Design, synthesis and cardiovascular evaluation of some aminoisopropanoloxy derivatives of xanthone

Article abstract of DOI:10.1016/j.bmc.2018.04.038

A series of aminoisopropanoloxy derivatives of xanthone has been synthesized and their pharmacological properties regarding the cardiovascular system has been evaluated. Radioligand binding and functional studies in isolated organs revealed that title compounds present high affinity and antagonistic potency for α₁-(compound 2 and 8), β-(compounds 1, 3, 4, 7), α₁/β-(compounds 5 and 6) adrenoceptors. Furthermore, compound 7, the structural analogue of verapamil, possesses calcium entry blocking activity. The title compounds showed hypotensive and antiarrhythmic properties due to their adrenoceptor blocking effect. Moreover, they did not affect QRS and QT intervals, and they did not have proarrhythmic potential at tested doses. In addition they exerted anti-aggregation effect. The results of this study suggest that new compounds with multidirectional activity in cardiovascular system might be found in the group of xanthone derivatives.

Full text of DOI:10.1016/j.bmc.2018.04.038

Accepted Manuscript
Design, synthesis and cardiovascular evaluation of some aminoisopropanoloxy
derivatives of xanthone
M. Kubacka, N. Szkaradek, S. Mogilski, K. Pańczyk, A. Siwek, A. Gryboś, B.
Filipek, P. Żmudzki, H. Marona, A.M. Waszkielewicz
PII:
S0968-0896(17)32186-7
https://doi.org/10.1016/j.bmc.2018.04.038
BMC 14321
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 November 2017
10 April 2018
16 April 2018
Please cite this article as: Kubacka, M., Szkaradek, N., Mogilski, S., Pańczyk, K., Siwek, A., Gryboś, A., Filipek,
B., Żmudzki, P., Marona, H., Waszkielewicz, A.M., Design, synthesis and cardiovascular evaluation of some
aminoisopropanoloxy derivatives of xanthone, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/
10.1016/j.bmc.2018.04.038
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design, synthesis and cardiovascular evaluation of some
aminoisopropanoloxy derivatives of xanthone
M. Kubackaa, N. Szkaradekb*, S. Mogilskia, K. Pańczykb, A. Siwekc, A. Grybośc, B. Filipeka,
P. Żmudzkid, H. Maronab, A.M. Waszkielewiczb
aDepartment of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical
College, Medyczna 9, 30-688 Krakow, Poland
bDepartment of Bioorganic Chemistry, Chair of Organic Chemistry, Faculty of Pharmacy,
Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland
cDepartment of Pharmacobiology, Faculty of Pharmacy, Jagiellonian University Medical
College, Medyczna 9, 30-688 Krakow, Poland.
dDepartment of Medicinal Chemistry, Faculty of Pharmacy, Jagiellonian University Medical
College, Medyczna 9, 30-688 Krakow, Poland.
* corresponding author: nszkarad@cm-uj.krakow.pl
Abstract
A series of aminoisopropanoloxy derivatives of xanthone has been synthetized and their
pharmacological properties regarding the cardiovascular system has been evaluated.
Radioligand binding and functional studies in isolated organs revealed that title compounds
present high affinity and antagonistic potency for α1-(compound 2 and 8), β- (compounds 1, 3,
4, 7), α1/β- (compounds 5 and 6) adrenoceptors. Furthermore, compound 7, the structural
analogue of verapamil, possesses calcium entry blocking activity. The title compounds
showed hypotensive and antiarrhythmic properties due to their adrenoceptor blocking effect.
Moreover, they did not affect QRS and QT intervals, and they did not have proarrhythmic
potential at tested doses. In addition they exerted anti-aggregation effect. The results of this
study suggest that new compounds with multidirectional activity in cardiovascular system
might be found in the group of xanthone derivatives.
1

Keywords: xanthone, synthesis, cardiovascular, adrenoceptor, hypertension, arrhythmia
1. Introduction
Xanthone (dibenzo-γ-pyrone, 9H-xanthen-9-one) derivatives comprise of wide group
of natural and synthetic compounds. Beneficial effects of xanthones rich extracts from fruits
were used in natural medicine in many asian countries. Scientific works in this field started in
the sixties of the last century. Da Re et al. described central stimulating and analeptic
1
activities of aminoalkyl xanthone derivatives , meanwhile Pfister et al. presented Xanoxate
Sodium which was effective in the passive cutaneous anaphylaxis (PCA) test after p.o.
2
administration in mice. Since then, synthetic xanthone derivatives have become the subject
of intensive researches. There are many papers describing their rich pharmacological activity
profiles, and beneficial activities, such as antitumor3,4, antioxidant5, antituberculotic6,
antimalarial7 or cardiovascular protective8. Some efforts have been also made in our
Laboratory of Bioorganic Chemistry. We have proved antituberculotic9, anticonvulsant10,
antimicrobial11, anticancer12 and cardiovascular13 properties of xanthone derivatives designed
and synthesized by our group.
Our former research related to structure-activity relationship in the group of xanthone
derivatives resulted in promising results. One of our previously published xanthone
derivatives active in the cardiovascular system was 4-(2-hydroxy-3-(4-(2-
methoxyphenyl)piperazine-1-yl)propoxy)-9H-xanthen-9-one hydrochloride and its
S
enantiomer MH-3.14,15 It revealed nanomolar affinity to α1-adrenoreceptors as well as
antiarrhythmic activity in i.v. adrenaline induced model of arrhythmia in rats (ED50=0.81
mg/kg).
These encouraging results prompted us to synthesis of new xanthone derivatives. We
were wondering if changing of the aromatic rings to xanthone skeleton in the structure of
known cardiovascular drugs would improve their effectiveness. For that reason, most of the
synthesized xanthones contain some pharmacophore moieties of cardiovascular drugs.
Compound 6 is a racemic analog of carvedilol, where xanthone is used in place of carbazole.
Compounds 1 and 3 are xanthone analogs of nadolol or carteolol. Whereas 7 is
xanthonoxyisopropyl derivative of homoveratrilamine (3,4-dimethoxyphenethylamine), which
is also present in the structure of verapamil. The reference cardiovascular drugs are presented
in Figure 1. Commercially available carvedilol and verapamil are equimolar mixtures of its R-
and S- enantiomers. Taking into account stereochemistry of approved drug nadolol, it is
2

equimolar mixture of four stereoisomers: 1-R, 2-S, 3-R; 1-R, 2-S, 3-S; 1-S, 2-R, 3-R; 1-S, 2-R,
3-S (see Figure 1).
2. Materials and methods
2.1. Synthesis
The synthesis of the title compounds involved several steps. The first step was
Ulmann's condensation of the appropriate substituted chlorobenzoic acid and phenol. The
reaction was carried out for few hours in oil at about 200°C in the presence of Cu/Cu2O
catalyst. The next step was cyclization in the presence of concentrated H2SO4. In case of
methoxy derivatives of phenol, concomitant demethylation occured (synthesis of compounds
2-8). We noticed that longer refluxing time of the mixture led to more effective
demethylation. The optimal refluxing time was 48 hours. Unreacted acid was washed out with
NaHCO3 and recovered by precipitating using diluted HCl. As a result of cyclization 3-
chloro-5-methyl-9H-xanthen-9-one, 3-hydroxy-9H-xanthen-9-one and 4-hydroxy-9H-
xanthen-9-one were obtained. Hydroxyxanthones could directly underwent further reactions
while 3-chloro-5-methyl-9H-xanthen-9-one should underwent some more modifications to
obtain 3-hydroxy-5-methyl-9H-xanthone-9-one. 3-Chloro-5-methyl-9H-xanthen-9-one firstly
underwent methanolysis (sodium methoxide was prepared by slow dissolution of sodium in
an excess of methanol - it is exothermic reaction that cooling of the reacting environment was
necessary; to that mixture, equimolar amount of 3-chloro-5-methyl-9H-xanthen-9-one was
added and refluxed for 48 hours; next steps included distillation to dryness, addition of water
and H2SO4 and filtration of the precipitate). 3-Methoxy-5-methyl-9H-xanthen-9-one was
treated tenfold excess of concentrated H2SO4 to obtain desirable 3-hydroxy-5-methyl-9H-
xanthen-9-one. Physico-chemical properties of 3-hydroxy-5-methyl-9H-xanthone-9-one11, 3-
hydroxy-9H-xanthen-9-one16 and 4-hydroxy-9H-xanthen-9-one17 were described previously
as mentioned. Appropriate derivatives of hydroxyxanthone were conducted into oxiranes.
Each hydroxyxanthone was dissolved in water solution of equimolar amount of NaOH during
heating. After cooling, 50 % excess of epichlorohydrine was gradually added during stirring.
After few hours, reacting mixture was filtered, washed with alkalized water (to remove
unreacted hydroxyxanthone) and cool water. Crude products were recrystallized from ethanol.
Physico-chemical properties of 5-methyl-3-((oxiran-2-yl)methoxy)-9H-xanthen-9-one11, 3-
((oxiran-2-yl)methoxy)-9H-xanthen-9-one18 and
4-((oxiran-2-yl)methoxy)-9H-xanthen-9-
one17 were described previously as indicated. Oxirane derivatives were taken to aminolysis
3

performed in heated 250 mL flasks, in n-propanol using 100 % excess of adequate amine.
After 48 h of heating, n-propanol was distilled off and crude amine products were bit by bit
dissolved in heated, about 1 % solution of HCl. After cooling, 10 % solution of NaOH was
added to each solutions and precipitates were filtered, washed with water and dried. All of the
title compounds were then converted in hydrochloride salts using excess of ethanol saturated
with HCl. The crude products were recrystallized from ethanol/acethone (3/1).
Physicochemical properties of the final compounds are mentioned below or were subject of
our former publications. Compounds 4 and 7 have been published previously for their
microbiological (anti-Helicobacter) activity.11 Compound 3 was formerly achieved by
Crowther et al.19 as a base which was published for its β-adrenergic properties, and the
hydrochloride was also evaluated in our laboratory by Szkaradek et al. for its anticancer
properties. 12
4

Table 1. Structures and binding properties of the title compounds 1-8.
Compd. Position
R1
Position R2
Formula
Ki
IC50 [nM]
α2
Mol. mass
α1
β1
3
3
5
CH3 C21H26NO4Cl
391.89
–
–
784.5 18.5
1
2
1570.0 36.5
R,S xHCl
H
C25H27N3O4Cl
355.6 5.7
–
–
814.0 30.8
2
504.41
R,S xHCl
R,S xHCl
R,S xHCl
4
4
H
H
C20H24NO4Cl
377.86
–
–
–
5.4 0.5
3
4
10.8 1.2
C24H24NO5Cl 1870.0 31.
179.0 5.5
441.91
0
359.0 11.0
4676.0 78.
0
5

4
4
4
4
H
H
H
H
C25H26NO5Cl 349.8 13.6
–
–
–
–
426.0 16.0
852.0 31.0
5
6
7
8
455.93
875.5 34.2
R,S xHCl
R,S xHCl
R,S xHCl
R,S xHCl
C25H26NO6Cl
471.93
109.5 2.3
274.0 6.3
178.5 8.5
357.0 17.0
C26H28NO6Cl
485.96
–
106.3 3.9
213.0 7.7
C25H27N3O4Cl
171.0 3.6
1418.0 89.5
2835.0 179.0
400.0 19.4
2
504.41
– No binding at 10-5 M
6

2.2. Experimental protocol
Reagents:
methoxybenzylamine, o-cresol were purchased from Sigma–Aldrich Chemie (Steinheim,
Germany), 2-(2-methoxyphenoxy)ethylamine was obtained from TCI Chemicals
2,3-dimethoxyphenethylamine,
(R,S)-epichlorohydrin,
2-
(Zwijndrecht, Belgia), whereas reagents: 2-chlorobenzoic acid, 2,4-dichlorobenzoic acid, 2-
methoxyphenethylamine, 2-methoxyphenol, 3-methoxyphenol, 1-(2-pirydyl)-piperazine, t-
butylamine were provided by Lancaster Synthesis (Frankfurt amMain, Germany). Solvents
were commercially available materials of reagent grade. Melting points (mp) are uncorrected
and were determined using a Buchi SMP-20 apparatus (Buchi Labortechnik, Flawil,
Switzerland). The 1H-NMR were obtained in CDCl3 (for bases) or DMSO-d6 (for
hydrochloride salts) with a Varian Mercury-VX 300 NMR spectrometer (Varian Inc., Palo
Alto, CA, USA) or a Bruker AMX spectrometer (Brucker, Karlsruhe, Germany) with 500.13
MHz with TMS or DMSO respectively as an internal standard. Spectral data contains:
chemical shifts in ppm, multiplicities, constant couplings in Hz, number of protons, protons'
positions. Multiplicities were abbreviations: s (singlet), bs (broad singlet), d (doublet), dd
(doublet of doublets), ddd (double doublet of doublets), t (triplet), m (multiplet). The
homogeneity of obtained compounds was confirmed by the thin-layer chromatography (TLC),
carried out on precoated plates (silica gel, 60 F-254 Merck) using the solvent system of
methanol/ethyl acetate (1/1); the obtained corresponding spots were visualized under UV
light. 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 Waters TQD
mass spectrometer (electrospray ionization mode ESI-tandem quadrupole). All the analyses
were carried out using an Acquity UPLC BEH C18, 1.7µm, 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.
R,S-3-[3-(Tert-butylamino)-2-hydroxypropoxy]-5-methyl-9H-xanthen-9-one
hydrochloride (1)
1
C21H26NO4Cl; M=391.89; mp 259-261 °C; Rf=0.24 (CH3OH/ethyl acetate 1/1); H NMR
(DMSO-d6, 300 MHz): 8.90 - 9.11 (m, 1H, -NHH+-), 8.47 - 8.72 (m, 1H, -NHH+-), 8.09 (d,
J=8.8, 1H, Ar-H1), 7.94 - 8.02 (m, 1H, Ar-H8), 7.69 (d, J=8.2, 1H, Ar-H6), 7.33 (t, J=7.6,
7

1H, Ar-H7), 7.20 (d, J=2.3, 1H, Ar-H4), 7.07 (dd, J=8.8, J=2.3, 1H, Ar-H2), 5.92 - 6.05 (m,
1H, -OH), 4.25 - 4.33 (m, 1H, >CH-), 4.17 - 4.24 (m, 2H, Ar-O-CH2-), 3.13 (bs, 1H, -CHH-
NH-), 2.89 - 3.00 (m, 1H, -CHH-NH-), 2.51 (s, 3H, Ar-CH3), 1.31 (s, 9H, -C-(CH3)3); 13C
NMR (CDCl3, 75 MHz): 176.22 (C=O), 164.18 (C8b), 157.88 (C8a), 156.11 (C4a), 134.28
(C4b), 128.19 (C3), 126.56 (C4), 123.84 (C1), 121.84 (C8), 117.67 (C2), 115.82 (C6), 113.50
(C7), 100.81 (C5), 71.22 (-OCH2-), 68.32 (=CH-OH), 50.43 (-CH2-NH-), 44.58 (-NH-C≤),
29.14 ((-CH3)3); Mbase=355.43 LC-MS [M+H]+ m/z 356.28, 98.99 %.
R,S-3-{2-hydroxy-3-[4-(pyridin-2-yl)piperazin-1-yl]propoxy}-9H-xanthen-9-one
hydrochloride (2)
C25H25N3O4; M=467.94; mp 255-257 (base: 168-170) °C; Rf=0.87 (CH3OH/ethyl acetate 1/1);
1H NMR (CDCl3, 500.13 MHz) for base: 8.17 (ddd, J=7.9, J=1.8, J=0.5, 1H, Ar-H8), 8.12
(ddd, J=4.9, J=2.0, J=0.9, 1H, Pyr-H6'), 8.10 (d, J=8.9, 1H, Ar-H1), 7.84 (ddd, J=8.4, J=7.1,
J=1.8, 1H, Ar-H6), 7.58 (ddd, J=8.4, J=1.0, J=0.5, 1H, Ar-H5), 7.53 (ddd, J=8.6, J=7.1,
J=2.1, 1H, Pyr-H4'), 7.47 (ddd, J=7.9, J=7.1, J=1.0, 1H, Ar-H7), 7.19 (d, J=2.3, 1H, Ar-H4),
7.08 (dd, J=8.9, J=2.3, 1H, Ar-H2), 6.82 (ddd, J=8.7, J=0.8, J=0.8, 1H, Pyr-H3'), 6.64 (ddd,
J=7.0, J=4.8, J=0.8, 1H, Pyr-H5'), 5.09 (d, J=4.7, 1H, -OH), 4.24 (dd, J=9.6, J=2.8, 1H, -O-
CHH-), 4.10 (dd, J=9.6, J=6.1, 1H, -O-CHH-), 4.05 - 4.11 (m, 1H, =CH-O-), 3.43 - 3.52 (m,
4H, piperazine), 2.53 - 2.64 (m, 5H, piperazine + -CHH-N<), 2.46 (dd, J=12.7, J=6.1, 1H, -
CHH-N<); 13C NMR (CDCl3, 75 MHz): 176.27 (C=O), 164.12 (C8b), 159.38 (C8a), 157.91
(C4a), 156.17 (C4b), 147.96 (C3), 137.55 (C4), 134.34 (C1), 128.30 (C8), 126.62 (C2),
123.90 (C6), 121.89 (C7), 117.70 (C5), 115.97 (C2'), 113.57 (C6'), 113.52 (C4'), 107.14
(C3'), 100.91 (C5'), 70.78 (-O-CH2-), 65.37 (=CH-OH), 60.39 (-CH2-N=), 53.14 ((-CH2)2=N-
), 45.29 (-N=(CH2-)2); Mbase=431.48; LC-MS [M+H]+ m/z 432.33, 100 %.
R,S-4-{2-hydroxy-3-[(2-methoxyphenethyl)amino]propoxy}-9H-xanthen-9-one
hydrochloride (5)
1
C25H26NO5Cl; M=455.93; mp 206-208 °C; Rf=0.30 (CH3OH/ethyl acetate 1/1); H NMR
+
(DMSO-d6, 300 MHz): 8.88 - 9.19 (m, 2H, -NH2 -), 8.19 (dd, J=7.6, 1.8, 1H, Ar-H8), 7.84 -
7.92 (m, 1H, Ar-H6), 7.73 - 7.78 (m, 1H, Ar-H5), 7.70 (d, J=7.6, 1H, Ar-H1), 7.58 (dd, J=7.9,
1.5, 1H, Ar-H3), 7.45 - 7.52 (m, 1H, Ar-H2), 7.35 - 7.43 (m, 1H, Ar-H7), 7.15 - 7.26 (m, 2H,
Ph-H3', Ph-H5'), 6.97 (d, J=8.2, 1H, Ph-H6'), 6.84 - 6.91 (m, 1H, Ph-H4'), 6.02 (d, J=5.3, 1H,
-OH), 4.31 - 4.42 (m, J=4.7, 1H, >CH-), 4.20 - 4.26 (m, 2H, Ar-O-CH2-), 3.77 (s, 3H, Ph-
13
OCH3), 3.17 (bs, 4H, -CH2-NH-CH2-), 2.94 - 3.04 (m, 2H, Ph-CH2-); C NMR (DMSO-d6,
8

75 MHz): 176.39 (C=O), 157.55 (C8b), 155.75 (C8a), 147.92 (C4a), 146.46 (C4b), 135.94
(C4), 130.32 (C1), 128.72 (C8), 126.38 (C3), 125.48 (C6), 124.96 (C7), 124.41 (C2), 122.47
(C5), 121.36 (C2'), 120.92 (C1'), 118.88 (C3'), 118.49 (C5'), 117.47 (C4'), 111.26 (C6'), 71.72
(-OCH2-), 65.26 (-OCH3), 55.79 (=CH-OH), 50.05 (-CH2-NH-), 47.35 (-NH-CH2-), 26.74 (-
CH2-Ar); Mbase 419.47; LC-MS [M+H]+ m/z 420.30, 99.28 %.
R,S-4-(2-hydroxy-3-{[2-(2-methoxyphenoxy)ethyl]amino}propoxy)-9H-xanthen-9-one
hydrochloride (6)
1
C25H26NO6Cl; M=471.93; mp 127-128 °C; Rf=0.30 (CH3OH/ethyl acetate 1/1); H NMR
+
(DMSO-d6, 500.13 MHz): 9.21 (bs, 2H, NH2 ), 8.20 (ddd, J=8.0, J=1.8, J=0.5, 1H, H-8); 7.86
(ddd, J=8.5, J=7.1, J=1.8, 1H, H-6); 7.77 (dd, J=8.1, J=1.5, 1H, H-1); 7.67 (ddd, J=8.5,
J=1.1, J=0.5, 1H, H-5); 7.61 (dd, J=8.1, 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=8.1, J=8.1, 1H, H-2); 7.06-6.86 (m, 4H, H-Ar); 6.08 (bs, 1H, OH); 4.84-
4.42 (m, 1H, C-H); 4.36-4.32 (m, 2H, Ar’-O-CH2); 4.29 (dd, J=10.3, J=5.0, 1H, Ar-O-CHH-
R); 4. 25 (dd, J=10.3, J=5.2, 1H, Ar-O-CHH-R); 3.72 (s, 3H, Ar’-O-CH3); 3.47 (dd, J=12.8,
J=3.5, 1H, NH-CHH-CH); 3.45 (dd, J=6.8, J=4.8, 2H, Ar’-OCH2-CH2-N); 3.31 (dd, J=12.8,
J=9.3, 1H, NH-CHH-CH); 13C NMR (DMSO-d6, 75 MHz): 175.92 (>C=O); 155.24 (C-4b);
149.36 (C-2’); 147.44 (C-4); 147.02 (C-4a); 146.00 (C-1’); 135.43 (C-6); 125.87 (C-8);
124.47 (C-7); 123.92 (C-2); 122.18 (C-anisole); 121.99 (C-8b); 120.86 (C-anisole); 120.64
(C-8a); 118.35 (C-5); 118.06 (C-3); 117.05 (C-1); 114.84 (C-anisole); 112.25 (C-anisole);
71.34 (Ar-O-CH2-R); 64.84 (Ar’-O-CH2); 64.75 (-CH); 55.37 (-OCH3); 50.02 (NH-CH2-
CHOH); 46.24 (NH-CH2-CH2-O-Ar’); Mbase=435.25; LC-MS [M+H]+ m/z 436.25, 100 %.
R,S-4-{2-hydroxy-3-[4-(pyridin-2-yl)piperazin-1-yl]propoxy)-9H-xanthen-9-one
hydrochloride (8)
1
C25H27N3O4Cl2; M=504.41; mp 251-253 °C; Rf=0.83 (CH3OH/ethyl acetate 1/1); H NMR
(DMSO-d6, 300 MHz): 11.27 (bs, 1H, -NH+<), 8.18 (dd, J=8.2, 1.8, 1H, Ar-H8), 8.10 (dd,
J=5.9, 1.2, 1H, Pyr-H6'), 7.93 - 8.04 (m, 1H, Ar-H6), 7.83 - 7.92 (m, 1H, Pyr-H4'), 7.70 - 7.80
(m, 2H, Ar-H1, Ar-H3), 7.59 (dd, J=7.9, 1.5, 1H, Ar-H5), 7.43 - 7.52 (m, 1H, Ar-H7), 7.34 -
7.40 (m, 2H, Ar-H2, Pyr-H3'), 6.98 (t, J=6.4, 1H, Pyr-H5'), signal from -OH not visible, 4.48
- 4.69 (m, 3H, -O-CH2-CH<), 4.16 - 4.30 (m, 2H, -CH2-pip), 3.59 - 3.89 (m, 4H, (CH2)2>N-
Pyr), 3.29 - 3.56 (m, 4H, (CH2)2>N-CH2-); 13C NMR (DMSO-d6, 75 MHz): 176.40 (C=O),
155.73 (C8b), 153.99 (C8a), 147.88 (C4a), 146.46 (C4b), 143.89 (C4), 139.46 (C1), 135.94
(C8), 126.35 (C3), 125.00 (C6), 124.41 (C7), 122.44 (C2), 121.33 (C5), 119.05 (C2'), 118.66
9

(C6'), 117.56 (C4'), 114.29 (C3'), 112.65 (C5'), 71.92 (-O-CH2-), 64.24 (=CH-OH), 59.00 (-
CH2-N=), 51.81 ((-CH2)2=N-), 50.80 (-N=(CH2-)2); Mbase=431.48; LC-MS [M+H]+ m/z
432.33, 99.62 %.
2.3. Pharmacology
2.3.1. General information
The experiments were carried out using male Wistar rats (Krf:(WI) WU), 200 – 250
g). The animals were housed in constant temperature facilities exposed to 12:12 h light/dark
cycles and were maintained on a standard pellet diet with tap water given ad libitum. All
procedures were conducted according to guidelines of ICLAS (International Council on
Laboratory Animal Science) and approved by the Local Ethics Committee of the Jagiellonian
University in Kraków (resolution no. 168/2012, 14.11.2012) and by the Second Ethics
Committee (resolution no 106/2016, 14.06.2016) in Krakow, Poland.
2.3.2. Radioligand binding
Binding experiments were conducted in 96-well microplates in a total volume of 250
µl of appropriate buffers. Reaction mix included 50 µl solution of tested compound, 50 µl of
radioligand and 150 µl of tissue suspension. Specific assay conditions for each receptors are
shown in the Table 2. Adrenergic receptors were prepared from rat cortex tissue. The
radioactivity was measured in MicroBeta TriLux 1450 - liquid scintillation counter
(PerkinElmer, USA). Radioligand binding data were analyzed using iterative curve fitting
routines (GraphPAD/Prism, Version 5.0 – San Diego, CA, USA). Ki values were calculated
from the Cheng and Prusoff equation. The concentrations of analyzed compounds ranged
from 10-10 to 10-5 M.
Table 2. Radioligand binding assay conditions
Blank
(nonspecific)
10µM
Incubation
conditions
Receptor
Radioligand
[3H]8-prazosin
[3H]-clonidine
[3H]- CGP-12177
Buffer
50 mM Tris-HCl pH
7,6
50 mM Tris-HCl pH
7,6
50 mM Tris-HCl pH
7,6
30 min, 30 °C
α1
α2
β1
phentolamine
10µM clonidine
1µM propranolol
25 min, 30 °C
60 min, 37 °C
10

2.3.3. Functional bioassays
2.3.3.1. α1-adrenoceptors
Isolated rat aorta was used in order to test the antagonistic activity of investigated
compounds for α1-adrenoceptors. The male Wistar rats weighing 200–350 g were anesthetized
with thiopental sodium (75 mg/kg i.p.) and the aorta was dissected, cleaned, denuded of
endothelium, cut and mounted as described earlier.20 Briefly, aorta rings were incubated in 30
mL chambers filled with a Krebs-Henseleit solution (NaCl 118 mM, KCl 4.7 mM, CaCl2 2.25
mM, MgSO4 1.64 mM, KH2PO4 1.18 mM, NaHCO3 24.88 mM, glucose 10 mM, C3H3O3Na
2.2 mM, EDTA 0.05 mM) at 37 °C and pH 7.4 with constant oxygenation (O2/CO2, 19:1) and
connected to an isometric FDT10-A force displacement transducer (BIOPAC Systems, Inc.,
COMMAT Ltd.,Turkey). The aorta rings were stretched and maintained at optimal tension of
2 g and allowed to equilibrate for 2 h. After the incubation period the cumulative
concentration-response curve to phenylephrine was obtained. Following the first
phenylephrine curve, aorta rings were incubated with the tested compounds for 20 min and
the next cumulative concentration curve was constructed.
2.3.3.2. β2-adrenoceptors
Isolated guinea-pig trachea was used in order to test the antagonist activity of
investigated compounds for β2-adrenoceptors. The male guinea-pigs were anesthetized with
urethane (1.4 g/kg i.p.) and the trachea was dissected, cleaned, cut into approximately 4 mm
long rings. Trachea rings were horizontally suspended between two stainless steel hooks
(diameter 0.15 mm). One hook was attached to the bottom of the chamber and the other to an
isometric FDT10-A force displacement transducer (BIOPAC Systems, Inc., COMMAT Ltd.,
Turkey), coupled to a MP100 analyser (BIOPAC Systems, Inc., COMMAT Ltd., Turkey) and
processed by Acqknowledge software (BIOPAC). The trachea rings were incubated in 30 mL
chambers filled with a Krebs-Henseleit solution of above composition at 37 °C and pH 7.4
with constant oxygenation (O2/CO2, 19:1) and subjected to a 2 g initial optimal tension and
allowed to equilibrate for 2 h. The trachea rings were conctracted to submaximal tension with
carbachol (10 µM) in the presence of phentolamine (10 µM). Once the plateau was attained,
concentration-relaxation curves were obtained by addition of cumulative doses of isporenaline
(0.001 - 3 µM) in the absence and in the presence of test compounds. Tissues were incubated
with antagonists for 20 min.
11

2.3.3.3. Voltage dependent calcium channels
In order to investigate the calcium entry blocking properties of the compounds they
were tested on isolated rat aorta precontracted with KCl. Rats were anaesthetized with
thiopental sodium (60 mg/kg i.p.) and the thoracic aorta was dissected, cleaned, denuded of
endothelium, cut and mounted as described earlier and placed in a Krebs-Henseleit solution of
the above composition. The aortic rings were stretched and maintained at optimal tension of 2
g and allowed to equilibrate for 2 h. The aortic rings were conctracted to submaximal tension
with KCl (60 mMol/l). The depolarising solution KCl (60 mMol/l) was obtained by equimolar
substitution of NaCl for KCl. The KCl-induced contraction was elicited in the presence of
phentolamine (10 µMol/l) to avoid a possible participation of noradrenaline released by
depolarization.21 Once the plateau was attained, concentration-relaxation curves were
obtained by addition of cumulative doses of test compounds to the precontracted preparations.
Concentration-response curves were analyzed using GraphPad Prism 5.0 software
(GraphPad Software Inc., San Diego, CA, USA). Contractile responses to vasoconstrictor (in
the presence or absence of tested compounds) are expressed as a percentage of the maximal
vasoconstrictor effect (Emax = 100 %), reached in the concentration-response curves obtained
before incubation with the tested compounds. Data are the means SEM of at least 4 separate
experiments. From the EC50 values of the agonist in the presence and absence of different
antagonist concentrations, concentration ratios were calculated. Schild plots were constructed,
and where the slope was not significantly different from unity, the pA2 values were
determined.22 When Schild analysis could not be performed the affinity was estimated with
the equation pKB = log(concentration ratio–1)-log(molar antagonist concentration), where the
concentration ratio is the ratio of equieffective agonist concentrations in the absence and in
the presence of the antagonist.
2.3.3.4. Measurement of blood pressure and heart rate
Rats were anesthetized with thiopental (60 mg/kg i.p.). The right carotid artery was
cannulated with polyethylene tube filled with heparin in saline to facilitate pressure
measurements using a Datamax apparatus (Columbus Instruments). The studied compounds
were injected into the caudal vein after a 15 min stabilization period at a volume equivalent to
the 1 mL/kg in a wide range of doses.
12

2.3.3.5. β-adrenergic responses in vivo
In order to examine the β-adrenoceptors blocking properties of studied compound, the
effect on the cardiovascular response to isoprenaline (0.5 µg/kg) was investigated. This was
done in separate series of experiments in rats, and the compounds were given i.v. in the lowest
effective doses. Isoprenaline was administered i.v. and the resultant tachycardia recorded as
the control. A single dose of studied compound was then administered. After 10 min, a further
injection of isoprenaline was given.
2.3.3.6. The effect on normal electrocardiogram
In vivo electrocardiographic investigations were carried out using an ASPEL
ASCARD B5 apparatus, 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 i.v. administration of the compounds. The
√RR 23
QTc was calculated according to the formula of Bazzett: QTc = QT/
.
2.3.3.7. Prophylactic antiarrhythmic activity in adrenaline-induced arrhythmia
The arrhythmia was evoked in rats anesthetized with thiopental (60 mg/kg, i.p.) by
intravenous injection of adrenaline into the caudal vein (20 µg/kg, in a volume of 1 mL/kg).
The tested compounds were administered i.v. and p.o. 15 min or 60 min, respectively, before
adrenaline administration. The criterion of antiarrhythmic activity was the lack of premature
beats and inhibition of cardiac arrhythmia in comparison to the control group and the ED50
values with their 95 % confidence limits were calculated by computer log-probit analysis
according to Litchfield and Wilcoxon.24
2.3.3.8. In vitro whole blood aggregation tests
In vitro aggregation tests were conducted using freshly collected whole blood with
Multiplate platelet function analyzer (Roche Diagnostic), the five-channel aggregometer
based on measurements of electric impedance. Blood was drawn from carotid of rats with
hirudin blood tube (Roche Diagnostic). 300 µl of hirudin anticoagulated blood was mixed
with 300 µl prewarmed isotonic saline solution containing studied compound or vehicle
13

(deionized water or DMSO 0.1 %) and preincubated for 3 min at 37 C with continuous
stirring. Collagen (COLtest, Roche Diagnostic) was diluted using deionized water.
Aggregation was induced by adding collagen with final concentration of 1.6 µg/mL or
adrenaline and sub-threshold concentration of collagen (3 µM + 0.9 µg/mL). Activated
platelet function was recorded for 6 min. The Multiplate software analyzed the area under the
curve of the clotting process of each measurement and calculated the mean values.
3. Results
3.1. Radioligand binding study
Radioligand binding studies revealed that compounds 1, 3, 4 and 7 have high affinity
for β1-adrenoceptors with no significant binding to α1-, α2-adrenoceptors, with compound 3
showing the highest affinity (Ki = 5.4 nM), comparable to affinities presented by commonly
used beta-blockers.25 Compounds 2 and 8 bound selectively to α1-adrenoceptors, whereas
compounds 5 and 6, similar to carvedilol, presented high affinity to both α1- and β1-
adrenoceptors. None of the compounds bound to α2-adrenoceptors significantly. The affinities
described by Ki values (nM) are shown in Table 1.
3.1. Functional bioassays results
3.1.1. α1-adrenoceptors
Compounds with high affinity for α1-adrenoceptors (2, 5, 6, 8) were evaluated in
biofunctional studies in isolated rat aorta for their intrinsic activity. The tested compounds 2,
5, 6, 8 attenuated the contractions induced by phenylephrine, shifting the response to the right,
what indicate an interaction with α1-adrenoceptors in the rat aorta. The antagonistic potencies
reported as pA2 or pKB estimates (Table 3 column A, Figure 2) ranged between 7.521 and
8.317 what let us consider the compounds 2, 5, 6, 8 as potent α1-adrenoceptor antagonists.
3.1.2. β2-adrenoceptors
Compounds with high affinity for β1-adrenoceptor (1, 3, 4, 5, 6, 7) were also tested for
their antagonist potency at β2-adrenoceptor in guinea-pig trachea. These compounds reduced
the bronchodilatant effect evoked by isoprenaline, shifting the concentration-response curve
to the right without reducing the maximal response, what suggests that they are competitive
antagonists of β2-adrenoceptors present in guinea-pig trachea (Figure 3). The antagonistic
14

potencies reported as pA2 or pKB estimates (Table 3, column B, Figure 3) ranged between
6.184 (compound 1) and 9.509 (compound 3).
Table 3. Antagonist potency of tested compounds at α1-adrenoceptors in rat aorta and β2-
adrenoceptors in guinea-pig ileum
pKB /pA2 SEM (slope SEM)
Compound
α1
β2
pA2 = 6.184 0.08
s = 1.07 0.08
1
-
2
pKB = 8.202 0.12
-
pA2 = 9.509 0.03
s = 0.96 0.01
3
-
pA2 = 6.943 0.06
s = 0.90 0.01
4
-
5
pKB = 8.280 0.20
pKB = 8.344 0.12
-
pKB = 7.669 0.14
pA2 = 7.742 0.03
s = 0.99 0.01
6
pA2 = 7.383 0.16
s = 1.02 0.08
7
pA2 = 7.521 0.17
s = 1.13 0.18
8
-
propranolol
-
pA2 = 8.570 +- 0.17 26
pKB values were calculated according to relationship pKB= log (concentration ratio–1) – log
(molar antagonist concentration). pA2 values were obtained from the linear regression of
Schild plot. Each value was the mean SEM of 4-8 experimental results.
3.1.3. Voltage-dependent calcium channels
In order to investigate the calcium entry blocking properties of compound 7, it was
tested in isolated rat aorta contracted with depolarising KCl solution. As a reference
compound we used verapamil as compound 7 is derivative of homoveratrilamine (3,4-
dimethoxyphenethylamine), the moiety present in the structure of verapamil as well.
Compound 7 and verapamil relaxed KCl (60 mMol/l)-precontracted aortic rings in a dose-
dependent manner (Figure 4) by 95%. The IC50 value for compound 7 was 10.3 1.0 µM,
whereas verapamil, a voltage-dependent calcium channel blocker, inhibited contraction
induced by KCl depolarisinig solution with the IC50 value of 32.9 7.4 nM. On the basis of
this result we may state that compound 7 possesses weak voltage-dependent calcium channel
blocking properties.
3.1.4. β-adrenergic responses in vivo
15

In order to confirm the β-adrenoceptors blocking properties of studied compounds in
vivo, their effect on isoprenaline induced tachycardia was investigated in rats. Injection of
compounds 1, 4, 5, 6 and 7 (5 mg/kg) showed a significant decrease of isoprenaline (0.5
µg/kg) induced tachycardia. Compound 3 as well as carvedilol, administered at 1 mg/kg, also
inhibited the tachycardia effects induced by isoprenaline and were more potent than other
compounds (Figure 5), what is in line with their affinity for β-adrenoceptors.
Summarizing the studies so far, we obtained the xanthone derivatives and among them
compounds 2 and 8 are potent and selective α1-adrenoceptor antagonist, compounds 1, 3, 4, 7
are non selective β1/β2-blockers, and compounds 5 and 6, which present similar to carvedilol
receptor profile, being α1/β-adrenoreceptor antagonists. In addition, compound 7 possesses
voltage dependent calcium channel blocking properties. Compound 3 is the most potent β-
blocker, what is in line with Crowther et al.19 where that derivative was tested for β-
adrenoceptor blocking effect as a free base. In our studies we tested that structure as a
hydrochloride.
3.1.5. The effect on the blood pressure
In the next step we evaluated the hypotensive activity of studied compounds. The
investigated substances were tested starting from the dose of 5 mg/kg and that dose was
reduced until the hypotensive activity disappeared. The strong and significant hypotensive
effect was shown for compounds with α1-adrenolytic properties: compounds 2 and 8, the
selective α1-adrenoceptor antagonists as well as compounds 5 and 6, α1/β-adrenoceptor
antagonists, which were active in a wide range of doses and significantly diminished blood
16

pressure. The lowest effective dose was 1 mg/kg for compounds 2, 5 and 6 and 0.5 mg/kg for
compound 8 (Table 4). Compound 2 at the dose of 1 mg/kg significantly reduced systolic and
diastolic blood pressure by 8-10 % since the 40th min of the observation. Compound 5 at the
dose of 1 mg/kg significantly diminished systolic blood pressure by 5-11 % starting from 30
min after administration, and diastolic blood pressure by 7 % at the last 10 min of the
observation period. Compound 6 at the dose of 1 mg/kg 10 min after injection, significantly
reduced systolic blood pressure by 6-13 % and diastolic blood pressure by 7-14 % starting
from the 30 min after administration. Compound 8 at the dose 0.5 mg/kg 30 min after
injection significantly reduced systolic and diastolic blood pressure by 7-11 % and 5-9 %,
respectively.
Among beta-blockers with no α1-adrenolytic properties compound 3, administered at
a dose as low as 1 mg/kg, induced a significant fall in arterial blood pressure within 30-60
min after its administration. It reduced systolic blood pressure by 10 % and diastolic blood
pressure by 10-13 %. Compound 7, beta-blocker and verapamil analogue, revealed also strong
hypotensive activity. Compound 7 at the dose of 2.5 mg/kg 5-10 min after injection
significantly decreased systolic and diastolic blood pressure by 9-12 % and 8-16 %,
respectively. This was probably due to its calcium channels blocking ability in addition to
beta-blockade. Non-selective beta-blockers (compound 1 and 4) showed a weak hypotensive
effect after a single i.v. administration. In contrary, these structures increased blood pressure
in the first 20 min after administration. Compound 4 decreased systolic and diastolic blood
pressure in 60 min after its administration, by around 7 % , whereas compound 1 did not. This
may be explained by the differences in affinity for β1-adrenoceptors, with compound 4 having
higher affinity and stronger reducing cardiac output.
Table 4 The hypotensive activity of compounds 1-8 in normotensive anesthetized rats after
i.v. administration.
Blood
pressure
(mmHg)
Time of observation (min)
Dose
mg/kg
Compd
0
5
10
20
30
60
Systolic
148.4 3.6
119.4 3.2
166.6 7.5***
129.2 5.1**
159.2 7.5*
160.0 6.2*
155.8 5.5
144.0 4.6
1
5
Diastolic
Systolic
Diastolic
Systolic
125.0 5.7
110.3 7.5
80.5 5.3
136.0 5.8
126.0 5.4
108.8 8.2*
80.3 6.1
123.8 5.5
108.7 7.5*
79.5 5.5
114.0 4.2
102.5 6.7***
74.7 7.2**
122.7 5.7***
119.2 8.1
86.5 5.5
114.7 6.3
81.0 5.9
5
1
1
2
3
131.0 5.6
127. 2 5.8
135.7 6.5
104.8 5.9
138.3 7.5
107.5 6.2
Diastolic
Systolic
Diastolic
107.0 5.2
142.6 7.2
109.5 3.5
103.8 5.5
131.6 5.2
103.7 2.0
100.2 5.2
128.2 5.2
100.0 3.0***
94.2 4.8***
123.0 4.5***
96.7 2.9***
136.8 2.2
111.7 2.4
146.4 6.2
111.0 1.8
17

Systolic
Diastolic
Systolic
Diastolic
Systolic
Diastolic
Systolic
Diastolic
Systolic
Diastolic
135.3 7.3
101.3 5.9
137.6 3.3
107.8 1.7
141.8 6.4
108.7 4.9
137.0 9.3
104.0 8.8
122.4 2.7
88.4 3.3
149.3 8.1**
107.2 4.9
147.7 4.1*
107.0 3.7
133.0 4.3
100.6 4.4
126.0 3.2
96.0 4.2
125.3 1.2*
94.3 2.3*
4
5
5
5
1
5
1
146.2 11.5
108.6 7.8
147.8 6.0
114.8 4.6*
121.6 10.1
89.7 10.2
119.6 4.6
85.4 3.1
133.4 13.3
101.8 11.2
147.0 6.5
114.2 4.8
106.5 18.2**
73.0 17.6***
114.8 4.2*
82.8 4.0
128.2 15.2
98.8 13.4
141.0 6.1
111.5 4.7
97.7 7.9***
71.5 18.5**
114.8 3.9*
84.0 3.5
126.0 14.2
97.2 13.1
135.0 5.5*
107.7 4.2
94.5 19.1***
77.2 17.5**
112.4 3.5***
81.6 3.9*
117.0 12.6**
89.6 13.7
126.3 4.9***
101.2 3.7**
106.2 8.5***
80.0 9.3**
106.8 3.9***
75.6 5.1***
6
7
148.6 4.7
115.8 3.4
148.5 2.6
116.2 1.2
135.4 4.5
101.4 4.7***
143.7 2.9
135.6 2.6
105.0 0.7*
135.0 2.5**
101.5 2.1***
127.0 2.4***
97.4 2.4***
132.0 1.1***
99.7 1.7***
128.0 3.4***
96.2 2.8***
130.7 2.5***
97.7 2.9***
129.8 2.7**
98.6 2.7***
133.0 2.1**
100.7 2.0***
Systolic
Diastolic
Systolic
Diastolic
5
2.5
107.0 2.3*
146.2 3.5
115.2 4.4
156.5 3.8
122.7 3.0
147.7 5.9
116.0 4.6
136.8 1.8
110.8 2.3
146.2 1.7
111.7 2.6
146.7 4.6
116.3 4.4
131.4 3.1
106.0 3.2
146.2 2.1**
113.5 2.8*
144.0 4.4
114.3 3.8
129.8 3.1*
102.6 5.1
142.5 2.1**
133.0 2.2**
143.0 4.7
114.0 3.3
126.6 3.4**
99.8 4.4*
126.4 4.1**
96.6 5.0**
Systolic
Diastolic
Systolic
Diastolic
Systolic
Diastolic
5
1
140.0 1.8***
110.5 2.3***
137.7 4.7***
110.3 3.7**
138.7 2.3***
106.7 2.6***
132.0 3.2***
105.7 4.2***
8
0.5
The data are the means of 5-6 experiments S.E.M. Statistical analyses were performed using
a one-way ANOVA test; *p<0.05; **p<0.01; ***p<0.001.
3.1.6. The effect on normal electrocardiogram
In vivo ECG study showed that the compounds marked no significant effect on QRS
and QT intervals (Table 5). Selective α1-adrenolytics (compounds 2 and 8) did not influence
also the heart rate significantly. The rest of the compounds (1, 3 - 7), with beta-blocking
properties, decreased the heart rhythm and induced bradycardia. In the case of compound 4
and 7 the bradycardia was accompanied with PQ interval prolongation.
Table 5 Effects of an i.v. injection of the investigated compounds on the heart rate and ECG
intervals in anesthetized rat (75 mg/kg i.p.)
Time of observation (min)
Compound
Dose (mg/kg)
Parameters
0
1
5
15
Beats/min
PQ (ms)
360.0 14.2
55.0 1.7
16.7 0.7
57.7 1.4
141.2 1.5
337.0 8.7
60.5 1.5
18.0 0.0
59.0 1.0
139.8 1.2
310.0 4.5*
59.5 0.5
18.2 0.6
59.5 1.7
135.2 3.7
339.2 15.7
55.5 2.2
18.5 1.0
58.5 1.0
138.9 3.2
Cmpd 1
5
QRS (ms)
QT (ms)
QTc (ms)
Beats/min
PQ (ms)
324.0 4.9
55.0 1.0
20.7 2.9
340.7 13.0
55.3 1.3
18.7 1.3
313.0 6.8
54.7 1.8
20.0 2.3
295.3 7.0
56.0 1.7
21.3 1.3
Cmpd 2
5
QRS (ms)
18

QT (ms)
QTc (ms)
Beats/min
PQ (ms)
59.0 1.0
136.7 1.9
320.6 8.1
56.8 1.8
19.4 0.9
59.2 0.5
137.7 2.0
323.0 11.1
55.7 0.9
20.0 1.1
59.0 1.0
140.2 3.3
307.2 9.4
57.6 1.5
19.6 0.8
59.2 0.5
134.5 3.1
58.3 0.7
133.3 1.7
295.6 5.2**
58.0 1.7
59.0 1.0
133.9 1.4
290.6 5.9***
58.0 1.3
Cmpd 3
1
QRS (ms)
QT (ms)
QTc (ms)
Beats/min
PQ (ms)
19.0 0.9
19.2 0.8
59.2 0.5
59.6 0.4
132.2 1.9
131.0 2.1
246.0 10.2*** 243.7 12.3*** 265.0 13.6***
60.7 0.7
22.0 1.1
65.5 5.0
151.4 8.1
356.0 19.6
52.0 1.1
18.0 0.0
60.5 1.4
149.3 1.8
63.3 3.3*
22.7 0.7
64.7 2.9
129.8 4.4
336.0 9.8**
52.5 1.4
19.0 0.6
61.0 1.7
148.2 1.9
57.7 1.2
21.3 0.7
66.7 3.3
139.3 1.8
390.5 11.8
51.0 1.7
17.0 0.0
60.0 1.1
148.5 2.0
Cmpd 4
5
QRS (ms)
QT (ms)
QTc (ms)
Beats/min
PQ (ms)
59.3 0.7
137.5 1.3
374.5 14.7
52.0 1.1
18.0 0.0
61.0 1.7
146.2 2.0
Cmpd 5
5
QRS (ms)
QT (ms)
QTc (ms)
Beats/min
PQ (ms)
355.7 14.5
57.0 1.5
18.7 0.7
60.0 0.0
145.8 3.0
307.0 17.5
56.0 2.3
19.0 1.7
70.0 5.8
157.2 7.6
296.0 18.6*
58.0 2.0
19.3 0.7
67.3 2.7
148.3 2.7
308.7 10.5
57.3 1.4
18.7 0.7
63.3 3.3
131.1 2.5
Cmpd 6
5
QRS (ms)
QT (ms)
QTc (ms)
Beats/min
PQ (ms)
353.3 17.7
55.3 0.7
19.3 0.7
59.3 0.7
262.7 15.9*** 254.7 12.7***
265.7 7.3***
59.3 0.7*
21.3 1.3
62.7 1.3***
22.8 2.1
58.0 1.1**
22.7 2.7
66.7 6.7
138.4 7.7
Cmpd 7
5
QRS (ms)
QT (ms)
QTc (ms)
61.3 1.3
63.3 3.3
141.3 2.3
128.1 1.5
132.7 4.2
Beats/min
PQ (ms)
365.5 15.4
57.0 2.4
18.0 0.0
58.2 0.2
143.7 2.8
368.2 16.8
58.2 6.1
18.5 1.0
58.7 0.7
145.4 3.4
356.0 11.1
58.0 2.9
18.0 0.0
59.5 1.5
144.8 2.6
343.0 3.5
59.0 2.1
19.2 0.5
60.2 0.6
144.0 1.7
Cmpd 8
5
QRS (ms)
QT (ms)
QTc (ms)
The data are the means of 4-6 experiments S.E.M. Statistical analyses were performed using
b
c
d
a one-way ANOVA test, post hoc Dunnet test; aP<0.05; P<0.02; P<0.01; P<0.001
compared to the initial values
3.1.7. Prophylactic antiarrhythmic activity in adrenaline-induced arrhythmia
In the next stage the compounds were tested for their prophylactic antiarrhythmic
effect in the adrenaline-induced model of arrhythmia. In anesthetized rats intravenous
19

injection of adrenaline (20 µg/kg) caused ventricular extrasystoles in 100 % of the animals,
which led to the death of approximately 80 % of the animals. The studied compounds
administered 15 min i.v. prior to adrenaline reduced mortality but only compounds 3, 5 and 8
decreased the number of extrasystoles as well. Table 6 shows the preventive effect on
extrasystoles and the ED50 values (a dose producing a 50 % inhibition of premature
ventricular beats) obtained for the studied and reference compounds.
Table 6 Prophylactic antiarrhythmic activity in adrenaline-induced arrhythmia
Compound
ED50 i.v. (mg/kg)
1.63 (0.88 – 3.02)
2.20 (1.78 – 2.72)
4.82 (4.19 – 5.53)
0.36 (0.16 - 0.80) 27
1.05 (0.64 – 1.64) 28
3
5
8
carvedilol
propranolol
Each value was obtained from 3 experimental groups. The ED50 values and their confidence
limits were calculated according to the methods of Litchfield and Wilcoxon.
3.1.8. Anti-aggregation effect
As xanthone derivatives may exert protective effects in cardiovascular system in many
aspects we also tested the obtained structures for their anti-aggregation activity. Figure 6
shows that compounds 1 - 6 and 8 were able to attenuate aggregation induced with collagen
dose-dependently. The IC50 values ranged between 33 µM (compounds 3 and 5) and 88.4 µM
(compound 8), (Table 7). Compound 7 and carvedilol were not active.
Two compounds with the strongest antiplatelet effect (compound 3 and 5) were tested
additionally in aggregation test induced by 3 µM of adrenaline in the presence of a sub-
threshold concentration of collagen (0.9 µg/mL) (Figure 7). This aggregation was attenuated
with compounds 3 and 5 in a dose-dependent manner, however the obtained IC50 values were
higher than in the aggregation induced by collagen (Table 7). This suggests that adrenergic
mechanisms are not specific for the anti-aggregation effect produced by tested xanthone
derivatives.
20

Table 7. Potencies of studied and reference compounds in inhibiting whole rat blood
aggregation in vitro induced by collagen (1.6 µg/mL) and adrenaline and sub-threshold
concentration of collagen (3 µM + 0.9 µg/mL).
IC50 (µM)
Compound
Collagen
Collagen + adrenaline
nt
62.7 18.6
77.0 20.8
33.0 3.3
86.8 8.9
33.4 12.8
49.5 8.4
na
1
nt
2
82.3 6.9
3
nt
4
44.8 3.2
5
nt
nt
nt
na
6
7
8
64.1 11.4
na
carvedilol
IC50 (the concentration inhibiting the whole rat blood aggregation in vitro by 50%), nt – not
tested, na- not active up to 100 µM.
4. Discussion
In our work we described the synthesis of aminoisopropanoloxy derivatives of
xanthone and the evaluation of their pharmacological profile regarding the cardiovascular
system. On the basis of radioligand binding and functional studies we can state that the
synthesized group of xanthone derivatives revelaed sympatholytic activity. The exact activity
profile of these compounds coincided with the activity profile of the parent drugs. Therefore,
compounds 1, 3 (nadolol analogues containing t-butylamine moiety) and 4 are non-selective
beta blockers, whereas compound 7 (verapamil derivative with homoveratrilamine
substituent) is a beta-blocker with calcium channels blocking properties. Moreover, analogues
of carvedilol (5 and 6) acted as vasodilating beta-blockers, being α1 and β1/β2-adrenoceptor
antagonists. They possessed 2-(2-methoxyphenoxy)ethylamine in their structure, as their
parent drug. In the end compounds 2 and 8 may be classified as selective α1-adrenoceptors
antagonists. Taking into account their structure they comprised heterocyclic nitrogen cores as
commercially available α1-adrenoceptors antagonists such as prazosine, docsazosine,
urapidil. Overall observation was that compounds with heterocyclic nitrogen fragments
revealed affinity to α1-adrenoceptors, compounds with aliphatic amine were β-blockers,
whereas 2-methoxyphenyl as substituent predestined compounds to affinity to both types of
adrenoreceptores.
In the present study we tested the obtained aminoisopropanoloxy derivatives of
xanthone for hypotensive activity. All compounds apart from 1 caused a significant decrease
21

in blood pressure. In the case of compounds 2 and 8 it was the result of their α1-adrenoceptors
blocking properties. Compound 8 showed the strongest hypotensive properties (the lowest
active dose: 0.5 mg/kg), what is in line with the affinity for α1-adrenoceptors. Blockade of α1-
adrenoceptors prevents the binding of noradrenaline, what leads to vasodilation and reduced
peripheral resistance. α1-adrenoceptors antagonists are still valuable therapeutic option in the
treatment of hypertension, however the side effects of currently available α1-adrenolytics
concerning reflex tachycardia, postural hypotension, or dizziness make it necessary to develop
drugs with more favourable properties.29,30 Compounds classified as non-selective beta-
blockers (1, 4) did not show significant hypotensive activity, instead they cause a temporary
increase in blood pressure, however compound 3, the most potent β-blocker among the
presented group of xanthone derivatives, induced a fall in blood pressure. The pressor effect
of non-selective beta-blockers is associated with the increase in total peripheral resistance due
to their inhibitory action on vasodilatory β2-adrenoceptors.30,31 Besides the direct
hemodynamic effects beta-blockers are suggested to lower blood pressure due to the reduction
in renin secretion and the reduction in central sympathetic activity and vasomotor tone.30,31
Moreover, the effect on pre-junctional beta-receptors has also been postulated, leading to a
diminished norepinephrine release.30 Compound 7 contains homoveratrilamine, a structural
fragment of verapamil, revealed beta-blocking and calcium channels blocking properties, as
well as significant hypotensive activity. This was probably due to its calcium channels
blocking ability in addition to beta-blockade. Compound 7 in functional bioassay relaxed
aortic rings depolarized with K+ what suggests its Ca2+ channel blocking activity.
The hypotensive activity of compounds 5 and 6 may be related to their α1-
adrenoceptor blocking properties and vasorelaxant effect as well as β–blocking activity and
cardiac output reduction. The above mechanism of action is similar to that of carvedilol, and
allows to classify compounds 5 and 6 to beta-blockers with vasodilating properties.
Vasodilating beta-blockers as carvedilol are especially valuable agents in hypertension; they
reduce the heart rate and cardiac output, relax large arteries and produce vasodilatation of
small arteries.30 Moreover, vasodilating properties of beta-blockers contribute to the reversal
of arterial remodeling and lack of a worsening of glycemic and lipidic control which makes
them more favourable than non-vasodilating beta-blockers.32
Apart from antihypertensive, sympatholytics are antiarrhythmic agents. They inhibit
the chronotropic, inotropic, and vasoconstrictor responses to the catecholamines, and they are
especially valuable in atrial arrhythmias, ischemic heart disease and post-myocardial
infarction.33,34 In order to establish whether the compounds under consideration are potential
22

candidates for effective antiarrhythmic drugs, we assessed their protective antiarrhythmic
effect in the adrenaline-induced model of arrhythmia. We also evaluated their influence on
ECG components. All compounds reduced mortality in adrenaline-induced arrhythmia but
only the compounds 3, 5 and 8 diminished the number of extrasystoles as well. The activity of
compounds 3 and 5 was comparable to that of carvedilol or propranolol, whereas the activity
of compound 8 was weaker. Compounds 3 and 5 are xanthone analogues of known beta-
blockers carteolol, nadolol and carvedilol and revealed high affinity and antagonistic activity
for β- and α1/β-adrenoceptors respectively, what is responsible for their high antiarrhythmic
effect. The heart of rat contains comparable density of α1- and β-adrenoceptors and blockade
of α1-adrenoceptor contribute to calcium level stabilization and exert protective
antiarrhythmic effect.27,28 Therefore, not only beta-blockers but also α1-adrenolytics may have
potential in the treatment of arrhythmias. In our study compound 8, selective α1-adrenoceptor
blocker, revealed high antiarrhythmic activity.
Antiarrhythmic agents have proarrhythmic potential, thus we evaluated the influence
of studied compounds on ECG components. In vivo ECG study showed that the compounds,
apart from compound 4 and 7, marked no significant effect on PQ, QRS and QT intervals. In
addition, compounds 2 and 8, with α1-adrenolytic properties did not influence the heart rate,
what indicates that they not induce a reflex tachycardia, the side effect of α-adrenolytics, and
the limitation in their use. The rest of compounds with beta-blocking properties significantly
decreased heart rate proved that blockade of β-adrenoceptors resulted in the negative
chronotropic effect. This effect was less pronounced for compounds with α1/β-blocking
properties (compounds 5 and 6). α1-adrenoceptor blockade and vasodilatation reduce the
decrease in heart rhythm owing to the β-adrenoceptor blocking activity of the mentioned
compounds. However, in the case of compound 4 the obtained bradycardia due to β-
adrenoceptor blockade was even accompanied with PQ interval prolongation. Similar
observations were made in the case of compound 7, negative chronotropic effect was
accompanied with PQ interval prolongation. Beta-blockers at high doses, and calcium
channels blockers are known to increase atrioventricular conduction time, what manifests in
PQ interval prolongation in ECG record.35 Compound 7 in addition to beta-blockade possess
calcium channels blocking activity what explains the observed changes in ECG record.
None of the compound prolonged the QT interval or alter QRS complex, what suggests that
they did not influence ventricular depolarization and repolarization, therefore we can assume
that they did not have proarrhythmic potential at tested doses.
23

Xanthone derivatives have been reported to possess antiplatelet activity.36 In our study
the tested substances (except compound 7) counteracted the aggregation induced by natural
aggregation agonist – collagen. Moreover, their anti-aggregation effect is probably not due to
adrenoceptor blocking properties as the inhibition of adrenaline potentiated-, collagen induced
aggregation required higher concentration of studied compounds when compared to collagen
induced aggregation.
In conclusion, the results of this study is that new compounds with hypotensive and
antiarrhythmic properties have been found in the group of xanthone derivatives. Their
cardiovascular activity is due to α1- (compound 2 and 8), α1/β- (compounds 5 - 6), β
(compounds 1, 3, 4) and β-adrenoceptor blocking properties and calcium channel blocking
activity (compound 7). Moreover, they did not affect QRS and QT intervals. We were
wondering if changing of the aromatic rings to xanthone skeleton in the structure of known
cardiovascular drugs would change their effectiveness. Based on our experiments, we can
state that the presence of the xanthone core slightly decreases hypotensive and/or
antiarrhythmic activity relative to the parent compounds. On the other hand, studied
xanthones revealed additional anti-aggregation activity of which e.g. carvedilol was deprived
of. In this light, our new xanthone derivatives can be taken into consideration as potential
cardioprotective substances.
Acnowledgements
The study was supported by the research grants of the Ministry of Science and Higher
Education (K/DSC/004307, K/ZDS/005487 and K/ZDS/006235).
Bibliography
1.
Da Re P, Mancini V, Tòth E, Cima L. Xanthone derivatives with centrally stimulating
and analeptic activities. Arzneimittelforschung. 1968;18(6):718-720.
Pfister JR, Ferraresi RW, Harrison IT, et al. Xanthone-2-carboxylic acids, a new series
of antiallergic substances. J Med Chem. 1972;15(10):1032-1035.
2.
3.
Wang Q, Ma C, Ma Y, Li X, Chen Y, Chen J. Structure–activity relationships of
diverse xanthones against multidrug resistant human tumor cells. Bioorg Med Chem
Lett. 2017;27(3):447-449.
4.
Liu C, Zhang M, Zhang Z, et al. Synthesis and anticancer potential of novel xanthone
derivatives with 3,6-substituted chains. Bioorganic Med Chem. 2016;24(18):4263-
24

4271.
5.
6.
7.
8.
9.
Santos CMM, Freitas M, Ribeiro D, et al. 2,3-Diarylxanthones as strong scavengers of
reactive oxygen and nitrogen species: A structure-activity relationship study. Bioorg
Med Chem. 2010;18(18):6776-6784.
Pickert M, Schaper KJ, Frahm a W. Substituted xanthones as antimycobacterial
agents, Part 2: Antimycobacterial activity. Arch Pharm (Weinheim). 1998;331(5):193-
197.
Kelly JX, Winter R, Peyton DH, Hinrichs DJ, Riscoe M. Optimization of Xanthones
for Antimalarial Activityꢀ: the 3 , 6-Bis- -Diethylaminoalkoxyxanthone Series.
Antimicrob Agents Chemother. 2002;46(1):144-150.
Wang L-W, Kang J-J, Chen I-J, Teng C-M, Lin C-N. Antihypertensive and
vasorelaxing activities of synthetic xanthone derivatives. Bioorg Med Chem.
2002;10(3):567-572.
Szkaradek N, Stachura K, Waszkielewicz AM, Cegła M, Szneler E, Marona H.
Synthesis and antimycobacterial assay of some xanthone derivatives. Acta Pol Pharm.
2008;65(1):21-28.
10. Szkaradek N, Gunia A, Waszkielewicz AM, et al. Anticonvulsant evaluation of
aminoalkanol derivatives of 2- and 4-methylxanthone. Bioorganic Med Chem.
2013;21(5):1190-1198.
11. Klesiewicz K, Karczewska E, Budak A, Marona H, Szkaradek N. Anti-Helicobacter
pylori activity of some newly synthesized derivatives of xanthone. J Antibiot (Tokyo).
2016;69(11):825-834.
12. Szkaradek N, Sypniewski D, Waszkielewicz AM, et al. Synthesis and in vitro
evaluation of anticancer potential of new aminoalkanol derivatives of xanthone.
Anticancer Agents Med Chem. 2016;16(12):1587-1604.
13. Szkaradek N, Rapacz A, Pytka K, Filipek B, Siwek A, Cegła M. Synthesis and
preliminary evaluation of pharmacological properties of some piperazine derivatives of
xanthone. Bioorg Med Chem. 2013;21:514-522.
14. Schlicker E, Pędzińska-Betiuk A, Kozłowska H, et al. MH-3: Evidence for non-
competitive antagonism towards the low-affinity site of β1-adrenoceptors. Naunyn
Schmiedebergs Arch Pharmacol. 2014;387(8):743-752.
15. Szkaradek N, Rapacz A, Pytka K, et al. Cardiovascular activity of the chiral xanthone
derivatives. Bioorganic Med Chem. 2015;23(20):6714-6724.
16. Liu J, Zhang J, Wang H, et al. Synthesis of xanthone derivatives and studies on the
25

inhibition against cancer cells growth and synergistic combinations of them. Eur J Med
Chem. 2017;133:50-61.
17. Marona H, Szkaradek N, Kubacka M, et al. Synthesis and evaluation of some xanthone
derivatives for anti-arrhythmic, hypotensive properties and their affinity for adrenergic
receptors. Arch Pharm (Weinheim). 2008;341(2):90-98.
18. Da Re, P.; Primofiore, G. P.; Bertelli A. β-Adrenergic blocking agents of the chromone
and xanthone groups. II. Propanolol type derivatives. J Med Chem. 1972;115(8):868-
869.
19. Crowther AF, Howe R, McLoughlin BJ, et al. β-Adrenergic blocking agents. 12.
Heterocyclic compounds related to propranolol. J Med Chem. 1972;15(3):260-266.
20. Marona H, Kubacka M, Filipek B, et al. Synthesis, α-adrenoceptors affinity and α 1-
adrenoceptor antagonistic properties of some 1,4-substituted piperazine derivatives.
Pharmazie. 2011;66(10):733-739.
21. Chulia S, Moreau J, Naline E, et al. The effect of S- ( + ) -boldine guinea-pig aorta on
the alpha1-adrenoceptor of the guinea-pig aorta. Br J Pharmacol. 1996;119:1305-1312.
22. Arunlakshana O, Schild HO. Some quantitative uses of drug antagonists. Br J
Pharmacol. 1959;14(1):48-58.
23. De Clerck F, Van de Water A, D’Aubioul J, et al. In vivo measurement of QT
prolongation, dispersion and arrhythmogenesis: application to the preclinical
cardiovascular safety pharmacology of a new chemical entity. Fundam Clin
Pharmacol. 2002;16(2):125-140.
24. Litchfield JT, Wilcoxon F. A simplified method of evaluating dose-effect experiments.
J Pharmacol Exp Ther. 1949;96(2):99-113.
25. Lowe MD, Lynham J a, Grace A a, Kaumann AJ. Comparison of the affinity of beta-
blockers for two states of the beta 1-adrenoceptor in ferret ventricular myocardium. Br
J Pharmacol. 2002;135(2):451-461.
26. Rapacz A, Sapa J, Bednarski M, Filipek B. Antiarrhythmic activity of some xanthone
derivatives with β1 -adrenoceptor affinities in rats. Eur J Pharmacol. 2014;738:14-21.
27. Pytka K, Lustyk K, Zmudzka E, et al. Chemically homogenous compounds with
antagonistic properties at all α1-adrenoceptor subtypes but not β1-adrenoceptor
attenuate adrenaline-induced arrhythmia in rats. Front Pharmacol. 2016;7(AUG):1-14.
28. Kubacka M, Mogilski S, Filipek B, Marona H. Antiarrhythmic properties of some1,4-
disubstituted piperazine derivatives with α1 -adrenoceptor affinities. Eur J Pharmacol.
2013;720:237-246.
26

29. Kubacka M, Mogilski S, Filipek B, Marona H. The hypotensive activity and alpha1-
adrenoceptor antagonistic properties of some aroxyalkyl derivatives of 2-
methoxyphenylpiperazine. Eur J Pharmacol. 2013;698(1-3):335-344.
30. Laurent S. Antihypertensive drugs. Pharmacol Res. 2017;124:116-125.
31. Prichard BNC, Cruickshank JM, Graham BR. Beta-adrenergic blocking drugs in the
treatment of hypertension. Blood Press. 2001;10(5-6):366-386.
32. Ram CVS. Beta-blockers in hypertension. Am J Cardiol. 2010;106(12):1819-1828.
33. Messerli FH, Bangalore S, Yao SS, Steinberg JS. Cardioprotection with beta-blockers:
Myths, facts and Pascal’s wager. J Intern Med. 2009;266(3):232-241.
34. Stroe AF, Gheorghiade M. Carvedilol: beta-blockade and beyond. Rev Cardiovasc
Med. 2004;5 Suppl 1:18-27.
35. Merentie M, Lipponen JA, Hedman M, et al. Mouse ECG findings in aging, with
conduction system affecting drugs and in cardiac pathologies: Development and
validation of ECG analysis algorithm in mice. Physiol Rep. 2015;3(12):e12639 1-13.
36. Lin K-W, Fang S-C, Hung C-F, et al. Synthesis, antiplatelet and vasorelaxing activities
of xanthone derivatives. Arch Pharm (Weinheim). 2009;342(1):19-26.
27

Fig. 1.

Fig. 2