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Monatshefte für Chemie - Chemical Monthly

, Volume 149, Issue 5, pp 969–976 | Cite as

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

  • Ružena Čižmáriková
  • Andrej Némethy
  • Ladislav Habala
  • Eva Račanská
  • Jindra Valentová
  • Katarína Hroboňová
Original Paper
  • 318 Downloads

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 substituents 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, alkoxymethyl, and oxirane intermediates. The purity of the target compounds was checked by TLC and their structures were confirmed by the interpretation of the IR, UV, 1H NMR, and 13C NMR spectra. The pharmacological evaluation of the obtained compounds confirmed their vasodilatory and specific antiisoprenaline activities. All evaluated compounds at conc. 10−6 mol dm−3 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 evaluated compounds at conc. 10−7 and 10−6 mol dm−3. The calculated pA2 values of specific antagonistic effect against isoprenaline, related to their apparent β-adrenolytic activity, ranged between 6.54 and 7.57. The value for the standard compound carvedilol was 8.15 ± 0.22. The majority of the evaluated compounds at conc. 10−6–10−7 mol dm−3 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

Keywords

Aryloxyaminopropanols Vasodilatory Antiisoprenaline Beta-adrenolytic Chirality Drug research 

Introduction

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 β-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 α- and β-adrenolytic efficacy that exhibits antihypertensive activity and a high-antioxidant potency [4, 5, 6].

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 α1- and β-adrenergic receptors [9, 10]. Some piperazine derivatives show antidepressant-like- [11] and H1-antihistaminic 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 α1-receptor blockade, while the affinity for α2-and β-adrenoceptors was very weak [13, 14]. Naftopidil also blocks Ca2+ channels, inhibits serotonin-induced platelet aggregation, and reduces serotonin uptake by thrombocytes [15]. Other results demonstrated its use in benign prostatic hyperplasia [16].

Since compounds of the aryloxyaminopropanol type possess a stereogenic center in their structure, chiral HPLC technique was used for the separation of individual enantiomers. Chiral stationary phases used for enantioseparation 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 pharmacological evaluation and the study of HPLC enantioseparation 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 orientated towards their vasodilatory and specific antiisoprenaline properties, which are inherent to all clinically used β-blockers. Chiral columns, based on amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak AD) and native teicoplanin (Chirobiotic T), were used to separate the individual enantiomers.

Results and discussion

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 β-blockers exhibited higher affinity for β1-receptors in comparison with celiprolol and acebutolol 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 vasodilatory effect. The target compounds 15 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 14) or 2-methoxyethanol (compound 5) and solid NaHCO3, yielding (3-alkoxymethyl-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 intermediates in ethanol reacted with pyrrolidine and N-methylpiperazine to attach the basic heterocyclic moiety. An exception was the compound 6, which could be 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 2:1 ratio (base:acid) and crystallized from ethyl acetate or propan-2-ol [23]. The yields in the final synthetic step ranged between 34 and 65%. The prepared aryloxyaminopropanols in the form of their fumarates were white solids. General procedures for each of the four synthetic steps are given in the experimental part.
Table 1

The prepared compounds

The purity of the products was tested by TLC and their structures were investigated by spectral methods (UV, IR, 1H and 13C NMR). In the IR spectra, the stretching vibrations of the OH (3402–3460 cm−1), C=O (1668–1675 cm−1), C=C (1600–1602 cm−1), and ArOalk (1259–1268 cm−1) groups could be observed. The UV spectra display three bands corresponding to π → π* transitions. The signals in 1H and 13C NMR spectra correspond to the expected structure of the compounds, showing clearly the successful attachment of the heterocyclic moiety. The NMR spectra are available online in the supplementary material for this article.

The effect of the target compounds on α-adrenergic receptors was studied in isolated, phenylephrine reconstituted aorta of Wistar rats. The described vasodilatory effect of the investigated compounds was comparable to or stronger than that of carvedilol (EC50 = 4.97 ± 1.36 × 10−7 mol dm−3). Experimental results (Table 2) show that all compounds at conc. 10−6 mol dm−3 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 β-antagonism, the heterocyclic moiety (pyrrolidine or N-methylpiperazine) in the basic part of aryloxyaminopropanols may have additional vasodilatory (apparently α-adrenolytic) effect.
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 compounds

Compound

Conc./mol dm−3

% Inhibition

EC50/mol dm−3

1

10−6

8.20 ± 3.10

2.82 ± 1.65 × 10−10

2

10−6

8.4 ± 3.20

9.18 ± 0.39 × 10−8

3

10−6

21.6 ± 6.36

5.98 ± 0.66 × 10−8

4

10−6

33.7 ± 11.35

1.01 ± 0.39 × 10−7

5

10−6

16.1 ± 2.24

5.05 ± 1.72 × 10−8

6

10−6

10.44 ± 7.50

4.86 ± 1.42 × 10−8

Carvedilol

10−7

73.14 ± 9.58

4.97 ± 1.36 × 10−7

The ability of the compounds to inhibit positive chronotropic effect of the β-adrenergic agent isoprenaline at conc. 10−7 and 10−6 mol dm−3 was evaluated on spontaneously beating rat atria. The specific antiisoprenaline activity was expressed as pA2 values, corresponding to the dissociation constants of the receptor–antagonist complex.

A comparison of the pA2 values indicated that the most effective compound was carvedilol (8.15 ± 0.22), while the generally inferior pA2 values of the evaluated compounds were in the range from 6.54 to 7.57.

All the evaluated compounds, at used concentrations, decreased the spontaneous heart rate of isolated rat’s atria (except compd. 2 at conc. 10−7 mol dm−3) with maximum effect at 20th min after their administration (Table 3).
Table 3

Influence of the evaluated compounds on heart rate of spontaneously beating rat’s atria and calculated pA2 values expressing their apparent β-adrenolytic potency

Compound

Conc./mol dm−3

5 min

10 min

15 min

20 min

pA2

1

10−7

100.0 ± 2.81

98.0 ± 2.91

96.0 ± 2.40

92.0 ± 1.95

7.53 ± 0.21

2

10−7

115.0 ± 3.29

110.0 ± 3.10

108.0 ± 3.62

100.0 ± 3.71

7.57 ± 0.50

3

10−6

101.3 ± 0.33

100.3 ± 0.33

98.4 ± 0.95

97.4 ± 0.70

6.57 ± 0.13

4

10−6

99.3 ± 0.42

98.9 ± 1.13

97.4 ± 0.96

96.8 ± 0.76

6.60 ± 0.19

5

10−6

101.0 ± 1.38

99.6 ± 1.59

98.4 ± 1.42

96.8 ± 1.66

6.54 ± 0.14

6

10−6

96.7 ± 1.23

97.8 ± 0.78

98.2 ± 0.98

98.5 ± 0.87

6.99 ± 0.23

Carvedilol

10−7

98.8 ± 0.79

96.3 ± 1.35

95.4 ± 1.05

94.6 ± 1.52

8.15 ± 0.22

Enantioseparation of the studied compounds was performed on a Chiralpak AD column based on derivatized amylose (amylose tris(3,5-dimethylcarbamate) chiral stationary 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 column with α = 1.17–1.24 and RS = 0.82–3.64, whereas 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 were separated with α = 1.05–1.06 and RS = 1.41–1.55.
Table 4

HPLC enantioseparation on Chirobiotic T (T) and Chiralpak AD (AD-H) columns

Compound

k 1

k 2

α

R S

Columns

1

9.50

10.01

1.05

1.41

T

1

10.7

11.14

1.04

0.82

AD-H

2

5.49

5.49

1.0

0

T

2

2.10

2.47

1.17

2.30

AD-H

3

4.99

5.28

1.06

1.55

T

3

1.64

1.64

1.00

0

AD-H

4

27.90

27.90

1.00

0

T

4

2.05

2.57

1.22

3.33

AD-H

5

5.85

6.85

1.17

1.94

AD-H

6

8.25

10.22

1.24

3.64

AD -H

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).
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 cm3/min

Conclusion

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 compounds were derived from 4-hydroxyphenylethanone and 4-hydroxyphenylpropan-1-one, with pyrrolidin-1-yl and 4-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 substances were successfully synthesized in a four-step synthesis and in satisfactory yield. The structure and purity of the compounds was established by appropriate analytical techniques, first and foremost by 1H and 13C NMR. The compounds were then subjected to biological testing. It could be shown that all evaluated compounds at the conc. 10−6 mol dm−3 exhibited vasoconstrictor effect of phenylephrine on isolated rat aorta. The specific antiisoprenaline activity was expressed as pA2 values, which were in the range from 6.54 to 7.57.

The compounds prepared as racemates could be enantioseparated using chiral HPLC. The results of the enantioseparation 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.

Experimental

All HPLC grade solvents were obtained from Merck (Germany). All reactions were carried out using commercial grade reagents and solvents. Diethyl ether was dried by refluxing over potassium hydroxide and sodium followed by distillation.

The melting points were determined using a Kofler Micro Hot Stage instrument. The purity of prepared compounds was assessed using Silufol® UV 254 (Merck) sheets 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 recorded using Nicolet 6700 (Thermo Scientific). 1H NMR and 13C NMR were recorded on the Varian Gemini 2000 Spectrometer operating at 300 MHz for protons and 75 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-chloromethyl-4-hydroxyphenyl)alkanones

To a sulfonation flask set up with mechanical stirrer, contact thermometer, and powder funnel, 0.15 mol of 4-hydroxyphenylalkanone and 90 cm3 of concentrated HCl were added. The temperature was raised to 45–50 °C and 7.5 g of paraformaldehyde was gradually added. The mixture was subsequently stirred at the same temperature and the reaction was allowed to proceed for 4.5 h. Following the precipitation, the solid product was collected, washed with water, and crystallized from benzene or ethyl acetate.

General procedures for (3-ethoxymethyl-4-hydroxyphenyl)alkanones

To a sulfonation flask equipped with mechanical stirrer, reflux condenser, and thermometer, 0.12 mol of (3-chloromethyl-4-hydroxyphenyl)alkanone and 100 cm3 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. Then, NaHCO3 was filtered off and ethanol was removed from the filtrate by distillation. The residue was crystallized from hexane or cyclohexane.

General procedures for [3-alkoxymethyl-4-(3-heterocyclo-2-hydroxypropoxy)phenyl]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-(alkoxymethyl)-4-(oxirane-2-ylmethoxy)phenyl]alkanones were extracted into diethylether and isolated as oils.

General procedures for final products

In the last step, 0.08 mol of the oxirane intermediate and 0.16 mol pyrrolidine or N-methylpiperazine in 150 cm3 ethanol were heated for 4 h under reflux. The solvent and the unreacted amine were distilled off, the residue was diluted with 50 cm3 water and the basic product was then taken into diethyl ether. The final products as salts were prepared by adding fumaric acid into an ether solution of the base.

(2RS)-Bis[1-[3-[4-acetyl-2-(ethoxymethyl)phenoxy]-2-hydroxypropyl]pyrrolidinium] fumarate (1, C40H58O12N2)

Yield: 43%; m.p.: 128–131 °C (ethyl acetate); Rf = 0.83; IR (solid): \(\bar{\nu }\) = 3426 (OH), 1675 (C=O), 1601 (C=C), 1268 (ArOalk) cm−1; UV–Vis (methanol): λmax (log ε) = 218 (3.28), 270 (3.18) nm; 1H NMR (300 MHz, DMSO-d6): δ = 1.22–1.27 (t, J = 7.5 Hz, 3H, OCH2CH3), 2.05–2.10 (m, 4H, CH 2 pyr3,4 ), 2.54 (s, 3H, COCH3), 3.29–3.41 (m, 6H, NCH2, CH 2 pyr2,5 ), 3.57–3.64 (q, J = 7 Hz, 2H, OCH2CH3), 4.12–4.14 (d, 2H, OCH2CH), 4.35–4.37 (m, 1H, OCH2CH), 4.59 (s, 2H, ArCH2), 6.66 (s, 2H, CHfum), 7.05–7.07 (d, J = 9 Hz, 1H, CHAr6), 7.94–8.00 (m, 2H, CHAr3,5) ppm; 13C NMR (75 MHz, DMSO-d6): δ = 15.7 (OCH2CH3), 24.1 (Cpyr3,4), 26.6 (COCH3), 55.8 (CHCH2N), 58.9 (Cpyr2,5), 67.0 (OCH2CH3), 67.3 (CHCH2N), 68.4 (ArCH2), 71.8 (ArOCH2), 112.3 (CAr6), 128.6 (CAr5), 130.6 (CAr3), 131.5 (CAr2), 131.7 (CAr4), 137.3 (CHfum), 161.7 (CAr1), 174.6 (COO), 199.5 (CO) ppm.

(2RS)-Bis[1-[3-[4-acetyl-2-(ethoxymethyl)phenoxy]-2-hydroxypropyl]-4-methylpiperazinium] fumarate (2, C42H64O12N4)

Yield: 53%; m.p.: 145–148 °C (ethyl acetate); Rf = 0.46; IR (solid): \(\bar{\nu }\) = 3435 (OH), 1670 (C=O), 1602 (C=C), 1259 (ArOalk) cm−1; UV–Vis (methanol): λmax (log ε) = 218 (3.36), 270 (3.02) nm; 1H NMR (300 MHz, DMSO-d6): δ = 1.22–1.27 (m, 3H, OCH2CH3), 2.55 (s, 3H, COCH3), 2.68–2.72 (m, 9H, CH 2 pip2,6 , CHCH2N, NCH3), 3.03–3.07 (m, 4H, CH 2 pip3,5 ), 3.57–3.64 (q, J = 7 Hz, 2H, COCH2CH3), 4.07–4.17 (m, 3H, OCH2CH), 4.59 (s, 2H, ArCH2), 6.67 (s, 2H, CHfum), 7.05–7.07 (d, J = 9 Hz, 1H, CHAr6), 7.94–8.00 (m, 2H, CHAr3,5) ppm; 13C NMR (75 MHz, DMSO-d6): δ = 15.7 (OCH2CH3), 26.6 (COCH3), 44.3 (NCH3), 52.6 (CH 2 pip2,6 ), 54.9 (CH 2 pip3,5 ), 61.1 (CHCH2N), 67.4 (CH), 68.4 (OCH2CH3), 68.4 (ArCH2), 72.3 (ArOCH2), 112.3 (CAr6), 128.6 (CAr5), 130.7 (CAr3), 131.3 (CAr2), 131.6 (CAr4), 136.9 (CHfum), 162.1 (CAr1), 173.4 (COO), 199.5 (CO) ppm.

(2RS)-Bis[1-[3-[2-(ethoxymethyl)-4-propanoylphenoxy]-2-hydroxypropyl]pyrrolidinium] fumarate (3, C42H62O12N2)

Yield: 51%; m.p.: 130–133 °C (ethyl acetate); Rf = 0.70; IR (solid): \(\bar{\nu }\) = 3460 (OH), 1668 (C=O), 1602 (C=C), 1268 (ArOalk) cm−1; UV–Vis (methanol): λmax (log ε) = 218 (3.38), 268 (3.27) nm; 1H NMR (300 MHz, DMSO-d6): δ = 1.14–1.19 (t, J = 7 Hz, 3H, COCH2CH3), 1.22–1.27 (t, J = 7 Hz, 3H, OCH2CH3), 2.05–2.09 (m, 4H, CH 2 pyr3,4 ), 2.97–3.04 (q, J = 7 Hz, 2H, COCH2CH3), 3.29–3.11 (m, 6H, NCH2, CH 2 pyr2,5 ), 3.57–3.64 (q, J = 7 Hz, 2H, OCH2CH3), 4.11–4.13 (d, 2H, OCH2CH), 4.33–4.35 (m, 1H, OCH2CH), 4.60 (s, 2H, ArCH2), 6.66 (s, 2H, CHfum), 7.08–7.05 (d, J = 9 Hz, 1H, CHAr6), 7.95–8.01 (m, 2H, CHAr3,5) ppm; 13C NMR (75 MHz, DMSO-d6): δ = 8.9 (COCH2CH3), 15.7 (OCH2CH3), 24.1 (Cpyr3,4), 32.5 (COCH2CH3), 55.8 (CHCH2N), 58.9 (Cpyr2,5), 67.0 (OCH2CH3), 67.2 (CHCH2N), 68.5 (ArCH2), 71.8 (ArOCH2), 112.4 (CAr6), 128.6 (CAr5), 130.5 (CAr3), 131.2 (CAr4), 131.3 (CAr2), 137.2 (CHfum), 161.6 (CAr1), 174.3 (COO), 202.1 (CO) ppm.

(2RS)-Bis[1-[3-[2-(ethoxymethyl)-4-propanoylphenoxy]-2-hydroxypropyl]-4-methylpiperazinium] fumarate (4, C44H68O12N4)

Yield: 51%; m.p.: 173–175 °C (ethyl acetate); Rf = 0.51; IR (solid): \(\bar{\nu }\) = 3432 (OH), 1670 (C=O), 1602 (C=C), 1259 (ArOalk) cm−1; UV–Vis (methanol): λmax (log ε) = 218 (3.38), 270 (3.20) nm; 1H NMR (300 MHz, DMSO-d6): δ = 1.15–1.19 (t, J = 6 Hz, 3H, COCH2CH3), 1.22–1.27 (t, J = 7.5 Hz, 3H, OCH2CH3), 2.72–2.92 (m, 9H, CH 2 pip2,6 , CHCH2N, NCH3), 2.97–3.02 (q, J = 5 Hz, 2H, COCH2CH3), 3.18–3.22 (m, 4H, CH 2 pip3,5 ), 3.57–3.64 (q, J = 7 Hz, 2H, OCH2CH3), 4.12–4.17 (m, 3H, OCH2CH), 4.59 (s, 2H, ArCH2), 6.71 (s, 2H, CHfum), 7.06–7.08 (d, J = 6 Hz, 1H, CHAr6), 7.96–8.01 (m, 2H, CHAr3,5) ppm; 13C NMR (75 MHz, DMSO-d6): δ = 9.5 (COCH2CH3), 15.6 (OCH2CH3), 32.3 (COCH2CH3), 43.8 (NCH3), 52.1 (CH 2 pip2,6 ), 54.7 (CH 2 pip3,5 ), 60.7 (CHCH2N), 67.2 (CH), 68.2 (OCH2CH3), 68.4 (ArCH2), 72.0 (ArOCH2), 112.2 (CAr6), 128.4 (CAr5), 130.4 (CAr3), 131.0 (CAr4), 131.1 (CAr2), 135.8 (CHfum), 161.9 (CAr1), 170.0 (COO), 202.0 (CO) ppm.

(2RS)-Bis[1-[3-[2-[(2-methoxyethoxy)methyl]-4-propanoylphenoxy]-2-hydroxypropyl]-4-methylpiperazinium] fumarate (5, C44H68O12N4)

Yield: 34%; m.p.: 173–175 °C (ethyl acetate); Rf = 0.24; IR (solid): \(\bar{\nu }\) = 3402 (OH), 1675 (C=O), 1601 (C=C), 1261 (ArOalk) cm−1; UV–Vis (methanol): λmax (log ε) = 218 (3.38), 270 (3.20) nm; 1H NMR (300 MHz, DMSO-d6): δ = 2.37 (s, 3H, COCH3), 2.51–2.56 (m, 9H, CH 2 pip2,6 , CHCH2N, NCH3), 3.24–3.32 (m, 4H, CH 2 pip3,5 ), 3.39 (s, 3H, OCH3), 3.61–3.65 (m, 4H, CH2CH2O), 3.98–4.07 (m, 3H, OCH2CH), 4.54 (s, 2H, ArCH2), 6.61 (s, 2H, CHfum), 7.0–7.1 (d, J = 7 Hz, 1H, CHAr5), 7.90–7.93 (m, 2H, CHAr2,6) ppm; 13C NMR (75 MHz, DMSO-d6): δ = 26.8 (COCH3), 43.4 (NCH3), 51.3 (CH 2 pip2,6 ), 53.1 (CH 2 pip3,5 ), 58.6 (OCH3), 60.2 (CHCH2N), 66.7 (CH), 67.2 (CH2OCH2), 69.9 (ArOCH2), 71.4 (ArCH2), 71.8 (CH2OCH3), 111.5 (CAr5), 127.4 (CAr6), 128.7 (CAr2), 129.9 (CAr3), 130.3 (CAr1), 134.9 (CHfum), 160.2 (CAr4), 173.4 (COO), 196.8 (CO) ppm.

(2RS)-Bis[1-[3-(4-acetylphenoxy)-2-hydroxypropyl]-4-methylpiperazinium] fumarate (6, C36H52O10N4)

Yield: 65%; m.p.: 197–200 °C (ethyl acetate); Rf = 0.52; IR (solid): \(\bar{\nu }\) = 3326 (OH), 1671 (C=O), 1600 (C=C), 1261 (ArOalk) cm−1; UV–Vis (methanol): λmax (log ε) = 218 (3.48), 272 (3.49) nm; 1H NMR (300 MHz, DMSO-d6): δ = 2.40 (s, 3H, COCH3), 2.49–2.67 (m, 13H, CH2N, CH 2 pip2,3,5,6 , NCH3), 3.95–4.08 (m, 3H, OCH2CH), 6.60 (s, 2H, CHfum), 7.02–7.05 (d, J = 9 Hz, 2H, CHAr2,6), 7.91–7.94 (d, J = 9 Hz, 2H, CHAr3,5) ppm; 13C NMR (75 MHz, DMSO-d6): δ = 26.9 (COCH3), 44.6 (NCH3), 52.4 (CH 2 pip2,6 ), 54.1 (CH 2 pip3,5 ), 60.6 (CHCH2N), 66.8 (CH), 71.6 (ArOCH2), 110.0 (CAr2,6), 114.8 (CAr4), 131.0 (CAr3,5), 136.4 (CHfum), 163.0 (CAr1), 173.6 (COO), 199.6 (CO) ppm.

Pharmacological evaluation

β-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 pharmacological evaluation represents the mean ± SEM from 5 to 7 experiments.

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.

HPLC analysis

HPLC studies were carried out on the HPLC system AGILENT 1200 with a quaternary pump and a diode detector, using the chiral stationary phases (Chiralpak AD) based on amylase tris(3,5-dimethylphenylcarbamate) (0.46 × 25). The mobile phases consisted of hexane/ethanol/methanol/diethylamine 87/11/11/0.1, v/v/v/v. Samples for analyses were prepared as approximately 1 mg/cm3 solution in methanol. Compounds were diluted in the ratio 1:4 (1–4) and 1:10 (5–6). Separations were carried out at the flow rate of 0.8 cm3/min and the column temperature was maintained at 25 °C. Chromatograms were scanned at the wavelength 267 ± 8 nm.

Second HPLC studies were performed with a Hewlett-Packard (series 1100) HPLC system consisting of a quaternary pump equipped with an injection valve (Rheodyne) and diode array detector. The macrocyclic chiral stationary phase was Chirobiotic T (250 × 4 mm LD-particle size 5 µm Advanced Separation technologies. Inc. USA). The mobile phase was a mixture of methanol/acetonitrile/acetic acid/triethylamine 45/55/0.3/0.2) v/v/v/v. The separation was carried out at the flow rate of 1 cm3/min and the column temperature was 23 °C. The chromatograms were scanned at 270 nm. The injection volume was 20 mm3. The analyte was dissolved in methanol (concentration 1 mg/cm3).

Chromatographic characteristics

The separation factor was expressed as α = k1/k2, where k1, k2 are retention factors for the first and second eluting enantiomers. The retention factors k’ were calculated as follows: \(k_{ 1} \, = \,(t_{ 1} - t_{0} )/t_{0}\) and \(k_{2} \, = \,(t_{2} - t_{0} )/t_{0}\), where t0, t1, and t2 are the dead elution time and elution times of enantiomers 1 and 2. The stereochemical resolution factor (RS) of the first and second eluting enantiomer was calculated as the ratio of the difference between the retention times t1 and t2 to the arithmetic sum of the two peaks’ widths w1 and w2: \(R_{\text{S}} \, = \,2(t_{2} - t_{1} )/(w_{1} \, + \,w_{2} )\).

Notes

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. APVV-0516-12; Vedecká Grantová Agentúra MŠVVaŠ SR a SAV (VEGA 1/0346/16).

Supplementary material

706_2018_2185_MOESM1_ESM.pdf (929 kb)
Supplementary material 1 (PDF 928 kb)

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Chemical Theory of Drugs, Faculty of PharmacyComenius University in BratislavaBratislavaSlovak Republic
  2. 2.Department of Pharmacology and Toxicology, Faculty of PharmacyComenius University in BratislavaBratislavaSlovak Republic
  3. 3.Faculty of Chemical and Food Technology, Institute of Analytical ChemistrySlovak University of Technology in BratislavaBratislavaSlovak Republic

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