Synthesis and Biological Evaluation of New Combined α/β-Adrenergic Blockers

Article abstract of DOI:10.1002/ardp.201600394

The synthesis, characterization, and pharmacological evaluation of new aryloxyaminopropanol compounds based on substituted (4-hydroxyphenyl)ethanone with alterations in the alkoxymethyl side chain in position 2 and with 2-methoxyphenylpiperazine in the ba

Full text of DOI:10.1002/ardp.201600394

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Arch. Pharm. Chem. Life Sci. 2017, 350, e1600394
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Full Paper
SynthesisandBiologicalEvaluationofNewCombined a/b-Adrenergic
Blockers
1
2
2
2
ꢀ2
ꢀ
ꢁꢀ
ꢁ
ꢀ
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Andrej Nemethy
, Peter Vavrinec , Diana Vavrincova-Yaghi , Diana Cepcova , Svetozar Misuth ,
ꢀ
ꢀ2
ꢁ
ꢁ
ꢀ
ꢀ1
ꢁ
ꢀ2
Eva Kral’ova , Ruzena Cizmarikova , and Eva Racanska
ꢁ
1
Department of Chemical Theory of Drugs, Faculty of Pharmacy, Comenius University in Bratislava,
Bratislava, Slovakia
2
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University in Bratislava,
Bratislava, Slovakia
The synthesis, characterization, and pharmacological evaluation of new aryloxyaminopropanol
compounds based on substituted (4-hydroxyphenyl)ethanone with alterations in the alkoxymethyl
side chain in position 2 and with 2-methoxyphenylpiperazine in the basic part of the molecule are
reported. For the in vitro pharmacological evaluation, isolated aorta and atria from normotensive
Wistar rats were used. Compared to naftopidil, compounds with ethoxymethyl, propoxymethyl,
butoxymethyl, and methoxyethoxymethyl substituent displayed similar a1-adrenolytic potency.
Compounds with methoxymethyl, ethoxymethyl, and propoxymethyl substituent caused a significant
decrease in both spontaneous and isoproterenol-induced beating of isolated rat atria. Naftopidil and
the tested substances containing a butoxymethyl and methoxyethoxymethyl substituent had no effect
on the spontaneous or isoproterenol-induced beating. The tested substance that had the most
pronounced effect was the compound with a propoxymethyl substituent. Its antihypertensive efficacy
was investigated in vivo on spontaneously hypertensive rats (SHRs). The systolic blood pressure was
found to be significantly lower in SHRs subjected to the treatment for 2 weeks than in untreated
SHRs. Naftopidil had no significant effect.
Keywords: 4-Hydroxyphenylethanone / a/b-Blockers / Isolated aorta / Isolated atria / Spontaneously
hypertensive rats
Received: December 26, 2016; Revised: March 29, 2017; Accepted: April 6, 2017
DOI 10.1002/ardp.201600394
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Adrenergic receptors belong to the family of G-protein
coupled receptors. They are divided into three subclasses: a1,
Introduction
Hypertension is a major human health problem of our times.
Several mechanisms can be used to reduce high blood pressure.
One of the main approaches is decreasing sympathetic
neurotransmission via antagonization of adrenoceptors [1].
a2, and b, and further into several subtypes (i.e., a1A, a1B, a1D,
a2A, a2B, a2C, b1, b2, b3). The classification is based on structural
similarity, localization, and pharmacology as well as on
cloning techniques [2, 3]. The a-adrenoceptors participate
in the functioning of the central and peripheral nervous
system [4], while b-adrenoceptors are mainly involved in the
physiological processes in the cardiovascular system, respira-
tory tract, and in fat tissue [5]. In particular, a1-receptors play a
role in vascular smooth muscle contraction, increasing of
blood pressure, dilation of the pupil, the human prostate
smooth muscle contraction [6], as well as in regulation of
cerebral microcirculation [7].
ꢀ
Correspondence: Andrej Nemethy, Department of Chemical
Theory of Drugs, Faculty of Pharmacy, Comenius University in
ꢁ
Bratislava, Kalinciakova 8, 83232 Bratislava, Slovakia.
E-mail: nemethy@fpharm.uniba.sk
Fax: þ421250117357
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a1-Antagonists have been successfully applied in the therapy
of hypertension over the past two decades [3]. However, both
b- and a-adrenergic mechanisms are involved in the control of
blood pressure; a-mediated vasoconstriction regulates the
vascular tone, and b-mediated responses stimulate the heart
directly or indirectly by renin release, thus affecting vascular
smooth muscle tone [8].
The combination of a1- and b-adrenoceptor antagonists in
the therapy of hypertension also makes sense in terms of
hemodynamics. a1-Adrenoceptor blockade leads to vasodila-
tation and in this way counteracts elevated peripheral
vascular resistance which is the most common hemodynamic
derangement found in established essential hypertension.
One drawback of this approach is the possibility of induction
of reflex tachycardia [9]. The reflex cardiostimulation can be
conveniently suppressed by simultaneous antagonization of
b-adrenoceptors [10]. The blockade of b-adrenoceptors
does not implicate a decrease in vascular resistance; the
observed hypotensive response seems to be based on a
reduction in cardiac output [9]. Adverse consequences of
b-blockade, on the other hand, predominantly affect the
a-antagonization [8].
Combined a/b-adrenoceptor blockade can be achieved
either by simultaneous administration of both types of
adrenoceptor antagonists or by employing drugs that
possess a- and b-adrenoceptor antagonistic activity in the
same molecule [9]. Hybrid drugs consist of different
pharmacophoric groups which are linked together in one
molecule. The advantage of hybrid drugs is stable pharma-
cokinetics, ensuring that the concentration of pharmaco-
phores during the entire treatment remains in balance.
While concomitantly applied drugs compete with each other
for plasma-protein binding sites and in this way influence
each other’s bioavailability, the total plasma-protein bound
fraction and the free drug concentration for hybrid drugs
remain constant [11].
The structural fragment of piperazine occurs in molecules of
several potent a-receptor antagonists; 2-methoxyphenylpi-
perazine is part of the molecules of urapidil and naftopidil [3].
It is worth mentioning that naftopidil contains an arylo-
xyaminopropanol structure (Fig. 1), but like its metabolites O-
desmethyl-naftopidil, (phenyl)hydroxy-naftopidil and (naph-
thyl)hydroxy-naftopidil, it possesses no affinity to a2- or b-
adrenoceptors [12, 13]. Several authors have already tried,
with varying degrees of success, to connect the aryloxyami-
nopropanol structure with a 2-methoxyphenylpiperazine
moiety in order to enhance the hypotensive effect. In the
aromatic part of aryloxyaminopropanols, fragments of b1-
selective betaxolol, the oxypropanol derivative of labetalol,
and benzothiazine derivatives were investigated [14–17].
Likewise, the combination of vasodilating and b-antagonistic
effects in the treatment of hypertension was studied in hybrid
structures that connect the dihydropyridine pharmacophore
of calcium channel blockers and the aryloxyaminopropanol
structure [18, 19].
This work investigated the biological activity of newly
synthesized aryloxyaminopropanols based on substituted (4-
hydroxyphenyl)ethanone with modified alkoxymethyl side
chain in position 2 and with 2-methoxyphenylpiperazine in
the basic part of the molecule (Fig. 1).
The synthesis and characterization of the compounds are
described. For the biological evaluation, isolated aorta and
atria from normotensive Wistar rats were used. The antihy-
pertensive efficacy of selected compound was tested on
spontaneously hypertensive Wistar rats (SHR).
Results and discussion
Chemistry
The target substances were synthesized (as racemates) by a
four-step synthesis (Scheme 1). In the first step, the (4-
hydroxyphenyl)ethanone (a) was chloromethylated in the
second position on the aromatic ring by using paraformalde-
hyde and concentrated HCl to give (3-chloromethyl-4-
In this work, we decided to combine the aryloxyaminopro-
panol structure of b-blockers with a 2-methoxyphenylpiper-
azine moiety that provides the a1-antagonization.
Figure 1. Structures of naftopidil and the synthe-
tized compounds.
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Scheme 1. Synthetic route for title compounds A1–5. Reagents and conditions: (i) paraformaldehyde, conc. HCl, 4.5 h, 45–50°C; (ii)
Alk-OH (1: methanol; 2: ethanol; 3: propan-1-ol; 4: butan-1-ol; 5: 2-methoxyethanol), NaHCO3, 6 h, 40–50°C; (iii) chloromethylox-
irane, KOH, 4 h, 50–55°C; (iv) 2-methoxyphenylpiperazine, EtOH, 2 h, 30°C, 4 h reflux; (v) diethylether, fumaric acid.
hydroxyphenyl)ethanone (b). The chloromethyl derivative (b)
wastreatedwithappropriatealcoholstoform(3-alkoxymethyl-
4-hydroxyphenyl)ethanones (c1–5) according to the published
method [20]. Subsequently theprepared intermediates reacted
with chloromethyloxirane to form 1-[3-(alkoxymethyl)-4-(oxir-
ane-2-ylmethoxy)phenyl]ethanones (d1–5) [21]. Oxirane deriv-
atives were treated with 2-methoxyphenylpiperazine in
ethanol to give bases of the final compounds, which
were subsequently transformed into salts with fumaric acid
(A1–5). The fumarates were crystallized from ethyl acetate or
propan-2-ol.
and efficacy (Fig. 2 and Table 1). The a-adrenolytic effect was
shown by a rightward shift in the dose–response curves for
agonist phenylephrine in the presence of the tested
substances. The values of pD2 (ꢀlog of EC50) calculated from
the agonist dose–response curve for phenylephrine in the
presence of all tested substances were significantly lower than
the values of pD2 for phenylephrine in the presence of the
vehicle (DMSO).
The compounds A2, A3, A4, and A5 displayed similar
potency to naftopidil, while the potency of compound A1 was
lower than that of naftopidil, A2, A3, A4, and A5. The pD2
values for phenylephrine in the presence of A2, A3, A4, and
A5 were not significantly different from the values for
naftopidil, while the pD2 values for A1 were significantly
higher than the values for naftopidil, A2, A3, A4, and A5.
When comparing the efficacy of the tested compounds, we
observed that A3 and A4 had higher efficacy than naftopidil.
This was shown by the maximal response of aortic rings to
phenylephrine (Emax) in the presence of A3 or A4, which was
significantly lower than the Emax of rings in the presence of
naftopidil. Furthermore, compounds A1–A4 lowered Emax
significantly more than did the vehicle-treated aortic rings,
while Emax values in the presence of compound A5 and
naftopidil were not significantly different from Emax values
obtained for phenylephrine in the presence of vehicle
(DMSO).
The structure of the prepared substances was determined
1
by the interpretation of FTIR, H NMR, 13C NMR spectra, and
elemental analysis. The purity of the products was verified by
TLC. The FTIR spectra of prepared compouþnds showed
–
–
–
characteristic functional groups, such as R NH , C O, C C
–
3
aryl, COOꢀ, Ar–O–C, C–O–C. In the NMR spectral data, the
chemical shifts are consistent with the proposed structures.
Pharmacology
a-Adrenolytic activity
In order to assess the a-adrenolytic activity of the tested
compounds, we performed experiments on aortic rings and
constructed dose–response curves for phenylephrine in the
presence and absence of the tested compounds. The tested
substances displayed a-adrenolytic activity of varying potency
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beating was comparable across the substances investigated,
with no significant effect among A1, A2 and A3 (9.0 ꢁ 2.4%
for A1, 11.3 ꢁ 0.9 for A2 and 5.5 ꢁ 1.5 for A3).
Antihypertensive activity
In order to investigate the antihypertensive effect of
substance A3, which had the most pronounced effect on
isolated atria, we experimented on SHRs by treating them
with either the A3 compound or naftopidil, administered in
once daily doses of 30 mg/kg by oral gavage (Fig. 4). Systolic
blood pressure was found to be significantly lower in SHRs
subjected to the treatment for 2 weeks than in the untreated
SHRs (172 ꢁ 8 vs. 195 ꢁ 10, p < 0.05). The systolic blood
pressure of naftopidil-treated SHRs was not significantly
different from either the vehicle-treated or A3-treated rats
(195 ꢁ 10).
Figure 2. Dose–response curves of aortic rings for phenyleph-
rine (PE) in the presence of tested substances (A1, A2 A3, A4, A5,
naftopidil) and in the absence of tested substances (vehicle ¼
DMSO). Average values were calculated as a percentage of KCl-
mediated constriction. Data are presented as mean ꢁ SEM
ꢂp < 0.05.
Discussion
This work describes the synthesis, characterization, and
pharmacological evaluation of new hybrid a/b-adrenoceptor
antagonists. The structure of newly synthesized compounds
contains the aryloxyaminopropanol structure of b-blockers
and a 2-methoxyphenylpiperazine moiety that provides the
a1-antagonization. For the in vitro pharmacological evalua-
tion, isolated aorta and atria from normotensive Wistar rats
were used.
b-Adrenolytic activity
In order to assess the b-adrenolytic properties, we performed
experiments on spontaneously isolated and isoproterenol
stimulated beating rat atria in the presence of the tested
compounds (Fig. 3). We observed that A1, A2, and A3 caused a
significant decrease in both spontaneously and isoproterenol-
induced beating of isolated rat atria. Substances A4, A5, and
naftopidil had no effect on the spontaneous or isoproterenol-
induced beating. Compound A3 had the most pronounced
effect among the tested substances. After incubating atrial
strips with the tested substances, the spontaneous beating
rate of A3 was reduced by 19.1 ꢁ 0.6% (p < 0.05 vs. A1,
A2), which was significant compared to A1 (7.2 ꢁ 0.9%) and
A2 (12.9 ꢁ 4.3%). The inhibition of isoproterenol-induced
a-Adrenolytic activity
We observed that substances A2, A3, and A4 decreased the
maximal response of aortic rings to a-adrenergic receptor
stimulation by phenylephrine (Emax). Emax reduction is a
typical feature of non-competitive antagonism where even
maximal concentration of the agonist cannot displace the
antagonist from the receptor, thus causing a decrease in the
maximal response. Unfortunately, there are insufficient data
to demonstrate whether naftopidil is a competitive or non-
competitive antagonist. In our study, however, naftopidil did
not cause a change in Emax, and so the decrease of Emax in the
Table 1. The effect of tested substances expressed as pD2 (ꢀlogEC50) and Emax obtained from individual dose–response
curves for phenylephrine in the presence (A1, A2, A3, A4, A5, naftopidil) or absence (vehicle–DMSO) of tested
substances.
pD2
Mean
p < 0.05 vs.
Emax
Mean
p < 0.05 vs.
A4
A3
A5
NAFT
A2
A1
DMSO
5.95 ꢁ 0.07
5.96 ꢁ 0.11
5.99 ꢁ 0.06
6.02 ꢁ 0.08
6.08 ꢁ 0.08
6.26 ꢁ 0.05
6.80 ꢁ 0.12
A1, DMSO
A1, DMSO
A1, DMSO
A1, DMSO
A1, DMSO
A2, A3, A4, A5, NAFT, DMSO
versus all
A4
A3
A2
A1
A5
NAFT
DMSO
59 ꢁ 11
84 ꢁ 16
A1, A5, NAFT, DMSO
NAFT, A5, DMSO
DMSO
A4, DMSO
A3, A4
A3, A4
A1, A2, A3, A4
92 ꢁ 14
111 ꢁ 17
124 ꢁ 12
129 ꢁ 16
151 ꢁ 10
Data are presented as mean ꢁ SEM. Differences with p < 0.05 were considered significantly different.
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Figure 3. The effect of the tested substances (A1, A2, A3, A4, A5, and naftopidil) or DMSO (vehicle) on spontaneously beating
isolated rat atria (spont) and after stimulation with isoproterenol in concentration 10ꢀ8 M (ISO 10ꢀ8). Data are presented as
mean ꢁ SEM. Differences with p < 0.05 were considered significantly different.
case of A2, A3, and A4 (derived from naftopidil) must have
been caused by modification in the side carbon chain of the
phenylethanone moiety. In the case of A1 and A5, we did not
observe a decrease in Emax, meaning that in the structure of
substance A1, the single carbon substituent is probably too
small to ensure a stronger binding to the a-adrenoceptor.
Structure A5, on the other hand, having a side chain length of
four atoms, contains oxygen in the side chain, making it more
flexible in comparison with the more rigid four-carbon side
chain of A4, which could cause different behavior in the
environment of the a-adrenoceptor.
b-Adrenolytic activity
Compounds A1, A2,andA3causedasignificantdecreaseinboth
spontaneously and isoproterenol-induced beating of isolated
rat atria and thus their supposed b-adrenolytic properties could
be manifested. Substances A4, A5, and naftopidil had no effect
on spontaneous or isoproterenol-induced beating. Compound
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The evaluation of b-antagonistic activity showed that
compounds A1, A2, and A3 caused a significant decrease in
both spontaneously and isoproterenol-induced beating of
isolated rat atria.
Compound A3 had the most pronounced effect among
tested substances. Systolic blood pressure was found to be
significantly lower in SHRs subjected to the treatment for
2 weeks than in untreated SHRs. Naftopidil had no significant
effect.
Experimental
Chemistry
General
The melting point was determined using a Kofler hot stage
microscope and was quoted uncorrected. The purity of the
newly prepared compounds was assessed using TLC silica gel
plates Silufol1 UV 254 (Merck). The solvent system used was
ethyl acetate/diethylamine (9.5:0.5 v/v). FTIR spectra were
recorded using Nicolet 6700 (Thermo Scientific). NMR spectra
were recorded with the Varian Gemini 2000 spectrometer
operating at 300 MHz for 1H NMR and 75 MHz for 13C NMR,
using Si(CH3)4 as the reference. Chemical shifts are reported in
ppm (d). In reporting the NMR multiplicities, we used the
following abbreviations: s, singlet; d, doublet; dd, doublet of
doublet; t, triplet; q, quartet; m, multiplet. Elemental analysis
was performed using FLASH 2000 Organic Elemental Analyzer
(Thermo Scientific). All reactions were carried out using
commercial grade reagents and solvents. Diethylether was
dried by refluxing over potassium hydroxide and sodium
followed by distillation.
Figure 4. Effect of tested substances on systolic blood pressure
of SHRs treated with A3 or naftopidil or vehicle (DMSO), with
the SBP measured using a tail cuff. Data are presented as
average ꢁ SEM; ꢂp < 0.05.
A3 had the most pronounced effect among the tested
substances, and its antihypertensive efficacy was investigated
invivoonspontaneouslyhypertensiveWistarrats.Thestructural
difference between molecules of naftopidil and the compound
A3 isinthe aromaticmoiety.If1-naphtholinthearomaticpart is
combined with 2-methoxyphenylpiperazine in the basic part of
its molecule, naftopidil loses its ability to antagonize the
b-receptor. Replacing naphthol with substituted phenyletha-
none provides the b-lytic efficacy, albeit with methoxyphenyl-
piperazine in the basic part.
Systolic blood pressure (SBP)
The InChI codes of the investigated compounds together
with some biological activity data are provided as Supporting
Information.
It is apparent from our study that replacing naphthol with
substituted phenylethanone in A3 had an effect on blood
pressure, because naftopidil had no effect on the SBP of
treated SHRs. Naftopidil and A3 were also shown to have
a-adrenolytic properties in vitro, but with no effect on blood
pressure. Unfortunately, there are no data showing whether
naftopidil can decrease blood pressure in SHRs, and so we
cannot compare our results with other studies. It has,
however, been shown in other studies that b-blockers can
decrease blood pressure of SHRs in experimental settings [22,
23], which is in line with our results. We, therefore, conclude
that the substituted phenylethanone in the aromatic moiety
was the prominent one in blood pressure reduction in SHRs.
General procedure for the preparation of (3-chloromethyl-
4-hydroxyphenyl)ethanone (b)
The mixture of (4-hydroxyphenyl)ethanone (20.41 g; 0.15 mol)
and 90 mL of concentrated HCl was heated to 45–50°C and
paraformaldehyde (7.5 g; 0.25 mol) was gradually added with
stirring. The reaction was allowed to proceed for 4.5 h. The
solid product was filtered, washed with water, and crystal-
lized from toluene. Yield: 71%; m.p. 155–157°C; 1H NMR
(CD3OD): d 2.53 (s, 3H, CH3); 4.68 (s, 2H, CH2); 6.86–6.89 (d,
J ¼ 8.7 Hz, 1H, Ar5H); 7.83–7.86 (dd, J ¼ 2.4, J ¼ 11.1 Hz, 1H,
Ar6H); 7.98 (s, 1H, Ar2H).
General procedure for the preparation of (3-
alkoxymethyl-4-hydroxyphenyl)-ethanones (c1–5)
Conclusion
This study describes the synthesis, characterization, and
pharmacological evaluation of new hybrid a/b-adrenoceptor
antagonists. Compared to naftopidil, compounds A2, A3,
A4, and A5 displayed similar potency to antagonize the
a1-adrenergic receptors, while compound A1 showed lower
potency.
The solution of (3-chloromethyl-4-hydroxyphenyl)ethanone
(b) (25.84 g; 0.14 mol) in 60 mL of appropriate alcohol
(1: methanol; 2: ethanol; 3: propan-1-ol; 4: butan-1-ol; 5:
2-methoxyethanol) was heated to 50°C while stirring. Sodium
bicarbonate (23.52 g; 0.28 mol) was added gradually over 1 h
and the reaction was allowed to proceed for 6 h at a constant
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temperature 50°C. After removal of redundant NaHCO3 by
filtration, alcohol was distilled off and the product was
crystallized from cyclohexane. Yields: 65–73%; c3: 1H NMR
(CD3OD): d 0.93–0.98 (t, J ¼ 7.5 Hz, 3H, CH2CH3); 1.58–1.68 (m,
2H, CH2CH3); 2.52 (s, 3H, COCH3); 3.48–3.52 (t, J ¼ 6.6 Hz, 2H,
OCH2CH2); 4.54 (s, 2H, Ar-CH2); 6.83–6.85 (d, J ¼ 8.4 Hz, 1H,
Ar5H); 7.79–7.83 (dd, J ¼ 2.2, J ¼ 10.8 Hz, 1H, Ar6H); 7.96 (s, 1H,
Ar2H).
1H NMR (DMSO-d6) d 2.50 (s, 3H, COCH3); 2.54–2.98 (m, 10H,
CH2N, pip2,3,5,6); 3.37 (s, 3H, CH2OCH3); 3.77 (s, 3H, ArOCH3);
4.01–4.07 (m, 2H, ArOCH2); 4.11–4.13 (m, 1H, CH2CHOH); 4.48
(s, 2H, ArCH2); 6.60 (s, 2H, fumar); 6.86–6.94 (m, 4H, pip-Ar);
7.10–7.13 (d, J ¼ 9.3 Hz, 1H, Ar5H); 7.9–7.93 (m, 2H, Ar2,6H);
13C NMR (DMSO-d6) d 26.39, 49.88, 53.64, 55.30, 58.00, 60.76,
66.16, 68.23, 71.31, 111.11, 111.88, 117.89, 120.82, 122.40,
126.74, 128.07, 129.40, 129.86, 134.19, 141.13, 151.95, 159.81,
166.33, 196.37. Anal. calcd. for C48H64N4O10.C4H4O4, Mr
973.12; % C 64.12, % H 6.99, % N 5.75, found % C 63.84,
% H 6.71, % N 5.45.
General procedure for the preparation of 1-[3-
(alkoxymethyl)-4-(oxirane-2-ylmethoxy)phenyl]ethanones
(d1–5)
To a stirred solution of (3-alkoxymethyl-4-hydroxyphenyl)-
ethanones (c1–5) (0.15 mol; c1: 27.03 g; c2: 29.13 g; c3: 31.24 g;
c4: 33.34 g; c5: 33.64 g) in 3 mol (235 mL) of (ꢁ)-chloromethyl-
oxirane, 0.17 mol of 85% KOH (9.54 g; water: 1.7 mL) was
added. The mixture was stirred at 50–55°C under nitrogen for
4 h. The inorganic salts were filtered off and the (ꢁ)-chlor-
omethyloxirane was distilled off under reduced pressure.
Distilled water (50 mL) was added and the residue was
extracted three times with diethylether (3 ꢃ 100 mL). The
organic phase was separated and dried with anhydrous
Na2SO4. The filtered solution was concentrated under
reduced pressure and the remaining oil was used without
previous purification in the next reaction step. Yields: 64–
84%. d1: 1H NMR (CDCl3): d 2.52 (s, 3H, COCH3); 2.75–2.78 (d,
J ¼ 9 Hz, 1H, CH2oxirane); 2.93–2.97 (m, 1H, CH2oxirane); 3.02–
3.07 (m, 1H, CHoxirane); 3.28 (s, 3H, OCH3); 3.96–3.99
(d, J ¼ 9 Hz, 1H, CH2CH); 4.06–4.09 (d, J ¼ 9 Hz, 1H, CH2CH);
4.41 (s, 2H, ArCH2); 6.84–6.86 (d, J ¼ 8.4 Hz, 1H, Ar5H); 7.78–
7.83 (m, 1H, Ar6H); 7.95 (s, 1H, Ar2H).
(2RS)-bis-1-[3-(4-Acetyl-2-ethoxymethyl)phenoxy-2-
hydroxypropyl]-4-(2-methoxyphenyl)piperazinium
fumarate (A2)
Yield: 65%, m.p. 138–140°C, ꢀFTIR cmꢀ1: 2586 (R3NHþ), 1672
–
–
–
(C O), 1599 (C C), 1355 (COO ), 1242 (Ar–O–C), 1022 (C–O–C);
–
1H NMR (DMSO-d6) d 1.17–1.22 (t, J ¼ 7.1 Hz, 3H, CH2CH3); 2.50
(s, 3H, COCH3); 2.60–2.98 (m, 10H, CH2N, pip2,3,5,6); 3.53–3.60
(q, J ¼ 7.0 Hz, 2H, CH2CH3); 3.77 (s, 3H, ArOCH3); 4.02–4.13 (m,
3H, CH2CHOH); 4.52 (s, 2H, ArCH2); 6.60 (s, 2H, fumar); 6.85–
6.95 (m, 4H, pip-Ar); 7.09–7.12 (d, J ¼ 9.3 Hz, 1H, Ar5H); 7.89–
7.92 (m, 2H, Ar2,6H); 13C NMR (DMSO-d6) d 15.16, 26.38, 49.81,
53.60, 55.30, 60.71, 65.47, 66.11, 66.22, 71.26, 111.06, 111.89,
117.90, 120.82, 122.42, 127.12, 128.01, 129.42, 129.84, 134.24,
141.10, 151.95, 159.79, 166.43, 196.38. Anal. calcd. for
C
50H68N4O10.C4H4O4, Mr 1001.17; % C 64.72, % H 7.19, % N
5.59, found % C 64.34, % H 7.12, % N 5.46.
(2RS)-bis-1-[3-(4-Acetyl-2-propoxymethyl)phenoxy-2-
hydroxypropyl]-4-(2-methoxyphenyl)piperazinium
fumarate (A3)
General procedure for the preparation of (2RS)-bis-1-[3-(4-
acetyl-2-alkoxymethyl)phenoxy-2-hydroxypropyl]-4-(2-
methoxyphenyl)piperazinium fumarates (A1–5)
Yield: 63%, m.p. 148–150°C, ꢀFTIR cmꢀ1: 2582 (R3NHþ), 1674
–
–
–
(C O), 1599 (C C), 1355 (COO ), 1241 (Ar–O–C), 1024 (C–O–C);
–
1H NMR (DMSO-d6) d 0.89–0.94 (t, J ¼ 7.3 Hz, 3H, CH2CH3);
1.58–1.60 (m, 2H, CH2CH3); 2.50 (s, 3H, COCH3); 2.60–2.95 (m,
10H, CH2N, pip2,3,5,6); 3.44–3.49 (t, J ¼ 7.5 Hz, 2H, OCH2CH2);
3.78 (s, 3H, ArOCH3); 4.01–4,12 (m, 3H, CH2CHOH); 4.52 (s, 2H,
ArCH2); 6.50 (s, 2H, fumar); 6.86–6.97 (m, 4H, pip-Ar); 7.09–
7.12 (d, J ¼ 9.0 Hz, 1H, Ar5H); 7.89–7.92 (m, 2H, Ar2,6H);
13C NMR (DMSO-d6) d 10.63, 22.50, 26.37, 43.48, 48.00, 50.09,
55.29, 55.35, 66.36, 71,36, 71.76, 111.08, 111.89, 117.87,
120.82, 123.12, 127.14, 127.98, 129.39, 129.81, 141.24, 151.96,
159.84, 167.26, 196.37. Anal. calcd. for C52H72N4O10.C4H4O4,
Mr 1029.22; % C 65.29, % H 7.38, % N 5.44, found % C 65.02,
% H 7.19, % N 5.14.
The solution of the oxirane derivative (d1–5) (80 mmol; d1:
18.90 g; d2: 20.02 g; d3: 21.14 g; d4: 22.27 g; d5: 22.43 g) and 1-
(2-methoxyphenyl)piperazine (16.34 g; 85 mmol) in ethanol
(150 mL) was kept at 30°C for 2 h and was then heated for an
additional 5 h at reflux temperature. The solvent was distilled
off.Distilledwater(50 mL)wasaddedtotheresidue,whichwas
then washed three times with diethylether (3 ꢃ 100 mL). The
combined organic layers were washed with water, separated,
and dried with K2CO3. The filtered solution was concentrated
under reduced pressure and the remaining oil was crystallized
from hexane to afford the final base. The salts were prepared
by quantitative reaction of anhydrous ether solution of base
andanhydrousethersolutionof fumaricacid. The crystalswere
collected by filtration and recrystallized from ethyl acetate or
propan-2-ol to give fumarate salt as a white solid.
(2RS)-bis-1-[3-(4-Acetyl-2-butoxymethyl)phenoxy-2-
hydroxypropyl]-4-(2-methoxyphenyl)piperazinium
fumarate (A4)
Yield: 59%, m.p. 129–131°C, ꢀFTIR cmꢀ1: 2578 (R3NHþ), 1674
–
–
–
(2RS)-bis-1-[3-(4-Acetyl-2-methoxymethyl)phenoxy-2-
hydroxypropyl]-4-(2-methoxyphenyl)piperazinium
fumarate (A1)
(C O), 1599 (C C), 1355 (COO ), 1242 (Ar–O–C), 1021 (C–O–C);
–
1H NMR (DMSO-d6) d 0.87–0.92 (t, J ¼ 7.3 Hz, 3H, CH2CH3);
1.33–1.41 (m, 2H, CH2CH3); 1.54–1.59 (m, 2H, CH2CH2CH3); 2.50
(s, 3H, COCH3); 2.58–2.98 (m, 10H, CH2N, pip2,3,5,6); 3.48–3.53
(t, J ¼ 6.5 Hz, 2H, OCH2CH2); 3.77 (s, 3H, ArOCH3); 4.02–4.12 (m,
Yield: 61%, m.p. 156–158°C, ꢀFTIR cmꢀ1: 2583 (R3NHþ), 1669
–
–
–
(C O), 1598 (C C), 1354 (COO ), 1242 (Ar–O–C), 1024 (C–O–C);
–
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3H, CH2CHOH); 4.52 (s, 2H, ArCH2); 6.61 (s, 2H, ArCH2); 6.85–
6.94 (m, 4H, pip-Ar); 7.09–7.12 (d, J ¼ 9.6 Hz, 1H, Ar5H); 7.90–
7.92 (m, 2H, Ar2,6H); 13C NMR (DMSO-d6) d 13.79, 18.93, 26.36,
31.35, 49.86, 53.63, 55.29, 60.76, 66.15, 66.42, 69.84, 71.27,
111.07, 111.89, 117.89, 120.82, 122.41, 127.15, 127.99, 129.41,
129.81, 134.10, 141.11, 151.95, 159.80, 166.18, 196.36. Anal.
calcd. for C54H76N4O10.C4H4O4, Mr 1057.27; % C 65.82, % H
7.56, % N 5.29, found % C 65.56, % H 7.42, % N 5.23.
After washout and another 30 min of stabilization, the aortic
rings were incubated either with the tested substances
(10ꢀ7 M final concentration) or dimethyl sulphoxide (DMSO,
0.5% final concentration; vehicle). Subsequently, the contrac-
tility of the aortic rings was measured as the contractile
response to cumulative concentrations of phenylephrine
(1 ꢃ 10ꢀ8M ꢀ 1 ꢃ 10ꢀ5M) [24].
Preparation of atrial strips and organ baths;
(2RS)-bis-1-[3-(4-Acetyl-2-methoxyethoxymethyl)-
phenoxy-2-hydroxypropyl]-4-(2-methoxyphenyl)-
piperazinium fumarate (A5)
measurements
Under brief anesthesia with 2.5 % isoflurane in O2, the rat
heart was quickly removed. The atria were isolated and
mounted in an organ bath, were attached to a fixed point at
the loop end and to the tension-transducer, and were
submerged in oxygenated (1.5 L/min 95% O2 and 5% CO2)
modified Krebs–Henseleit solution (Naþ 137 mM, Kþ 6 mM,
Yield: 61%, m.p. 136–138°C, ꢀFTIR cmꢀ1: 2577 (R3NHþ), 1678
–
–
–
(C O), 1599 (C C), 1360 (COO ), 1240 (Ar–O–C), 1025 (C–O–C);
–
1H NMR (DMSO-d6) d 2.52 (s, 3H, COCH3); 2.68–3,01 (m, 10H,
CH2N, pip2,3,5,6); 3.28 (s, 3H, CH2OCH3); 3.51–3.54 (m, 2H,
CH2CH2OCH3); 3.63–3.66 (m, 2H, CH2CH2OCH3); 3.771 (s, 3H,
ArOCH3); 4.04–4.10 (m, 3H, CH2CHOH); 4.56 (s, 2H, ArCH2);
6.61 (s, 2H, fumar.); 6.86–6.95 m (m, 4H, pip-Ar); 7.10–7.12 (d,
J ¼ 8.7 Hz, 1H, Ar5H); 7.90–7.95 (m, 2H, Ar2,6H); 13C NMR
(DMSO-d6) d 26.37, 49.50, 53.42, 55.31, 58.15, 60.43, 65.83,
66.71, 69.49, 71.16, 71.30, 111.09, 111.90, 117.94, 120.83,
122.53, 126.94, 128.16, 129.47, 129.83, 134.15, 140.93, 151.95,
159.75, 166.28, 196.39. Anal. calcd. for C52H72N4O12.C4H4O4,
Mr 1061.22; % C 63.32, % H 7.16, % N 5.27, found % C 63.05,
% H 6.98, % N 5.08.
Mg2þ 1.3 mM, Ca2þ 2.2 mM, Clꢀ 134 mM, HCO3 15,4 mM,
ꢀ
ꢀ
H2PO4 1.2 mM, glucoseꢀ 5.5 mM) at 30°C [25].
After 30min of stabilization, a response for isoproterenol
(1 ꢃ 10ꢀ8 M) was obtained on spontaneously beating atria.
After washout, the tested substances were added to the organ
bath in the concentration 10ꢀ7 M and the atria were incubated
for 30min. The response for isoproterenol (1ꢃ 10ꢀ8 M) was
thenobtainedinthepresenceoftestedsubstancesorDMSOasa
vehicle.
Antihypertensive effect of tested substances—in vivo
experiment
Pharmacological evaluation
Animals
SHRs were randomized into three groups. First group
comprised untreated SHRs (SHR, n ¼ 8), the second group
comprised SHRs treated with A3 at doses of 30 mg/kg of body
weight (SHRþA3, n ¼ 8), and the third comprised SHRs treated
with naftopidil at doses of 30 mg/kg (SHRþNAFT, n ¼ 5).
After a period of acclimatization, the rats were given tested
substances by oral gavage, once daily. The drugs were
suspended in methylcellulose. After 2 weeks of treatment,
the rats’ blood pressure was measured using a tail cuff.
Thereafter rats were sacrificed in isoflurane anesthesia by
exsanguination.
All animal care and experimental procedures were in
accordance with the NIH Guide for the Care and Use of
Laboratory Animals and approved by the local Committee for
Animals. Experiments were performed on 67 male Wistar rats
(12–14 weeks old, 280–320 g) and 21 spontaneously hyper-
tensive rats. The animals were housed under standard
conditions of temperature (21–24°C), humidity (40–60%),
and light (12 h/12 h light/dark cycles) at the animal facilities of
Comenius University in Bratislava, Slovakia. All animals had
free access to food and drinking water throughout the study.
Tissue preparation and contraction studies in rat aortic
rings
Statistical analysis
The contractility of aortic rings was calculated as
a
Under brief anesthesia with 2.5% isoflurane in O2, the thoracic
aorta was quickly removed, cleared of fat and connective
tissue, and cut into segments (rings of approximately 2 mm).
The rings were mounted in a 10 mL organ baths with Krebs
solution (pH 7.5) containing(in mmol/L): NaCl (120.4), KCl (5.9),
CaCl2 (2.5), MgCl2 (1.2), NaH2PO4 (1.2), glucose (11.5), NaHCO3
(25.0). The solution was kept at 37°C and continuously bubbled
with 95% O2 and 5% CO2. Prior to isotonic measurements of
vascular contractility, arteries were allowed to equilibrate for
40 min. Totestforviabilityofsmoothmusclecells,arterieswere
twice pre-constricted with KCl (60 mM). After washout, the
aortic rings were examined for full characterization of the
antagonist properties of the tested substances on aorta
contractions mediated by phenylephrine.
percentage of KCl constriction. The results are expressed
as mean ꢁ SEM. Differences between the experimental
groups were calculated using paired t-test (atrial strips),
ANOVA with LSD post-hoc test (aortic rings), ANOVA, and
LSD post-hoc test (SHR experiment). Differences were
considered significant at p < 0.05.
This publication utilizes research results of the CEBV project,
ITMS: 26240120034. This work was supported by the Slovak
Research and Development Agency under the contract nos.
APVV-0516-12, VEGA 1/0223/15 and VEGA 1/0247/17.
The authors have declared no conflicts of interest.
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