloride (Sigma, Aldrich Chemie
Gmbh); thiopental sodium (Biochemie Gmbh, Vienna); [3H]prazosin,
[3H]clonidine (Amersham). Other reagents were of analytical grade from
4.2.1. Radioligand binding test
The compounds were evaluated on their affinity for ?1- and ?2-
adrenoceptors by determining for each compound its ability to displace
[3H]prazosin or [3H]clonidine from specific binding sites on rat cere-
bral cortex. [3H]prazosin (19.5 Ci/mmol, ?1-adrenergic receptor) and
were homogenised in 20 volumes of ice-cold 50mM Tris-HCl buffer (pH
7.6), and centrifuged at 20 000 x g for 20min (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
pension, 30?l of [3H]prazosin (0.2nM) or [3H]clonidine (2nM) solution
and 30?l buffer containing from seven to eight concentrations (10−11–
10−4M) of tested compounds. For measuring unspecific binding, phento-
lamine −10?M (in the case of [3H]prazosin) and clonidine −10?M (in the
filtration over glass fiber filters (Whatman GF/C) using a vacum manifold
(Millipore). The filters were then washed 2 times with the assay buffer and
placed in scintillation vials with liquid scintillation coctail. Radioactivity
was measured in WALLAC 1409 DSA – liquid scintillation counter. All
assays were done in duplicates.
Radioligand binding data were analyzed (Maj et al. 1985) using iterative
curve fitting routines (GraphPAD/Prism, Version 3.0 – San Diego, CA,
USA). Ki values were calculated from the Cheng and Prusoff equation.
4.2.2. Functional bioassay
Isolated rat aorta was used in order to test the antagonistic activity of inves-
tigated compounds for ?1-adrenoceptors. The male Wistar rats weighing
200–350g were anaesthetized with thiopental sodium (75mg/kg ip) and the
aorta was dissected and placed in a Krebs-Henseleit solution and cleaned of
surrounding fat tissues. The thoracic aorta was denuded of endothelium and
cut into approximately 4mm long rings. The aorta rings were incubated in
30ml chambers filled with a Krebs-Henseleit solution (NaCl 118mM, KCl
4.7mM, CaCl22.25mM, MgSO41.64mM, KH2PO41.18mM, NaHCO3
24.88mM, glucose 10mM, C3H3O3Na 2.2mM, EDTA 0.05mM) at 37◦C
and pH 7.4 with constant oxygenation (O2/CO2, 19:1). Two stainless steel
pins were inserted through the lumen of each arterial segment: one pin was
attached to the bottom of the chamber and the other to an isometric FDT10-
A force displacement transducer (BIOPAC Systems, Inc., COMMAT Ltd.,
Turkey). The aortae rings were stretched and maintained at optimal tension
of 2g and allowed to equilibrate for 2h. The lack of endothelium was con-
rings precontracted by noradrenaline (0.1?M).
Cumulative concentration-response curves to phenylephrine (0.003 to
3?M) were obtained by the method of van Rossum (1963). Following the
first phenylephrine curve, aortae rings were incubated with one of three
concentrations of tested compounds (one concentration of the antagonist
was used in each arterial ring in every experiment) for 20min and the next
Pharmazie 66 (2011)
ORIGINAL ARTICLES Download full-text
cumulative concentration curve to phenylephrine was constructed. In order
to avoid fatigue of the aortae preparation, a 60min recovery period was
allowed between phenylephrine curves.
Concentration-response curves were analysed using GraphPad Prism 4.0
software (GraphPad Software Inc., San Diego, CA, USA). Contractile
responses to vasoconstrictor (in the presence or absence of tested com-
pounds) are expressed as a percentage of the maximal phenylephrine
before incubation with the tested compounds. Data are the means±SEM of
at least 4 separate experiments. From the EC50values of the agonist in the
presence and absence of different antagonist concentrations, concentration-
ratios were calculated. Schild plots were constructed, and where the slope
was not significantly different from unity, the pA2values were determined
(Arunlakshana and Schild 1959).
Acknowledgments: The work was supported by the Medical College of
Jagiellonian University (Grant No. K/ZBW/000399 and K/ZDS/000951).
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