Synthesis, pharmacological activity, and chromatographic enantioseparation of new heterocyclic compounds of the aryloxyaminopropanol type derived from 4-hydroxyphenylalkanones

Article abstract of DOI:10.1007/s00706-018-2185-z

Abstract: In the paper, a series of six pharmacologically active compounds (β-adrenolytics) derived from 4-hydroxyphenylethanone and 4-hydroxyphenylpropan-1-one are reported. The compounds incorporate pyrrolidin-1-yl and 4-methylpiperazin-1-yl substituent

Full text of DOI:10.1007/s00706-018-2185-z

Monatshefte für Chemie - Chemical Monthly
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ORIGINAL PAPER
Synthesis, pharmacological activity, and chromatographic
enantioseparation of new heterocyclic compounds
of the aryloxyaminopropanol type derived from
4
-hydroxyphenylalkanones
1
Andrej N e´ methy1 • Ladislav Habala1 • Eva Ra cˇ ansk a´ 2 • Jindra Valentov a´ 1 •
ˇ
•
Ru zˇ ena Ci zˇ m a´ rikov a´
Katar ´ı na Hrobo nˇ ov a´
3
Received: 16 November 2017 / Accepted: 14 March 2018
Ó Springer-Verlag GmbH Austria, part of Springer Nature 2018
Abstract
In the paper, a series of six pharmacologically active compounds (b-adrenolytics) derived from 4-hydroxyphenylethanone and
4
-hydroxyphenylpropan-1-one are reported. The compounds incorporate pyrrolidin-1-yl and 4-methylpiperazin-1-yl sub-
stituents in the hydrophilic part of the molecule and ethoxymethyl and methoxyethoxymethyl side chains on the aromatic ring
in the lipophilic moiety. They were prepared by a four-step synthesis from 4-hydroxyalkanones via chloromethyl, alkox-
ymethyl, and oxirane intermediates. The purity of the target compounds was checked by TLC and their structures were
1
13
confirmed by the interpretation of the IR, UV, H NMR, and C NMR spectra. Thepharmacological evaluation of the obtained
-
6
compounds confirmed their vasodilatory and specific antiisoprenaline activities. All evaluated compounds at conc. 10
-
3
mol dm inhibited vasoconstrictory effect of phenylephrine (8.22–33.7%) on isolated rat aorta. The ability to inhibit positive
chronotropic effect of isoprenaline was observed on isolated spontaneously beating rat’s atria after pre-treatment with the
-3
evaluated compounds at conc. 10 and 10 mol dm . The calculated pA values of specific antagonistic effect against
-
7
-6
2
isoprenaline, related to their apparent b-adrenolytic activity, ranged between 6.54 and 7.57. The value for the standard
-
6
-7
-3
compound carvedilol was 8.15 ± 0.22. The majority of the evaluated compounds at conc. 10 –10 mol dm also showed
negative chronotropic effect on the basic heart rate of atria. Enantioseparation of the prepared compounds was performed by
chiral HPLC on an amylose tris(3,5-dimethylphenylcarbamate) column (Chiralpak AD) and a native teicoplanin column
(
Chirobiotic T). The chromatographic characteristics as retention, separation, and resolution factors were reported.
Graphical abstract
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00706-018-2185-z) contains supplementary
material, which is available to authorized users.
2
3
ˇ
Ru zˇ ena Ci zˇ m a´ rikov a´
cizmarikova@fpharm.uniba.sk
&
Department of Pharmacology and Toxicology, Faculty of
Pharmacy, Comenius University in Bratislava, Bratislava,
Slovak Republic
1
Department of Chemical Theory of Drugs, Faculty of
Pharmacy, Comenius University in Bratislava, Kalin cˇ iakova
Faculty of Chemical and Food Technology, Institute of
Analytical Chemistry, Slovak University of Technology in
Bratislava, Bratislava, Slovak Republic
8
, 83232 Bratislava, Slovak Republic
123

ˇ
R. Ci zˇ máriková et al.
Keywords Aryloxyaminopropanols Á Vasodilatory Á Antiisoprenaline Á Beta-adrenolytic Á Chirality Á Drug research
Introduction
Results and discussion
In the group of various drugs acting on the cardiovascular
system, compounds with a built-in heterocycle moiety in
the lipophilic and/or in the hydrophilic part of the molecule
exhibit significant biological activity. One of the most
famous b-adrenolytics with indole in the lipophilic part of
the molecule is pindolol [1, 2] and its prodrug form
bopindolol [3]. Carvedilol with incorporated carbazole is a
clinically used drug with combined a- and b-adrenolytic
efficacy that exhibits antihypertensive activity and a high-
antioxidant potency [4–6].
Previously, we reported several aryloxyaminopropanol
derivatives with isopropyl and tert-butyl groups [23] and
with the 3,4-dimethoxyphenethyl moiety, respectively [17].
These newly prepared b-blockers exhibited higher affinity
for b -receptors in comparison with celiprolol and acebu-
1
tolol used as standard compounds.
In the present work, we prepared a series of compounds
with pyrrolidine or N-methylpiperazine moieties in the
hydrophilic part of the molecule and an acyl substituent on
the aromatic ring, in anticipation of an additive vasodila-
tory effect. The target compounds 1–5 were prepared by an
established four-step synthetic procedure (Scheme 1,
Table 1). In the first step, (4-hydroxyphenyl)alkanones
reacted with paraformaldehyde and 36% HCl to give (3-
chloromethyl-4-hydroxyphenyl)alkanones in 70–75% yield
[24]. The isolated chloromethyl derivatives were treated
with dry ethanol (compounds 1–4) or 2-methoxyethanol
Derivatives with phenylpiperazine moiety in the
hydrophilic part of the molecule are under consideration as
potential antihypertensive agents [7, 8]. Their activity is
based on their interaction with the binding site of the a1-
and b-adrenergic receptors [9, 10]. Some piperazine
derivatives show antidepressant-like- [11] and H -antihis-
1
taminic effects [12].
The structure of naftopidil was obtained by replacing the
propan-2-amino (isopropylamino) group in the molecule of
propranolol with the 2-methoxyphenylpiperazine-1-yl
substituent. The antagonistic effect was shifted towards
selective a -receptor blockade, while the affinity for a -and
(compound 5) and solid NaHCO , yielding (3-alkox-
3
ymethyl-4-hydroxyphenyl)alkanones in 50–60% yield.
These intermediates then produced 1-[3-alkoxymethyl-4-
(oxirane-2-yl)phenyl]alkanones by reaction with oxirane
(54–66% yield) [25]. In the fourth step, oxirane interme-
diates in ethanol reacted with pyrrolidine and N-
methylpiperazine to attach the basic heterocyclic moiety.
An exception was the compound 6, which could be
1
2
b-adrenoceptors was very weak [13, 14]. Naftopidil also
2
?
blocks Ca channels, inhibits serotonin-induced platelet
aggregation, and reduces serotonin uptake by thrombocytes
[
15]. Other results demonstrated its use in benign prostatic
hyperplasia [16].
Scheme 1
Since compounds of the aryloxyaminopropanol type
possess a stereogenic center in their structure, chiral HPLC
technique was used for the separation of individual enan-
tiomers. Chiral stationary phases used for enantiosepara-
tion of aryloxyaminopropanols are based on teicoplanin
and vancomycin [17, 18], cyclodextrin [19], ovomucoid
[
20], and on derivatives of cellulose and amylose [21, 22].
The aim of this work was the preparation, basic phar-
macological evaluation and the study of HPLC enan-
tioseparation of new aryloxyaminopropanol derivatives
with pyrrolidin-1-yl and 4-methylpiperazin-1-yl substituent
in the hydrophilic part of molecule. The pharmacological
in vitro evaluation of the prepared compounds was orien-
tated towards their vasodilatory and specific antiisopre-
naline properties, which are inherent to all clinically used
b-blockers. Chiral columns, based on amylose tris(3,5-
dimethylphenylcarbamate) (Chiralpak AD) and native tei-
coplanin (Chirobiotic T), were used to separate the indi-
vidual enantiomers.
i. paraformaldehyde, 36% HCl, 4-5 h, 45-50 °C; ii. ethanol, 2-
methoxyethanol, NaHCO
3
; iii. chloromethyloxirane, KOH, 4 h, 50-55 °C;
iv. pyrrolidine, N-methylpiperazine / diethylether, fumaric acid.
1
23

Synthesis, pharmacological activity, and chromatographic enantioseparation of new…
Table 1 The prepared
compounds
R1
R2
R3
Compound
1
-CH
3
3
2
2
-CH
2
OCH
2
CH
3
2
3
4
5
6
-CH
-CH
-CH
-CH
-CH
CH
2
OCH
2
CH
3
CH
3
3
-CH
2
OCH
2
CH
3
CH
CH
-CH
-H
2
OCH
2
CH
3
3
3
2
OCH
2
CH
2
OCH
3
prepared, because of absence of the alkoxymethyl group,
by a simplified two-step procedure. The basic intermediates
were finally transformed into salts with fumaric acid in a
aryloxyaminopropanols may have additional vasodilatory
(apparently a-adrenolytic) effect.
The ability of the compounds to inhibit positive chron-
2
:1 ratio (base:acid) and crystallized from ethyl acetate or
otropic effect of the b-adrenergic agent isoprenaline at
conc. 10 and 10 mol dm was evaluated on sponta-
neously beating rat atria. The specific antiisoprenaline
-
7
-6
-3
propan-2-ol [23]. The yields in the final synthetic step
ranged between 34 and 65%. The prepared ary-
loxyaminopropanols in the form of their fumarates were
white solids. General procedures for each of the four syn-
thetic steps are given in the experimental part.
activity was expressed as pA values, corresponding to the
2
dissociation constants of the receptor–antagonist complex.
A comparison of the pA values indicated that the most
2
The purity of the products was tested by TLC and their
structures were investigated by spectral methods (UV, IR,
effective compound was carvedilol (8.15 ± 0.22), while
the generally inferior pA values of the evaluated com-
2
1
13
H and C NMR). In the IR spectra, the stretching
pounds were in the range from 6.54 to 7.57.
-
vibrations of the OH (3402–3460 cm ), C=O
1
All the evaluated compounds, at used concentrations,
decreased the spontaneous heart rate of isolated rat’s atria
-
1668–1675 cm ), C=C (1600–1602 cm ), and ArOalk
1
-1
(
-
1259–1268 cm ) groups could be observed. The UV
1
-7
-3
(
(except compd. 2 at conc. 10 mol dm ) with maximum
effect at 20th min after their administration (Table 3).
Enantioseparation of the studied compounds was per-
formed on a Chiralpak AD column based on derivatized
amylose (amylose tris(3,5-dimethylcarbamate) chiral sta-
tionary phase) and on Chirobiotic T column based on
native teicoplanin. As shown in Table 4, compounds 2 and
4 were separated to the baseline on the Chiralpak-AD
*
spectra display three bands corresponding to p ? p
1
13
transitions. The signals in H and C NMR spectra cor-
respond to the expected structure of the compounds,
showing clearly the successful attachment of the hetero-
cyclic moiety. The NMR spectra are available online in the
supplementary material for this article.
The effect of the target compounds on a-adrenergic
receptors was studied in isolated, phenylephrine reconsti-
tuted aorta of Wistar rats. The described vasodilatory effect
of the investigated compounds was comparable to or
column with a = 1.17–1.24 and R = 0.82–3.64, whereas
S
on the Chirobiotic T column no separation was obtained.
Using the mobile phase methanol/acetonitrile/acetic acid/
triethylamine 45/55/0.3/0.2) v/v/v/v on a Chirobiotic T
column, compounds 1 and 3 with the pyrrolidine moiety
stronger
4.97 ± 1.36 9 10
Table 2) show that all compounds at conc. 10
than
that
of
carvedilol
(EC50
-
7
-3
=
mol dm ). Experimental results
-
6
(
were separated with a = 1.05–1.06 and R = 1.41–1.55.
S
-
3
mol dm on isolated aorta inhibited the vasoconstrictor
effect of phenylephrine (8.2–33.7%). The inhibitory effect
seems to be non-competitive, involving several potential
mechanisms. Besides b-antagonism, the heterocyclic moi-
ety (pyrrolidine or N-methylpiperazine) in the basic part of
These results show that the Chirobiotic T column was
most suitable for compounds 1 and 3 with the pyrrolidine
moiety, while for compounds 2, 4, 5, and 6 the Chiralpak-
AD-H column showed the best efficiency (Table 4, Fig. 1).
123

ˇ
R. Ci zˇ máriková et al.
Table 2 Inhibitory effect of the
evaluated compounds and
carvedilol on isolated aorta of
rats (% inhibition ± SEM) and
mean effective concentration of
phenylephrine (EC50 ± SEM)
in the presence of the
-3
-3
Compound
Conc./mol dm
% Inhibition
EC50/mol dm
-6
10
-10
1
8.20 ± 3.10
8.4 ± 3.20
2.82 ± 1.65 9 10
-
-
-
6
6
6
-8
2
10
10
10
10
10
10
9.18 ± 0.39 9 10
-8
3
21.6 ± 6.36
33.7 ± 11.35
16.1 ± 2.24
10.44 ± 7.50
73.14 ± 9.58
5.98 ± 0.66 9 10
-7
4
1.01 ± 0.39 9 10
compounds
-6
-8
5
5.05 ± 1.72 9 10
-
-
6
7
-8
6
4.86 ± 1.42 9 10
-7
Carvedilol
4.97 ± 1.36 9 10
Table 3 Influence of the evaluated compounds on heart rate of spontaneously beating rat’s atria and calculated pA
apparent b-adrenolytic potency
2
values expressing their
-3
Compound
Conc./mol dm
5 min
10 min
15 min
20 min
pA
2
-
-
-
-
-
-
-
7
7
6
6
6
6
7
1
10
10
10
10
10
10
10
100.0 ± 2.81
115.0 ± 3.29
101.3 ± 0.33
99.3 ± 0.42
101.0 ± 1.38
96.7 ± 1.23
98.8 ± 0.79
98.0 ± 2.91
110.0 ± 3.10
100.3 ± 0.33
98.9 ± 1.13
99.6 ± 1.59
97.8 ± 0.78
96.3 ± 1.35
96.0 ± 2.40
108.0 ± 3.62
98.4 ± 0.95
97.4 ± 0.96
98.4 ± 1.42
98.2 ± 0.98
95.4 ± 1.05
92.0 ± 1.95
100.0 ± 3.71
97.4 ± 0.70
96.8 ± 0.76
96.8 ± 1.66
98.5 ± 0.87
94.6 ± 1.52
7.53 ± 0.21
7.57 ± 0.50
6.57 ± 0.13
6.60 ± 0.19
6.54 ± 0.14
6.99 ± 0.23
8.15 ± 0.22
2
3
4
5
6
Carvedilol
the compounds was established by appropriate analytical
Table 4 HPLC enantioseparation on Chirobiotic T (T) and Chiralpak
AD (AD-H) columns
1
13
techniques, first and foremost by H and C NMR. The
compounds were then subjected to biological testing. It
could be shown that all evaluated compounds at the conc.
Compound
k
1
k
2
a
R
S
Columns
1
1
2
2
3
3
4
4
5
6
9.50
10.01
11.14
5.49
1.05
1.04
1.0
1.41
0.82
0
T
-6
-3
10
mol dm
exhibited vasoconstrictor effect of
10.7
5.49
AD-H
T
phenylephrine on isolated rat aorta. The specific antiiso-
prenaline activity was expressed as pA values, which were
2
2.10
4.99
1.64
27.90
2.05
5.85
8.25
2.47
1.17
1.06
1.00
1.00
1.22
1.17
1.24
2.30
1.55
0
AD-H
T
in the range from 6.54 to 7.57.
5.28
The compounds prepared as racemates could be enan-
tioseparated using chiral HPLC. The results of the enan-
tioseparation were dependent on the chiral column used,
thus the compounds with the pyrrolidin-1-yl moiety were
most efficiently separated on a Chirobiotic T column,
whereas for compounds with the 4-methylpiperazin-1-yl
substituent the most effective column was the Chiralpak
AD.
1.64
AD-H
T
27.90
2.57
0
3.33
1.94
3.64
AD-H
AD-H
AD -H
6.85
10.22
Conclusion
Experimental
The aim of this paper was the study of the relationship
between structure and activity of six newly prepared
compounds of the aryloxyaminopropanol type. The com-
pounds were derived from 4-hydroxyphenylethanone and
All HPLC grade solvents were obtained from Merck
(Germany). All reactions were carried out using commer-
cial grade reagents and solvents. Diethyl ether was dried by
refluxing over potassium hydroxide and sodium followed
by distillation.
4
4
-hydroxyphenylpropan-1-one, with pyrrolidin-1-yl and
-methylpiperazin-1-yl in the hydrophilic part of molecule
and with ethoxymethyl or methoxyethoxymethyl side chain
on the aromatic ring in the lipophilic moiety. The sub-
stances were successfully synthesized in a four-step syn-
thesis and in satisfactory yield. The structure and purity of
The melting points were determined using a Kofler
Micro Hot Stage instrument. The purity of prepared com-
Ò
pounds was assessed using Silufol UV 254 (Merck) sheets
1
23

Synthesis, pharmacological activity, and chromatographic enantioseparation of new…
Fig. 1 Representative chromatograms illustrating the enantiomeric resolution of the compounds 1, 2, and 4 on the Chiralpak AD CSP (compound
3
shows no resolution on this column). Mobile phase: hexane/ethanol/methanol/ethylethanamine (87/11/11/0.0.1, v/v/v/v), flow rate 0.8 cm /min
3
3
in the solvent system ethyl acetate/diethylamine (9.5/0.5
v/v). UV spectra were measured on the spectrophotometer
GENESYS 10 s UV–Vis in methanol. IR spectra were
chloromethyl-4-hydroxyphenyl)alkanone and 100 cm of
dried ethanol were added. The temperature was raised to
40–50 °C and 19.2 g of solid sodium hydrogen carbonate
(0.23 mol) was added. The mixture was stirred for 6 h.
1
recorded using Nicolet 6700 (Thermo Scientific). H NMR
1
3
and C NMR were recorded on the Varian Gemini 2000
Spectrometer operating at 300 MHz for protons and
Then, NaHCO was filtered off and ethanol was removed
3
from the filtrate by distillation. The residue was crystal-
lized from hexane or cyclohexane.
7
5 MHz for carbons. Elemental analysis was carried out on
a FLESH 2000 (Thermo Scientific) analyzer and the results
were within 0.3% of the theoretical values.
General procedures for [3-alkoxymethyl-4-(3-
heterocyclo-2-hydroxypropoxy)phenyl]alkanones
General procedures for (3-chloromethyl-4-
hydroxyphenyl)alkanones
(3-Alkoxymethyl-4-hydroxyphenyl)alkanone (0.15 mol)
with 3 mol of chloromethyloxirane and 0.17 mol of 85%
KOH were heated at 50–55 °C for 4 h. The unreacted
chloromethyloxirane was removed in vacuum and [4-
(oxiran-2-ylmethoxy)phenyl]ethanone or 1-[3-(alkoxy-
methyl)-4-(oxirane-2-ylmethoxy)phenyl]alkanones were
extracted into diethylether and isolated as oils.
To a sulfonation flask set up with mechanical stirrer,
contact thermometer, and powder funnel, 0.15 mol of
3
-hydroxyphenylalkanone and 90 cm of concentrated HCl
4
were added. The temperature was raised to 45–50 °C and
.5 g of paraformaldehyde was gradually added. The
7
mixture was subsequently stirred at the same temperature
and the reaction was allowed to proceed for 4.5 h. Fol-
lowing the precipitation, the solid product was collected,
washed with water, and crystallized from benzene or ethyl
acetate.
General procedures for final products
In the last step, 0.08 mol of the oxirane intermediate and
3
0.16 mol pyrrolidine or N-methylpiperazine in 150 cm
ethanol were heated for 4 h under reflux. The solvent and
the unreacted amine were distilled off, the residue was
3
diluted with 50 cm water and the basic product was then
General procedures for (3-ethoxymethyl-4-
hydroxyphenyl)alkanones
taken into diethyl ether. The final products as salts were
prepared by adding fumaric acid into an ether solution of
the base.
To a sulfonation flask equipped with mechanical stirrer,
reflux condenser, and thermometer, 0.12 mol of (3-
123

ˇ
R. Ci zˇ máriková et al.
Ar6
Ar5
Ar3
(
2RS)-Bis[1-[3-[4-acetyl-2-(ethoxymethyl)phenoxy]-2-hy-
(ArOCH ), 112.4 (C ), 128.6 (C ), 130.5 (C ), 131.2
2
Ar4
Ar2
fum
(C ), 131.3 (C ), 137.2 (CH ), 161.6 (C ), 174.3
-
(COO ), 202.1 (CO) ppm.
Ar1
droxypropyl]pyrrolidinium] fumarate (1, C H O N )
Yield: 43%; m.p.: 128–131 °C (ethyl acetate); R = 0.83;
IR (solid): mꢀ = 3426 (OH), 1675 (C=O), 1601 (C=C), 1268
4
0 58 12 2
f
(
2RS)-Bis[1-[3-[2-(ethoxymethyl)-4-propanoylphenoxy]-2-hy-
droxypropyl]-4-methylpiperazinium] fumarate (4, C H O N )
Yield: 51%; m.p.: 173–175 °C (ethyl acetate); R = 0.51; IR
solid): mꢀ = 3432 (OH), 1670 (C=O), 1602 (C=C), 1259
ArOalk) cm ; UV–Vis (methanol): k (log e) = 218
3.38), 270 (3.20) nm; H NMR (300 MHz, DMSO-d ):
-
1
(
ArOalk) cm ; UV–Vis (methanol): k
(log e) = 218
3.28), 270 (3.18) nm; H NMR (300 MHz, DMSO-d ):
max
1
44 68 12 4
(
6
f
d = 1.22–1.27 (t, J = 7.5 Hz, 3H, OCH CH ), 2.05–2.10
(
2
3
(
(
(
pyr3,4
m, 4H, CH2
pyr2,5
), 2.54 (s, 3H, COCH ), 3.29–3.41 (m, 6H,
3
-1
max
NCH , CH
), 3.57–3.64 (q, J = 7 Hz, 2H, OCH CH ),
2
2
2
3
1
6
4
.12–4.14 (d, 2H, OCH CH), 4.35–4.37 (m, 1H, OCH
2 2-
fum
d = 1.15–1.19 (t, J = 6 Hz, 3H, COCH CH ), 1.22–1.27 (t,
2
3
CH), 4.59 (s, 2H, ArCH ), 6.66 (s, 2H, CH ), 7.05–7.07
2
pip2,6
J = 7.5 Hz, 3H, OCH CH ), 2.72–2.92 (m, 9H, CH
2
,
Ar6
d, J = 9 Hz, 1H, CH ), 7.94–8.00 (m, 2H, CH
Ar3,5
2
3
(
1
) ppm;
CHCH N, NCH ), 2.97–3.02 (q, J = 5 Hz, 2H, COCH
2
3
3
2-
C NMR (75 MHz, DMSO-d ): d = 15.7 (OCH CH ),
6
2
3
pip3,5
CH ), 3.18–3.22 (m, 4H, CH
2
), 3.57–3.64 (q, J = 7 Hz,
pyr3,4
3
2
4.1 (C
pyr2,5
C
), 26.6 (COCH ), 55.8 (CHCH N), 58.9
3 2
2
H, OCH CH ), 4.12–4.17 (m, 3H, OCH CH), 4.59 (s, 2H,
2 3 2
fum
(
), 67.0 (OCH CH ), 67.3 (CHCH N), 68.4 (ArCH ),
2 3 2 2
ArCH ), 6.71 (s, 2H, CH ), 7.06–7.08 (d, J = 6 Hz, 1H,
2
Ar6
Ar5
Ar3
7
1
1
1.8 (ArOCH ), 112.3 (C ), 128.6 (C ), 130.6 (C ),
2
Ar6
CH ), 7.96–8.01 (m, 2H, CH
Ar3,5
13
) ppm; C NMR
Ar2
31.5 (C ), 131.7 (C ), 137.3 (CH ), 161.7 (C ),
Ar4
fum
Ar1
(
75 MHz, DMSO-d ): d = 9.5 (COCH CH ), 15.6 (OCH
-
74.6 (COO ), 199.5 (CO) ppm.
6
2
3
2-
pip2,6
CH ), 32.3 (COCH CH ), 43.8 (NCH ), 52.1 (CH
2
),
3
2
3
3
pip3,5
(2RS)-Bis[1-[3-[4-acetyl-2-(ethoxymethyl)phenoxy]-2-hydrox-
54.7 (CH2
), 60.7 (CHCH N), 67.2 (CH), 68.2 (OCH
2 2-
Ar6
ypropyl]-4-methylpiperazinium] fumarate (2, C H O N )
Yield: 53%; m.p.: 145–148 °C (ethyl acetate); R = 0.46; IR
(
(
(
CH ), 68.4 (ArCH ), 72.0 (ArOCH ), 112.2 (C ), 128.4
42
64 12
4
3
Ar5
2
Ar3
2
Ar4
Ar2
(C ), 130.4 (C ), 131.0 (C ), 131.1 (C ), 135.8
Ar1
(CH ), 161.9 (C ), 170.0 (COO ), 202.0 (CO) ppm.
f
fum
-
solid): mꢀ = 3435 (OH), 1670 (C=O), 1602 (C=C), 1259
-
1
ArOalk) cm ; UV–Vis (methanol): k
3.36), 270 (3.02) nm; H NMR (300 MHz, DMSO-d ):
(log e) = 218
max
(
2RS)-Bis[1-[3-[2-[(2-methoxyethoxy)methyl]-4-
propanoylphenoxy]-2-hydroxypropyl]-4-methylpiper-
azinium] fumarate (5, C H O N ) Yield: 34%; m.p.:
73–175 °C (ethyl acetate); R = 0.24; IR (solid): mꢀ = 3402
OH), 1675 (C=O), 1601 (C=C), 1261 (ArOalk) cm
1
6
d = 1.22–1.27 (m, 3H, OCH CH ), 2.55 (s, 3H, COCH ),
2
2
3
3
pip2,6
2
44 68 12 4
.68–2.72 (m, 9H, CH
, CHCH N, NCH ), 3.03–3.07
2 3
1
pip3,5
m, 4H, CH2
f
(
), 3.57–3.64 (q, J = 7 Hz, 2H, COCH CH ),
2 3
-1
(
;
4
.07–4.17 (m, 3H, OCH CH), 4.59 (s, 2H, ArCH ), 6.67 (s,
2 2
UV–Vis (methanol): k
(log e) = 218 (3.38), 270 (3.20)
fum
H, CH ), 7.05–7.07 (d, J = 9 Hz, 1H, CH ), 7.94–8.00
Ar6
max
2
1
nm; H NMR (300 MHz, DMSO-d ): d = 2.37 (s, 3H,
COCH ), 2.51–2.56 (m, 9H, CH
3
pip3,5
.24–3.32 (m, 4H, CH2
Ar3,5
13
) ppm; C NMR (75 MHz, DMSO-d6):
6
(
m, 2H, CH
pip2,6
2
, CHCH N, NCH ),
2 3
d = 15.7 (OCH CH ), 26.6 (COCH ), 44.3 (NCH ), 52.6
(
2
3
pip3,5
3
3
3
), 3.39 (s, 3H, OCH ), 3.61–3.65
3
pip2,6
CH2
), 54.9 (CH2
), 61.1 (CHCH N), 67.4 (CH), 68.4
2
Ar6
(
m, 4H, CH CH O), 3.98–4.07 (m, 3H, OCH CH), 4.54 (s,
2 2 2
fum
(
OCH CH ), 68.4 (ArCH ), 72.3 (ArOCH ), 112.3 (C ),
2
3
Ar5
2
2
2
H, ArCH ), 6.61 (s, 2H, CH ), 7.0–7.1 (d, J = 7 Hz, 1H,
2
Ar3
Ar2
Ar4
1
28.6 (C ), 130.7 (C ), 131.3 (C ), 131.6 (C ), 136.9
Ar5
CH ), 7.90–7.93 (m, 2H, CH
Ar2,6
13
) ppm; C NMR
fum
Ar1
-
(
CH ), 162.1 (C ), 173.4 (COO ), 199.5 (CO) ppm.
2RS)-Bis[1-[3-[2-(ethoxymethyl)-4-propanoylphenoxy]-2-
hydroxypropyl]pyrrolidinium] fumarate (3, C H O N )
(
75 MHz, DMSO-d ): d = 26.8 (COCH ), 43.4 (NCH ),
6
3
3
pip2,6
pip3,5
(
51.3 (CH2
), 53.1 (CH2
), 58.6 (OCH ), 60.2
3
(CHCH N), 66.7 (CH), 67.2 (CH OCH ), 69.9 (ArOCH ),
2
4
2
62 12
2
2
2
2
Ar5
71.4 (ArCH ), 71.8 (CH OCH ), 111.5 (C ), 127.4
2 2 3
Yield: 51%; m.p.: 130–133 °C (ethyl acetate); R = 0.70;
IR (solid): mꢀ = 3460 (OH), 1668 (C=O), 1602 (C=C), 1268
f
Ar6
(C ), 128.7 (C ), 129.9 (C ), 130.3 (C ), 134.9
Ar2
Ar3
Ar1
-
1
fum
Ar4
-
(
ArOalk) cm ; UV–Vis (methanol): k
(log e) = 218
3.38), 268 (3.27) nm; H NMR (300 MHz, DMSO-d ):
(CH ), 160.2 (C ), 173.4 (COO ), 196.8 (CO) ppm.
2RS)-Bis[1-[3-(4-acetylphenoxy)-2-hydroxypropyl]-4-
methylpiperazinium] fumarate (6, C H O N ) Yield:
max
1
(
6
(
d = 1.14–1.19 (t, J = 7 Hz, 3H, COCH CH ), 1.22–1.27 (t,
J = 7 Hz, 3H, OCH CH ), 2.05–2.09 (m, 4H, CH
2
3
pyr3,4
2
36 52 10 4
),
.97–3.04 (q, J = 7 Hz, 2H, COCH CH ), 3.29–3.11 (m,
2
3
6
5%; m.p.: 197–200 °C (ethyl acetate); R = 0.52; IR
solid): mꢀ = 3326 (OH), 1671 (C=O), 1600 (C=C), 1261
ArOalk) cm ; UV–Vis (methanol): k
3.48), 272 (3.49) nm; H NMR (300 MHz, DMSO-d ):
f
2
6
2
3
(
(
(
pyr2,5
2
H, NCH , CH
2
), 3.57–3.64 (q, J = 7 Hz, 2H, OCH2-
-1
(log e) = 218
max
CH ), 4.11–4.13 (d, 2H, OCH CH), 4.33–4.35 (m, 1H,
3
fum
OCH CH), 4.60 (s, 2H, ArCH ), 6.66 (s, 2H, CH ),
2
1
6
2
2
d = 2.40 (s, 3H, COCH ), 2.49–2.67 (m, 13H, CH N, C-
, NCH ), 3.95–4.08 (m, 3H, OCH CH), 6.60 (s,
2
fum
H, CH ), 7.02–7.05 (d, J = 9 Hz, 2H, CH
Ar6
.08–7.05 (d, J = 9 Hz, 1H, CH ), 7.95–8.01 (m, 2H,
3
2
7
pip2,3,5,6
H2
Ar3,5
13
) ppm; C NMR (75 MHz, DMSO-d ): d = 8.9
3
CH
6
pyr3,4
Ar2,6
2
7
),
(
(
(
COCH CH ), 15.7 (OCH CH ), 24.1 (C
2
), 32.5
), 67.0
3
2
3
Ar3,5
13
) ppm; C NMR
.91–7.94 (d, J = 9 Hz, 2H, CH
pyr2,5
COCH CH ), 55.8 (CHCH N), 58.9 (C
2
2
3
(
75 MHz, DMSO-d ): d = 26.9 (COCH ), 44.6 (NCH ),
6
3
3
OCH CH ), 67.2 (CHCH N), 68.5 (ArCH2), 71.8
3
2
2
1
23

Synthesis, pharmacological activity, and chromatographic enantioseparation of new…
pip2,6
pip3,5
5
2.4 (CH2
), 54.1 (CH2
), 60.6 (CHCH N), 66.8
Chromatographic characteristics
2
Ar4
Ar2,6
(
(
(
CH), 71.6 (ArOCH ), 110.0 (C
Ar3,5
), 114.8 (C ), 131.0
2
fum
Ar1 -
), 136.4 (CH ), 163.0 (C ), 173.6 (COO ), 199.6
C
The separation factor was expressed as a = k /k , where k ,
1
2
1
CO) ppm.
k2 are retention factors for the first and second eluting
enantiomers. The retention factors k’ were calculated as
follows: k ¼ ðt À t Þ=t and k ¼ ðt À t Þ=t , where t ,
Pharmacological evaluation
1
1
0
0
2
2
0
0
0
t , and t are the dead elution time and elution times of
1
2
b-Adrenolytic activity of the synthesized compounds was
evaluated on isolated rat’s atria and expressed as pA2
values against tachycardia, induced by isoprenaline,
according to [26]. The vasodilatory activity was evaluated
on phenylephrine-induced contraction of rat aortal strips.
The inhibitory effect on phenylephrine-induced contraction
of isolated aorta was expressed as % of inhibition ± SEM
and mean effective concentration of phenylephrine in the
presence of tested compounds at previously determined
concentration (EC50 ± SEM) [27]. Each value of phar-
macological evaluation represents the mean ± SEM from
enantiomers 1 and 2. The stereochemical resolution factor
(R ) of the first and second eluting enantiomer was cal-
S
culated as the ratio of the difference between the retention
times t and t to the arithmetic sum of the two peaks’
2
1
widths w and w : R ¼ 2ðt À t Þ=ðw þ w Þ.
S
2
1
1
2
1
2
Acknowledgements 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 no.
ˇ
ˇ
APVV-0516-12; Vedeck a´ Grantov a´ Agent u´ ra MSVVaS SR a SAV
(VEGA 1/0346/16).
5
to 7 experiments.
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23