Synthesis and preliminary evaluation of pharmacological properties of some piperazine derivatives of xanthone

Article abstract of DOI:10.1016/j.bmc.2012.11.014

A series of 9 piperazine derivatives of xanthone were synthesized and evaluated for cardiovascular activity. The following pharmacological experiments were conducted: the binding affinity for adrenoceptors, the influence on the normal electrocardiogram, the effect on the arterial blood pressure and prophylactic antiarrhythmic activity in adrenaline induced model of arrhythmia (rats, iv). Three compounds revealed nanomolar affinity for α₁- adrenoceptor which was correlated with the strongest cardiovascular (antiarrhythmic and hypotensive) activity in animals' models. The most promising compound was 4-(3-(4-(2-methoxyphenyl)piperazine-1-yl)propoxy)-9H-xanthen-9-one hydrochloride (12) which revealed antiarrhythmic activity with ED₅₀ value of 0.69 mg/kg in adrenaline induced arrhythmia (rats, iv). Other synthesized xanthone derivatives, that is, (R,S)-4-(2-hydroxy-3-(4-(2- methoxyphenyl)piperazine-1-yl)propoxy)-9H-xanthen-9-one hydrochloride (10) and (R,S)-4-(2-acetoxy-3-(4-(2-methoxyphenyl)piperazine-1-yl)propoxy) -9H-xanthen-9-one hydrochloride (11) also acted as potential antiarrhythmics in adrenaline induced model of arrhythmia in rats after intravenous injection (ED₅₀ = 0.88 mg/kg and 0.89 mg/kg, respectively). These values were lower than values obtained for reference drugs such as propranolol and urapidil, but not carvedilol. Results were quite promising and suggested that in the group of xanthone derivatives new potential antiarrhythmics and hypotensives might be found.

Full text of DOI:10.1016/j.bmc.2012.11.014

Bioorganic & Medicinal Chemistry 21 (2013) 514–522
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and preliminary evaluation of pharmacological properties
of some piperazine derivatives of xanthone
Natalia Szkaradek a, , Anna Rapacz b, Karolina Pytka b, Barbara Filipek b,c, Agata Siwek d, Marek Cegła e,
⇑
Henryk Marona a
a Department of Bioorganic Chemistry, Chair of Organic Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Krakow, Poland
b Department of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical College, Krakow, Poland
c Laboratory of Pharmacological Screening, Faculty of Pharmacy, Jagiellonian University Medical College, Krakow, Poland
d Department of Cytobiology and Histochemistry, Laboratory of Pharmacobiology, Faculty of Pharmacy, Jagiellonian University Medical College, Krakow, Poland
e Chair of Organic Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Krakow, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 June 2012
Revised 4 November 2012
Accepted 5 November 2012
Available online 24 November 2012
A series of 9 piperazine derivatives of xanthone were synthesized and evaluated for cardiovascular
activity. The following pharmacological experiments were conducted: the binding affinity for adreno-
ceptors, the influence on the normal electrocardiogram, the effect on the arterial blood pressure and
prophylactic antiarrhythmic activity in adrenaline induced model of arrhythmia (rats, iv). Three com-
pounds revealed nanomolar affinity for
a1-adrenoceptor which was correlated with the strongest car-
diovascular (antiarrhythmic and hypotensive) activity in animals’ models. The most promising
compound was 4-(3-(4-(2-methoxyphenyl)piperazine-1-yl)propoxy)-9H-xanthen-9-one hydrochloride
(12) which revealed antiarrhythmic activity with ED50 value of 0.69 mg/kg in adrenaline induced
arrhythmia (rats, iv). Other synthesized xanthone derivatives, that is, (R,S)-4-(2-hydroxy-3-(4-(2-
methoxyphenyl)piperazine-1-yl)propoxy)-9H-xanthen-9-one hydrochloride (10) and (R,S)-4-(2-acet-
oxy-3-(4-(2-methoxyphenyl)piperazine-1-yl)propoxy)-9H-xanthen-9-one hydrochloride (11) also acted
as potential antiarrhythmics in adrenaline induced model of arrhythmia in rats after intravenous injec-
tion (ED50 = 0.88 mg/kg and 0.89 mg/kg, respectively). These values were lower than values obtained for
reference drugs such as propranolol and urapidil, but not carvedilol.
Keywords:
Antiarrhythmic
Hypotensive
Adrenoceptor binding affinity
Xanthone
Synthesis
Results were quite promising and suggested that in the group of xanthone derivatives new potential
antiarrhythmics and hypotensives might be found.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Sympathetic nervous system is not the only one responsible for
cardiac rhythm disturbances. It’s worth noticing that the action
Cardiovascular diseases including coronary heart disease, cere-
brovascular disease, hypertension, peripheral artery disease, rheu-
matic heart disease, congenital heart disease and heart failure are
caused by disorders of the heart and blood vessels. Even though
there are many modern and effective drugs, cardiovascular dis-
eases still remain the main cause of death, being responsible for
30% of all global deaths.1
Arrhythmias account for nearly one quarter of all cardiovascu-
lar-related deaths. The majority of them is due to cardiac rhythm
disturbances which can be caused by many different conditions.
The key role in the pathogenesis of abovementioned cardiac dys-
functions, plays activation of sympathetic nervous system. There-
fore, it is not surprising that adrenergic receptors antagonists are
effective in the treatment of this disease and are widely used.
potential of heart cells also depends on the proper interplay of
ion channels, especially sodium, potassium, and calcium.2 Thus,
taking into account the variety of arrhythmias’ types, their diverse
aetiology and numerous side effects of antiarrhythmic drugs (such
as tiredness, changes in mood, sleep disturbances, dry mouth, blur-
ry vision or impotence) there is still a need to search for new
agents that can improve heart function with minimal side effects.3
Many efforts in this field were made in the group of xanthone
derivatives. The first investigation of xanthones date back to
1990. Valenti and co-workers studied
a series of xanthone
1,4-dihydropiridine derivatives, xanthone analogues of nifedipine.
The tests that had been performed proved negative inotropic and
chronotropic properties as well as selectivity on pacemaker activ-
ity of these compounds, although precise mechanism of their ac-
tion was not clearly explained.4
Another, serious cardiovascular disorder is hypertension. Most
cases of hypertension are so-called essential or primary hyperten-
sion, with unknown aetiology. Only 5% of cases, named secondary
⇑
Corresponding author at present address: 9 Medyczna Str., 30-688 Krakow,
Poland. Tel.: + 48 12 620 55 76; fax: +48 12 620 54 05.
E-mail address: nszkarad@cm-uj.krakow.pl (N. Szkaradek).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.014

N. Szkaradek et al. / Bioorg. Med. Chem. 21 (2013) 514–522
515
hypertension, have known aetiology and are associated with for
example, arteriosclerosis or hyperthyreosis. Nowadays, the num-
ber of patients suffering from hypertension is still increasing, par-
tially due to current lifestyle: lack of physical activity, overweight
or obesity, using tobacco, too much sodium and too little potas-
sium in diet, too little vitamin D in diet, drinking too much alcohol
and stress. Other risk factors are age and family predispositions.
Taking into account the global scale of this disorder, searching
for new potential hypotensive agents seems to be reasonable.5
Xanthone itself was proved to posses vasorelaxating properties
in thoracic aorta isolated from rats.6 These data suggested that
xanthone-induced vasorelaxation was endothelium independent
and the mechanism might involve an increase in intracellular cyc-
lic adenosine 30,50-monophosphate (cAMP) and the blockade of
Ca2+ channels. Other research on a series of aminoalkanolic xan-
thone derivatives confirmed hypotensive activity of all tested
compounds. It was noticed that, an oxypropanolamine side chain
substituted at the C-3 position of the xanthone nucleus signifi-
cantly enhanced the hypotensive activity. The most potent seemed
to be 3-(3N-iso-propylamine-2-hydroxypropoxy)-9H-xanthen-
9-one (xanthonolol), which lowered systolic blood pressure by
about 32–7% in dependence of dosage (from 5 to 0.1 mg/kg). Its
mechanism of action was calcium channel dependant and re-
quired the blockade of beta adrenergic receptors.7 Xanthonolol
possess typical, b-blocker moiety (3-amine-2-hydroxypropan-
1-yloxy) characteristic for cardiovascular drugs such as proprano-
lol and carvedilol.
(1:4). These intermediates are characterised in Experimental
protocols.
Compounds 10 and 12–18 were obtained by amination of respec-
tive parent compounds with appropriate amines in n-propanol (for
10, 15–18) or toluene in a presence of K2CO3 (for 12–14), as de-
scribed previously.13,16 In addition, compound 10 underwent acety-
lation to get compound 11. Acetylation was performed using acetic
anhydride, according to well known procedures. All resulted bases
were converted into hydrochloride salts using an excess of ethanol
saturated with HCl. The crude products were recrystallized from
acetone/ethanol (1:3). These procedures are summarized in Scheme
1. Physicochemical properties of compounds 10–18 are summa-
rized in Experimental protocol. In order to optimize synthesis of
the tested compounds, compound 10 was obtained also using alter-
native method including (R,S)-4-(3-chloro-2-hydroxypropoxy)-9H-
xanthen-9-one as intermediate (see Scheme 1). This pathway of the
synthesis was conducted analogously to the earlier described proce-
dure.12 Nevertheless, this method seems to be less potent, so other
compounds were obtained using oxirane derivatives as intermedi-
ates. Structures of the tested compounds are presented in Table 1.
3. Pharmacology
3.1. Animals and experimental conditions
The studies were carried out on normotensive male Wistar rats
weighing 170–350 g (Source: Animal House, Faculty of Pharmacy,
Jagiellonian University Medical College, Krakow, Poland, stocks
name: KRF: WI(WU)). The animals were kept in plastic cages in a
room at a temperature of 20 4 °C, under 12/12 h light/dark cycle
(light on from 7 a.m. to 7 p.m.). They were fed with granulated feed
(standard laboratory pellets) and had free access to water. The con-
trol and study groups consisted of six animals each. All procedures
were conducted according to the Animal Care and Use Committee
guidelines, and approved by the Ethical Committee of Jagiellonian
University, Krakow.
The newest communications exhibited interesting properties of
xanthone-amine derivatives without typical b-blocker structure
and bearing relatively long (4–5 methylene groups) alkane linker
connecting pharmacophore structures.8
Our Laboratory of Bioorganic Chemistry, Chair of Organic
Chemistry (previously Department of Technology and Biotechnol-
ogy of Drugs) has also documented experience in searching for
hypotensive, aminoalkanolic xanthone derivatives.9,10 The stron-
gest hypotensive effects were observed for compounds containing
piperazine moiety. These compounds showed both in vitro (via
binding affinity evaluations) and in vivo activity.10 Piperazine moi-
ety was mentioned as cardiovascular pharmacophore elsewhere.11
Basing on literature survey, herein we report in vitro and in vivo
cardiovascular activity of some new structures. Structures were
designed to combine xanthone and piperazine rings. Some of them
possessed typical b-blocker moiety (3-amine-2-hydroxypropan-1-
yloxy), others were devoided of hydroxyl group analogously to
structures described by Lin et al.8 and were evaluated to estimate
role of hydroxyl group. Piperazine like amines were also diversi-
fied. Most of them contained methoxyphenylpiperazine—struc-
3.2. Drugs
The tested compounds 10–18 were synthesized at the Depart-
ment of Bioorganic Chemistry UJ CM. Adrenaline (Polfa, Warsaw,
Poland), propranolol (Fluka Chemie AG, Seelze, Germany),
[3H]CGP-12177 (NEN-Du Pont, Warsaw, Poland), [3H]prazosin
(Amersham, Uppsala, Sweden), sodium heparin (Polfa, Warsaw,
Poland), thiopental sodium (Biochemie Gmbh, Vienna, Austria)
were dissolved in saline. The tested compounds were administered
intravenously (iv) at a constant volume of 1 mL/kg and were inves-
tigated at the wide range of doses starting at a dose of 10 mg/kg.
tural element of urapidil (typical
a1-adrenoceptor antagonist),
others contain benzylpiperazine, phenoxyethylpiperazine, cinnam-
ylpiperazine to ascertain pharmacophoric effect of methoxyphen-
ylpiperazine or piperazine itself. We also introduced chlor
substituent into xanthone ring to enhance lipophilicity and exam-
ine its effect on cardiovascular system.
3.3. The effect on the normal electrocardiogram
Electrocardiographic investigations were carried out using
ASPEL ASCARD B5 apparatus, (Aspel SA, Zabierzów, Poland) stan-
dard lead II and paper speed of 50 mm/s. The ECG was recorded
just prior to and also 1, 5, and 15 min following the administration
of the compounds.
2. Chemistry
The synthetic route that was used to synthesize of starting
materials is outlined in Scheme 1. The detailed description of the
method and physico-chemical properties of 4-((oxiran-2-yl)-
methoxy)-9H-xanthen-9-one, 3-chloro-5-((oxiran-2-yl)methoxy)-
9H-xanthen-9-one and 4-(3-bromopropoxy)-9H-xanthen-9-one
were described elsewhere.12–15 3-Chloro-5-(3-bromopropoxy)-9H-
xanthen-9-one was obtained analogously to 4-(3-bromopropoxy)-
9H-xanthen-9-one from 3-chloro-5-hydroxy-9H-xanthen-9-one.
The crude products were recrystallized from n-hexane/toluene
3.4. Adrenaline-induced arrhythmia
Prophylactic antiarrhythmic activity was determined according
to the method of Szekeres and Papp17 The arrhythmia was evoked
in rats under anesthesia with thiopental (75 mg/kg, ip) by iv injec-
tion of adrenaline (20 lg/kg). The studied compounds were admin-
istered via iv route 15 min before adrenaline administration. The
criteria of antiarrhythmic activity were the lack of premature beats

516
N. Szkaradek et al. / Bioorg. Med. Chem. 21 (2013) 514–522
Scheme 1. General synthetic route of the tested compounds 1–18.
and inhibition of cardiac arrhythmia in comparison to the control
group. The ED50 value was calculated according to the method of
Litchfield and Wilcoxon.18
(70.5 Ci/mmol, a
2-adrenergic receptor) and [3H]CGP-12177 (48 Ci/
mmol, b1-adrenergic receptor) were used. The membrane prepara-
tion and the assay procedure were carried out according to the
published procedure19 with slight modifications. The brains of
the rats were homogenized in 20 volumes of ice-cold 50 mM
Tris–HCl buffer (pH 7.6) and centrifuged at 20,000ꢀg for 20 min
(0–4 °C). The cell pellet was resuspended in Tris–HCl buffer and
centrifuged again.
3.5. The effect on the arterial blood pressure
Male Wistar normotensive rats were anesthetized with thio-
pental (75 mg/kg) by intraperitoneally injection. The right carotid
artery was cannulated with polyethylene tub filled with heparin
in saline to facilitate pressure measurements using a Datamax
apparatus (Columbus Instruments, Columbus OH, USA). The stud-
ied compounds were injected into the caudal veins of rats after a
5 min stabilization period, at a constant volume of 1 mL/kg.
The final incubation mixture (final volume 300
240 L membrane suspension, 30
L of a [3H]prazosin (0.2 nM),
[3H]clonidine (2 nM) or [3H]CGP-12177 (0.2 nM) solution and
30 buffer containing from seven to eight concentrations
(10ꢁ11–10ꢁ4 M) of investigated compounds. For measuring unspe-
M (in the case of [3H]prazosin),
M clonidine (in the case of [3H]clonidine) and propranolol
M (in the case of [3H]CGP-12177), were applied. Radioactivity
lL) consisted of
l
l
l
L
cific binding, phentolamine ꢁ10
l
3.6. Adrenoceptor radioligand binding assay
10 l
ꢁ1
l
The experiments were conducted on the rat cerebral cortex.
was measured in a WALLAC 1409 DSA liquid scintillation counter
[3H]prazosin (19.5 Ci/mmol, 1-adrenergic receptor), [3H]clonidine
a
(Perkin Elmer, USA). All assays were done in duplicates. Radioligand

N. Szkaradek et al. / Bioorg. Med. Chem. 21 (2013) 514–522
517
Table 1
Table 2
Chemical structures of the tested compounds 10–18
Affinity of compounds 10–18 to a1-, a2- and b1-adrenoceptors
O
Compound
Ki SEM (
lM)
a1-Adrenoceptors
a
2-Adrenoceptors
b1-Adrenoceptors
10
11
12
13
14
15
16
17
18
0.018 0.001
0.050 0.009
0.004 0.001
1.100 0.200
0.082 0.006
0.677 0.025
1.100 0.100
0.506 0.020
0.614 0.035
4.260 0.100
2.100 0.100
0.695 0.097
—
5.500 0.200
0.634 0.055
3.400 0.300
0.450 0.046
7.700 0.800
7.6 3.30
11.2 0.50
3.2 0.30
31.6 2.10
4.5 0.40
5.2 0.10
43.0 2.2
—
R"
R
O
N
R'
O
N
Compound
R
R0
R00
Form
HCl
27.6 0.20
2
10
H
OH
Ki SEM values was obtained from 2 to 3 experiments in duplicates.
O
CH3
CH3
CH3
O
10 mg/kg (compd 13, 17, 18). Table 3 shows effects of the tested
compounds on the heart rate and ECG parameters. Among all stud-
ied compounds only 13, 15 and 17 significantly decreased the
number of cardiac beats per minute ꢁ13 by 6%, 15 by 10%, 17 by
16%. Compounds 13 and 15 also significantly prolonged the QT
interval and QRS complex.
2
O
CH3
11
H
HCl
O
O
2
12
13
14
H
H
Cl
H
H
H
HCl
HCl
HCl
4.3. Anti-arrhythmic activity
O
Prophylactic anti-arrhythmic activity of the tested compounds
was evaluated using anesthetized rats in the adrenaline-induced
model of arrhythmia. Rapid intravenous injection of adrenaline at
CH3
4
2
a dose of 20 lg/kg caused reflex bradycardia (100%), atrioventricu-
lar disturbances, extrasystoles (100%), which led to the death of
approximately 76% of animals of the control group within 15 min
of the observation. The tested compounds 10–13, 15 and 7 injected
intravenously 15 min before adrenaline, diminished the occur-
rence of heart-rhythm disturbances.
Among studied compounds, the strongest prophylactic anti-
arrhythmic activity was shown by compounds 10–12. Their ED50
values (a dose producing 50% inhibition of premature ventricular
beats) are presented in Table 4. These data show that compound
12 was the most active in this test. This compound as well as com-
pounds 11 and 12 prevented the appearance of other adrenaline-
induced arrhythmia symptoms (bradycardia, blocks, bigeminy)
and significantly reduced mortality. Tested compounds were more
active in this experiment than reference drugs propranolol and
urapidil, but not than carvedilol.
O
CH3
15
16
H
OH
OH
2HCl
2HCl
Cl
O
17
18
Cl
Cl
OH
OH
2HCl
2HCl
binding data were analyzed using iterative curve fitting routines
with GraphPAD/Prism, Version 3.0 (GraphPad ware, San Diego,
CA, USA). Ki Values were calculated using the Cheng and Prusoff
equation.20
4.4. Influence on blood pressure
The hypotensive activity of compounds 10–12 and 15–18 was
determined after their iv administration to anaesthetized, normo-
tensive rats. The results are shown in Tables 5 and 6. Four com-
pounds (10–12 and 17) significantly decreased systolic and/or
diastolic blood pressure in anaesthetized, normotensive rats. Par-
ticularly potent hypotensive effect was observed after application
of compounds 10 and 12. Intravenous injections of compound 10
at the dose of 0.31 mg/kg significantly reduced systolic (by 19%)
and diastolic (by 17%) blood pressure in normotensive rats and
the hypotensive effect lasted throughout the observation. At a low-
er dose (0.16 mg/kg), compound 10 significantly reduced the blood
pressure from the 20th minute of observation (by 14–15%). Com-
pound 11 at the dose of 1.25 mg/kg significantly reduced the sys-
tolic blood pressure by 17–15% and diastolic blood pressure by
19–13%. At a lower dose (0.625 mg/kg), the compound 11 signifi-
cantly reduced systolic blood pressure by 12–10% and diastolic
blood pressure by 19–13%. At a dose of 0.31 mg/kg, compound
11 did not have any significant influence on blood pressure (data
not shown). Compound 12 revealed hypotensive activity at the
4. Pharmacological results
4.1. Radioligand binding assay
The pharmacological profile of the new compounds was evalu-
ated by radioligand binding assays. The results obtained are
presented in Table 2. Compounds 10–12 and 14 displaced
[3H]prazosin from cortical binding sites in low concentration range
(Ki = 0.004–0.082
[3H]clonidine binding and inhibited [3H]CGP12177 binding to cor-
tical b1-adrenoceptors with M-range.
lM). All tested compounds moderately inhibited
l
4.2. The influence on the normal electrocardiogram
Effect on ECG intervals and the heart rate was determined for a
dose of 2.5 mg/kg (compd 10–12), 5 mg/kg (compd 15, 16) or

518
N. Szkaradek et al. / Bioorg. Med. Chem. 21 (2013) 514–522
Table 3
The effect of an intravenous injection of the tested compounds on the heart rate and ECG intervals in anaesthetized male Wistar rats
Compound
Dose (mg/kg)
Parameters
Time of observation (min)
10
0
5
15
10
2.5
PQ (ms)
QRS (ms)
QT (ms)
Beats per min
PQ (ms)
QRS (ms)
QT (ms)
Beats per min
PQ (ms)
QRS (ms)
QT (ms)
Beats per min
PQ (ms)
QRS (ms)
QT (ms)
Beats per min
PQ (ms)
QRS (ms)
QT (ms)
Beats per min
PQ (ms)
QRS (ms)
QT (ms)
Beats per min
PQ (ms)
QRS (ms)
QT (ms)
Beats per min
PQ (ms)
QRS (ms)
QT (msms)
Beats per min
43.2 1.2
20.0 0.3
68.0 1.1
330.7 14.2
48.0 2.0
19.0 0.6
59.3 0.7
375.0 0.0
42.0 0.5
19.9 0.7
68.5 1.4
322.7 12.7
41.6 3.0
20.4 0.4
78.2 1.2
333.3 10.5
39.6 2.0
21.8 0.3
73.2 0.7
341.8 11.7
41.7 1.6
20.0 0.0
70.4 2.4
345.8 13.7
29.84 1.72
19.12 1.14
50.80 2.12
338.13 7.72
39.3 0.4
18.7 0.9
78.5 1.9
323.0 20.1
44.3 0.4
19.6 0.5
69.7 1.1
341.2 23.3
48.0 2.0
43.3 0.5
19.8 0.5
68.2 1.1
315.2 16.4
48.0 2.0
19.0 0.6
60.0 0.0
369.0 5.95
43.2 0.7
20.6 0.4
42.8 0.8
20.2 0.5
68.4 1.2
303.3 15.3
48.0 2.0
11
12
13
15
16
17
18
2.5
2.5
10
5
19.0 0.6
19.0 0.6
61.80 1.3
375.0 0.0
43.0 0.6
20.4 0.4
70.0 2.3
60.00 0.0
366.3 8.72
43.5 0.7
20.3 0.2
71.8 1.8
69.8 1.9
314.4 14.2
43.5 2.8
306.7 17.4
43.7 2.1
295.7 12.4
43.7 2.9
22.8 0.5*
79.4 1.5
23.6 0.6**
80.2 1.3*
317.4 12.2
41.7 3.1
24.2 0.6**
82.4 0.7*
311.4 11.1*
41.7 3.1
323.0 12.4
41.5 2.9
23.4 0.4**
76.4 0.9*
328.0 8.6
45.7 2.5
23.5 0.4**
77.0 0.9**
311.3 9.0*
46.0 2.2
24.0 0.4***
77.6 0.9**
309.0 7.0*
45.2 2.1
5
20.2 0.1
73.6 2.9
20.2 0.3
74.7 2.9
20.3 0.2
74.6 0.2
317.3 10.5
33.12 1.80
18.80 0.95
54.40 1.23
303.69 10.41
41.9 0.6
19.2 0.8
74.0 1.3
295.3 20.7
324.3 8.5
33.60 2.11
20.32 0.54
50.48 1.77
242.43 54.15
41.1 0.5
19.6 0.9
72.8 2.9
285.0 23.9
329.3 5.8
32.81 2.42
20.40 0.62
53.20 1.25
283.41 10.33***
40.9 0.6
20.1 0.7
78.8 2.5
268.8 24.8
10
10
The data are means of six experiments SEM.
Statistical analyses were performed using one-way ANOVA test:
*
p <0.05.
p <0.02.
p <0.01.
**
***
particular: design, synthesis, physico-chemical analysis, antiar-
rhythmic and hypotensive activity estimation and measuring of
binding affinity for adrenoceptors.
Table 4
The prophylactic anti-arrhythmic activity in adrenaline induced
arrhythmia in anesthetized rats
In the first step the affinity for adrenoceptors was determined
Compound
ED50 (mg/kg)
using a radioligand binding assay. The highest affinity for a1
adrenoceptors revealed compounds: 12 > 10 > 11 > 14, containing
2-methoxyphenylpiperazine moiety in their structure.
The antiarrhythmic effects of novel compounds were examined
in rats using the model of adrenaline-induced arrhythmia. The
most active were compounds 10–12, administered iv 15 min be-
fore arrhythmogen, which prevented or attenuated the symptoms
of adrenaline-induced arrhythmia. Data reported in Table 4 sug-
gest that all compounds show more preferable therapeutic indexes
than propranolol (a non-selective beta-adrenoceptor blocker) and
-
10
11
12
0.88 (0.38–2.00)
0.87 (0.41–1.82)
0.69 (0.31–1.58)
1.05 (0.64–1.73)
1.26 (0.97–1.64)
0.25 (0.12–0.53)
Propranolol
Urapidila
Carvedilolb
Route of administration—iv.
Each value was obtained from three experimental groups. Each
group consisted of six animals. The ED50 values and their con-
fidence limits were calculated according to the method of
Litchfield and Wilcoxon18
.
a
urapidil (a1-adrenoceptor antagonist and an 5-HT1A receptor ago-
nist), but not carvedilol (a non-selective beta-adrenoceptor blocker
Ref. 28.
Ref. 29.
b
with alpha 1-blocking properties).
Hypotensive activity of investigated compounds was deter-
mined after iv administration to normotensive anesthetized rats.
The most active were compounds 10–12, which showed a signifi-
cant hypotensive effect throughout the whole observation period.
The hypotensive effect was probably the result of strong
doses of 0.625–0.16 mg/kg and reduced both systolic (17–6%) and
diastolic (16–14%) blood pressure. Compound 17 at a dose of
1.25 mg/kg significantly reduced only systolic blood pressure from
the 20th minute of observation (by 7–10%). Compounds 15, 16 and
18 were found to be inactive.
a1-adrenoceptors blockade. Compounds 15–17 were less active.
Compound 18 slightly increased blood pressure.
5. Discussion
Results obtained via adrenoceptor binding assay correlated
with in vivo antiarrhythmic and hypotensive evaluation. Com-
pounds 10–12, which were the most potent substances in hypo-
tensive tests, displaced [3H]prazosine from cortical binding sites
The aim of this study was to evaluate cardiovascular activity of
new compounds-derivatives of xanthone. This study included in

N. Szkaradek et al. / Bioorg. Med. Chem. 21 (2013) 514–522
519
Table 5
Hypotensive activity of compounds 10, 11 and 12 in anesthetized normotensive rats after iv administration
Compound
Dose mg/kg
Blood pressure (mmHg)
Time of observation (min)
20
0
5
10
30
60
10
0.31
0.16
1.25
0.625
0.625
0.31
0.16
Systolic
Diastolic
Systolic
Diastolic
Systolic
Diastolic
Systolic
Diastolic
Systolic
Diastolic
Systolic
Diastolic
Systolic
Diastolic
144.0 3.7
118.2 3.2
131.8 3.9
115.3 3.9
140.2 3.5
108.2 5.7
139.4 8.2
105.0 4.1
138.5 1.5
116.2 4.5
135.3 5.8
116.3 5.7
124.2 3.3
110.2 3.8
122.8 2.3***
102.8 3.8**
124.0 2.6
109.2 2.8
119.0 2.8****
92.8 4.5*
121.8 2.8***
103.0 3.7**
124.8 2.9
110.2 3.3
118.4 3.1****
93.6 5.0*
123.8 3.1**
96.4 2.7
120.6 2.3***
102.0 3.2***
118.5 3.3**
103.8 4.0*
117.0 2.9****
92.2 5.5*
118.6 1.2***
100.6 2.1***
115.7 3.5***
101.8 4.1*
117.6 3.5****
90.8 5.3*
116.0 1.2***
98.4 1.6***
115.2 2.9***
101.5 3.3**
11
12
116.6 4.2****
89.6 5.5**
123.2 3.5***
92.0 3.3***
129.5 1.5*
112.0 2.3
116.0 6.3**
97.2 6.7*
124.8 3.6**
95.6 3.2
122.2 5.0***
95.4 3.5*
120.6 3.5***
93.2 2.1**
129.5 2.5*
111.7 4.0
122.0 2.9***
101.0 4.1*
122.0 3.9*
107.2 5.9
109.6 4.1*
97.0 4.8*
125.5 3.2**
106.5 4.4
122.8 4.5
107.7 5.7
114.0 4.5
99.2 4.8
129.5 3.6*
110.5 4.8
121.0 4.0*
105.7 5.4
113.4 5.9*
101.0 5.0
119.5 3.3*
103.3 4.8
111.4 4.6*
100.0 4.3
108.2 5.1*
95.2 5.1*
The data are means of five to six experiments SEM.
Statistical analyses were performed using one-way ANOVA test:
*
p <0.05.
p <0.02.
p <0.01.
p <0.001.
**
***
****
Table 6
Hypotensive activity of compounds 15–18 in anesthetized normotensive rats after iv administration
Compound
Dose mg/kg
Blood pressure (mmHg)
Time of observation (min)
20
0
5
10
30
60
15
16
17
18
2.5
Systolic
Diastolic
Systolic
Diastolic
Systolic
Diastolic
Systolic
Diastolic
137.6 4.4
115.4 5.9
144.2 4.1
118.4 6.7
144.2 4.1
117.2 5.4
139.8 4.3
114.0 5.1
132.6 5.0
110.8 5.7
125.6 4.7
105.0 4.8
2.5
136.20 2.08
114.40 1.57
123.80 2.44
108.40 5.08
143.0 4.2
125.00 8.55
103.20 7.49
117.20 3.53
102.60 3.65
147.2 4.6
133.00 2.95
110.60 1.69
115.20 7.84
101.20 7.10
149.5 4.5
130.80 1.68
110.40 1.22
110.80 9.52*
97.40 8.45
152.5 2.5
129.80 2.03
110.80 1.02
113.20 6.97***
100.60 5.36
152.2 2.4
126.80 1.02
108.60 0.93
114.80 3.90***
103.60 3.81
151.2 0.9
1.25
2.5
124.0 4.0
128.7 4.8
132.2 4.9
134.5 2.5
134.2 2.0
134.2 1.3
The data are means of five to six experiments SEM.
Statistical analyses were performed using one-way ANOVA test:
*
p <0.05.
p <0.01.
***
in low concentration range (Ki = 18, 50 and 4 nM), whereas other
these two
functionally distinct. To date, the data imply that
are associated with cardiac growth and 1A-adrenoceptors with
contractility and possibly with cardioprotection. It seems that both
1-adrenoceptors subtypes can provide protection under ischae-
a
1-adrenoceptors subtypes expressed in the heart are
compounds moderately inhibited [3H]prazosine binding with
a
1B-adrenoceptors
lM-range. It is well known that
a
1-adrenoceptors play key role
a
in the stimulation of smooth muscle contraction. They stimulate
the contraction of veins and arteries and in consequence increase
a
the resistance in circulatory blood flow. In that way,
tors are able to regulate blood pressure. As 1-receptors are impor-
tant for maintenance of peripheral vascular resistance, 1-receptor
a1-adrenocep-
mia/reperfusion conditions but involve different mechanisms.23
Taking into account structure-activity relationship, it can be sta-
ted that the most active compounds contained 2-methoxyphenyl-
piperazine moiety (10–12 and 14). Compound 13 bearing
4-methoxyphenylpiperazine moiety was less potent in in vivo tests
than its 2-substituted analogues and also inhibited [3H]prazosine
a
a
antagonists may be used to relax blood vessels in hypertensive pa-
tients and to reduce symptoms of benign prostate hyperplasia.
Classic
a1-antagonists, like prazosin, doxazosin and terazosin
proved to be a highly effective antihypertensive drugs and remain
important options in the treatment regimen available for
hypertension.21
The above mentioned compounds are also potential antiarrhyth-
mic agents. In human heart, b1- and b2-adrenoceptors coexist,
whereby b1-adrenoceptors predominate; in general, the ratio
b1-:b2-adrenoceptors is about 70%:30% in the atria and 80%:20% in
the ventricles. On the other hand 15–20% of adrenoceptors in hu-
binding with
placed [3H]prazosine from cortical binding sites in nM-range. In
vivo tests indicated that compounds 10–12 were 1-receptor
antagonists. The binding affinity for 2-receptors and b1-adreno-
ceptors of all of the tested compounds were in M-range and re-
vealed any structural relationship. Introduction of the chlor
substituent in the xanthone ring also diminished binding affinity
for a1-adrenoceptors (compare compounds 12 with 14 and 15 with
lM-range in contrast to compounds 10–12 that dis-
a
a
l
man heart are different subtypes of
a
1-adrenoceptors, especially
1-blocking drugs, such
16 in Table 2) suggesting that enhancing of the lipophilicity was not
desirable. From the synthesized compounds, 12 seemed to be the
most potent. This compound was devoided of hydroxyl group and
contained 3-aminepropan-1-yloxy linker instead of typical b-block-
er: 3-amine-2-hydroxypropan-1-yloxy linker. This observation
a
1A and a1B a
.
22 In previously published data
as prazosin and phentolamine, have been shown to be effective
against ischemia-induced arrhythmias in a variety of animal mod-
els. Other studies using transgenic animals have demonstrated that

520
N. Szkaradek et al. / Bioorg. Med. Chem. 21 (2013) 514–522
proved in vitro screenings that evaluated structures acting via a1
adrenoceptors rather than b1-adrenoceptors.
-
Waters TQD mass spectrometer (electrospray ionization mode
ESI-tandem quadrupole). All the analyses were carried out using
an Acquity UPLC BEH C18, 1.7
lm, 2.1 ꢀ 100 mm column. A flow
rate of 0.3 mL/min and a gradient of (5–95)% B over 10 min and
then 100% B over 2 min was used. Eluent A: water/0.1% HCO2H;
eluent B: acetonitrile/0.1% HCO2H. LC/MS data were obtained by
scanning the first quadrupole in 0.5 s in a mass range from 50 to
1000 Da; eight scans were summed up to produce the final spec-
trum. The purity of obtained compounds was confirmed by the
thin-layer chromatography (TLC), carried out on precoated plates
(silica gel, 60 F-254, Merck, Darmstadt, Germany) using the solvent
systems mentioned below. The obtained corresponding spots were
visualized under UV light.
6. Conclusion
In conclusion, we have synthesized a series of 9 piperazine like
derivatives of xanthone. All compounds were in vitro screened for
their affinity for chosen adrenoceptors. Then, in vivo antiarrhyth-
mic and hypotensive activity of the structures was tested. Three
compounds revealed nanomolar affinity for
a1-adrenoceptor as
well as the highest cardiovascular efficacy in animals’ models.
The most promising compound was 4-(3-(4-(2-methoxyphenyl)
piperazine-1-yl)propoxy)-9H-xanthen-9-one hydrochloride (12)
which revealed ED50 value of 0.69 mg/kg in adrenaline induced
arrhythmia (rats, iv). Other synthesized xanthone derivatives,
that is, (R,S)-4-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazine-1-
yl)propoxy)-9H-xanthen-9-one hydrochloride (10) and (R,S)-4-(2-
acetoxy-3-(4-(2-methoxyphenyl)piperazine-1-yl)propoxy)-9H-
xanthen-9-one hydrochloride (11) were also proved to act as anti-
arrhythmics and showed ED50 values in adrenaline induced
arrhythmia (rats, iv) of 0.88 and 0.89 mg/kg, respectively. These
values were lower than values obtained for reference drugs such
as propranolol and urapidil. The pharmacological results and bind-
ing studies suggested that the antiarrhythmic and hypotensive
activity of these compounds were related to their adrenolytic prop-
erties. More extensive pharmacological studies that are in progress
will expand the knowledge of the other possible mechanisms of
cardiovascular action of the tested compounds (i.e. antioxidant,
vasorelaxant effect via calcium entry blocking activity or the im-
pact on NO pathway).
The synthetic rout of the tested compounds and starting mate-
rials is outlined in Scheme 1. Synthesis of the compounds men-
tioned above was multistage. In the first stage there was
synthesized xanthone skeleton. This synthesis included Ullmann’s
condensation of appropriate chlorobenzoic acid and phenol deriv-
ative.24,25 The reaction was performed for few hours, in oil, in the
195–200 °C, in the presence of Cu/Cu2O catalyst. After condensa-
tion, intermediate product underwent cyclization with concomi-
tant demethylation in the presence of concentrated H2SO4. It was
noticed that, the longer the mixture was heated, the more effective
demethylation was achieved. The optimal refluxing time was
2 days. Then reacting mixture was poured into crashed ice, filtered
and washed to neutral reaction. Unreacted acid was washed with
NaHCO3. The detailed methodology and physicochemical proper-
ties of 4-hydroxy-9H-xanthen-9-one and 3-chloro-5-hydroxy-9H-
xanthen-9-one were described in.14,15 Then synthesis underwent
two different pathways to obtain bromopropoxy or oxirane xan-
thone’s derivatives. 4-(3-Bromopropoxy)-9H-xanthen-9-one and
3-chloro-5-(3-bromopropoxy)-9H-xanthen-9-one were synthe-
sized by the reaction of appropriate hydroxyxanthone with 3-chlo-
ropropanol in acetone, in the presence of the big excess of K2CO3.
After that, solvent was distilled and unreacted hydroxyxanthone
was elutriated using NaOH solution. Then obtained intermediate
product was taken to bromination using PBr3 in CHCl3. Subse-
quently, CHCl3 was distilled and unreacted intermediate product
was washed with NaHCO3. Crude product was recrystallized from
n-hexane/toluene (1:6). The procedure and physicochemical prop-
erties of 4-(3-hydroxypropoxy)-9H-xanthen-9-one and 4-(3-
bromopropoxy)-9H-xanthen-9-one were mentioned formerly.14
3-Chloro-5-(3-hydroxypropoxy)-9H-xanthen-9-one and 3-Chloro-
5-(3-bromopropoxy)-9H-xanthen-9-one are new and their proper-
ties are summarized below. The oxirane derivatives of xanthone
were obtained in the reaction of appropriate hydroxyxanthone
with epichlorohydrine according to procedures described earlier.13
Epichlorohydrine was used in double excess. Reacting mixture was
stirred during night, at the room temperature. After that, precipi-
tate was filtered and unreacted substrates were washed with 10%
NaOH solution. Physicochemical properties of 4-((oxiran-2-yl)-
methoxy)-9H-xanthen-9-one were described in 14, whereas char-
acteristic of 3-chloro-5-((oxiran-2-yl)methoxy)-9H-xanthen-9-
one was published in.15
The results of the tested substances are quite encouraging,
therefore further modification of their structures might lead to dis-
covering of new potential drugs. Potential pathways of modifica-
tions will include: changes in the longevity of alkane linker,
changes in the place of substitution in the xanthone ring and intro-
duction of other substituents in the xanthone ring.
7. Experimental protocols
Reagents: benzylpiperazine, epichlorohydrine, phenoxyethyl-
piperazine were purchased from Sigma–Aldrich Chemie (Steinheim,
Germany), whereas reagents: 2-chlorobenzoic acid, 3-chloro-1-pro-
panol, cinnamylpiperazine, 2,4-dichlorobenzoic acid, 2-methoxy-
phenol, 2-methoxyphenylpiperazine, 4-methoxyphenylpiperazine,
phosphorus tribromide were provided by Lancaster Synthesis
(Frankfurt am Main, Germany). Solvents were commercially avail-
able materials of reagent grade.
Melting points (mp) are uncorrected and were determined
using a Büchi SMP-20 apparatus (Büchi Labortechnik, Flawil,
Switzerland). The infrared spectra were recorded on potassium
bromide pellets using a Jasco FT/IR 410 spectrometer (Jasco Inc.,
Easton, MD, USA). The 1H NMR were recorded on a Bruker AMX
spectrometer (Brucker, Karlsruhe, Germany) with 500.13 MHz or
Varian Mercury-VX 300 NMR spectrometer (Varian Inc., Palo Alto,
CA, USA) using signal from DMSO in DMSO-d6 and CHCl3 in CDCl3
as an internal standard. The results are presented in the following
format: chemical shift d ppm), multiplicity, J values in Hertz (Hz),
number of protons, proton’s position. Multiplicities were shown
as the abbreviations: s (singlet), bs (broad singlet), d (doublet),
dd (doublet of doublets), t (triplet), ddd (double doublet of
doublets), qu (quintet), m (multiplet). Elemental analyses were
performed on an Elementar Vario EL III (Elementar Analysansys-
teme, Hanau, Germany). For mass spectrometry analysis samples
were prepared in acetonitrile/water (10/90 v/v) mixture. The LC/
MS system consisted of a Waters Acquity UPLC, coupled to a
Compounds 10 and 12–18 were obtained by the amination of
respective parent compound with appropriate amines in n-propa-
nol (for 10, 15–18) or toluene in a presence of K2CO3 (for 12–14), as
described previously.13,16 After aminolysis, solvents were distilled.
Resulted amines were dissolved in diluted HCl, cleaned with char-
coal and precipitated using NaOH solution. In addition, compound
10 underwent acetylation to get compound 11. Compound 10 was
dissolved in the smallest volume of acetic anhydride and stirred
with concomitant heating for 4 h. Then, reacting mixture was
pulled into the crushed ice, neutralized with NaHCO3 and precipi-
tated compound 11 was filtered. All resulted bases of compounds
10–18 were converted into hydrochloride salts using an excess of

N. Szkaradek et al. / Bioorg. Med. Chem. 21 (2013) 514–522
521
ethanol saturated with HCl. The crude products were recrystallized
from acetone/ethanol (1:3). Physicochemical properties of com-
pounds 10–18 are summarized below. In order to optimize synthe-
sis of the tested compounds, compound 10 was obtained also using
alternative method including (R,S)-4-(3-chloro-2-hydroxyprop-
oxy)-9H-xanthen-9-one as intermediate (see Scheme 1). (R,S)-4-
(3-Chloro-2-hydroxypropoxy)-9H-xanthen-9-one was obtained in
the reaction of equimolar amounts of 4-hydroxy-9H-xanthen-9-
one and epichlorohydrine in propan-1-ol in the presence of pyri-
dine. The reaction mixture was stirred for 1 h on water bath, then
the unsoluble precipitate was filtered off. From the cooled filtrate
the crude product was separated, filtered off and recrystallized
from toluene/heptane (5:1) analogously to.12,26 Nevertheless, this
method seems to be less potent, so other compounds were ob-
tained using oxirane derivatives as intermediates. Structures of
the tested compounds are presented in Table 1.
for C27H29O5N2Cl: C, 65.25; H, 5.88; N, 5.63. Found: C, 64.94; H,
6.17; N, 5.60. IR (KBr) v (cmꢁ1): 3361, 2929, 1662, 1462, 1334,
1265, 1245, 1223, 10627, 930, 748. 1H NMR (DMSO-d6, 300 MHz)
d ppm: 8.21 (dd, J = 8.0, J = 1.5, 1H, H-8), 7.93–7.88 (m, 1H, H-6),
7.78 (dd, J = 7.8, J = 1.4, 1H, H-1), 7.76 (d, J = 7.2, 1H, H-5), 7.60
(dd, J = 8.1, J = 1.4, 1H, H-3), 7.50 (t, J = 8.0, 1H, H-7), 7.40 (t,
J = 8.1, 1H, H-2), 7.02–6.90 (m, 4H, H-Ph), 6,12–6.08 (m, 1H,
-OH), 4.58–4.48 (m, 1H, CH), 4.24–4.23 (m, 1H, Ar–O–CH2–), 3.79
(s, 3H, Ar–O–CH3), 3.67–3.03 (m, 10H, –N–CH2–). LC–MS: calcd
for [M+H]+: C27H29O5N2 m/z: 461.20, found 461.45. RF = 0.85
(mathanol/ethyl acetate (1:1)).
7.5. (R,S)-4-(2-Acetoxy-3-(4-(2-methoxyphenyl)piperazine-1-
yl)propoxy)-9H-xanthen-9-one hydrochloride (compound 11)
(R,S)-4-(2-Acetoxy-3-(4-(2-methoxyphenyl)piperazine-1-
yl)propoxy)-9H-xanthen-9-one hydrochloride (compound 11) was
obtained as a white solid (yield 70%), mp 214–216 °C. Anal. calcd
for C29H31O6N2Cl: C, 64.62; H, 5.80; N, 5.20. Found: C, 64.83; H,
6.02; N, 5.57. IR (KBr) v (cmꢁ1): 3430, 2935, 1648, 1594, 1500,
1449, 1275, 1229, 1082, 758. LC–MS: calcd for [M+H]+:
7.1. 3-Chloro-5-(3-hydroxypropoxy)-9H-xanthen-9-one
(compound 4)
3-Chloro-5-(3-hydroxypropoxy)-9H-xanthen-9-one (compound
4) was obtained as white solid (yield 70%), mp 113–115 °C. Anal.
calcd for C16H13O4Cl: C, 63.06; H, 4.30. Found: C, 62.99; H, 4.52.
IR (KBr) v (cmꢁ1): 3412, 2935, 1668, 1460, 1271, 1223, 1064,
753. 1H NMR (CDCl3, 300 MHz) d ppm: 8.25 (d, J = 8.5, 1H, H-8),
7.86 (dd, J = 7.1, J = 2.4, 1H, H-1), 7.57 (d, J = 2.0, 1H, H-5), 7.34
(dd, J = 8.6, J = 2.0, 1H, H-7), 7.31–7.24 (m, 2H, H-2, H-3), 4.33 (t,
J = 5.9, 2H, Ar–O–CH2–), 3.99 (t, J = 5.4, 2H, –CH2–OH), 2.20 (qu,
J = 5.9, 2H, –CH2–CH2–CH2–). LC–MS: calcd for [M+H]+: C16H14O4Cl
m/z: 305.05, found 305.16. RF = 0.60 (toluene/acetone (5:3)).
C29H31O6N2 m/z: 503.21, found 503.33. RF = 0.79 (mathanol/ethyl
acetate (1:1)). Base of compound 2: 1H NMR (CDCl3, 300 MHz) d
ppm: 8.35 (dd, J = 8.0, J = 1.3, 1H, H-8), 7.94–7.26 (m, 6H, H–Ar),
6.99–6.84 (m, 4H, H-Ph), 5.53–5.50 (m, 1H, CH), 4.45 (d, J = 10.5,
J = 3.6, 1H, Ar–O–CHH–), 4.32 (d, J = 10.5, J = 5.9, 1H, Ar–O–CHH–),
3.86 (s, 3H, Ar–O–CH3), 3.13–3.00 (m, 4H, –N–CH2– (pip)a), 2.87–
2.74 (m, 6H, –N–CH2– (pip)e + –N–CH2–R), 2.12 (s, 3H, –C–CH3).
7.6. 4-(3-(4-(2-Methoxyphenyl)piperazine-1-yl)propoxy)-9H-
7.2. 3-Chloro-5-(3-bromopropoxy)-9H-xanthen-9-one
xanthen-9-one hydrochloride (compound 12)
(compound 6)
4-(3-(4-(2-Methoxyphenyl)piperazine-1-yl)propoxy)-9H-xan-
then-9-one hydrochloride (compound 12) was obtained as a white
solid (yield 80%), mp 210–212 °C. Anal. calcd for C27H29O4N2Cl: C,
67.42; H, 6.08; N, 5.82. Found: C, 66.86; H, 6.51; N, 5.79. IR (KBr)
v (cmꢁ1): 3421, 3023, 2427, 1665, 1607, 1493, 1467, 1340, 1276,
1065, 746. 1H NMR (DMSO-d6, 500.13 MHz) d ppm: 8.21 (ddd,
J = 8.0, J = 1.8, J = 0.5, 1H, H-8), 7.89 (ddd, J = 8.7, J = 7.1, J = 1.8,
1H, H-6), 7.75 (dd, J = 8.0, J = 1.5, 1H, H-1), 7.71 (ddd, J = 8.5,
J = 1.1, J = 0.5, 1H, H-5), 7.55 (dd, J = 8.0, J = 1.5, 1H, H-3), 7.50
(ddd, J = 8.0, J = 7.1, J = 1.1, 1H, H-7), 7.41 (dd, J = 8.0, J = 8.0, 1H,
H-2), 6.99–6.84 (m, 4H, Ar–H), 4.27 (t, J = 6.4, 2H, Ar–O–CH2–),
3.77 (s, 3H, Ar–O–CH3), 3.06–2.92 (m, 4H, –N–CH2– (pip)a), 2.69–
2.53 (m, 6H, –N–CH2– (pip)e + –N–CH2–R), 2.05 (qu, 2H, R–CH2–
R). LC–MS: calcd for [M+H]+: C27H29O4N2 m/z: 445.20, found
445.36. RF = 0.70 (methanol/ethyl acetate (1:1)).
3-Chloro-5-(3-bromopropoxy)-9H-xanthen-9-one (compound
6) was obtained as white solid (yield 65%), mp 140–142 °C. Anal.
calcd for C16H12O3ClBr: C, 52.27; H, 3.29. Found: C, 52.84; H,
3.22. IR (KBr) v (cmꢁ1): 2933, 1644, 1428, 1273, 1064, 933, 755.
1H NMR (CDCl3, 300 MHz) d ppm: 8.25 (d, J = 8.7, 1H, H-8), 7.88
(dd, J = 6.8, J = 2.8, 1H, H-1), 7.60 (d, J = 1.5, 1H, H-5), 7.36–7.24
(m, 3H, H-7, H-2, H-3), 4.29 (t, J = 5.9, 2H, Ar–O–CH2–), 3.73 (t,
J = 6.4, 2H, –CH2–Br), 2.46 (qu, J = 6.2, 2H, –CH2–CH2–CH2–). LC–
MS: calcd for [M+H]+: C16H13O3ClBr m/z: 366.97, found 367.12.
RF = 0.87 (mathanol/ethyl acetate (1:3)).
7.3. (R,S)-4-(3-Chloro-2-hydroxypropoxy)-9H-xanthen-9-one
(compound 7)
(R,S)-4-(3-Chloro-2-hydroxypropoxy)-9H-xanthen-9-one (com-
pound 7) was obtained as white solid (yield 70%), mp 158–160 °C
(Lit. 158–159 °C).27 Anal. calcd for C16H13O4Cl: C, 63.06; H, 4.30.
Found: C, 63.00; H, 4.67. IR (KBr) v (cmꢁ1): 3455, 2930, 1642,
1420, 1271, 1053, 934, 749. 1H NMR (CDCl3, 300 MHz) d ppm:
8.33 (dd, J = 8.0, J = 1.5, 1H, H-8), 7.93 (dd, J = 5.6, J = 4.1, 1H, H-
1), 7.75–7.22 (m, 5H, H-2, H-3, H-5, H-6, H-7), 4.42–4.32 (m, 1H,
@CH–), 4.31–4.24 (m, 2H, Ar–O–CH2–), 3.95–3.83 (m, 2H, –CH2–
Cl), 2.94 (d, J = 5.9, 1H, –OH). LC–MS: calcd for [M+H]+: C16H14O4Cl
m/z: 305.05, found 305.16. RF = 0.75 (methanol/ethyl acetate (1:3)).
7.7. 4-(3-(4-(4-Methoxyphenyl)piperazine-1-yl)propoxy)-9H-
xanthen-9-one hydrochloride (compound 13)
4-(3-(4-(4-Methoxyphenyl)piperazine-1-yl)propoxy)-9H-xan-
then-9-one hydrochloride (compound 13) was obtained as a
white solid (yield 65%), mp 214–216 °C. Anal. calcd for
C
27H29O4N2Cl: C, 67.42; H, 6.08; N, 5.82. Found: C, 67.28; H,
6.22; N, 5.48. LC–MS: calcd for [M+H]+: C27H29O4N2 m/z: 445.20,
found 445.36. RF = 0.67 (methanol/ethyl acetate (1:1)). Base of
compound 4: IR (KBr) v (cmꢁ1): 3413, 2919, 2850, 1672, 1607,
1514, 1451, 1261, 1226, 1071, 749. 1H NMR (CDCl3, 300 MHz) d
ppm: 8.35 (dd, J = 8.0, J = 1.5, 1H, H-8), 7.92–7.26 (m, 6H, H–Ar),
6.93–6.83 (m, 4H, H-Ph), 4.26 (t, J = 6.4, 2H, Ar–O–CH2–), 3.77
(s, 3H, Ar–O–CH3), 3.31–3.15 (m, 4H, –N–CH2– (pip)a), 2.72–2.68
(m, 6H, –N–CH2– (pip)e + –N–CH2–R), 2.17 (qu, J = 6.4, 2H, R–
CH2–R).
7.4. (R,S)-4-(2-Hydroxy-3-(4-(2-methoxyphenyl)piperazine-1-
yl)propoxy)-9H-xanthen-9-one hydrochloride (compound 10)
(R,S)-4-(2-Hydroxy-3-(4-(2-methoxyphenyl)piperazine-1-yl)
propoxy)-9H-xanthen-9-one hydrochloride (compound 10) was
obtained as white solid (yield 80%), mp 218–220 °C. Anal. calcd

522
N. Szkaradek et al. / Bioorg. Med. Chem. 21 (2013) 514–522
7.8. 3-Chloro-5-(3-(4-(2-methoxyphenyl)piperazine-1-
yl)propoxy)-9H-xanthen-9-one hydrochloride (compound 14)
H-5), 7.75 (dd, J = 8.2, J = 1.3, 1H, H-1), 7.61 (dd, J = 8.0, J = 1.3,
1H, H-3), 7.54 (dd, J = 8.7, J = 1.8, 1H, H-7), 7.41 (t, J = 8.1, 1H, H-
2), 7.34–6.95 (m, 5H, H-Ph), 4.51 (bs, 1H, CH), 4.38 (bs, 2H,
–CH2–O–Ph), 4.22 (d, J = 4.4, 2H, Ar–O–CH2–), 4.04–3.02 (m, 12H,
–N–CH2–, overlap with signal from H2O in DMSO). LC–MS: calcd
for [M+H]+: C28H30O5N2Cl m/z: 509.18, found 509.31. RF = 0.55
(methanol).
3-Chloro-5-(3-(4-(2-methoxyphenyl)piperazine-1-yl)propoxy)-
9H-xanthen-9-one hydrochloride (compound 14) was obtained as
a
white solid (yield 70%), mp 270–272 °C. Anal. calcd for
C27H28O4N2Cl2: C, 62.91; H, 5.48; N, 5.44. Found: C, 62.90; H,
5.66; N, 5.41. LC–MS: calcd for [M+H]+: C27H28O4N2Cl m/z:
479.17, found 479.33. RF = 0.72 (methanol/ethyl acetate (1:1)).
Base of compound 5: IR (KBr) v (cmꢁ1): 3446, 2946, 2817, 1660,
1602, 1499, 1427, 1281, 1242, 1073, 757. 1H NMR (CDCl3,
300 MHz) d ppm: 8.28 (d, J = 8.5, 1H, H-8), 7.88–7.29 (m, 5H, H-
Ar), 7.03–6.86 (m, 4H, H-Ph), 4.26 (t, J = 6.4, 2H, Ar–O–CH2–),
3.87 (s, 3H, Ar–O–CH3), 3.14 (bs, 4H, –N–CH2– (pip)a), 2.74–2.69
(m, 6H, –N–CH2– (pip)e + –N–CH2–R), 2.17 (qu, J = 6.4, 2H, R–
CH2–R).
7.12. (R,S)-5-(3-(2-Benzylpiperazine-1-yl)-2-hydroxypropoxy)-
3-chloro-9H-xanthen-9-one dihydrochloride (compound 18)
(R,S)-5-(3-(2-Benzylpiperazine-1-yl)-2-hydroxypropoxy)-3-
chloro-9H-xanthen-9-one dihydrochloride (compound 18) was ob-
tained as a white solid (yield 65%), mp 286–288 °C. Anal. calcd for
C27H29O4N2Cl3: C, 58.76; H, 5.30; N, 5.08. Found: C, 58.84; H, 5.53;
N, 5.11. IR (KBr) v (cmꢁ1): 3389, 2994, 1657, 1601, 1492, 1428,
1271, 1220, 1074, 756. 1H NMR (DMSO-d6, 300 MHz) d ppm:
8.20–7.37 (m, 11H, H–Ar), 4.48 (bs, 1H, –CH–), 4.21 (d, J = 4.6,
2H, Ar–O–CH2–), 4.05–3.12 (m, 12H, –N–CH2–, overlap with signal
from H2O in DMSO). LC–MS: calcd for [M+H]+: C27H28O4N2Cl m/z:
579.17, found 579.33. RF = 0.63 (methanol:ethyl acetate (1:1)).
7.9. (R,S)-4-(3-(4-Cinnamylpiperazine-1-yl)-2-hydroxypropoxy)-
9H-xanthen-9-one dihydrochloride (compound 15)
(R,S)-4-(3-(4-Cinnamylpiperazine-1-yl)-2-hydroxypropoxy)-
9H-xanthen-9-one dihydrochloride (compound 15) was obtained
as a white solid (yield 75%), mp 256–258 °C. Anal. calcd for
Supplementary data
C29H32O4N2Cl2: C, 64.09; H, 5.94; N, 5.15. Found: C, 63.73; H,
6.19; N, 5.10. IR (KBr) v (cmꢁ1): 3300, 2977, 2361, 1604, 1572,
1492, 1450, 1340, 1275, 1073, 757. 1H NMR (DMSO-d6, 300 MHz)
d ppm: 8.21–7.32 (m, 12H, H–Ar), 6.87 (d, J = 15.9, 1H, @C<HPh),
6.43–6.33 (m, 1H, RH>C@), 4.49 (bs, 1H, –CH–), 4.22 (d, J = 4.6,
2H, Ar–O–CH2–), 4.08–3.11 (m, 12H, –N–CH2–, overlap with signal
from H2O in DMSO). LC–MS: calcd for [M+H]+: C29H31O4N2 m/z:
471.22, found 471.35. RF = 0.53 (methanol:ethyl acetate (1:1)).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.11.014.
References and notes
1. Mathers, C. D.; Boerma, T.; Ma Fat, D. Br. Med. Bull. 2009, 92, 7.
2. Sanguinetti, M. C.; Bennett, P. B. Circ. Res. 2003, 93, 491.
3. Bardage, C.; Isacson, D. G. Blood Press. 2000, 9, 328.
4. Valenti, P.; Chiarini, A.; Gasperi, F.; Budriesi, R. Arzneimittelforschung 1990, 40,
122.
5. Ford, E. S.; Cooper, R. S. Hypertension 1991, 18, 598.
6. Cheng, Y. W.; Kang, J. J. Eur. J. Pharmacol. 1997, 336, 23.
7. Wang, L. W.; Kang, J. J.; Chen, I. J.; Teng, C. M.; Lin, C. N. Bioorg. Med. Chem. 2002,
10, 567.
7.10. (R,S)-3-Chloro-5-(3-(4-cinnamylpiperazine-1-yl)-2-
hydroxypropoxy)-9H-xanthen-9-one dihydrochloride
(compound 16)
8. Lin, K. W.; Fang, S. C.; Hung, C. F.; Shieh, B. J.; Yang, S. C.; Tang, C. M.; Lin, C. N.
Arch. Pharm. 2009, 342, 19.
9. Librowski, T.; Czarnecki, R.; Jastrze˛bska-Wie˛sek, M.; Opoka, W.; Marona, H. Boll.
Chim. Farm. 2004, 143, 267.
10. Marona, H.; Librowski, T.; Cegła, M.; Erdog˘an, C.; Sahin, N. O. Acta Pol. Pharm.
2008, 65, 383.
11. Malawska, B.; Kulig, K.; Filipek, B.; Sapa, J.; Macia˛g, D.; Zygmunt, M.;
Antkiewicz-Michaluk, L. Eur. J. Med. Chem. 2002, 37, 183.
12. Marona, H.; Górka, Z.; Szneler, E. Pharmazie 1998, 53, 219.
13. Marona, H.; Szneler, E.; Filipek, B.; Sapa, J. Acta Pol. Pharm. 1997, 54, 63.
14. Marona, H.; Szkaradek, N.; Kubacka, M.; Bednarski, M.; Filipek, B.; Cegła, M.;
Szneler, E. Arch. Pharm. 2008, 341, 90.
15. Marona, H.; Szkaradek, N.; Rapacz, A.; Filipek, B.; Dybała, M.; Siwek, A.; Cegła,
M.; Szneler, E. Bioorg. Med. Chem. 2009, 17, 1345.
16. Marona, H. Pharmazie 1998, 53, 672.
17. Schmier, J.; Eichler, O. Handbook of Experimental Pharmacology In Szekeres, L.,
Papp, J. G., Eds.; Springer: Berlin–Heidelberg–New York, 1975; pp 131–182.
18. Litchfield, J. T.; Wilcoxon, F. J. Pharmacol. Exp. Ther. 1949, 96, 99.
19. Maj, J.; Klimek, V.; Nowak, G. Eur. J. Pharmacol. 1985, 119, 113.
20. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
21. Jensen, B. C.; O’Connell, T. D.; Simpson, P. C. J. Mol. Cell. Cardiol. 2011, 51, 518.
22. Brodde, O. E.; Bruck, H.; Leineweber, K. J. Pharmacol. Sci. 2006, 100, 323.
23. Woodcock, E. A. Clin. Exp. Pharmacol. Physiol. 2007, 34, 884.
24. Ullmann, F.; Kipper, H. Ber. Dtsch. Chem. Ges. 1905, 38, 2120.
25. Ullmann, F.; Zlokasoff, M. Ber. Dtsch. Chem. Ges. 1905, 38, 2111.
26. Crowther, A. F.; Howe, R.; McLoughlin, B. J.; Mallion, K. D.; Rao, B. S.; Smith, L.
H.; Turner, R. W. J. Med. Chem. 1972, 15, 260.
27. Howe, R. McLoughlin British Patent 1,058,822, 1967.
28. Groszek, G.; Bednarski, M.; Dybała, M.; Filipek, B. Eur. J. Med. Chem. 2009, 44,
809.
29. Kulig, K.; Spieces, C.; Sapa, J.; Caspers, C.; Filipek, B.; Malawska, B. Pharmacol.
Rep. 2010, 62, 68.
(R,S)-3-Chloro-5-(3-(4-cinnamylpiperazine-1-yl)-2-hydroxy-
propoxy)-9H-xanthen-9-one dihydrochloride (compound 16) was
obtained as a white solid (yield 75%), mp 252–254 °C. Anal. calcd
for C29H31O4N2Cl3: C, 60.27; H, 5.41; N, 4.85. Found: C, 60.57; H,
5.79; N, 4.89. 1H NMR (DMSO-d6, 300 MHz) d ppm: 8.18 (d,
J = 8.5, 1H, H-8), 7.92 (d, J = 1.8, 1H, H-5), 7.74 (dd, J = 8.0, J = 1.3,
1H, H-1), 7.60 (dd, J = 8.0, J = 1.3, 1H, H-3), 7.54 (dd, J = 8.5,
J = 1.8, 1H, H-7), 7.50–7.21 (m, 6H, H-2, Phenyl), 6.87 (d, J = 15.6,
1H, @C<HPh), 6.38 (dt, J = 15.6, J = 7.4, 1H, RH>C@), 4.48 (bs, 1H,
–CH–), 4.21 (d, J = 4.6, 2H, Ar–O–CH2–), 3.92–3.37 (m, 12H, –N–
CH2–, overlap with signal from H2O in DMSO). LC–MS: calcd for
[M+H]+: C29H30O4N2Cl m/z: 505.18, found 505.32. RF = 0.46 (meth-
anol:ethyl acetate (1:1)).
7.11. (R,S)-3-Chloro-5-(2-hydroxy-3-(4-(2-phenoxyethyl)-
piperazine-1-yl)propoxy)-9H-xanthen-9-one dihydrochloride
(compound 17)
(R,S)-3-Chloro-5-(2-hydroxy-3-(4-(2-phenoxyethyl)piperazine-
1-yl)propoxy)-9H-xanthen-9-one dihydrochloride (compound 17)
was obtained as a white solid (yield 70%), mp 246–248 °C. Anal.
calcd for C28H31O5N2Cl3: C, 57.79; H, 5.37; N, 4.81. Found: C,
57.28; H, 5.87; N, 4.78. IR (KBr) v (cmꢁ1): 3426, 2949, 1656,
1603, 1494, 1428, 1277, 1224, 1074, 756. 1H NMR (DMSO-d6,
300 MHz) d ppm: 8.19 (d, J = 8.7, 1H, H-8), 7.93 (d, J = 2.0, 1H,