Cardiovascular activity of the chiral xanthone derivatives

Article abstract of DOI:10.1016/j.bmc.2015.09.005

A series of 6 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, the effect on blood pressor response and prophylactic antiarrhythmic activity in adrenaline induced model of arrhythmia (rats, iv). Two compounds revealed nanomolar affinity for α₁-adrenoceptor which was correlated with the strongest cardiovascular (antiarrhythmic and hypotensive) activity in animals' models. They were enantiomers of previously described (R,S)-4-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)propoxy)-9H-xanthen-9-one hydrochloride and revealed similar antiarrhythmic potential in adrenaline induced model of arrhythmia in rats after intravenous injection (ED₅₀ = 0.53 mg/kg and 0.81 mg/kg, respectively). These values were lower than values obtained for reference drug urapidil. These compounds were more active in this experiment than urapidil (ED₅₀ = 1.26 mg/kg). The compound 5 administered iv at doses of 0.62-2.5 mg/kg at the peak of arrhythmia prevented and/or reduced the number of premature ventricular beats in a statistically significant manner. The ED₅₀ value was 1.20 mg/kg. The S-enantiomer (6) given at the same doses did not show therapeutic antiarrhythmic activity in this model. These compounds significantly decreased the systolic and diastolic blood pressure throughout the whole observation period in anesthetized, normotensive rats. The studied enantiomers showed higher toxicity than urapidil, but imperceptibly higher that another cardiovascular drugs, that is, carvedilol or propranolol. They were also evaluated for mutagenic potential in the Ames (Salmonella) test. It was found that at the concentrations tested the compounds were non mutagenic when compared to solvent control. 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.2015.09.005

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Cardiovascular activity of the chiral xanthone derivatives
b
b
b,c
a
Natalia Szkaradek a, , Anna Rapacz , Karolina Pytka , Barbara Filipek , Dorota Zelaszczyk ,
⇑
´
_
d
e
a
´
Przemysław Szafranski , Karolina Słoczynska , Henryk Marona
a Department of Bioorganic Chemistry, Chair of Organic Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, 9 Medyczna St., 30-688 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 Chair of Organic Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Krakow, Poland
e Department of Pharmaceutical Biochemistry, 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:
A series of 6 derivatives of xanthone were synthesized and evaluated for cardiovascular activity. The fol-
lowing pharmacological experiments were conducted: the binding affinity for adrenoceptors, the influ-
ence on the normal electrocardiogram, the effect on the arterial blood pressure, the effect on blood
pressor response and prophylactic antiarrhythmic activity in adrenaline induced model of arrhythmia
Received 15 July 2015
Revised 2 September 2015
Accepted 3 September 2015
Available online xxxx
(rats, iv). Two compounds revealed nanomolar affinity for
a1-adrenoceptor which was correlated with
the strongest cardiovascular (antiarrhythmic and hypotensive) activity in animals’ models. They were
enantiomers of previously described (R,S)-4-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)pro-
poxy)-9H-xanthen-9-one hydrochloride and revealed similar antiarrhythmic potential in adrenaline
induced model of arrhythmia in rats after intravenous injection (ED50 = 0.53 mg/kg and 0.81 mg/kg,
respectively). These values were lower than values obtained for reference drug urapidil. These com-
pounds were more active in this experiment than urapidil (ED50 = 1.26 mg/kg). The compound 5 admin-
istered iv at doses of 0.62–2.5 mg/kg at the peak of arrhythmia prevented and/or reduced the number
of premature ventricular beats in a statistically significant manner. The ED50 value was 1.20 mg/kg. The
S-enantiomer (6) given at the same doses did not show therapeutic antiarrhythmic activity in this model.
These compounds significantly decreased the systolic and diastolic blood pressure throughout the
whole observation period in anesthetized, normotensive rats. The studied enantiomers showed higher
toxicity than urapidil, but imperceptibly higher that another cardiovascular drugs, that is, carvedilol or
propranolol. They were also evaluated for mutagenic potential in the Ames (Salmonella) test. It was found
that at the concentrations tested the compounds were non mutagenic when compared to solvent control.
Results were quite promising and suggested that in the group of xanthone derivatives new potential
antiarrhythmics and hypotensives might be found.
Keywords:
Antiarrhythmic
Hypotensive
Adrenoceptor
Xanthone
Synthesis
Ó 2015 Published by Elsevier Ltd.
1. Introduction
One quarter of all cardiovascular-related deaths is caused by
arrhythmias. What is important, their etiology is diversified and
Cardiovascular diseases include broad spectrum of disorders, in
especial: coronary heart disease, cerebrovascular disease, hyper-
tension, peripheral artery disease, rheumatic heart disease, con-
genital heart disease and heart failure are caused by disorders of
the heart and blood vessels. They remain number one cause of
death worldwide, particularly among women (32% of all deaths).
The Global Burden of Disease study estimated that in 2004,
12.9 million of almost 59 million total worldwide deaths were
caused by them.1
associated with many different conditions. The key role in the
pathogenesis of abovementioned cardiac dysfunctions, plays acti-
vation of sympathetic nervous system. In that light, it is not sur-
prising that adrenergic receptors antagonists are effective in the
treatment of this disease and are widely used. Another important
fact is proper interplay of ion channels, especially sodium, potas-
sium, and calcium thus disturbances in their function may result
in arrhythmia occurence.2 Moreover, antiarrhythmic drugs are
commonly affected with adverse effects such as bradykardia, tired-
ness, dizziness or thyroid dysfunction.3 Taking into account the
variety of arrhythmias’ types, their diverse etiology and numerous
side effects of antiarrhythmic drugs there is still a need to search
⇑
Corresponding author. Tel.: +48 12 620 05 576; fax: +48 12 620 54 05.
E-mail address: nszkarad@cm-uj.krakow.pl (N. Szkaradek).
http://dx.doi.org/10.1016/j.bmc.2015.09.005
0968-0896/Ó 2015 Published by Elsevier Ltd.
Please cite this article in press as: Szkaradek, N.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.005

2
N. Szkaradek et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
for new agents that can improve heart function with minimal side
effects.
methoxyphenylpiperazine or piperazine itself. We also introduced
chlor substituent into xanthone ring to enhance lipophilicity and
examine its effect on cardiovascular system.
The second, serious cardiovascular disorder is hypertension.
Hypertension is one of the most common significant diseases,
affecting approximately 1.4 billion people worldwide. The preva-
lence of essential hypertension steadily increases with age. As
the world population ages, the prevalence of hypertension is
expected to increase even further. Despite extensive translational
and clinical research, concerted patient education with regard to
vascular risk factors and lifestyle modifications, and a serious effort
on the part of health care professionals over the last few decades,
only one third of hypertensive patients have blood pressure (BP)
controlled to recommended levels of: 140/90 mm Hg.4
Most cases of hypertension are so-called essential or primary
hypertension, with unknown etiology. Only 5% of cases, named
secondary hypertension, have known etiology and are associated
with e.g. arteriosclerosis or hyperthyreosis. Nowadays, the number
of patients suffering from hypertension is still increasing, partially
due to current lifestyle: lack of physical activity, overweight or
obesity, using tobacco, too much sodium and too little potassium
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 possess 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 xanthone
derivatives confirmed hypotensive activity of all tested com-
pounds. It was noticed that, an oxypropanolamine side chain sub-
stituted at the C-3 position of the xanthone nucleus significantly
enhanced the hypotensive activity. The most potent seemed to
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 3-((oxiran-2-yl)meth-
oxy)-9H-xanthen-9-one were described in Ref. 13, 4-((oxiran-2-yl)
methoxy)-9H-xanthen-9-one in Ref. 14, 3-chloro-5-((oxiran-2-yl)
methoxy)-9H-xanthen-9-one was published in Ref. 15, whereas
characteristic of (R)-4-((oxiran-2-yl)methoxy)-9H-xanthen-9-one
and (S)-4-((oxiran-2-yl)methoxy)-9H-xanthen-9-one is mentioned
below. The crude products were recrystallized from n-hexane/
toluene (1:4). These intermediates are characterized in Section 7.
Compounds 1–6 were obtained by amination of respective par-
ent compounds with appropriate amines in n-propanol, as
described previously.13,16 The last step of the synthesis was amina-
tion of the respective parent compound with appropriate amines in
n-propanol as described previously.17 After aminolysis, n-propanol
was distilled off. Resulted amines were dissolved in diluted HCl,
cleaned with charcoal and precipitated using NaOH solution. 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).
3. Pharmacology
3.1. Animals and experimental conditions
The studies were carried out on normotensive male Wistar rats
weighing 180–250 g and male Albino Swiss (CD-1) mice weighing
18–24 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 con-
stant temperature of 20 4 °C, under 12/12 h light/dark cycle (light
on from 7 a.m. to 7 p.m.). They had free access to food (standard
laboratory pellets) and water before experiments. The control
and study groups consisted of six to eight animals each. The exper-
iments were performed between 8 a.m. and 3 p.m. The animals
were killed by cervical dislocation immediately after the assay.
All the procedures were conducted according to the Animal Care
and Use Committee guidelines, and approved by the Local Ethics
Committee of the Jagiellonian University in Cracow (resolution
No. 118/2012).
be
3-(3N-i-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 mecha-
nism of action was calcium channel dependant and required the
blockade of b-adrenergic receptors. Xanthonolol possess typical,
b-blocker moiety (3-amine-2-hydroxypropan-1-yloxy) character-
istic for cardiovascular drugs such as propranolol and carvedilol.7
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 Chem-
istry (previously Department of Technology and Biotechnology of
Drugs) has been conducting searching for new hypotensive and/
or antiarrhythmic structures, especially in the group of xanthone
derivatives.9 The strongest hypotensive effects were observed for
compounds containing piperazine moiety. These compounds
showed both in vitro (via binding affinity evaluations) and
in vivo activity.10 Other researchers also confirmed pharma-
cophoric effect of piperazine moiety.11
Basing on literature survey, herein we report in vitro and in vivo
cardiovascular activity of some new derivatives. Two of the men-
tioned structures were enantiomers of previously described active
one.12 Next four compounds were analogs of our reported
aminoalkanolic structures, possessed typical b-blocker moiety
(3-amine-2-hydroxypropan-1-yloxy), others were devoided of hydro-
xyl group analogously to structures described by Lin et al.8 and were
evaluated to estimate role of hydroxyl group. Piperazine like amines
were also diversified. Most of them contained methoxyphenylpipe
3.2. Drugs
The tested compounds 1–6 were synthesized at the Department
of Bioorganic Chemistry UJ CM. The following drugs were used:
adrenaline, noradrenaline (Polfa, Poland), methoxamine (Sigma–
Aldrich, Germany), sodium heparin (Polfa, Poland), thiopental
sodium (Biochemie GmbH, Austria), [3H]CGP-12177 (NEN-Du Pont,
Poland), [3H]prazosin (NEN, Life Science Products, USA), [3H]cloni-
dine (NEN, Life Science Products, USA). Other chemicals used were
obtained from POCH (Polish Chemical Reagents, Poland). The
tested compounds were dissolved in saline and administered intra-
venously at a constant volume of 1 ml/kg (rats) or 10 ml/kg (mice).
Urapidil (Sigma–Aldrich, Germany) was used as a reference drug.
3.3. Radioligand binding assay
razine—structural element of urapidil (typical
a1-adrenoceptor
The experiments were conducted on the rat cerebral cortex.
antagonist), others contain benzylpiperazine, phenoxyethylpiper-
azine, cinnamylpiperazine to ascertain pharmacophoric effect of
[3H]prazosin (19.5 Ci/mmol, 1-adrenergic receptor), [3H]clonidine
a
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N. Szkaradek et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
Scheme 1. Synthesis of the tested compounds 1–4.
(70.5 Ci/mmol,
a
2-adrenergic receptor) and [3H]CGP-12177 (48
measured only for compounds 5, 6 that showed prophylactic
Ci/mmol, b1-adrenergic receptor) were used. The membrane
preparation and the assay procedure were carried out accordingly
as previously published.18 Radioactivity was measured in a WAL-
LAC 1409 DSA liquid scintillation counter (Perkin Elmer, USA). All
assays were done in duplicates. Radioligand binding data were
analyzed using iterative curve fitting routines with GraphPAD/
Prism, Version 4.0 (GraphPAD ware, San Diego, CA, USA). Ki values
were calculated using the Cheng and Prusoff equation.19
antiarrhythmic properties.
3.6. Influence on blood pressure in rats
Male Wistar normotensive rats were anesthetized with
thiopental (75 mg/kg) by ip injection. The right carotid artery
was cannulated with polyethylene tubing filled with heparin in
saline to facilitate pressure measurements using a Datamax appa-
ratus (Columbus Instruments, USA). The studied compounds were
administrated iv after a 15 min stabilization period at the dose
0.3 mg/kg, at a volume equivalent to the 1 ml/kg.
3.4. The effect on the normal electrocardiogram
Electrocardiographic investigations were carried out using
ASPEL ASCARD B5 apparatus, (Aspel SA, Poland) standard 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 com-
3.7. Influence on blood pressor response in rats
In separate series of experiments on anesthetized normotensive
rats, the effect of studied compounds, administered intravenously
at the dose of 1 mg/kg on a pressor response (increase blood pres-
pounds. The QTc was calculated according to the formula of Bazzet:
p
QTc = QT/ RR.20
sure) to adrenaline (2
amine (150
lg/kg), noradrenaline (2 lg/kg) and methox-
l
g/kg) was investigated according to the method
3.5. Prophylactic and therapeutic antiarrhythmic activity in
adrenaline-induced arrhythmia
described by Rapacz et al.23 Pressor response to adrenaline, nora-
drenaline and methoxamine injected iv was obtained before and
5 min after the tested compounds.
Antiarrhythmic activity was determined according to the
method of Szekeres and Papp.21 The arrhythmia was evoked in rats
anesthetized with thiopental (75 mg/kg, ip) by iv injection of adre-
3.8. Acute toxicity
naline into the caudal vein of rat (20 lg/kg, in a volume of 1 ml/kg).
The tested compounds were dissolved in 0.9% saline and
injected into a caudal vein of mouse (10 ml/kg). Each dose was
given to six animals. LD50 values were calculated according to
the method of Litchfield and Wilcoxon after a 24 h observation
period.22
The tested compounds were administered intravenously 15 min
before arrhythmogen—prophylactic activity or at the peak of
arrhythmia, immediately after administration of adrenaline—ther-
apeutic activity. The criterion of antiarrhythmic activity was the
lack of premature beats and inhibition of cardiac arrhythmia in
comparison with the control group. The ED50 value was calculated
according to the method of Litchfield and Wilcoxon.22 Prophylactic
antiarrhythmic activity was evaluated for all of the tested
compounds meanwhile therapeutic antiarrhythmic activity was
3.9. Bacterial tester strains
Salmonella typhimurium tester strains TA98, TA100, TA1535 and
TA1538 used for the assay were obtained from Dr. T. Nohmi,
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N. Szkaradek et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Division of Genetics and Mutagenesis, National Institute of Hygienic
Sciences, Tokyo, Japan.
the tested compounds were administrated immediately after an
arrhythmogenic substance.
3.10. Mutagenicity assay
4.4. Prophylactic anti-arrhythmic activity
The Ames mutagenicity assay was performed by a preincuba-
tion procedure with TA98, TA100, TA1535 and TA1538 Salmonella
typhimurium strains as described previously.24,25 Briefly, Salmonella
cultures were inoculated 12 h prior to performing the assay. Next,
Both enantiomers (5, 6) revealed high antiarrhythmic activity in
this model. This compounds administered 15 min before adrena-
line prevented and/or reduced in a statistically significant manner
the number of premature ventricular beats. The obtained ED50 val-
ues (a dose producing 50% inhibition of premature ventricular
beats) for tested compounds 5 and 6 were 0.53 and 0.81 mg/kg,
respectively (Table 3). These compounds were more active in this
experiment than urapidil (ED50 = 1.26 mg/kg). Other compounds
(1–4) did not prevent tested animals from heart-rhythm distur-
bances and dead in the dose of 10 mg/kg.
100
at different concentrations (0.4, 4.0, 40.0, 80.0 and 160.0
in DMSO), and 500 l of buffer solution were added to 2 ml of
ll of the overnight bacterial culture, 50
l
l of test compounds
lg/plate
l
the top agar containing trace amount of histidine. Subsequently,
the mixture was mixed and poured onto the surface on a glucose
minimal agar plates. Revertant S. typhimurium colonies were
scored on plates after incubation at 37 °C for 48 h. Positive control
(sodium azide, SA in TA100 and TA1535 strains and 4-nitro-o-
phenylenediamine, 4-NOPD in TA98 and TA1538 strains) and
negative control (DMSO) were performed alongside. Three inde-
pendent repetitions were performed for each substance. Addition-
ally, the mutagenic index (MI), that is, the ratio between the
number of revertants per plate with the test compound and the
number of revertants observed in the negative control was calcu-
lated. A sample was considered mutagenic when a dose–response
relationship was stated and a two-fold increase in the number
of revertants (MI P 2) was observed with at least one
concentration.26
4.5. Therapeutic anti-arrhythmic activity
Compound 5 administered iv at doses of 0.62–2.5 mg/kg at the
peak of arrhythmia prevented and/or reduced the number of pre-
mature ventricular beats in a statistically significant manner. The
ED50 value was 1.20 mg/kg. The S-enantiomer (6) given at the same
doses did not show therapeutic antiarrhythmic activity in this
model (Fig. 1).
4.6. Influence on blood pressure in rats
Compound 1 was tested at the dose of 2.5 mg/kg (in which it did
not cause cardiac arrhythmias). It reduced systolic and diastolic
blood pressure from the 20th minute of observation (by 15–12%).
Compound 2 was tested at the dose of 5 mg/kg and reduced the
systolic blood pressure by 14–6% and diastolic blood pressure by
21–11%. Compound 3 at the dose of 10 mg/kg significantly influ-
enced blood pressure only in 60 min of the test, whereas com-
pound 4 at the same dose did not have any significant influence
on blood pressure (data not shown). The results for these com-
pounds are presented in Table 4.
The hypotensive activity of compounds 5 and 6 was determined
after their iv administration in normotensive, anesthetized rats at
the dose of 0.3 mg/kg, in which racemic mixture revealed hypoten-
sive activity. At this dose both enantiomers revealed hypotensive
activity (Figs. 2 and 3). Compound 5 significantly decreased the
systolic by 8–14% and diastolic by 10–16% blood pressure through-
out the whole observation period in anesthetized, normotensive
rats. Compound 6 also significantly decreased the systolic by 8–
13% and diastolic by 10–17% blood pressure. In this experiment
activity of the tested enantiomers was similar.
4. Pharmacological results
4.1. Radioligand binding assay
The pharmacological profile of the new compounds was evalu-
ated by radioligand binding assays. Compounds 5 and 6 displaced
[3H]prazosin from cortical binding sites in low concentration range
(Ki values were 21.0 and 30.6 nM, respectively), the other
compounds in the range of >1000
not inhibit [3H]clonidine and [3H]CGP12177 binding to cortical
2–b1-adrenoceptors. The results have been presented in Table 1.
lM. Studied compounds did
a
4.2. The influence on the normal electrocardiogram
Effect on ECG intervals and the heart rate was determined in a
dose of 10.0 mg/kg (compounds 1–4) or 2.5 mg/kg (compounds 5,
6). Table 2 shows effects of the tested compounds on the heart rate
and ECG parameters. Among all studied compounds, compounds 1,
2, 4 significantly decreased the number of cardiac beats per min-
ute—1 by 27%, 2 by 32%, 4 by 14% and prolonged the PQ interval.
Compounds 2, 4 prolonged also QT interval and did not affect dura-
tion of QRS complex. Whereas, compound 1 elongated duration of
QRS complex and did not affect QT interval. Compounds 3, 5, 6 had
not any significant influence on heart rate, PR and QTc intervals, as
well as QRS complex.
4.7. Influence on blood pressor response in rats
Intravenous adrenaline (2
lg/kg), noradrenaline (2 lg/kg) and
methoxamine (150 g/kg) were given to rats to induce pressor
l
response (increase blood pressure). In the control group the
increases of blood pressure for adrenaline were from 68.5 3.3 to
85.5 8.2, for noradrenaline from 77.7 1.9 to 85.5 3.9 and for
methoxamine were from 68.7 7.5 to 101.2 9.7. Compound 5
administered iv at the dose of 1 mg/kg inhibited the blood pressure
increase elicited by adrenaline by 64.7% and methoxamine by 94.7,
but did not influence the effect of noradrenaline (F(5,25) = 42.540,
p <0.0001), (Fig. 4). Compound 6, administered iv at the same dose
also inhibited the blood pressure increase elicited by adrenaline by
72.9% and methoxamine by 86.7%, but did not influence the effect
of noradrenaline (F(5,25) = 33.180, p <0.0001), (Fig. 5). In our
4.3. Anti-arrhythmic activity in adrenaline-induced arrhythmia
In anesthetized rats, iv injections of adrenaline (20 lg/kg)
caused atrioventricular disturbances, ventricular and supraventric-
ular extrasystoles (in 100% of the animals, with a mean of 21
extrasystoles during the 15 min of observation), which led to death
of approximately 70% of the animals. To measure prophylactic
antiarrhythmic activity, the tested compounds were administrated
iv before adrenaline. To assess therapeutic antiarrhythmic activity,
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N. Szkaradek et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Table 1
Affinity for
a1-, a2- and b1-adrenoceptors in the rat cerebral cortex
Compound
Structure
a
1-Adrenoceptors Ki
a
2-Adrenoceptors Ki
b1-Adrenoceptors Ki
(nM) SEM
(nM) SEM
(nM) SEM
O
O
1
2900
>10,000
1100
Cl
OH
O
NH
CH3
CH3
O
O
2
3100
>10,000
2100
Cl
OH
CH3
NH CH3
O
CH3
O
3
4
39,500
2200
>10,000
>10,000
>10,000
1600
O
O
N
CH3
CH2OH
OH
O
O
O
OH
CH3
O
N
CH2OH
(R)-Enantiomer
5
9.2
5310
2900
4260
24,000
17,000
7600
O
O
OH
OH
OH
N
H
O
N
OCH3
(S)-Enantiomer
6
8.8
O
O
N
H
O
N
OCH3
(R,S)
18.0
O
N
O
N
N
OCH3
OCH3
CH3
N
N
O
NH
Urapidil
127.9
>10,000
—
N
H3C
O
Ki SEM values were obtained from 2 to 3 experiments in duplicates.
previous paper we reported that urapidil (reference compound)
also caused the same effect.27
4.9. Mutagenicity screening
Compounds 5 and 6 as well as their racemate were evaluated
for mutagenic potential in the Ames (Salmonella) test. It was found
that at the concentrations tested the compounds were non muta-
genic when compared to solvent control (Table 6).
4.8. Acute toxicity
The acute toxicity of tested compounds 5 and 6 was examined
in mice after intravenous administration according to Litchfield
and Wilcoxon.22 The LD50 values are given in Table 5. The studied
compounds showed higher toxicity than urapidil, but comparable
with another cardiovascular drugs i.e. carvedilol or propranolol.28
5. Discussion
The aim of this study was to evaluate cardiovascular activity of
new compounds—derivatives of xanthone. This study included in
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6
N. Szkaradek et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 2
The effect of an intravenous injection of the tested compounds on the heart rate and ECG intervals in anesthetized male Wistar rats
Compound
Dose (mg/kg)
Parameters
Time of observation (min)
10
0
5
15
1
10
PR (ms)
QRS (ms)
QTc (ms)
37.4 1.7
19.9 1.6
57.6 2.6
44.3 4.8
21.4 2.2
59.1 3.6
41.2 2.7
20.6 1.4
58.4 3.6
41.2 1.6
21.6 2.0
60.6 3.3
Beats per min
309.6 29.7
230.7 18.0
232.7 14.6
226.4 14.8
2
3
4
5
6
10
10
10
2.5
2.5
PR (ms)
QRS (ms)
QTc (ms)
43.8 3.1
18.3 0.4
53.0 1.4
51.6 1.8
19.4 0.4
52.1 2.0
251.8 6.1
46.1 2.1
19.4 0.3
55.5 2.0
239.1 4.8
46.5 2.7
19.5 0.7
57.1 1.5
233.2 5.6
Beats per min
342.5 12.6
PR (ms)
QRS (ms)
QTc (ms)
38.4 1.5
18.1 0.8
62.5 0.5
39.7 0.2
17.9 0.0
63.0 1.0
39.2 0.5
18.5 0.5
62.9 0.5
40.3 0.6
18.5 0.5
63.0 2.4
Beats per min
348.0 20.4
336.5 12.9
336.2 12.2
329.2 18.7
PR (ms)
QRS (ms)
QTc (ms)
28.2 2.5
22.5 2.3
46.3 1.8
34.9 1.9
20.5 0.8
51.4 1.3
304.3 8.6
34.4 2.3
20.2 0.6
51.7 1.8
35.6 2.3
21.2 1.6
51.9 1.0
300.3 9.7
Beats per min
351.5 11.3
302.7 10.0
PR (ms)
QRS (ms)
QTc (ms)
38.7 1.3
22.3 1.7
120.7 11.8
338.2 14.9
41.1 0.9
24.7 0.7
114.0 8.3
340.9 11.8
41.1 1.1
24.0 1.0
110.7 5.8
342.9 10.2
39.9 0.3
22.0 1.0
125.8 12.3
321.7 10.3
Beats per min
PR (ms)
QRS (ms)
QTc (ms)
49.7 4.2
21.7 1.1
120.1 8.4
363.1 11.9
47.3 5.6
22.2 2.9
118.2 10.1
366.0 11.1
45.5 5.9
22.3 3.3
120.1 9.7
366.5 11.5
45.1 5.8
22.8 3.4
120.7 9.2
351.1 12.2
Beats per min
The data are means of 5–6 experiments SEM.
Table 3
possess i-propylamine or t-butylamine moiety in their structure
(e.g., propranolol, alprenolol, metoprolol) encouraged us to synthe-
size their xanthonic analogs (compounds 1, 2). In addition there
were synthesized structures with tertiary amine moiety (com-
pounds 3, 4) being N-methyl analogs of previously described
aminoalcanolic derivatives of xanthone.10,16 The last two com-
pounds were enantiomers of formerly reported, active racemate.12
In the first step the affinity for adrenoceptors was determined
The prophylactic anti-arrhythmic activity in adrenaline induced arrhythmia in
anesthetized rats
Compound
ED50 iv (mg/kg)
5-R
6-S
(R,S)
Urapidil
0.53 (0.27–1.07)
0.81 (0.30–2.20)
0.88 (0.38–2.00)
1.26 (0.97–1.64)
using a radioligand binding assay. The highest affinity for a1
adrenoceptors revealed compounds: and 6, containing 2-
methoxyphenylpiperazine moiety in their structure.
-
Each value was obtained from three experimental groups. Each group consisted of
six animals. The ED50 values and their confidence limits were calculated according
to the log-probit method of Litchfield and Wilcoxon.22
5
The antiarrhythmic effects of novel compounds were examined
in rats using the model of adrenaline-induced arrhythmia. The
most active were compounds 5 and 6, administered iv 15 min
before arrhythmogen, which prevented or attenuated the symp-
toms of adrenaline-induced arrhythmia. Data reported in Table 3
suggest that these compounds possess preferable effective dose
than urapidil (a1-adrenoceptor antagonist and an 5-HT1A receptor
agonist) containing the same pharmacophore structure (2-
methoxyphenylpiperazine).
Hypotensive activity of investigated compounds was deter-
mined after iv administration to normotensive anesthetized rats.
The most active were compounds 5 and 6, which showed a signif-
icant hypotensive effect throughout the whole observation period.
The hypotensive effect was probably the result of strong
a1-adrenoceptors blockade.
Results obtained via adrenoceptor binding assay correlated
with in vivo antiarrhythmic and hypotensive evaluation. Com-
pounds 5 and 6, which were the most potent substances in
hypotensive tests, displaced [3H]prazosine from cortical binding
sites in low concentration range (Ki = 9.2 and 8.8 nM), whereas
Figure 1. Anti-arrhythmic activity of the compounds 5, 6.
other compounds inhibited [3H]prazosine binding with
lM-range.
It is well known that
a1-adrenoceptors play key role in the stimu-
particular: design, synthesis, physico-chemical analysis, antiar-
rhythmic and hypotensive activity estimation and measuring of
binding affinity for adrenoceptors. Fact that many of b-blockers
lation of smooth muscle contraction. They stimulate the contrac-
tion of veins and arteries and in consequence increase the
resistance in circulatory blood flow. In that way, a1-adrenoceptors
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N. Szkaradek et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
Table 4
Hypotensive activity of compounds 1–6 in anesthetized normotensive rats after iv administration
Compound
Dose mg/kg
Blood pressure (mmHg)
Time of observation (min)
20
0
5
10
30
60
1
2
3
4
5
6
2.5
5
Systolic
Diastolic
124.20 5.50
109.00 6.35
120.20 5.56
102.20 6.49
125.80 7.58⁄⁄
104.40 5.00⁄⁄
116.40 6.23
97.60 7.09
128.80 6.66⁄
108.20 3.64⁄
108.80 9.44⁄
92.80 9.18⁄
109.80 8.30⁄
94.60 8.27⁄
108.60 5.63⁄
93.80 6.64⁄⁄
Systolic
Diastolic
140.00 3.65
123.60 2.44
132.00 5.67
109.80 3.72
128.00 2.83
104.80 3.64⁄
120.00 6.83⁄⁄
98.00 7.54⁄⁄
122.25 5.07⁄
106.50 7.77⁄
10
10
0.3
0.3
Systolic
Diastolic
144.50 4.63
124.75 1.80
139.25 6.42
120.00 5.35
139.25 6.21
121.25 5.62
140.25 4.97
124.00 4.51
138.25 3.79
122.50 4.05
Systolic
Diastolic
141.20 4.81
121.10 3.51
133.60 5.43
112.20 4.78
118.2 3.34⁄⁄
93.60 5.53⁄⁄
132.20 6.34
112.40 5.60
121.2 1.88⁄
100.20 5.53
129.00 6.36
111.00 6.33
120.4 2.77⁄
98.80 6.32
115.0 2.95⁄
87.75 1.89⁄⁄
128.40 7.22
109.60 7.28
120.8 2.24⁄
98.00 6.69
114.4 2.99⁄⁄
88.50 1.44⁄⁄
124.60 8.79
106.60 9.16
115.0 4.09⁄⁄⁄
85.60 8.41⁄⁄⁄
112.2 4.02⁄⁄⁄
83.25 5.92⁄⁄
Systolic
Diastolic
130.0 2.00
105.8 6.26
Systolic
Diastolic
127.8 2.69
96.40 2.62
116.2 4.21
116.2 1.28
90.20 1.80⁄⁄
90.00 1.05⁄⁄
The values are expressed as mean of 5–6 experiments SEM. Statistical analysis: repeated measures ANOVA test, post hoc Dunnett test: *p <0.05, **p <0.01, ***p <0.001.
Figure 4. Influence of compound 5 on blood pressor response in rats.
proved to be a highly effective antihypertensive drugs and remain
important options in the treatment regimen available for
hypertension.29
Figure 2. Influence of compounds 5, 6 on systolic blood pressure in rats.
The above mentioned compounds are also potential antiar-
rhythmic 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
human heart are different subtypes of
a1-adrenoceptors, especially
a1A and 1B. In previously published data
a
a1-blocking drugs, such
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
these two
functionally distinct. To date, the data imply that
tors 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
a
1-adrenoceptors subtypes expressed in the heart are
a1B-adrenocep-
a
a
ischemia/reperfusion conditions but involve different mechanisms.
With respect to structure–activity relationship, it can be stated
that the most active compounds contained 2-methoxy
phenylpiperazine moiety (5 and 6). This substituent is well known
pharmacophoric structure present in some cardiovascular drugs
such as naftopidil, or urapidil. Present results show that enan-
tiomers 5 and 6 revealed similar effect to their already reported
racemate.12 In this case chirality was not the key factor determin-
ing cardiovascular activity occurence. Compounds 1, 2 possessing
i-propylamine or t-butylamine moiety were less potent in in vivo
tests (than their hydroxyl analogs described previously15) and
Figure 3. Influence of compounds 5, 6 on diastolic blood pressure in rats.
are able to regulate blood pressure. As
a1-receptors are important
for maintenance of peripheral vascular resistance,
a
1-receptor
antagonists may be used to relax blood vessels in hypertensive
patients and to reduce symptoms of benign prostate hyperplasia.
revealed
lM-range affinity to all evaluated adrenoceptors. It was
Classic
a1-antagonists, like prazosin, doxazosin and terazosin
unexpected taking into account structure of some well known
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8
N. Szkaradek et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
chosen adrenoceptors. Then, in vivo antiarrhythmic and hypoten-
sive activity of the structures was tested. Two compounds revealed
nanomolar affinity for 1-adrenoceptor as well as the highest car-
a
diovascular efficacy in animals’ models. The most promising com-
pounds were (R)-4-(2-hydroxy-3-(4-(2-methoxyphenyl)piper
zazine-1-yl)propoxy)-9H-xanthen-9-one hydrochloride (5) and
(S)-4-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazine-1-yl)pro-
poxy)-9H-xanthen-9-one hydrochloride (6). They proved to act as
anti-arrhythmics and showed ED50 values in adrenaline induced
arrhythmia (rats, iv) of 0.53 mg/kg and 0.81 mg/kg, respectively.
These values were lower than values obtained for reference drug
urapidil. The pharmacological results and binding studies sug-
gested that the antiarrhythmic and hypotensive activity of these
compounds were related to their adrenolytic properties. 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 impact on NO
pathway).
Figure 5. Influence of compound 6 on blood pressor response in rats.
Table 5
Acute toxicity in mice
Compound
LD50 iv (mg/kg)
5-R
6-S
26.6 (18.4–38.4)
21.6 (12.8–36.5)
13.8 (9.3–20.5)
203.0
(R,S)
Urapidil
The results of the tested substances are quite encouraging,
therefore further modifications of their structures might lead to
discovering of new potential drugs. Planned pathways of modifica-
tions will include three strategic points: xanthone ring, alcanolic
linker and amine moiety.
Each value was obtained from three experimental groups. Each group consisted of
six animals. The LD50 values and their confidence limits were calculated according
to the log-probit method of Litchfield and Wilcoxon.22
drugs such as propranolol, pindolol, nadolol and formerly
described xanthones with hypotensive activity.13 According to
the results obtained for compounds 3, 4 bearing N-methyl analogs
of aminoalkanols, one can state that introduction of the methyl
7. Experimental protocols
Reagents: (R,S)-epichlorohydrin, (R)-epichlorohydrin, (S)-
epichlorohydrin, N-methylaminoethanol were purchased from
Sigma–Aldrich Chemie (Steinheim, Germany), whereas reagents:
2-chlorobenzoic acid, 2,4-dichlorobenzoic acid, 2-methoxyphenol,
2-methoxyphenylpiperazine, 3-methoxyphenol, t-butylamine,
i-propylamine were provided by Lancaster Synthesis (Frankfurt
am Main, Germany). Solvents were commercially available materials
of reagent grade.
substituent in the amine moiety diminish binding affinity for a1
-
adrenoceptors (compare former publications treating aminoal-
canols10,16). This fact suggests that enhancing of the lipophilicity
was not desirable.
6. Conclusion
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
In conclusion, we have synthesized 6 new derivatives of xan-
thone. All compounds were in vitro screened for their affinity for
Table 6
Mutagenic evaluation of compound A54 and its enantiomers in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1538
Test item
Concentration (
lg per plate)
Number of revertants
Base-pair substitution types
TA1535
Frameshift types
TA1538
Mean SD and MI
TA100
TA98
Mean SD and MI
87 10
Mean SD and MI
Mean SD and MI
22
Negative control
7
2
3
8
3
5-R
0.4
4.0
40.0
80.0
160.0
73 3 (0.8)
74 3 (0.9)
83 2 (1.0)
81 8 (0.9)
73 3 (0.8)
9
6
6
6
4
4 (1.3)
2 (0.9)
2 (0.9)
3 (0.9)
2 (0.6)
23 4 (1.1)
19 4 (0.9)
21 2 (1.0)
20 2 (0.9)
18 3 (0.8)
9
3 (1.1)
13 5 (1.6)
7
9
2 (0.9)
5 (1.1)
11 2 (1.4)
6-S
0.4
4.0
40.0
80.0
160.0
74 8 (0.9)
70 5 (0.8)
72 4 (0.8)
87 7 (1.0)
88 5 (1.0)
5
2 (0.7)
21 4 (1.0)
19 2 (0.9)
25 3 (1.1)
18 1 (0.8)
27 6 (1.2)
5
5
7
8
6
1 (0.6)
2 (0.6)
3 (0.9)
3 (1.0)
2 (0.8)
10 5 (1.4)
12 2 (1.7)
8
4
5 (1.1)
1 (0.6)
(R,S)
0.4
4.0
40.0
80.0
160.0
87 3 (1.0)
75 2 (0.9)
78 5 (0.9)
86 6 (1.0)
74 4 (0.9)
10 5 (1.4)
24 3 (1.1)
20 4 (0.9)
26 3 (1.2)
17 1 (0.7)
25 7 (1.1)
6
6
4
5
6
2 (0.8)
3 (0.8)
2 (0.5)
1 (0.6)
2 (0.8)
5
9
6
5
2 (0.7)
3 (1.3)
2 (0.9)
2 (0.7)
Positive control
998 72
452 33
436 18
468 29
Mean values of three independent experiments standard deviation as well as mutagenic index (MI) are given. In S. typhimurium TA100 and TA1535 the positive control was
sodium azide (5.0 g/plate), whereas in TA98 and TA1538 assay systems 4-nitro-o-phenylenediamine (2.5 g/plate) was used; negative control—DMSO. Values in brackets
(MI) P2 indicate mutagenicity.
l
l
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N. Szkaradek et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
Scheme 2. Synthesis of the tested compounds 5, 6.
bromide pellets using a Jasco FT/IR 410 spectrometer (Jasco Inc.,
Easton, MD, USA). The 1H NMR were recorded on a Bruker AMX
spectrometer (Bruker, 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), br s (broad singlet), d (doublet), dd
(doublet of doublets), ddd (double doublet of doublets), dddd (dou-
ble doublet of double doublets), t (triplet), qu (quintet), m (multi-
plet). Elemental analyses were performed on an Elementar Vario EL
III (Elementar Analysansysteme, Hanau, Germany). For mass spec-
trometry 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 Waters TQD mass spectrometer (electrospray
ionization mode ESI-tandem quadrupole). All the analyses were
concomitant 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 properties of 3-hydroxy-9H-xanthen-9-one,32
4-hydroxy-9H-xanthen-9-one and 3-chloro-5-hydroxy-9H-xan-
then-9-one were described in Ref. 33,14,15. The oxirane deriva-
tives of xanthone were obtained in the reaction of appropriate
hydroxyxanthone with epichlorohydrin ((R,S) or (R) or (S)) accord-
ing to procedures described earlier.16 Epichlorohydrin was used in
double excess. Reacting mixture was stirred during night, at the
room temperature. After that, precipitate was filtered and unreacted
substrates were washed with 10% NaOH solution. Physicochemical
properties of 3-((oxiran-2-yl)methoxy)-9H-xanthen-9-one were
described in Ref. 13, 4-((oxiran-2-yl)methoxy)-9H-xanthen-9-one
in Ref. 14, 3-chloro-5-((oxiran-2-yl)methoxy)-9H-xanthen-9-one
was published in Ref. 15, whereas characteristic of (R)-4-((oxiran-
2-yl)methoxy)-9H-xanthen-9-one and (S)-4-((oxiran-2-yl)meth-
oxy)-9H-xanthen-9-one is mentioned below. It is worth noticing
that due to change in substituents’ priority using of (R)-epichlorohy-
drin leads to (S)-4-((oxiran-2-yl)methoxy)-9H-xanthen-9-one and
using of (S)-epichlorohydrin leads to (R)-4-((oxiran-2-yl)meth-
oxy)-9H-xanthen-9-one as shown in Scheme 2. The last step of the
synthesis was amination of the respective parent compound with
appropriate amines in n-propanol as described previously.17 After
aminolysis, n-propanol was distilled off. Resulted amines were dis-
solved in diluted HCl, cleaned with charcoal and precipitated using
NaOHsolution. All resultedbaseswereconverted intohydrochloride
salts using an excess of ethanol saturated with HCl. The crude prod-
ucts were recrystallized from acetone/ethanol (1:3). Physicochemi-
cal properties of compounds 1–6 are summarized below.
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; 8 scans were summed
up to produce the final spectrum. The purity of obtained com-
pounds was confirmed by the thin-layer chromatography (TLC),
carried out on precoated plates (silica gel, 60 F-254, Merck, Darm-
stadt, Germany) using the solvent systems mentioned below. The
obtained corresponding spots were visualized under UV light.
Specific rotation was measured on Jasco P-2000 polarimeter in
20 °C (1% w/v solutions in methanol or 1% w/v solution in chloro-
form, sodium light 589 nm).
The synthetic route of the tested compounds and starting mate-
rials is outlined in Schemes 1 and 2. Synthesis of the compounds
mentioned above was multistage. In the first stage there was syn-
thesized xanthone skeleton. This synthesis included Ullmann’s
condensation of appropriate chlorobenzoic acid and phenol deriva-
tive.30,31 The reaction was performed for few hours, in oil, in
the 195–200 °C, in the presence of Cu/Cu2O catalyst. After conden-
sation, intermediate product underwent cyclization with
7.1. (R)-(ꢁ)-4-((Oxiran-2-yl)methoxy)-9H-xanthen-9-one
This compound was obtained as white solid (yield 70%), mp
177–178 °C. Anal. Calcd for C16H12O4: C, 71.63; H, 4.51. Found: C,
71.88; H, 4.66. 1H NMR (CDCl3, 500.13 MHz) d (ppm): 8.21 (ddd,
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10
N. Szkaradek et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
J = 8.0, J = 1.8, J = 0.5, 1H, H-8), 7.89 (ddd, J = 8.5, J = 7.1, J = 1.8, 1H,
H-6), 7.77 (dd, J = 8.0, J = 1.5, 1 H, H-1), 7.74 (ddd, J = 8.5, J = 1.1,
J = 0.5, 1H, H-5), 7.56 (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), 7.39 (dd, J = 8.0, J = 8.0, 1H, H-2),
4.60 (dd, J = 11.6, J = 4.9, 1H, Ar-O-CHH-R), 4.09 (dd, J = 11.6,
J = 6.6, 1H, Ar-O-CHH-R), 3.49 (dddd, J = 6.6, J = 4.9, J = 4.3, J = 2.7,
1H, C-H), 2.92 (dd, J = 5.1, J = 4.3, 1H, CHH), 2.82 (dd, J = 5.1,
J = 2.7, 1H, CHH). LC–MS: Calcd for [M+H]+: C16H13O4 m/z:
3.69. Found: C, 59.99; H, 5.81; N, 3.78. 1H NMR (CDCl3, 300 MHz)
d (ppm): 10.24–9.99 (m, 1H, NH+), 8.20 (ddd, J = 7.9, J = 1.5,
J = 0.5, 1H, H-8), 7.90 (ddd, J = 8.5, J = 7.1, J = 1.5, 1H, H-6), 7.75
(dd, J = 8.0, J = 1.4, 1H, H-1), 7.74 (ddd, J = 8.5, J = 1.0, J = 0.5, 1H,
H-5), 7.60 (dd, J = 8.0, J = 1.4, 1H, H-3), 7.50 (ddd, J = 7.9, J = 7.1,
J = 1.0, 1H, H-7), 7.38 (dd, J = 8.0, J = 8.0, 1H, H-2), 6.13 (br s, 1H,
ACHAOH), 5.44 (br s, 1H, ACH2AOH), 4.55 (br s, 1H, ACH@),
4.27 (dd, J = 10.1, J = 5.1, 1H, ArAOACHH), 4.22 (dd, J = 10.1,
J = 5.4, 1H, ArAOACHH), 3.90–3.85 (m, 2H, ACH2AOH), 3.68–3.24
(m, 4H, 2⁄@NACH2A), 2.90 (s, 3H, @NACH3). Rf = 0.27 (methanol/
ethyl acetate (1:1)).
269.26, found 269.13.
(toluene/acetone (5:3)).
[a
]
D
20 = ꢁ8.14 (chloroform). Rf = 0.72
7.2. (S)-(+)-4-((Oxiran-2-yl)methoxy)-9H-xanthen-9-one
7.7. (R)-(+)-4-(2-Hydroxy-3-(4-(2-methoxyphenyl)piperazine-1-
yl)propoxy)-9H-xanthen-9-one hydrochloride (compound 5)
This compound was obtained as white solid (yield 65%), mp
178–179 °C. [a]
20 = 9.53 (chloroform). Rf = 0.72 (toluene/acetone
D
(5:3)).
This compound was obtained as a white solid (yield 60%), mp
234–236 °C. Anal. Calcd for C27H29O5N2Cl: C, 65.25; H, 5.88; N,
5.64. Found: C, 64.82; H, 6.04; N, 5.55. 1H NMR (DMSO-d6,
500.13 MHz) d(ppm): 10.43 (br s, 1H, ANH+), 8.22 (ddd, J = 8.0,
J = 1.7, J = 0.4, 1H, H-8), 7.92 (ddd, J = 8.7, J = 7.1, J = 1.7, 1H, H-6),
7.80 (dd, J = 8.0, J = 1.5, 1H, H-1), 7.79 (ddd, J = 8.7, J = 1.0, J = 0.4,
1H, H-5), 7.63 (dd, J = 8.1, J = 1.5, 1H, H-3), 7.52 (ddd, J = 8.0,
J = 7.1, J = 1.0, 1H, H-7), 7.42 (dd, J = 8.1, J = 8.0, 1H, H-2), 7.08–
6.88 (m, 4H, ArAH), 4.66–4.56 (m, 1H, @CHAOH), 4.42–4.32 (m,
2H, ArAOACH2A), 3.81 (s, 3H, ArAOACH3), 3.65–3.06 (m, 11H,
ANACH2A(pip), ACH2ANH+, AOH). LC–MS: calcd for [M+H]+:
7.3. 3-Chloro-5-[2-hydroxy-3-(propan-2-ylamino)propoxy]-2-
hydroxypropoxy]-3-chloro-9H-xanthen-9-one
(compound 1)
hydrochloride
This compound was obtained as white solid (yield 80%), mp
291–193 °C. 1H NMR (DMSO-d6, 300 MHz) d (ppm): 8.76 (br s,
2H, ANHH+), 8.18 (d, J = 8.4, 1H, H-8), 7.85 (d, J = 1.8, 1H, H-5),
7.74 (dd, J = 8.1, J = 1.5, 1H, H-1), 7.59 (dd, J = 8.1, J = 1.5, 1H, H-
3), 7.54 (dd, J = 8.6, J = 2.1, 1H, H-7), 7.41 (t, J = 7.8, 1H, H-2), 5.96
(br s, 1H, AOH), 4.34 (br s, 1H, @CHAOH), 4.24–4.22 (m, 2H,
ArAOACH2A), 3.40–3.06 (m, 3H, ACH2ANA, ACH@), 1.28 (dd,
J = 6.4, J = 2.7, 6H, A(CH3)2). Base of compound 1: Anal. Calcd for
C27H29O5N2 m/z: 461.52, found 461.32. [
a]
20 = 12.83 (methanol).
D
Rf = 0.78 (methanol/ethyl acetate (1:1)).
C
19H20O4NCl: C, 63.07; H, 5.57; N, 3.87. Found: C, 62.70; H, 5.79;
7.8. (S)-(ꢁ)-4-(2-Hydroxy-3-(4-(2-methoxyphenyl)piperazine-1-
yl)propoxy)-9H-xanthen-9-one hydrochloride (compound 6)
N, 3.51. Rf = 0.23 (methanol/ethyl acetate (1:1)).
7.4. 3-Chloro-5-[2-hydroxy-3-(2-methylpropan-2-ylamino)pro-
This compound was obtained as a white solid (yield 65%), mp
234–236 °C. Anal. Calcd for C27H29O5N2Cl: C, 65.25; H, 5.88; N,
5.63. Found: C, 65.45; H, 6.20; N, 5.75. 1H NMR spectra as for (R)
enantiomer. LC–MS: calcd for [M+H]+: C27H28O5N2 m/z: 461.52,
poxy]-2-hydroxypropoxy]-9H-xanthen-9-one
hydrochloride
(compound 2)
found 461.32. [
acetate (1:1)).
a]
D
20 = ꢁ10.57 (methanol). Rf = 0.78 (methanol/ethyl
This compound was obtained as white solid (yield 75%), mp
269–271 °C Anal. Calcd for C20H23O4NCl2: C, 58.26; H, 5.62; N,
3.40. Found: C, 58.07; H, 6.03; N, 3.41. 1H NMR (DMSO-d6,
300 MHz) d (ppm): 8.50 (br s, 2H, ANHH+), 8.19 (d, J = 8.4, 1H, H-
8), 7.80 (d, J = 1.8, 1H, H-5), 7.76 (dd, J = 8.1, J = 1.5, 1H, H-1),
7.60 (dd, J = 8.0, J = 1.2, 1H, H-3), 7.55 (dd, J = 8.7, J = 2.1, 1H, H-
7), 7.42 (t, J = 8.1, 1H, H-2), 5.92 (br s, 1H, AOH), 4.26 (br s, 3H,
@CHAOH, ArAOACH2A), 3.40–3.06 (m, 2H, ACH2ANA), 1.32 (s,
9H, A(CH3)3). Rf = 0.16 (methanol/ethyl acetate (1:1)).
Acknowledgements
The study was supported by the research Grants of the Ministry
of Science and Higher Education (K/DSC/001962 and
K/ZDS/005487).
Supplementary data
7.5. 3-{2-Hydroxy-3-[(2-hydroxyethyl)(methyl)amino]propoxy}-
9H-xanthen-9-one hydrochloride (compound 3)
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.09.005.
This compound was obtained as a white solid (yield 60%), mp
113–115 °C. Anal. Calcd for C19H22O5NCl: C, 60.08; H, 5.84; N,
3.69. Found: C, 60.00; H, 6.07; N, 3.69. 1H NMR (DMSO-d6,
300 MHz) d(ppm): 8.16 (dd, J = 8.0, J = 1.5, 1H, H-8), 8.10
(d, J = 8.7, 1H, H-1), 7.87–7.81 (m, 1H, H-6), 7.61 (d, J = 8.0, 1H,
H-5), 7.49–7.43 (m, 1H, H-7), 7.19 (d, J = 2.4, 1H, H-4), 7.07 (dd,
J = 8.9, J = 2.4, 1H, H-2), 6.03 (br s, 1H, @CHAOH), 5.32 (br s, 1H,
@CHA), 4.41 (br s, 1H, CH2AOH), 4.17 (d, J = 4.8, 2H, ArAOACH2A),
3.78 (d, J = 4.8, 2H, ACH2AOH), 3.50–3.14 (m, 4H, ACH2
ANACH2A), 2.88 (s, 3H, ACH3). Rf = 0.29 (methanol/ethyl acetate
(1:1)).
References and notes
1. WHO Rep. 2008.
2. Sanguinetti, M. C.; Bennett, P. B. Circ. Res. 2003, 93, 491.
3. Estrada, J. C.; Darbar, D. Eur. J. Clin. Pharmacol. 2008, 64, 1139.
4. Kherada, N.; Heimowitz, T.; Rosendorff, C. Curr. Hypertens. Rep. 2015, 17, 79.
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. Crowther, A. F.; Howe, R.; McLoughlin, B. J.; Mallion, K. B.; Rao, B. S.; Smith, L.
H.; Turner, R. W. J. Med. Chem. 1972, 15, 260.
8. Lin, K.-W.; Fang, S.-C.; Hung, C.-F.; Shieh, B.-J.; Yang, S.-C.; Teng, C.-M.; Lin, C.-
N. Arch. Pharm. (Weinheim) 2009, 342, 19.
9. Rapacz, A.; Sapa, J.; Bednarski, M.; Filipek, B. Eur. J. Pharmacol. 2014, 738, 14.
10. Marona, H.; Librowski, T.; Cegła, M.; Erdog˘an, C.; Sßahin, N. Ö. 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. Szkaradek, N.; Rapacz, A.; Pytka, K.; Filipek, B.; Siwek, A.; Cegła, M. Bioorg. Med.
Chem. 2013, 21, 514.
7.6. 4-{2-Hydroxy-3-[(2-hydroxyethyl)(methyl)amino]propoxy}-
9H-xanthen-9-one hydrochloride (compound 4)
This compound was obtained as a white solid (yield 65%), mp
163–165 °C. Anal. Calcd for C19H22O5NCl: C, 60.08; H, 5.84; N,
Please cite this article in press as: Szkaradek, N.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.005

N. Szkaradek et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
11
13. Da Re, P.; Primofiore, G. P.; Bertelli, A. J. Med. Chem. 1972, 115, 868.
14. Marona, H.; Szkaradek, N.; Kubacka, M.; Bednarski, M.; Filipek, B.; Cegla, M.;
Szneler, E. Arch. Pharm. (Weinheim) 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.; Szneler, E.; Filipek, B.; Sapa, J. Acta Pol. Pharm. 1997, 54, 63.
17. Marona, H. Pharmazie 1998, 53, 672.
18. Maj, J.; Klimek, V.; Nowak, G. Eur. J. Pharmacol. 1985, 119, 113.
19. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
20. White, J. P. M.; Cibelli, M.; Fidalgo, A. R.; Paule, C. C.; Noormohamed, F.; Urban,
L.; Maze, M.; Nagy, I. Anesthesiology 2010, 112, 729.
23. Rapacz, A.; Pytka, K.; Sapa, J.; Kubacka, M.; Filipek, B. Eur. J. Pharmacol. 2014,
735, 10.
24. Maron, D. M.; Ames, B. N. Mutat. Res. 1983, 113, 173.
25. Mortelmans, K.; Zeiger, E. Mutat. Res. 2000, 455, 29.
26. Santos, F. V.; Colus, I. M. S.; Silva, M. A.; Vilegas, W.; Varanda, E. A. Food Chem.
Toxicol. 2006, 44, 1585.
27. Kubacka, M.; Mogilski, S.; Filipek, B.; Marona, H. Eur. J. Pharmacol. 2013, 720,
237.
28. Groszek, G.; Bednarski, M.; Dybała, M.; Filipek, B. Eur. J. Med. Chem. 2009, 44,
809.
29. Jensen, B. C.; O’Connell, T. D.; Simpson, P. C. J. Mol. Cell. Cardiol. 2011, 51, 518.
30. Ullmann, F.; Kipper, H. Ber. Dtsch. Chem. Ges. 1905, 38, 2120.
31. Ullmann, F.; Zlokasoff, M. Ber. Dtsch. Chem. Ges. 1905, 38, 2111.
32. Chen, X.; Whissell, P.; Orser, B. A.; MacDonald, J. F. PLoS One 2011, 6, e21970.
33. Mauthner, N. Mat. es Termeszettudomanyi Ert. 1943, 62, 360.
21. Handbook of Experimental Pharmacology; Szekeres, L., Papp, J. G., Schmier,
J., Eichler, O., Eds.; Springer: Berlin, Heidelberg, New York, 1975; pp 131–
182.
22. Litchfield, J. T.; Wilcoxon, F. J. Pharmacol. Exp. Ther. 1949, 96, 99.
Please cite this article in press as: Szkaradek, N.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.005