Synthesis and determination of physicochemical properties of new 3-(4-arylpiperazin-1-yl)-2-hydroxypropyl 4-alkoxyethoxybenzoates

Article abstract of DOI:10.3390/molecules21121682

Nine new dihydrochloride salts of 3-(4-arylpiperazin-1-yl)-2-hydroxypropyl 4-alkoxyethoxybenzoates were designed and synthesized. The physicochemical properties such as lipophilicity index (log k_w) and dissociation constant (pK_a) wer

Full text of DOI:10.3390/molecules21121682

molecules
Communication
Synthesis and Determination of Physicochemical
Properties of New 3-(4-Arylpiperazin-1-yl)-2-
hydroxypropyl 4-Alkoxyethoxybenzoates
Pavlina Marvanova 1,*, Tereza Padrtova 1, Klara Odehnalova 1, Ondrej Hosik 1, Michal Oravec 2
and Petr Mokry 1,
*
1
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences,
Palackeho 1, 61242 Brno, Czech Republic; padrtova.tereza@seznam.cz (T.P.); odehnalovak@vfu.cz (K.O.);
f12058@vfu.cz (O.H.)
Global Change Research Institute CAS, Belidla 986/4a, 60300 Brno, Czech Republic; oravec.m@czechglobe.cz
Correspondence: marvanovap@gmail.com (Pa.M.); mokryp@vfu.cz (Pe.M.); Tel.: +420-541-562-928 (Pe.M.)
2
*
Academic Editor: Atanas G. Atanasov
Received: 27 September 2016; Accepted: 30 November 2016; Published: 7 December 2016
Abstract:
Nine new dihydrochloride salts of 3-(4-arylpiperazin-1-yl)-2-hydroxypropyl
4-alkoxyethoxybenzoates were designed and synthesized. The physicochemical properties
such as lipophilicity index (log kw) and dissociation constant (pKa) were experimentally determined
and compared to the software calculated data. The lipophilicity index was determined by means
of reversed-phase high performance liquid chromatography (RP-HPLC). The pKa values were
determined by means of capillary zone electrophoresis. The “drug-likeness” properties according to
the Lipinski Rule of Five and prediction of possible blood–brain barrier penetration were computed
and discussed.
Keywords: arylcarbonyloxyaminopropanols; phenylpiperazines; pKa determination; lipophilicity
index; Lipinski rules; blood–brain barrier
1. Introduction
Nowadays, many drugs used in clinical practice have acidic or basic functionalities in their
structures, antagonists of
prescribed and used cardiovascular drugs for more than 50 years, are still being improved with
additional features such as vasodilatory and antioxidant activity. The dual α1- and -blockers
β-adrenergic receptors (β-blockers) included. β-blockers, one of the most
β
(e.g., carvedilol, labetalol, vanylidilol) showed hemodynamic benefits, including reduction of
peripheral vascular resistance, improvement of cardiac output and left ventricular function, which
is why the vasodilatory activity is an enhancement to the antihypertensive effect of
offsets its drawback [ ]. The pharmacokinetic effect and toxicity of the newly prepared compound
can be predicted also from the determination of their physicochemical properties [4,5].
β-blockade and
1–3
The physicochemical parameters such as lipophilicity (log P), distribution coefficient (log D)
and dissociation constant (pKa) of an active pharmaceutical ingredient belong to crucial properties
used to estimate the absorption, distribution, metabolism, and excretion (ADME) of a compound
in biological systems as well as quantitative structure-activity and structure property relationship
(QSAR/QSPR) profiling [
predict these values, but the results of prediction can considerably differ depending on the algorithm
used [ ]. Therefore, predicted values should be replaced by experimental data in early stage of drug
development. Among others, the reversed-phase high performance liquid chromatography (RP-HPLC)
method is a successfully used tool for lipophilicity assessment [10 12]. One of the most predictive
methods for lipophilicity determination is realized by monitoring the logarithmic retention factor
6–8]. There are a number of commercially available programs that can
9
–
Molecules 2016, 21, 1682; doi:10.3390/molecules21121682
www.mdpi.com/journal/molecules

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(log ki) of the analyte with changing ratio of organic mobile phase fraction. Isocratic retention factors
are extrapolated to 100% water to obtain log kawpp values at a given pH. The dissociation constant pKa
can be commonly determined by means of potentiometric titration, UV-VIS spectroscopy, capillary
electrophoresis (CE), RP-HPLC or NMR spectroscopy [13
,14]. The determination by means of CE
proved to be a suitable technique [15 17], where the pKa values are determined from monitoring
–
dependence of effective mobility of the analyte on pH of the background electrolyte (BGE) using
non-linear regression analysis.
This contribution is focused on the synthesis and experimental determination of the physicochemical
properties of nine new 3-(4-arylpiperazin-1-yl)-2-hydroxypropyl 4-alkoxyethoxybenzoates. The benzene
ring (a part of 4-hydroxybenzoic acid) in an arylcarbonyloxyaminopropanol fragment is substituted
in the C4 position with a propoxyethoxy, isopropoxyethoxy or allyloxyethoxy chain. The basic
part of the molecule is formed with phenylpiperazine fragment that is either unsubstituted or
substituted with a methoxy moiety in the C2’ or C4’ position. All the compounds were prepared
as dihydrochloride salts to increase their solubility in water. The newly synthesized agents are
expected to have cardiovascular activity due to the presence of particular structures of pharmacophores
(arylcarbonyloxyaminopropanol moiety responsible for β-adrenolytic activity [18], phenylpiperazine
fragment providing α1-adrenolytic activity [19]), nevertheless, the biological activities of the discussed
compounds have not been determined so far.
2. Results and Discussion
2.1. Chemistry
The final products were prepared via multi-step synthesis as described in Scheme 1. Tosylate
intermediates T1a
the spectroscopic and physical data were in agreement with those reported in the literature [20
The reaction temperature (0 ◦C) and time (two to three hours) was crucial to obtain pure products
at good yields. Intermediates were already described in the literature but were prepared by a
different synthetic method, and their spectroscopic data were not published yet. Intermediates
were prepared from ethyl 4-hydroxybenzoate via reaction with the appropriate tosylate intermediates
T1a
The reaction was carried out in acetone with potassium carbonate at 65 ◦C for 12 h
–
c
and T2 were prepared according to the published procedure (Scheme 2), and
–
23].
1–3
4–6
–c.
and then stirred for at least 12 h at ambient temperature, changing the solvent (acetonitrile,
dimethylformamide) did not improve the purity nor the yield of the desired products. The hydrolysis
was then performed by using a 2 M solution of sodium hydroxide and the desired acids
were obtained by displacing with HCl, extracting to chloroform and evaporating. The final
products, hydrochloride salts 7a 8a–c 9a–c, were prepared according to the already published
synthesis [24]. Briefly, the appropriate acids were transformed into the potassium salt using
potassium hydroxide in a mixture of methanol and propan-2-ol (ratio 1:3). Compounds 4–6,
4–6
–
c,
,
1–3
(oxiran-2-yl)methyl 4-(2-alkoxyethoxy)benzoates were obtained by reaction of intermediate T2 and
potassium 4-(2-alkoxyethoxy)benzoate in dimethylformamide for 7 h at 70 ◦C. The oxirane ring was
then opened by appropriate phenylpiperazine, the reaction was carried out in propan-2-ol for one
hour at 80 ◦C and for 72 h at ambient temperature. The obtained bases were finally converted to their
dihydrochloride salts by extend of hydrochloric acid to enhance the solubility of the compounds in
water. The protonation of the phenylpiperazine nitrogens was not experimentally determined for the
discussed final compounds but the hypothesis is based on the measurement carried out for similar
structures [24].

Molecules 2016, 21, 1682
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Scheme 1. Synthesis of discussed compounds
acetone, reflux; ( ) NaOH, HCl, reflux; ( ) KOH, MeOH, propan-2-ol (IPA); (
(DMF), reflux; (e) arylpiperazine, IPA, reflux; (f) HCl, Et2O.
1–9a–c. Reagents and conditions: (a) T1a–c, K2CO3,
b
c
d)
T2; dimethylformamide
◦
Scheme 2. Synthesis of intermediates T1a–c and T2. Reagents and conditions: (a) TEA, DCM, 0 C.
2.2. Physicochemical Properties
The dissociation constants (pKa) of studied compounds were determined by means of CE.
The measured values and values predicted by chemical software (ACD Percepta ver. 2012, Advanced
Chemistry Development, Toronto, ON, Canada; MarvinSketch 6.0.6., ChemAxon Kft., Budapest,
Hungary) are shown in Table 1. The correlation coefficients of the experimentally determined
values were greater than 0.999 and standard deviation equal or less than 0.02 units (n = 4).
The experimental values are in the range 6.35–6.92 and the highest values were recorded for the
1-(2-methoxyphenylpiperazine) derivatives 7b, 8b, 9b. The clinically used β-blockers are usually
stronger bases [14], the values of studied compounds are lower due to the presence of piperazine
moiety instead of secondary amine group.
The lipophilicity indexes log k7w.4 were measured by means of RP-HPLC method. The log kw values
were then calculated according to Equation (2) given in Section 3.3. The correlation coefficients were
greater than 0.999 with covariance equal or less than 0.03 units (n = 2). All the measured, calculated
and predicted values are shown in Table 2.
The lipophilicity decreases in order from non-substituted through ortho- to para-substituted
derivatives, and also in order from propoxy- through isopropoxy- to allyloxy-derivatives.

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Table 1. Experimentally determined pKa in comparison with calculated values of final compounds.
a
c
R1
R
pKa (exp.)
pKa (calc.)
b
Comp.
pKa (calc.)
7a
7b
7c
8a
8b
8c
9a
9b
allyl
allyl
allyl
H
6.47 ± 0.02
6.88 ± 0.02
6.49 ± 0.02
6.49 ± 0.01
6.92 ± 0.01
6.35 ± 0.02
6.50 ± 0.02
6.79 ± 0.02
6.39 ± 0.01
6.60
6.70
6.70
6.60
6.70
6.70
6.60
6.70
6.70
7.70
7.40
7.63
7.70
7.40
7.63
7.70
7.40
7.63
2-MeO
4-MeO
H
2-MeO
4-MeO
H
isopropyl
isopropyl
isopropyl
propyl
propyl
propyl
2-MeO
4-MeO
9c
Mean and standard deviation of four measurements; b calculated by ACD Percepta, ver. 2012; c calculated by
a
MarvinSketch 6.0.6.
Table 2. Experimentally determined and calculated values of lipophilicity indexes (log k7w.4, log kw)
in comparison with software-calculated values of distribution coefficients (log D7.4) and lipophilicity
(log P) of final compounds.
log P c
7.4 a
c
R1
R
log kw
log D7.4
log kw
b
log D7.4
log P b
Comp.
7a
7b
7c
8a
8b
8c
9a
9b
9c
a
allyl
allyl
allyl
isopropyl
isopropyl
isopropyl
propyl
propyl
propyl
H
3.97 ± 0.03
3.78 ± 0.02
3.67 ± 0.02
4.21 ± 0.01
4.03 ± 0.02
3.90 ± 0.02
4.29 ± 0.03
4.11 ± 0.02
3.99 ± 0.02
3.97
3.47
3.40
4.03
3.38
3.35
4.01
3.49
3.41
3.15
3.17
3.04
3.19
3.21
3.08
3.30
3.32
3.19
4.01 ± 0.09
3.89 ± 0.08
3.71 ± 0.08
4.25 ± 0.05
4.15 ± 0.07
3.93 ± 0.06
4.34 ± 0.10
4.20 ± 0.09
4.03 ± 0.09
4.03
3.65
3.55
4.09
3.56
3.50
4.07
3.67
3.56
3.63
3.47
3.47
3.67
3.51
3.51
3.78
3.62
3.62
2-MeO
4-MeO
H
2-MeO
4-MeO
H
2-MeO
4-MeO
Mean and standard deviation of four measurements; b calculated by ACD Percepta, ver. 2012; c calculated by
MarvinSketch 6.0.6.
Moreover, other parameters, such as number of hydrogen bond donors (nOHNH), number of
hydrogen bond acceptors (nON), number of rotatable bonds (nRTB) and a polar surface area (PSA)
known as Lipinski criteria as well as blood–brain barrier (BBB) penetration (log BB), were estimated
and evaluated. All these parameters influence the toxicity and ADME of a drug in human body.
There is a great emphasis on the prediction of drug penetration through BBB in order to avoid
complications in the later phases of drug development due to undesirable neurotoxicity as a side
effect [25]. The penetration through the BBB has been negatively related to molecular properties such
as high drug lipophilicity, molecular weight, and the tendency to form hydrogen bonds quantified as

Molecules 2016, 21, 1682
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PSA [26–28]. As shown in Table 3, the discussed compounds do fulfill the Lipinski rules, except the
number of rotatable bonds, but the already clinically-used landiolol shows similarities. A large number
of rotatable bonds has been associated with poor oral bioavailability, particularly when associated
with a high polar surface area [29]. For prediction of the ability of our compounds to cross the BBB by
passive transport, we chose the model proposed by Vilar et al. [27]. Based on previously described
facts that compounds with log BB > 0.3 pass the BBB readily and compounds with log BB <
−
1 are
poorly distributed in the brain [30 31], Vilar et al. generated two distinct classification models for both
,
threshold values. These models were generated using in vivo log BB values of 307 compounds and
validated on 1457 molecules using their in silico physicochemical descriptors with the percentage of
good classified compounds around 80% and can be used also for prediction of log BB values of novel
compounds. According to the results calculated by model 1 (results shown in Table 3) none of our
compounds exceeded the threshold value of 0.3, suggesting that these compounds could not cross the
BBB readily. However the values calculated according to the model 2, which suggests poor penetration
through the BBB, are greater than the threshold value (log BB >
the category of compounds with log BB values comprised between 0.3 and
to the central nervous system (CNS); i.e., the studied compounds can possess low brain permeability.
These results should be taken into account during the further stages of drug development.
−
1). Therefore, studied drugs belong to
1, which still have access
−
Table 3. Calculated parameters related to toxicity and absorption, distribution, metabolism, and
excretion (ADME).
MW a
(<500)
nOHNH
(<5)
nON
(<10)
nRTB
(≤10)
Log BB Class c
(Model 1)
Log BB Class c
(Model 2)
b
PSApH 7.0
(≤140)
Comp.
7a
7b
7c
8a
8b
8c
9a
9b
9c
440.53
470.56
470.56
442.56
472.58
472.58
442.56
472.58
472.58
295.37
327.35
510.60
3
3
3
3
3
3
3
3
3
2
4
3
7
8
8
7
8
8
7
8
8
13
14
14
12
13
13
13
14
14
10
9
72.67
81.90
81.90
72.67
81.90
81.90
72.67
81.90
81.90
72.37
118.26
132.40
−0.29
−0.61
−0.70
−0.17
−0.48
−0.59
−0.12
−0.45
−0.54
−1.37 d
−3.52 d
−3.96 d
0.32
−0.01
−0.05
0.38
0.06
0.01
0.40
0.07
0.03
−0.15 d
−3.17 d
−3.65 d
esmolol
flestolol
landiolol
5
7
11
14
Calculated for free bases by ACD ChemSketch; b calculated by MarvinSketch; c calculated from experimental
(log kw) and calculated (PSA) data according to the models generated by Vilar et al. [27]; d data for calculation
were taken from PubChem. MW, molecular weight; nOHNH, number of hydrogen bond donors; nON, number
of hydrogen bond acceptors; nRTB, number of rotatable bonds; PSA, polar surface area; log BB, blood–brain
barrier penetration.
a
3. Experimental Section
3.1. General Information
All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Acros Organics
(Geel, Belgium) in sufficient purity. The solvents were purchased from Lach-Ner (Neratovice,
Czech Republic) and were dried or freshly distilled if necessary. Thin layer chromatography (TLC)
Kieselgel 60 F254 plates (Merck, Darmstadt, Germany) visualized by UV irradiation (254 nm) were
used to monitor the reactions and the purity of the prepared substances; the reversed-phase TLC RP-18
F254 plates (Merck) were used for the final compounds. The melting points were determined on a
Kofler hot-plate apparatus HMK (Franz Kustner Nacht KG, Dresden, Germany) and are uncorrected.
The purity of final compounds was analysed by a Dionex Ultimate 3000 (Thermo Fisher Scientific,
Waltham, MA, USA) HPLC system controlled through the Chromeleon® Chromatography Data
System (version 7.2, Thermo Fisher Scientific). The separation was performed on ZORBAX Extend-C18
(3.5
µm, 3
×
150 mm) column (Agilent Technologies, Waldbronn, Germany). Mobile phase consisted

Molecules 2016, 21, 1682
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of 0.02 M 3-morpholinopropanesulphonate buffer with addition of 0.15% of n-decylamine (pH = 7.4;
constituent A) and methanol to which 0.25% of n-octanol was added (constituent B). The total flow
rate was 0.4 mL/min, the injection volume was 1
µ
L, and the column temperature was maintained
◦
at 25 C. The detection wavelength of 254 nm was chosen. The purity of individual compounds
was calculated as the average of relative peak areas in the chromatograms of the sample solution
under isocratic conditions in the range 25:75–50:50 (A:B; v/v). The IR spectra were recorded on a
Nicolet Impact 410 FT-IR spectrometer (Thermo Fisher Scientific) using Attenuated Total Reflection
(ATR, ZnSe) instrumentation. The spectra were obtained by accumulation of 32 scans in the region
4000–600 cm−1. The instrument was controlled by Omnic v. 8.3 software (Thermo Fisher Scientific).
The following abbreviations are used for the characteristic infrared bands:
stretching vibration band; (C–H), C–H stretching vibration bands;
(NH+), hydrochloride salt of
piperazine N-H stretching vibration band; (C=O), ester C=O stretching vibration band; (arom.
C=C), aromatic ring C=C stretching vibration band; (C–N), piperazine C-N stretching vibration band;
ν(O–H), alcohol O–H
ν
ν
ν
ν
ν
ν
(C–O–C), ether C–O stretching vibration band. The 1D (1H, 13C{1H}, 13C-APT) and 2D (1H-1H COSY,
1H-13C HMQC and 1H-13C HMBC) NMR experiments for characterization of the final compounds were
performed using a JNM-ECZ4OOR FT-NMR spectrometer 9.39 T (399.78 MHz for 1H and 100.53 MHz
for 13C nucleus; JEOL RESONANCE Inc., Tokyo, Japan) equipped with a 5 mm High Sensitivity Pulse
Field Gradient Autotune
™
probe. Chemical shifts are reported in ppm, referenced to the chemical
shifts of residual solvent resonance (DMSO, 2.5 ppm for H and 39.5 ppm for 13C). The coupling
constants (J) are reported in Hz. The NMR spectra of intermediates were measured in DMSO-d6 on a
Gemini-2000 FT-NMR spectrometer (200 MHz for 1H and 50 MHz for 13C, Varian Comp., Palo Alto,
CA, USA). The following abbreviations are used to describe the NMR signal multiplicities: s, singlet; d,
doublet; t, triplet; q, quartet; dd, doublet of doublet; dt, doublet of triplet; dq, doublet of quartet; ddt,
doublet of doublet of triplet; m, multiplet.
1
High-resolution mass spectra of the final compounds were measured using a high-performance
liquid chromatograph Dionex UltiMate® 3000 (Thermo Fisher Scientific, West Palm Beach, FL, USA)
coupled with a LTQ Orbitrap XL™ Hybrid Ion Trap-Orbitrap Fourier Transform Mass Spectrometer
(Thermo Scientific) with injection into HESI II in the negative mode.
3.2. Synthesis
3.2.1. Synthesis of Tosylate Intermediates
p-Toluensulfonyl chloride (2.6 g, 0.014 mol) was slowly added to the mixture of appropriate
2-alkoxyethanol respectively (oxiran-2-yl)methanol (0.014 mol) and triethylamine (1.84 g, 0.018 mol) in
dichlormethane (20 mL) cooled at 0 ◦C. The mixture was then stirred for 1 h at ambient temperature.
The precipitate was filtered and the organic phase was washed with 2 M HCl, saturated solution
of Na2CO3 and water, dried over MgSO4, and then the solvent was evaporated. Product T2 was
recrystallized from petroleum ether/diethyl ether in ratio 5:1.
Allyloxyethyl 4-methylbenzensulfonate (T1a). Yield: 97%; Rf: 0.74 (ethyl acetate/petroleum ether 1:1);
1H-NMR (200 MHz, DMSO-d6)
δ
(ppm): 7.79 (d, J = 8.5 Hz, 2H, Ar-H2,6), 7.48 (d, J = 8.5 Hz, 2H,
Ar-H3,5), 5.79 (ddt, J = 17.2, 10.4, 5.4 Hz, 1H, -CH=), 5.27–5.07 (m, 2H, =CH2), 4.15–4.11 (m, 2H,
TsOCH2), 3.87 (dt, J = 5.3, 1.4 Hz, 2H, -CH2CH=), 3.56–3.52 (m, 2H, CH2CH2O), 2.42 (s, 3H, ArCH3);
13C-NMR (50 MHz, DMSO-d6)
δ (ppm): 144.77, 134.62, 132.40, 130.00, 127.47, 116.47, 70.78, 69.84, 66.93,
20.98. (CAS Registry Number 50563-72-9).
Isopropoxyethyl 4-methylbenzensulfonate (T1b). Yield: 85%; Rf: 0.70 (ethyl acetate/petroleum ether 1:1);
1H-NMR (200 MHz, DMSO-d6)
δ
(ppm): 7.78 (d, J = 8.5 Hz, 2H, Ar-H2,6), 7.48 (d, J = 8.5, 2H, Ar-H3,5),
4.10–4.06 (m, 2H, TsOCH2), 3.54–3.41 (m, 3H, CH2CH2O + -CH(CH3)2), 2.42 (s, 3H, ArCH3), 1.00 (d,
J = 6.0, 6H, -CH(C (ppm): 144.82, 132.47, 130.06, 127.57, 70.91,
3)2); 13C-NMR (50 MHz, DMSO-d6)
70.44, 64.85, 21.76, 21.05. (CAS Registry Number 51218-98-5).
H
δ

Molecules 2016, 21, 1682
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Propoxyethyl 4-methylbenzensulfonate (T1c). Yield: 86%; Rf: 0.72 (ethyl acetate/petroleum ether 1:1);
1H-NMR (200 MHz, DMSO-d6) (ppm): 7.78 (d, J = 8.5 Hz, 2H, Ar-H2,6), 7.48 (d, J = 8.5, 2H, Ar-H3,5),
δ
4.13–4.09 (m, 2H, TsOCH2), 3.53–3.49 (m, 2H, CH2CH2O), 3.23 (t, J = 6.4 Hz, 2H, -CH2CH2CH3), 2.42
(s, 3H, ArCH3), 1.50–1.32 (m, 2H, -CH2CH2CH3), 0.77 (t, J = 7.7 Hz, 3H, -CH3); 13C-NMR (50 MHz,
DMSO-d6)
δ (ppm): 144.85, 132.43, 130.08, 127.57, 71.71, 70.00, 67.39, 22.22, 21.05, 10.32. (CAS Registry
Number 52497-47-9).
(Oxiran-2-yl)methyl 4-methylbenzensulfonate (T2). Yield: 97%; Rf: 0.77 (ethyl acetate/petroleum ether
1:1); m.p.: 35–37 ◦C; 1H-NMR (200 MHz, DMSO-d6) δ (ppm): 7.80 (d, J = 8.4 Hz, 2H, Ar-H2,6), 7.49 (d,
J = 8.4 Hz, 2H, Ar-H3,5), 4.41 (dd, J = 11.5, 2.4 Hz, 1H, TsOCH2), 3.81 (dd, J = 11.5, 7.1 Hz, 1H, TsOCH2),
3.22–3.14 (m, 1H, CH-oxiran), 2.79–2.74 (m, 1H, CH2-oxiran), 2.60 (dd, J = 5.0, 2.6 Hz, 1H, CH2-oxiran),
2.42 (s, 3H, ArCH3); 13C-NMR (50 MHz, DMSO-d6)
δ (ppm): 144.99, 132.20, 130.10, 127.51, 71.58, 48.49,
43.63, 20.98. (CAS Registry Number 118712-54-2).
3.2.2. Synthesis of 4-Alkoxyethoxybenzoic Acid
A mixture of ethyl-4-hydroxybenzoate (3.32 g, 0.02 mol), tosylate intermediate T1a–c (0.02 mol)
and potassium carbonate (0.06 mol) in acetone (25 mL) was heated at 70 ◦C for 8 h and then stirred
for 12 h at room temperature. The reaction was monitored by means of TLC. The precipitation was
then filtered and acetone was evaporated. The residue was dissolved in ethyl acetate and washed with
2 M NaOH and water, dried over MgSO4, and then the solvent was evaporated. The crude product
heated for 2 h with 5 M NaOH (50 mL) at 100 ◦C. The reaction mixture was washed with chloroform
and neutralized with concentrated HCl. The resulting white precipitate was collected by filtration.
4-(2-Allyloxy)ethoxybenzoic acid (3a). Yield: 69%; Rf: 0.67 (ethyl acetate); m.p.: 120–123 ◦C; 1H-NMR
(200 MHz, DMSO-d6) δ
(ppm): 12.60 (bs, 1H, COOH), 7.88 (d, J = 9.0 Hz, 2H, Ar-H2,6), 7.02 (d, J = 9.0 Hz,
2H, Ar-H3,5), 5.89 (ddt, J = 17.3, 10.5, 5.3 Hz, 1H, -CH=), 5.26 (dq, J = 17.3, 1.8 Hz, 1H, =CH2), 5.18–5.11
(m, 1H, =CH2), 4.20–4.16 (m, 2H, ArOCH2), 4.01 (dt, J = 5.3, 1.6 Hz, 2H, -CH2CH=), 3.75–3.71 (m, 2H,
CH2Oallyl); 13C-NMR (50 MHz, DMSO-d6)
δ (ppm): 166.94, 162.05, 135.03, 131.30, 123.03, 116.52, 114.25,
71.10, 67.91, 67.37. (CAS Registry Number 153881-37-9).
4-(2-Isopropoxy)ethoxybenzoic acid (3b). Yield: 65%; Rf: 0.67 (ethyl acetate); m.p.: 128–130 ◦C; 1H-NMR
(200 MHz, DMSO-d6)
2H, Ar-H3,5), 4.15–4.10 (m, 2H, ArOCH2), 3.72–3.56 (m, 3H, -CH2OCH(CH3)2), 1.10 (d, J = 6.0 Hz, 6H,
-CH(CH3)2); 13C-NMR (50 MHz, DMSO-d6)
(ppm): 166.96, 162.13, 131.30, 122.96, 114.26, 71.07, 67.79,
65.74, 21.99. (CAS Registry Number 766552-53-8).
δ
(ppm): 12.61 (bs, 1H, COOH), 7.87 (d, J = 9.0 Hz, 2H, Ar-H2,6), 7.02 (d, J = 9.0 Hz,
δ
◦
◦
4-(2-Propoxy)ethoxybenzoic acid (3c). Yield: 69%; Rf: 0.70 (ethyl acetate); m.p.: 109–111 C (lit. 114–115 C);
1H-NMR (200 MHz, DMSO-d6) (ppm): 12.63 (bs, 1H, COOH), 7.85 (d, J = 9.0 Hz, 2H, Ar-H2,6), 7.02
δ
(d, J = 9.0 Hz, 2H, Ar-H3,5), 4.18–4.13 (m, 2H, ArOCH2), 3.72–3.68 (m, 2H, -CH2OPr), 3.40 (t, J = 6.6 Hz,
2H, -CH2CH2CH3), 1.60–1.43 (m, 2H, -CH2CH2CH3), 0.85 (t, J = 7.5 Hz, 3H, -CH2CH2CH3); 13C-NMR
(50 MHz, DMSO-d6)
δ (ppm): 166.96, 162.10, 131.30, 122.99, 114.25, 72.01, 68.35, 67.44, 22.38, 10.44.
(CAS Registry Number: 67132-01-8).
3.2.3. Synthesis of Oxirane-Intermediates
A mixture of 4-alkoxyethoxybenzoic acid 3a–c (0.028 mol) in methanol (75 mL) and KOH (2.2 g,
0.042 mol) in propan-2-ol (50 mL) was stirred for 1 h at room temperature, and after that, propan-2-ol
(175 mL) was added for the final ratio methanol/propan-2-ol 1:3. The resulting white precipitate was
collected by filtration and dried under low pressure. A mixture of appropriate potassium salt (5 g,
0.023 mol) with (oxiran◦-2-yl)methyl 4-methylbenzensulfonate T2 (3.5 g, 0.015 mol) in DMF (50 mL)
was heated for 7 h at 70 C. The reaction was monitored by means of TLC. The solvent was evaporated,
and the residue was dissolved in ethyl acetate and washed with water; the organic layer was dried
over MgSO4, and ethyl acetate was evaporated.

Molecules 2016, 21, 1682
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(Oxiran-2-yl)methyl 4-allyloxyethoxybenzoate (
4
). Yield: 88%; Rf: 0.79 (ethyl acetate/petroleum ether
1:1); 1H-NMR (200 MHz, DMSO-d6) (ppm): 7.93 (d, J = 8.9 Hz, 2H, Ar-H2,6), 7.07 (d, J = 8.9 Hz, 2H,
δ
Ar-H3,5), 5.90 (ddt, J = 17.3, 10.5, 5.3 Hz, 1H, -CH=), 5.27 (dq, J = 17.4, 1.8 Hz, 1H, =CH2), 5.15 (dq,
J = 10.5, 1.5, 1H, =CH2), 4.60 (dd, J = 12.4, 2.7 Hz, 1H, COOCH2), 4.21–4.19 (m, 2H, ArOCH2), 4.08–4.01
(m, 3H, COOCH2 + -CH2CH=), 3.75–3.73 (m, 2H, CH2Oallyl), 3.34–3.30 (m, 1H, CH-oxirane), 2.84–2.82
(m, 1H, CH2-oxirane), 2.73–2.71 (m, 1H, CH2-oxirane); 13C-NMR (50 MHz, DMSO-d6)
δ (ppm): 165.07,
162.54, 135.02, 131.34, 121.51, 116.51, 114.52, 71.09, 67.87, 67.46, 65.05, 49.07, 43.83.
(Oxiran-2-yl)methyl 4-isopropoxyethoxybenzoate (
5
). Yield: 84%; Rf: 0.78 (ethyl acetate/petroleum ether
1:1); 1H-NMR (200 MHz, DMSO-d6)
δ
(ppm): 7.92 (d, J = 8.7 Hz, 2H, Ar-H2,6), 7.06 (d, J = 8.7 Hz, 2H,
Ar-H3,5), 4.60 (dd, J = 12.4, 2.5 Hz, 1H, COOCH2), 4.16–4.14 (m, 2H, ArOCH2-), 4.05 (dd, J = 12.4, 6.4
Hz, 1H, COOCH2), 3.72–3.69 (m, 2H, -CH2OiPr), 3.67–3.58 (m, 1H, -CH(CH3)2), 3.34–3.30 (m, 1H,
CH-oxirane), 2.84–2.82 (m, 1H, CH2-oxirane), 2.73–2.71 (m, 1H, CH2-oxirane), 1.10 (d, J = 6.0 Hz, 6H,
-CH(CH3)2); 13C-NMR (50 MHz, DMSO-d6)
δ (ppm): 165.10, 162.62, 131.35, 121.46, 114.55, 71.09, 67.88,
65.71, 65.08, 49.09, 43.84, 21.98.
(Oxiran-2-yl)methyl 4-propoxyethoxybenzoate (
6
). Yield: 87%; Rf: 0.69 (ethyl acetate/petroleum ether
1:1); 1H-NMR (200 MHz, DMSO-d6)
δ
(ppm): 7.92 (d, J = 8.7 Hz, 2H, Ar-H2,6), 7.07 (d, J = 8.7 Hz, 2H,
Ar-H3,5), 4.60 (dd, J = 12.4, 2.7 Hz, 1H, COOCH2), 4.19–4.16 (m, 2H, ArOCH2), 4.05 (dd, J = 12.4, 6.4
Hz, 1H, COOCH2), 3.72–3.70 (m, 2H, -CH2OPr), 3.40 (t, J = 6.6 Hz, 2H, -CH2CH2CH3), 3.34–3.30 (m,
1H, CH-oxirane), 2.84–2.82 (m, 1H, CH2-oxirane), 2.73–2.71 (m, 1H, CH2-oxirane), 1.56–1.47 (m, 2H,
-CH2CH2CH3), 0.85 (t, J = 7.3 Hz, 3H, -CH2CH2CH3); 13C-NMR (50 MHz, DMSO-d6)
δ (ppm): 165.10,
162.60, 131.35, 121.49, 114.53, 72.03, 68.33, 67.54, 65.08, 49.09, 43.84, 22.38, 10.44.
3.2.4. Synthesis of Final Compounds
Intermediate 4–6 (0.004 mol) was added to the solution of the corresponding arylpiperazine
(0.004 mol) in propan-2-ol (15 mL). The reaction mixture was heated at 80 ◦C for 1 h and stirred for
◦
72 h at room temperature. After that, the reaction mixture was cooled at least for 48 h at
−
18 C.
Precipitating crude base was filtered and dissolved in diethyl ether or chloroform and transformed
to their dihydrochloride salt 7a 8a 9a by addition of an excess of saturated ethereal hydrogen
–c,
–c
,
–c
chloride. The precipitate was filtered and recrystallized from propan-2-ol, if necessary.
1-{3-[(4-Allyloxyethoxybenzoyl)oxy]-2-hydroxypropyl}-4-phenylpiperazinediium dichloride (7a). Yield: 23%;
Rf: 0.87 (acetone/toluene 3:1); Rf reverse-phase (rev.): 0.51 (0.1 M HCl/acetone 3:2); m.p.: 185–187 ◦C;
HPLC purity (pur.) 91.67% (254 nm); IR (ATR, cm−1):
ν
(O–H) = 3289,
ν
(C–H) = 2930,
ν
(NH+) = 2537,
1
ν
(C=O) = 1697,
ν
(arom. C=C) = 1603,
ν
(C–N) = 1248,
ν
(C–O–C) = 1175; H-NMR (399.78 MHz,
DMSO-d6)
δ
(ppm): 10.75 (bs, 2H, NH+pip), 8.00 (d, J = 8.7 Hz, 2H, HArCOO), 7.28–7.24 (m, 2H,
HAr-pip), 7.07 (d, J = 8.7 Hz, 2H, HArO), 7.01–6.97 (m, 2H, HAr-pip), 6.88–6.84 (m, 1H, HAr-pip), 5.90 (ddt,
J = 17.4, 10.5, 5.4 Hz, 1H, -CH=), 5.27 (dq, J = 17.4, 1.8 Hz, 1H, =CH2), 5.16 (dq, J = 10.5, 1.8 Hz, 1H,
=CH2), 4.52–4.45 (m, 1H, CHOH), 4.23–4.20 (m, 4H, COOCH2 + ArOCH2), 4.02 (dt, J = 5.4, 1.4 Hz,
2H, -CH2CH=), 3.84–3.61 (m, 6H, Hpip + CH2Oallyl), 3.44–3.15 (m, 6H, Hpip + CH2Npip); 13C-NMR
(100.53 MHz, DMSO-d6) δ (ppm): 165.21, 162.57, 149.60, 135.06, 131.62, 129.14, 121.67, 119.93, 116.62,
115.89, 114.43, 71.13, 67.92, 67.50, 66.18, 63.33, 58.13, 52.13, 50.75, 45.25, 45.14; HR-MS: C25H33N2O5
[M − H]− calculated 441.2384 m/z, found 441.2383 m/z.
1-{3-[(4-Allyloxyethoxybenzoyl)oxy]-2-hydroxypropyl}-4-(2-methoxyphenyl)piperazinediium dichloride (7b).
Yield: 39%; Rf: 0.83 (acetone/toluene 3:1); Rf (rev.): 0.57 (0.1 M HCl/acetone 3:2); m.p.: 178–180 ◦C;
HPLC pur. 89.14% (254 nm); IR (ATR, cm−1): ν(O–H) = 3318, ν(C–H) = 2967, ν(NH+) = 2351, ν(C=O)
= 1711, ν(arom. C=C) = 1603, ν(C–N) = 1252, ν δ
(C–O–C) = 1167; 1H-NMR (399.78 MHz, DMSO-d6)
(ppm): 10.86 (bs, 2H, NH+pip), 8.00 (d, J = 8.7 Hz, 2H, HArCOO), 7.07 (d, J = 8.7 Hz, 2H, HArO), 7.04–7.02
(m, 1H, HAr-pip), 7.00–6.97 (m, 2H, HAr-pip), 6.93–6.89 (m, 1H, HAr-pip), 5.89 (ddt, J = 17.4, 10.5, 5.4 Hz,
1H, -CH=), 5.27 (dq, J = 17.4, 1.8 Hz, 1H, =CH2), 5.15 (dq, J = 10.5, 1.8 Hz, 1H, =CH2), 4.52–4.45 (m,

Molecules 2016, 21, 1682
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1H, CHOH), 4.22–4.19 (m, 4H, COOCH2 + ArOCH2), 4.01 (dt, J = 5.4, 1.4 Hz, 2H, -CH2CH=), 3.80
(s, 3H, OCH3), 3.77–3.60 (m, 6H, Hpip + CH2Oallyl), 3.52–3.13 (m, 6H, Hpip + CH2Npip); 13C-NMR
(100.53 MHz, DMSO-d6) δ (ppm): 165.22, 162.57, 151.85, 139.00, 135.08, 131.65, 123.79, 121.70, 120.88,
118.41, 116.62, 114.44, 112.03, 71.14, 67.93, 67.51, 66.18, 63.36, 58.35, 55.44, 52.50, 51.25, 46.90, 46.74;
HR-MS: C26H35N2O6 [M − H]− calculated 471.2489 m/z, found 471.2484 m/z.
1-{3-[(4-Allyloxyethoxybenzoyl)oxy]-2-hydroxypropyl}-4-(4-methoxyphenyl)piperazinediium dichloride (7c).
Yield: 44%; Rf: 0.82 (acetone/toluene 3:1); Rf (rev.): 0.58 (0.1 M HCl/acetone 3:2); m.p.: 170–173 ◦C;
HPLC pur. 94.28 (254 nm); IR (ATR, cm−1):
(C=O) = 1710, (arom. C=C) = 1604,
DMSO-d6)
ν
(O–H) = 3335,
ν
(C–H) = 2978,
ν
(NH+) = 2377,
1
ν
ν
ν
(C–N) = 1250,
ν
(C–O–C) = 1166; H-NMR (399.78 MHz,
δ
(ppm): 10.96 (bs, 2H, NH+pip), 8.00 (d, J = 8.7 Hz, 2H, HArCOO), 7.10 (d, J = 9.1 Hz, 2H,
HAr-pip), 7.07 (d, J = 8.7 Hz, 2H, HArO), 6.91 (d, 2H, J = 9.1 Hz, HAr-pip), 5.89 (ddt, J = 17.3, 10.5, 5.3 Hz,
1H, -CH=), 5.27 (dq, J = 17.3, 1.7 Hz, 1H, =CH2), 5.15 (dq, J = 10.5, 1.7 Hz, 1H, =CH2), 4.51–4.46 (m, 1H,
CHOH), 4.23–4.19 (m, 4H, COOCH2 + ArOCH2), 4.01 (dt, J = 5.3, 1.7 Hz, 2H, -CH2CH=), 3.79–3.60 (m,
6H, Hpip + CH2Oallyl), 3.71 (s, 3H, OCH3), 3.53–3.25 (m, 6H, Hpip + CH2Npip); 13C-NMR (100.53 MHz,
DMSO-d6) δ (ppm): 165.23, 162.57, 154.69, 142.13, 135.08, 131.64, 121.69, 118.78, 116.62, 114.56, 114.44,
71.14, 67.93, 67.51, 66.20, 63.46, 58.04, 55.32, 51.82, 50.62, 47.26, 47.19; HR-MS: C26H35N2O6 [M
calculated 471.2489 m/z, found 471.2485 m/z.
−
H]−
1-{2-Hydroxy-3-[(4-isopropoxyethoxybenzoyl)oxy]propyl}-4-phenylpiperazinediium dichloride (8a). Yield: 22%;
Rf: 0.85 (acetone/toluene 3:1); Rf (rev.): 0.25 (0.1 M HCl/acetone 3:2); m.p.: 200–202 ◦C; HPLC pur.
95.12% (254 nm); IR (ATR, cm−1):
(arom. C=C) = 1603, (C–N) = 1252,
ν
(O–H) = 3287,
ν
ν
(C–H) = 2972,
ν
(NH+) = 2559,
ν
(C=O) = 1699,
(ppm):
ν
ν
(C–O–C) = 1180; 1H-NMR (399.78 MHz, DMSO-d6)
δ
10.69 (bs, 2H, NH+pip), 7.99 (d, J = 8.7 Hz, 2H, HArCOO), 7.28–7.24 (m, 2H, HAr-pip), 7.06 (d, J = 8.7 Hz,
2H, HArO), 7.02–7.00 (m, 2H, HAr-pip), 6.88–6.84 (m, 1H, HAr-pip), 4.51–4.44 (m, 1H, CHOH), 4.23–4.21
(m, 2H, COOCH2, 4.17–4.14 (m, 2H, ArOCH2), 3.84–3.57 (m, 7H, -CH2OCH(CH3)2 + Hpip), 3.43–3.15
(m, 6H, -CH2Npip + Hpip), 1.10 (d, J = 5.9 Hz, 6H, -CH(CH3)2); 13C-NMR (100.53 MHz, DMSO-d6)
δ
(ppm): 165.23, 162.64, 149.52, 131.62, 129.16, 121.62, 120.03, 115.95, 114.46, 71.13, 67.93, 66.18, 65.76,
63.33, 58.11, 52.14, 50.71, 45.31, 45.19, 22.03; HR-MS: C25H36N2O5 [M
found 443.2535 m/z.
−
H]− calculated 443.2540 m/z,
1-{2-Hydroxy-3-[(4-isopropoxyethoxybenzoyl)oxy]propyl}-4-(2-methoxyphenyl)piperazinediium dichloride (8b).
Yield: 25%; Rf: 0.82 (acetone/toluene 3:1); Rf (rev.): 0.29 (0.1 M HCl/acetone 3:2); m.p.: 154–156 ◦C;
HPLC pur. 98.87% (254 nm); IR (ATR, cm−1):
(C=O) = 1701, (arom. C=C) = 1603,
DMSO-d6)
ν
(O–H) = 3318,
ν
(C–H) = 2969,
ν
(NH+) = 2560,
1
ν
ν
ν
(C–N) = 1251,
ν
(C–O–C) = 1166; H-NMR (399.78 MHz,
δ
(ppm): 10.67 (bs, 2H, NH+pip), 7.99 (d, J = 8.7 Hz, 2H, HArCOO), 7.07 (d, J = 8.7 Hz, 2H,
HArO), 7.03–6.89 (m, 4H, HAr-pip), 4.49–4.44 (m, 1H, CHOH), 4.23–4.21 (m, 2H, COOCH2), 4.17–4.15 (m,
2H, ArOCH2), 3.80 (s, 3H, OCH3), 3.72–3.70 (m, 2H, -CH2OiPr), 3.68–3.40 (m, 5H, Hpip + -CH(CH3)2),
3.35–3.08 (m, 6H, -CH2Npip + Hpip), 1.11 (d, J = 6.4 Hz, 6H, -CH(CH3)2); 13C-NMR (100.53 MHz,
DMSO-d6) δ (ppm): 165.19, 162.62, 151.82, 139.23, 131.60, 123.57, 121.62, 120.84, 118.29, 114.44, 111.96,
71.11, 67.91, 66.12, 65.74, 63.27, 58.27, 55.40, 52.63, 51.16, 46.82, 46.67, 22.01; HR-MS: C26H37N2O6
[M − H]− calculated 473.2646 m/z, found 473.2640 m/z.
1-{2-Hydroxy-3-[(4-isopropoxyethoxybenzoyl)oxy]propyl}-4-(4-methoxyphenyl)piperazinediium dichloride (8c).
Yield: 34%; Rf: 0.81 (acetone/toluene 3:1); Rf (rev.): 0.23 (0.1 M HCl/acetone 3:2); m.p.: 193–195 ◦C;
HPLC pur. 95.60 (254 nm); IR (ATR, cm−1):
(C=O) = 1698, (arom. C=C) = 1601,
DMSO-d6)
ν
(O–H) = 3270,
ν
(C–H) = 2970,
ν
(NH+) = 2561,
1
ν
ν
ν
(C–N) = 1247,
ν
(C–O–C) = 1179; H-NMR (399.78 MHz,
δ
(ppm): 10.74 (bs, 2H, NH+pip), 7.99 (d, J = 8.7 Hz, 2H, HArCOO), 7.07–7.03 (m, 4H,
HArO + HAr-pip), 6.89 (d, J = 9.1 Hz, 2H, HAr-pip), 4.50–4.44 (m, 1H, CHOH), 4.23–4.21 (m, 2H, COOCH2),
4.17–4.14 (m, 2H, ArOCH2), 3.76–3.57 (m, 10H, -CH2OCH(CH3)2 + OCH3 + Hpip), 3.45–3.22 (m, 6H,
-CH2Npip + Hpip), 1.10 (d, J = 5.9 Hz, 6H, -CH(CH3)2); 13C-NMR (100.53 MHz, DMSO-d6)
δ
(ppm):
165.24, 162.63, 154.24, 142.82, 131.62, 121.63, 118.41, 114.48, 114.46, 71.13, 67.93, 66.17, 65.76, 63.38, 58.05,

Molecules 2016, 21, 1682
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55.28, 52.06, 50.69, 46.96, 46.87, 22.03; HR-MS: C26H37N2O6 [M
473.2641 m/z.
−
H]− calculated 473.2646 m/z, found
1-{2-Hydroxy-3-[(4-propoxyethoxybenzoyl)oxy]propyl}-4-phenylpiperazinediium dichloride (9a). Yield: 32%;
Rf: 0.85 (acetone/toluene 3:1); Rf (rev.): 0.20 (0.1 M HCl/acetone 3:2); m.p.: 197–199 ◦C; HPLC pur.
96.27% (254 nm); IR (ATR, cm−1):
(arom. C=C) = 1601, (C–N) = 1250,
ν
(O–H) = 3295,
ν
ν
(C–H) = 2958,
ν
(NH+) = 2351,
ν
(C=O) = 1702,
(ppm):
ν
ν
(C–O–C) = 1161; 1H-NMR (399.78 MHz, DMSO-d6)
δ
10.88 (bs, 2H, NH+pip), 7.99 (d, J = 8.7 Hz, 2H, HArCOO), 7.28–7.24 (m, 2H, HAr-pip), 7.06 (d, J = 8.7 Hz,
2H, HArO), 7.03–7.01 (m, 2H, HAr-pip), 6.89–6.85 (m, 1H, HAr-pip), 4.52–4.46 (m, 1H, CHOH), 4.23–4.21
(m, 2H, COOCH2), 4.19–4.17 (m, 2H, ArOCH2), 3.83–3.61 (m, 6H, -CH2OPr + Hpip), 3.44–3.15 (m,
8H, -CH2CH2CH3 + CH2Npip + Hpip), 1.55–1.47 (m, 2H, -CH2CH2CH3), 0.85 (t, J = 7.5 Hz, 3H,
-CH2CH2CH3); 13C-NMR (100.53 MHz, DMSO-d6)
δ (ppm): 165.23, 162.59, 149.42, 131.62, 129.17,
121.66, 120.17, 116.03, 114.44, 72.04, 68.35, 67.58, 66.21, 63.39, 58.16, 52.03, 50.79, 45.38, 45.26, 22.40,
10.50; HR-MS: C23H35N2O5 [M − H]− calculated 443.2540 m/z, found 443.2534 m/z.
1-{2-Hydroxy-3-[(4-propoxyethoxybenzoyl)oxy]propyl}-4-(2-methoxyphenyl)piperazinediium dichloride (9b).
Yield: 28%; Rf: 0.81 (acetone/toluene 3:1); Rf (rev.): 0.24 (0.1 M HCl/acetone 3:2); m.p.: 143–146 ◦C;
HPLC pur. 98.29% (254 nm); IR (ATR, cm−1):
(C=O) = 1700, (arom. C=C) = 1605,
DMSO-d6)
ν
(O–H) = 3290,
ν
(C–H) = 2966,
ν
(NH+) = 2350,
1
ν
ν
ν
(C–N) = 1247,
ν
(C–O–C) = 1170; H-NMR (399.78 MHz,
δ
(ppm): 10.70 (bs, 2H, NH+pip), 7.99 (d, J = 8.7 Hz, 2H, HArCOO), 7.07 (d, J = 8.7 Hz, 2H,
HArO), 7.03–6.89 (m, 4H, HAr-pip), 4.50–4.44 (m, 1H, CHOH), 4.23–4.21 (m, 2H, COOCH2), 4.20–4.18
(m, 2H, ArOCH2-), 3.80 (s, 3H, OCH3), 3.73–3.70 (m, 2H, -CH2OPr), 3.69–3.46 (m, 4H, Hpip), 3.41
(t, J = 6.6 Hz, 2H, -CH2CH2CH3), 3.32–3.09 (m, 6H, -CH2Npip + Hpip), 1.56–1.47 (m, 2H, -CH2CH2CH3),
0.86 (t, J = 7.3 Hz, 3H, -CH2CH2CH3); 13C-NMR (100.53 MHz, DMSO-d6)
δ
(ppm): 165.19, 162.59,
151.82, 139.21, 131.61, 123.60, 121.65, 120.85, 118.30, 114.43, 111.98, 72.03, 68.34, 67.57, 66.13, 63.29, 58.29,
55.40, 52.61, 51.20, 46.83, 46.69, 22.38, 10.47; HR-MS: C26H37N2O6 [M
found 473.2642 m/z.
−
H]− calculated 473.2646 m/z,
1-{2-Hydroxy-3-[(4-propoxyethoxybenzoyl)oxy]propyl}-4-(4-methoxyphenyl)piperazinediium dichloride (9c).
Yield: 25%; Rf: 0.81 (acetone/toluene 3:1); Rf (rev.): 0.22 (0.1 M HCl/acetone 3:2); m.p.: 173–175 ◦C;
HPLC pur. 95.52 (254 nm); IR (ATR, cm−1):
(C=O) = 1700, (arom. C=C) = 1601,
DMSO-d6)
ν
(O–H) = 3279,
ν
(C–H) = 2934,
ν
(NH+) = 2562,
1
ν
ν
ν
(C–N) = 1246,
ν
(C–O–C) = 1182; H-NMR (399.78 MHz,
δ
(ppm): 10.90 (bs, 2H, NH+pip), 7.99 (d, J = 8.7 Hz, 2H, HArCOO), 7.08–7.05 (m, 4H,
HArO + HAr-pip), 6.89 (d, J = 9.1 Hz, 2H, HAr-pip), 4.52–4.46 (m, 1H, CHOH), 4.23–4.21 (m, 2H,
COOCH2), 4.19–4.17 (m, 2H, ArOCH2), 3.78–3.63 (m, 9H, -CH2OPr + OCH3 + Hpip), 3.45–3.22 (m,
8H, -CH2CH2CH3 + -CH2Npip + Hpip), 1.56–1.47 (m, 2H, -CH2CH2CH3), 0.85 (t, J = 7.5 Hz, 3H,
-CH2CH2CH3); 13C-NMR (100.53 MHz, DMSO-d6)
δ (ppm): 165.22, 162.60, 154.49, 142.41, 131.63,
121.65, 118.61, 114.52, 114.44, 72.04, 68.35, 67.57, 66.19, 63.43, 58.05, 55.29, 51.89, 50.65, 47.13, 47.05,
22.40, 10.50; HR-MS: C26H37N2O6 [M − H]− calculated 473.2646 m/z, found 473.2643 m/z.
3.3. Determination of Physicochemical Parameters
The log kw7.4 values of synthetized compounds were measured by a Dionex Ultimate 3000 (Thermo
Fisher Scientific) HPLC system controlled through the Chromeleon® Chromatography Data System
(version 7.2). The separation was performed on ZORBAX Extend-C18 (3.5
(Agilent Technologies). Mobile phase consists of 0.02 M 3-morphol, that can be used with addition of
µ
m, 3
×
150 mm) column
0.15% of n-decylamine (pH = 7.4; constituent A) and methanol to which 0.25% of n-octanol was added
(constituent B). The total flow rate was 0.4 mL/min, the injection volume was 1
µ
L, and the column
◦
temperature was maintained at 25 C. The detection wavelength of 254 nm was chosen. One milligram
of the sample was dissolved in 1 mL of methanol and diluted to the concentration 0.1 mg/mL with
50% methanol. Retention factors of the compound were measured under isocratic conditions in the
range 20:80–50:50 (A:B; v/v), 5 values for each factor in duplicate. Linear regression analysis was
performed and log k7w.4 values were calculated according to Equation (1).

Molecules 2016, 21, 1682
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logk = −Sφ + logkw
where log k represents a logarithm of individual isocratic factor,
(1)
φ
is the organic phase concentration,
and S is a constant derived by linear regression analysis. The retention factor corresponding to the
neutral form (log kw) was estimated from the apparent log k7w.4 by using Equation (2).
ꢀ
ꢁ
logkw = logkawpp + log 1 + 10pKa−pH
(2)
The CE experiments were carried out with an Agilent 3D CE (Agilent Technologies) capillary
electrophoresis system equipped with an autosampler, automatic injector, photodiode array detector,
and an air cooling unit for the capillary. The instrument control and analysis were performed
with 3D-CE ChemStation software (Agilent Technologies). Uncoated fused-silica capillary (Agilent
Technologies) of 50 µm internal diameter, total length of 33.0 cm and effective length (to the detector)
of 24.5 cm was used. The capillary cassette temperature was maintained at 25 ◦C with air cooling.
Three milligrams of the sample were dissolved in 1 mL of 50% methanol and 10 times diluted with
appropriate background electrolyte before analysis. Samples were injected hydrodynamically at
40 mbar pressure for 4.0 s. Running voltage was 15 kV of positive polarity. UV detection was
performed at 254 nm. As a marker of electroosmotic flow, mesityl oxide was used. The effective
mobilities were recorded for BGE pH values in the range of 3.9 to 9.2 (ionic strength equal to 10 mM),
4 runs at 10 different buffer pH values. The effective mobilities (µeff) were calculated according to
Equation (3).
!
LtotLe f f
1
tmig
1
tEOF
µe f f
=
−
(3)
U
where tmig is the migration time of the analyte and tEOF is the migration time of a neutral marker
compound that is of the same velocity as the electroosmotic flow (EOF). Leff is distance from the injection
end of capillary to the detector and Ltot is the total length of the capillary over which the voltage U
was applied. SigmaPlot for Windows version 11.0 (Systat Software GmbH, Erkrath, Germany) was
employed for the nonlinear regression analysis of µeff/pH relationship having characteristic sigmoidal
shape with inflection point indicating pKa value.
The pH measurements were taken with a combination glass electrode (HI 1332B, HANNA Instruments,
Woonsocket, RI, USA), using a Thermo Orion 370 PerpHect® pH meter (ThermoFisher Scientific).
4. Conclusions
In conclusion, nine new potential dual
β- and α1-blockers were prepared. In addition, three
oxiran intermediates (compounds ) have not yet been described in the literature. In this study, the
4–6
lipophilicity indexes and the pKa values have been experimentally determined and compared with
software calculated values. Furthermore, we discussed the ADME properties and potential toxicity of
prepared compounds by computing the parameters involved in the Lipinski Rule of Five and the log
BB parameter describing the possibility of the compound to penetrate through the BBB.
Acknowledgments: This study was supported by IGA VFU Brno 323/2016/FaF, 318/2016/FaF. The HPLC/HRMS
system forms a part of the National Infrastructure CzeCOS (LM2015061); Michal Oravec was supported by the
National Sustainability Program (NPU I; Grant No. LO1415).
Author Contributions: Pavlina Marvanova, Ondrej Hosik, Tereza Padrtova, Petr Mokry—synthesis and structural
characterization of the compounds, writing of the paper. Michal Oravec—LC-HRMS analysis of the compounds.
Klara Odehnalova—determination of physicochemical parameters, writing of the paper.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2016, 21, 1682
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
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