Design, synthesis, and anticonvulsant activity of some derivatives of xanthone with aminoalkanol moieties

Article abstract of DOI:10.1111/cbdd.12842

A series of new xanthone derivatives have been synthesized and evaluated for their anticonvulsant properties in the maximal electroshock, subcutaneous metrazole tests and for neurotoxicity in the rotarod in mice, i.p. and rats, p.o. Compound 9: R,S-2-{2-[(1-hydroxybutan-2-yl]amino)ethoxy}-9H-xanthen-9-one and compound 12: R,S-2-{3-[(1-hydroxybutan-2-yl)amino]propoxy}-9H-xanthen-9-one exerted activity in rats, p.o. 2 and 4?h after administration, respectively. Therefore, metabolic stability of the compounds was evaluated with use of rat microsomes, resulting in half-life t_1/2 136 and 108?min, respectively, indicating that either the metabolites are very active or the parent compounds exert ADME properties other than metabolism which influence the late onset of activity.

Full text of DOI:10.1111/cbdd.12842

Received Date : 24-May-2016
Revised Date : 01-Jul-2016
Accepted Date : 06-Aug-2016
Article type : Research Article
Design, Synthesis and Anticonvulsant Activity of Some Derivatives of Xanthone with
Aminoalkanol Moieties
Anna M. Waszkielewicz1*, Karolina Słoczyńska2, Elżbieta Pękala2, Paweł Żmudzki3,Agata
Siwek4, Anna Gryboś4, and Henryk Marona1
1 Department of Bioorganic Chemistry, Chair of Organic Chemistry, Faculty of Pharmacy,
Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland
2 Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Jagiellonian University
Medical College, Medyczna 9, 30-688 Krakow, Poland
3 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jagiellonian University
Medical College, Medyczna 9, 30-688 Krakow, Poland
4 Department of Pharmacobiology, Faculty of Pharmacy, Jagiellonian University Medical
College, Medyczna 9, 30-688 Krakow
* corresponding author: awaszkie@cm-uj.krakow.pl
Abstract
A series of new xanthone derivatives have been synthesized and evaluated for their
anticonvulsant properties in the maximal electroshock (MES), subcutaneous metrazole
(ScMet) tests as well as for neurotoxicity in the rotarod (TOX) in mice, i.p. and rats, p.o.
Compound 9: R,S-2-{2-[(1-hydroxybutan-2-yl]amino)ethoxy}-9H-xanthen-9-one and
compound 12: R,S-2-{3-[(1-hydroxybutan-2-yl)amino]propoxy}-9H-xanthen-9-one exerted
activity in rats, p.o. 2 and 4 h after administration, respectively. Therefore, metabolic stability
of the compounds was evaluated with use of rats microsomes, resulting in half-life t1/2 136
and 108 min, respectively, indicating that either the metabolites are very active, or the parent
compounds exert other ADME properties other than metabolism which influence the late
onset of activity.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.12842
This article is protected by copyright. All rights reserved.

Keywords: aminoalkanols, MES, seizures, status epilepticus, metabolic stability, rat
microsomes, xanthone, 9H-xanthen-9-one
Epilepsy constitutes a set of neurological disorders which are characterized by recurrent
seizures. Its incidence in overall population reaches 50/100 000/year, prevalence is in the
range 5-10/1000 and remission is observed in 70% of cases. Moreover, epilepsy carries an
increased risk of premature death especially in patients with chronic epilepsy. In spite of
large progress in pharmacotherapy, 30% of patients experience pharmacoresistant seizures
(1). According to the above, premises for drug discovery efforts in this field exist.
One of the premises for search of new drugs in the group of aminoalkanol derivatives is
activity of cardiovascular drugs on the central nervous system in terms of stabilizing cell
membrane potential and thus preventing seizures. Among such drugs one can find
propranolol, which is currently used for some seizures in children (2). On the other hand, the
antiarrhythmic drug mexiletine, prevents seizures, but simultaneously lowers seizure
threshold and increases patients’ susceptibility towards seizures (3, 4). It is worth mentioning
that unlike propranolol, the structure of mexiletine constitutes aroxyalkylamine and it does
not contain aminoalkanol moiety. The properties of these two drugs combined have been
premises for our interest in aroxyalkylaminoalkanols in terms of potential drug candidates for
the treatment of seizures (5).
Aminoalkanol derivatives and their anticonvulsant activity have been subject of our research
for many years, with variously substituted aroxyalkyl or aroxyacyl moiety. Such aroxy group
formerly constituted 2,3-dimethyl-, 2,4-dimethyl, 2,5-dimethyl- 2,6-dimethyl-, as well as 2-
chloro-5-methyl-, 4-chloro-2-methyl, 4-chloro-3-methyl, or 2-chloro-6-methylphenol (6-9).
Among the published results, the most active compounds were found (R)-2-{[2-(2,6-
dimethylphenoxy)ethyl]amino}propan-1-ol
(I)
(6)
and
(S)-2-{[2-(2,6-
dimethylphenoxy)ethyl]amino}butan-1-ol hydrochloride (II) (Fig. 1) (10).
We also have published derivatives of xanthone exhibiting anticonvulsant activity in maximal
electroshock MES seizures in mice or rats (11), with aminoalkanol moieties such as R,S-6-
chloro-2-{[(1-hydroxypropan-2-yl)amino]methyl}-9H-xanthen-9-one (III) (12), (S)-6-chloro-
2-{[(1-hydroxybutan-2-yl)amino]methyl}-9H-xanthen-9-one (IV) (12), as well as without
aminoalkanol moieties, such as 2-[(4-benzylpiperazin-1-yl)methyl]-6-methoxy-9H-xanthen-
9-one (V) (Fig. 1) (13). So far, according to our experience, position of substituents in the
This article is protected by copyright. All rights reserved.

xanthone rings plays crucial role and the most active compounds III-V contain substituents at
positions C2 and C6 of the xanthone structure. Since propranolol contains vast naphthoxy
moiety, the use of xanthonoxy moiety as aroxy part of the structures was the next step of
structure design within our research.
Due to the above premises, the aim of this study was to synthesize and perform
pharmacological examination of anticonvulsant activity of aroxyalkylaminoalkanol
derivatives, where aroxy moiety constituted xanthonoxy group, and the position of oxygen
substituent in the xanthone ring is C2, C3, or C4, expecting that position C2 might be
favorable.
During the research, in case of compounds 9 and 12 administered to rats, where activity
appeared 2 and 4 h after administration, respectively, the pharmacological results drew our
attention also to possible activity of a metabolite. Therefore, as a consequence we examined
potential metabolism of the compounds with use of rat microsomes.
During our former studies we showed some binding to serotonergic receptors for xanthone
derivatives exhibiting anticonvulsant activity (13), therefore we evaluated this mechanism of
action for a promising compounds 8a, 9, and 12.
Methods and Materials
Chemistry
The aminoalkanols used for synthesis of compounds 8-10, 12-13, 15, and 17 were racemic
(Table 1). 1-Aminobutan-2-ol was achieved according to formerly published procedures (10).
Other reagents were purchased from Alfa Aesar GmbH&Co KG (Karlsruhe, Germany) and
Merck Sp. z o.o. (Warszawa, Poland) and solvents were commercially available materials of
reagent grade.
Melting points (mp) were determined using a Büchi SMP-20 apparatus and are uncorrected.
Analyses of C, H, N were within 0.4% of the theoretical values. Analytical TLC was
carried out on precoated plates (silica gel, 60 F-254 Merck). Spots were visualized with UV
light.
The theoretical values of partition coefficient LogP was calculated with programs
ChemBioDraw Ultra 12.0 (CambridgeSoft) and with Molinspiration online toolkit (14) for
This article is protected by copyright. All rights reserved.

base forms (Table 1). 1H NMR spectra for compounds 1-2, 4-7, 11-12, and 16 were recorded
at Faculty of Pharmacy, Jagiellonian University Medical College (Krakow, Poland) with a
Varian Mercury-VX 300 NMR spectrometer at 29˚C. Chemical shifts were referenced against
solvent lock signal. Standard Varian pulse sequences were used for 2D experiments. 1H NMR
and 13C NMR spectra for compounds 3, 8-10, 13-15, 16a, and 17 were recorded at Faculty of
Chemistry, Jagiellonian University (Krakow, Poland), on a Bruker AVANCE III 600
1
13
(resonance frequencies 600.20 MHz for H and 150.94 MHz for C) equipped a 5-mm
1
probehead: PABBO with z-gradient or TBI with XYZ gradients. The H spectra were
recorded with 16 scans, 1 s relaxation delay, 4 s acquisition time, 128 kW FID size, with
13
1
16234 Hz spectral width. The C spectra were recorded with WALTZ-16 H broadband
decoupling, a few thousands scans, 2 s relaxation delay, 0.9 s acquisition time, 64 kW FID
size, 36 057 Hz spectral width. Standard pulse sequences from Bruker library were used for
2D spectra. Gradient enhanced sequences were used for the homo- and heteronuclear 2D
experiments. All processing and analysis were performed using Bruker's TopSpin 3.0 software suite.
Results are presented in the following format: chemical shift δ (ppm), multiplicity, J values in
Hertz (Hz), number of protons, protons’ position. Multiplicities are showed as the
abbreviations: s (singlet), bs (broad singlet), d (doublet), dd (doublet of doublets), ddd
(doublet of doublets of doublets), t (triplet), qn (quintet), m (multiplet).
The IR spectra were recorded on a Jasco FT/IR 410 spectrometer (KBr pellets). 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, 1.7 lm, 2.1 x 100 mm column. A flow rate of
0.3 mL/min and a gradient of (5–95)% B over 10 min and then 100% B over 2 min was used.
Eluent A: water/0.1% HCO2H; eluent B: acetonitrile/0.1% HCO2H. LC/MS data were
obtained by scanning the first quadrupole in 0.5 s in a mass range from 50 to 1000 Da; eight
scans were summed up to produce the final spectrum.
The title 2N-[(xanthonoxy)alkyl]aminoalkan-1-ols are presented in Table 1.
This article is protected by copyright. All rights reserved.

General procedures for preparation of the starting materials
The following starting materials have been achieved according to formerly published
procedures (15) (Scheme 1) and their properties were published as well: 2-(2-
hydroxyethoxy)-9H-xanthen-9-one and 4-(2-hydroxyethoxy)-9H-xanthen-9-one (15), 2-(2-
chloroethoxy)-9H-xanthen-9-one (16), 2-(3-hydroxypropoxy)-9H-xanthen-9-one (17), 4-(3-
hydroxypropoxy)-9H-xanthen-9-one (18).
2-Hydroxyxanthone was obtained by Ullmann’s (19) condensation of o-chlorobenzoic acid
with p-methoxyphenol in the form of sodium salts (catalytic amount of Cu/Cu2O) in liquid
paraffin (ca. 210 °C). The obtained 2-carboxy-4’-methoxydifenyl ether was subject to
cyclization and demethylation with 80% H2SO4 (10-fold excess) for 3 h on a boiling water
bath. After pouring into ice, filtration and washing with water and 10% NaHCO3, the crude
product was dissolved in 10% NaOH, and the dissolved product was 2-hydroxy-9H-xanthen-
9-one (yield 85-90%). Some remaining portion of 2-methoxy-9H-xanthen-9-one which was
not soluble in NaOH, was subject to demethylation with 80% H2SO4. The final product was
recrystallized from xylene resulting in white powder, mp 236-238 °C (235-236 °C (20).
The same method was used for achievement of 4-hydroxy-9H-xanthen-9-one using o-
chlorobenzoic acid and o-methoxyphenol as starting materials. The resulting product was
recrystallized from xylene and achieved as white powder, mp 239-241 °C (230-233 °C (21),
234-236 °C (22).
3-Hydroxy-9H-xanthen-9-one (23) necessary for synthesis of 3 was obtained by methanolysis
of 3-chloro-9H-xanthen-9-one to 3-methoxy-9H-xanthen-9-one and subsequent
demethylation of the product using 80% H2SO4. The product was recrystallized from xylene
resulting in white powder, mp 238-240 °C (241-242 °C (24)).
The starting materials 1-2, 4, 6-7 were achieved according to two methods A and B. In
method A, reaction of appropriate 2-, 3-, or 4-hydroxy-9H-xanthen-9-one with chloroalkanol
was performed (resulting in e.g. 3) (16), and the product was subject to chlorination using
SOCl2. Alternative method B constitutes reaction between respective 2-, 3-, or 4-hydroxy-
9H-xanthen-9-one and 2-bromo-1-chloroethane or 3-bromo-1-chloropropane (Scheme 1).
Method A
2-(2-Chloroethoxy)-9H-xanthen-9-one (1) was obtained from 2-(2-hydroxyethoxy)-9H-
xanthen-9-one (mp 151-153 °C) (15). The first step was the reaction between 2-
This article is protected by copyright. All rights reserved.

hydroxyxanthone with redistilled 2-chloroethanol in acetone in the presence of anhydrous
K2CO3 and catalytic amount of TEBA and KI. The mixture was refluxed for 48 h and filtered
hot. Then the solvent was evaporated. Water and 5% NaOH was added to the residue and the
mixture was stirred. The insoluble precipitate was filtered off and washed with water. The
resulting solid was recrystallized from EtOH. The next step was chlorination with SOCl2 in
toluene under reflux. The crystallization of the product was performed with use of EtOH or n-
hexene/toluene 3:1.
Additionally, 3-(2-bromoethoxy)-9H-xanthen-9-one (5) was achieved by bromination of 3-
(2-hydroxyethoxy)-9H-xanthen-9-one (3) with PBr3 in CHCl3 solution as an alternative
alkylating agent to 1. After distillation of solvent and washing with water, crystallization was
performed from EtOH.
Method B
2-(2-Chloroethoxy)-9H-xanthen-9-one (1) was obtained also alternatively by condensation of
2-hydroxy-9H-xanthen-9-one with 2-bromo-1-chloroethane in acetone in the presence of
anhydrous K2CO3 and catalytic amount of TEBA. The mixture was refluxed for 24 h and
filtered hot. Then the solvent was evaporated to dryness. Water and 5% NaOH was added to
the residue and the mixture was stirred. The insoluble precipitate was filtered off and washed
with water. The product was crystallized with use of EtOH or n-hexane/toluene 1:1 and was
used for synthesis of compounds 8-10.
The same methods were used for the formation of compounds 2, 4, and 6-7 and in case of 3
the compound was result of the condensation of 3-hydroxy-9H-xanthen-9-one with 2-
chloroethanol. The product was crystallized with use of EtOH or n-hexane/toluene 1:1.
General procedures for preparation of the title compounds
Compounds 8-17 were obtained by aminolysis of appropriate compounds 1-7 in toluene in
the presence of K2CO3, according to previously published procedures (25) or in DMF in
presence of TEA and catalytic amount of KI. The achieved bases were recrystallized from n-
heptane/toluene (3:1). Some portions of amines were converted to hydrochlorides with use of
EtOH saturated with gaseous HCl in EtOH solution and the achieved products were
recrystallized from mixture acetone/EtOH 1:3, resulting in compounds 8a-9a, and 14a-17a.
This article is protected by copyright. All rights reserved.

2-(2-chloroethoxy)-9H-xanthen-9-one (1)
C15H11O3Cl; M=274.70; mp 180-182 °C; Rf=0.89 (CH3OH/ethyl acetate 1/1);
0.00
65.58
4.04
Ncalc/found
=
/
0.00, Ccalc/found
=
/
65.43, Hcalc/found
=
/
3.90; 1H NMR (CDCl3): δ (ppm) 8.34 (dd,
J=8.2, J=1.8, 1H, Ar-H6), 7.76-7.68 (m, 2H, Ar-H5, Ar-H8), 7.52-7.44 (m, 2H, Ar-H7, Ar-
H3), 7.42-7.34 (m, 2H, Ar-H4, Ar-H1), 4.39-4.33 (m, 2H, -O-CH2-), 3.87 (t, J=5.9, 2H, -
CH2-Cl); LC-MS [M+H]+ m/z: 275.18, 100%.
2-(3-chloropropoxy)-9H-xanthen-9-one (2)
C16H13O3Cl; M=288.73; mp 136-138 °C; Rf=0.91 (CH3OH/ethyl acetate 1/1);
0.00
66.56
4.54
Ncalc/found
=
/
0.00, Ccalc/found
=
/
66.89, Hcalc/found
=
/
4.45; 1H NMR (CDCl3): δ (ppm) 8.35 (dd,
J=8.1, J=1.7, 1H, Ar-H6), 7.75-7.68 (m, 2H, Ar-H5, Ar-H8), 7.51-7.30 (m, 4H, Ar-H7, Ar-
H3, Ar-H4, Ar-H1), 4.24 (t, J=5.9, 2H, -O-CH2-), 3.77 (t, J=6.4, 2H, -CH2-Cl), 2.29 (qn,
J=6.1, 2H, -CH2-CH2-CH2); LC-MS [M+H]+ m/z: 289.09, 98.43%.
3-(2-hydroxyethoxy)-9H-xanthen-9-one (3)
C15H12O4; M=256.26; mp 158-160 °C (mp 236-238 °C (26)); Rf=0.84 (CH3OH/ethyl acetate
0.00
70.31
4.72
1/1); Ncalc/found
=
/
0.00, Ccalc/found
=
/
70.52, Hcalc/found
=
/
4.68; 1H NMR (DMSO-d6): δ (ppm)
8.17 (ddd, J=7.9, J=1.7, J=0.4, 1H, H-8), 8.10 (d, J=8.9, 1H, H-1), 7.85 (ddd, J=8.5, J=7.1,
J=1.7, 1H, H-6), 7.62 (ddd, J=8.5, J=1.0, J=0.4, 1H, H-5), 7.47 (ddd, J=7.9, J=7.1, J=1.0,
1H, H-7), 7.15 (d, J=2.4, 1H, H-4), 7.06 (dd, J=8.9, J=2.4, 1H, H-2), 4.97 (bs, 1H, -OH), 4.19
13
(t, J=4.8, 2H, Ar-O-CH2-), 3.78 (t, J=4.8, 2H, -CH2-OH); C NMR: 174.51 (C=O), 164.09
(C3), 157.16 (C4a), 155.25 (C4b), 134.65 (C6), 127.20 (C1), 125.51 (C8), 123.94 (C7),
120.86 (C8a), 117.53 (C5), 114.50 (C8b), 113.63 (C2), 100.68 (C4), 70,22 (Ar-O-CH2-),
58,95 (-CH2-OH); LC-MS [M+H]+ m/z: 257.24, 97.88%.
3-(2-chloroethoxy)-9H-xanthen-9-one (4)
C15H11O3Cl; M=274.70; mp 163-164 °C; Rf=0.88 (CH3OH/ethyl acetate 1/1);
65.59
4.04
0.00
Ccalc/found
=
/
65.88; Hcalc/found
=
/
3.95; N calc
/
=
/
0.00; 1H NMR (CDCl3): δ (ppm) 8.31 (dd,
found
J=7.6, J=1.8, 1H, Ar-H6), 8.25 (d, J=8.8, 1H, Ar-H1), 7.73-7.65 (m, 1H, Ar-H8), 7.44 (d,
J=8.2, 1H, Ar-H5), 7.40-7.33 (m, 1H, Ar-H7), 6.95 (dd, J=8.8, J=2.3, 1H, Ar-H2), 6.87 (d,
J=2.3, 1H, Ar-H4), 4.34 (t, J=5.9, 2H, -O-CH2-), 3.87 (t, J=5.9, 2H, -CH2-Cl); LC-MS
[M+H]+ m/z: 275.18, 100%.
This article is protected by copyright. All rights reserved.

3-(2-bromoethoxy)-9H-xanthen-9-one (5)
1
C15H11O3Br, M=319.15, mp 172-174 °C; Rf=0.84 (CH3OH/ethyl acetate 1/1); H NMR
(CDCl3): δ (ppm) 8.32 (dd, J=8.2, 1.8 Hz, 1H, Ar-H6), 8.29 - 8.24 (m, 1H, Ar-H1), 7.73 -
7.65 (m, 1H, Ar-H8), 7.45 (d, J=8.2 Hz, 1H, Ar-H5), 7.41 - 7.33 (m, 1H, Ar-H7), 6.96 (dd,
J=9.1, 2.6 Hz, 1H, Ar-H2), 6.89 (d, J=2.3 Hz, 1H, Ar-H4), 4.45 - 4.38 (m, 2H, Ar-O-CH2-),
3.70 (t, J=6.2 Hz, 2H, -CH2-Br); LC-MS [M+H]+ m/z: 320.99, 96.78%.
4-(2-chloroethoxy)-9H-xanthen-9-one (6)
C15H11O3Cl; M=274.70; mp 149-150 °C (no mp (16)); Rf=0.91 (CH3OH/ethyl acetate 1/1);
0.00
65.59
4.04
Ncalc/found
=
/
0.00, Ccalc/found
=
/
65.39, Hcalc/found
=
/
3.91; 1H NMR (CDCl3): δ (ppm) 8.33 (dd,
J=8.0, J=1.8, 1H, Ar-H6), 8.00-7.93 (m, 1H, Ar-H8), 7.78-7.70 (m, 1H, Ar-H5), 7.59 (dd,
J=8.5, J=0.5, 1H, Ar-H2), 7.43-7.35 (m, 1H, Ar-H1), 7.30-7.25 (m, 2H, Ar-H3, Ar-H8), 4.43
(t, J=5.9, 2H, -O-CH2-), 3.95 (t, J=5.9, 2H, -CH2-Cl); LC-MS [M+H]+ m/z: 275.25, 100%.
4-(3-chloropropoxy)-9H-xanthen-9-one (7)
C16H13O3Cl; M=288.73; mp 117-119 °C; Rf=0.88 (CH3OH/ethyl acetate 1/1);
0.00
66.56
4.54
Ncalc/found
=
/
0.00, Ccalc/found
=
/
66.93, Hcalc/found
=
/
4.40; 1H NMR (CDCl3): δ (ppm) 8.34 (dd,
J=7.6, J=1.8, 1H, Ar-H6), 7.96-7.89 (m, 1H, Ar-H8), 7.77-7.69 (m, 1H, Ar-H5), 7.60-7.55
(m, 1H, Ar-H1), 7.42-7.35 (m, 1H, Ar-H2), 7.30-7.25 (m, 2H, Ar-H3, Ar-H7), 4.32 (t, J=5.9,
2H, Ar-O-CH2-), 3.92-3.85 (m, 2H, -CH2-Cl), 2.39 (quin, J=6.0, 2H, -CH2-CH2-CH2-); LC-
MS [M+H]+ m/z: 289.21, 100%.
The title compounds
R,S-2-{2-[(2-hydroxybutyl)amino]ethoxy}-9H-xanthen-9-one hydrochloride (8a)
C19H22NO4Cl; M=363.84; mp 216-218 °C (8 - base mp 86-88 °C); base Rf=0.90
4.28
(CH3OH/ethyl
acetate
1/1);
(base C19H21NO4;
M=327.38
Ncalc/found
=
/
,
3.93
69.70
6.47
1
+
Ccalc/found
=
/
69.54, Hcalc/found
=
/
6.12); H NMR (DMSO-d6): δ (ppm) 9.00 (bs, 2H, NH2 ),
8.21 (ddd, J=8.0, J=1.7, J=0.5, 1H, H-8), 7.89 (ddd, J=8.5, J=7.1, J=1.7, 1H, H-6), 7.71 (dd,
J=9.1, J=0.4, 1H, H-4), 7.68 (ddd, J=8.5, J=1.0, 0.5, 1H, H-5), 7.65 (dd, J=3.1, J=0.4, 1H,
H-1), 7.60 (dd, J=9.4, J=3.1, 1H, H-3), 7.49 (ddd, J=8.0, J=7.1, J=1.0, 1H, H-7), 5.36 (d,
J=5.2, 1H, -OH), 4.45 (ddd, J=11.0, J=5.2, J=5.0, 2H, Ar-O-CH2-), 3.81-3.75 (m, 1H, >CH-
), 3.43 (dd, J=5.2, J=5.0, 2H, -CH2-N), 3.13 (dd, J=12.8, J=3.0, 1H, N-CHH-CH), 2.88 (dd,
This article is protected by copyright. All rights reserved.

J=12.8, J=9.7, 1H, N-CHH-CH), 1.51-1.36 (m, 2H, -CH2-CH3), 0.91 (t, J=7.4, 3H, -CH3);
13C NMR: 175.68 (C=O), 155.44 (C-4b), 154.11 (C-2), 150.50 (C4a), 135.42 (C-6), 125.85
(C-8), 124.89 (C-3), 124.23 (C-7), 121.39 (C-8b), 120.38 (C-8a), 119.86 (C-4), 118.12 (C-5),
107.19 (C-1), 67.02 (>CH-), 63.82 (Ar-O-CH2-), 52.22 (-N-CH2-CH<), 45.82 (-CH2-N),
27.54 (-CH2- (Et)), 9.48 (-CH3); LC-MS [M+H]+ m/z: 328.36, 100%.
R,S-2-{2-[(1-hydroxybutan-2-yl]amino)ethoxy}-9H-xanthen-9-one (9)
C19H21NO4; M=327.38; mp 105-106 °C (9a – hydrochloride mp 211-213 °C); Rf=0.39
1
(CH3OH/ethyl acetate 1/1); H NMR (DMSO-d6): δ (ppm) 8.20 (dd, J=8.0; J=1.7, 1H, H-8);
7.87 (ddd, J=8.7; J=7.1; J= 1.7, 1H, H-6); 7.65 (dd, J=8.7; J=1.0, 1H, H-5); 7.64 (d, J=9.1,
1H, H-4); 7.57 (d, J=3.1, 1H, H-1); 7.49 (dd, J=9.1; J=3.1, 1H, H-3); 7.48 (ddd, J=8.0; J=7.1;
J=1.0, 1H, H-7); 4.45 (bs, 1H, -OH); 4.17-4.10 (m, 2H, Ar-O-CH2-); 3.40 (dd, J=10.8; J=
4.5, 1H, -CHH-OH); 3.28 (dd, J=10.8; J=6.3, 1H, -CHH-OH); 2.95 (t, J=5.7, 2H, -CH2-N);
2.49-2.43 (m, 1H, >CH-); 1.83 (bs, 1H, -NH-); 1.44-1.31 (m, 2H, -CH2- (Et)); 0.86 (t, J=7.5,
3H, -CH3); 13C NMR: 175.66 (C=O); 155.42 (C-4b); 154.98 (C-2); 150.13 (C4a); 135.25 (C-
6); 125.83 (C-8); 124.86 (C-3); 124.09 (C-7); 121.40 (C-8b); 120.39 (C-8a); 119.68 (C-4);
118.07 (C-5); 106.50 (C-1); 68.64 (Ar-O-CH2-); 62.64 (-CH2-OH); 60.01 (>CH); 45.46 (-
CH2-N); 23.55 (-CH2- (Et)); 9.92 (-CH3); LC-MS [M+H]+ m/z: 328.36, 98.91%.
R,S-2-{2-[(2-hydroxy-2-phenylethyl)amino]ethoxy}-9H-xanthen-9-one (10)
3,73
C23H21NO4; M=375.42; mp 158-160 °C; Rf=0.47 (CH3OH); Ncalc/found
=
/
,
3,60
73,58
5,64
Ccalc/found
=
/
73,78, Hcalc/found
=
/
5,61; IR (KBr, cm-1): 3966, 3869, 3444, 3301, 3078, 2939,
1
2867, 2371, 2347, 1929, 1894, 1751, 1699, 1648, 1218; H NMR (DMSO-d6): δ (ppm) 8.21
(ddd, J=8.0, J=1.7, J=0.5, 1H, H-8), 7.88 (ddd, J=8.5, J=7.2, J=1.7, 1H, H-6), 7.49 (d,
J=9.4, 1H, H-4), 7.67 (ddd, J=8.5, J=1.1, J=0.5, 1H, H-5), 7.65 (d, J=3.1, 1H, H-1), 7.60
(dd, J=9.4, J=3.1, 1H, H-3), 7.47 (ddd, J=8.0, J=7.2, J=1.1, 1H, H-7), 7.37-7.35 (m, 2H,
Ar’-H2), 7.33-7.29 (m, 2H, Ar’-H3), 7.24-7.21 (m, 1H, Ar’-H4), 5.29 (d, J=5.2, 1H, -OH),
4.64 (ddd, J=8.1, J=5.2, J=4.7, 1H, >CH-), 4.19-4.10 (m, 2H, Ar-O-CH2-), 2.97 (t, J=5.5,
2H, -CH2-N), 2.72 (ddd, J=12.0, J=8.1, 1H, N-CHH-CH), 2.71 (ddd, J=12.0, J=4.7, 1H, N-
CHH-CH), 2.01 (bs, 1H, NH); 13C NMR: 175.68 (C=O), 155.43 (C-4b), 154.98 (C-2), 150.16
(C-4a), 144.48 (C-1’), 135.28 (C-6), 127.81 (C-3’, C-5’), 126.66 (C-4’), 125.85 (C-8), 125.80
(C-2’, C-6’), 124.88 (C-3), 124.12 (C-7), 121.42 (C-8b), 120.41 (C-8a), 119.71 (C-4), 118.08
(C-5), 106.51 (C-1), 71.53 (>CH-), 68.28 (Ar-O-CH2-), 57.48 (-N-CH2-CH<), 47.78 (-CH2-
N); LC-MS [M+H]+ m/z: 376.36, 96.69%.
This article is protected by copyright. All rights reserved.

2-{3-[(1-hydroxy-2-methylpropan-2-yl)amino]propoxy}-9H-xanthen-9-one (11)
1
C20H23NO4; M=341.40; mp 127-129 °C; Rf=0.28 (CH3OH/ethyl acetate 1/1); H NMR
(DMSO-d6): δ (ppm) 8.18 (dd, J=7.9, J=1.5, 1H, Ar-H6), 7.90-7.81 (m, 1H, Ar-H8), 7.67-
7.58 (m, 2H, Ar-H5, Ar-H7), 7.54 (d, J=2.9, 1H, Ar-H3), 7.42 - 7.49 (m, 2H, Ar-H1, Ar-H4),
4.33 - 4.53 (m, 1H, NH), 4.13 (t, J=6.4, 2H, -O-CH2-CH2-), 3.30 (s, 1H, OH), 3.15 (d, J=4.1,
2H, -CH2-OH), 2.61 (t, J=6.7, 2H, -CH2-NH-), 1.82 (qn, J=6.4, 2H, -CH2-CH2-CH2-), 0.91 (s,
6H, >C(-CH3)2); LC-MS [M+H]+ m/z: 342.46, 98.88 %.
R,S-2-{3-[(1-hydroxybutan-2-yl)amino]propoxy}-9H-xanthen-9-one (12)
1
C20H23NO4, M=341.40; mp 110-112 °C; Rf=0.25 (CH3OH/ethyl acetate 1/1); H NMR
(CDCl3): δ (ppm) 8.35 (dd, J=8.0, J=1.5, 1H, Ar-H), 7.75-7.68 (m, 2H, Ar-H), 7.51-7.30 (m,
4H, Ar-H), 4.18 (t, J=6.2, 2H, -O-CH2), 3.64 (dd, J=10.6, J=4.0, 1H, -CHH-OH), 3.30 (dd,
J=10.5, J=6.7, 1H, -CHH-OH), 2.97-2.74 (m, 2H, -CH2-NH-), 2.64-2.52 (m, 1H, NH-CH-),
2.01 (qn, J=6.4, 2H, -CH2-CH2-CH2-), 1.62-1.34 (m, 2H, -CH2-CH3), 0.99-0.86 (m, 3H, -
CH2-CH3); LC-MS [M+H]+ m/z: 342.39, 100%.
R,S-3-{2-[(1-hydroxybutan-2-yl)amino]ethoxy}-9H-xanthen-9-one (13)
4,28
C19H21NO4; M=327.38; mp 140-142 °C; Rf=0.36 (CH3OH); Ncalc/found
=
/
,
4,30
69,71
=6,47
69,58, Hcalc/found
Ccalc/found
=
/
/
6,41; IR (KBr, cm-1): 3427, 3290, 3148, 2964, 2930, 2857,
1658, 1605, 1598, 1497, 1474, 753; 1H NMR (DMSO-d6): δ (ppm) 8.17 (ddd, J=7.9, J= 1.7,
J=0.4, 1H, H-8), 8.10 (d, J=8.9, 1H, H-1), 7.84 (ddd, J=8.5, J=7.1, J=1.7, 1H, H-6), 7.62
(ddd, J=8.5, J=1.0, J=0.4, 1H, H-5), 7.47 (ddd, J=7.9, J=7.1, J=1.0, 1H, H-7), 7.15 (d,
J=2.4, 1H, H-4), 7.06 (dd, J=8.9, J=2.4, 1H, H-2), 4.58 (bs, 1H, -OH), 4.19 (m, 2H, Ar-O-
CH2-), 3.40 (dd, J=10.8, J=4.6, 1H, -CHH-OH), 3.30 (bs, 1H, NH (+H2O), 3.28 (dd, J=10.8,
J=6.3, 1H, -CHH-OH), 2.95 (t, J=5.7, 2H, -CH2-N), 2.49-2.43 (m, 1H, >CH-), 2.28 (s, 3H,
13
Ar-CH3), 1.96 (bs, 1H, -NH-), 1.44-1.31 (m, 2H, -CH2- (Et)), 0.85 (t, J=7.5, 3H, -CH3); C
NMR: 174.97 (C=O), 164.28 (C3), 157.44 (C4a), 155.50 (C4b), 135.04 (C6), 127.53 (C1),
125.77 (C8), 124.29 (C7), 121.02 (C8a), 117.82 (C5), 114.74 (C8b), 113.95 (C2), 100.89
(C4), 68.79 (Ar-O-CH2-), 62.47 (-CH2-OH), 59.91 (CH), 45.16 (-CH2-N), 23.39 (-CH2- (Et)),
9.91 (-CH3); LC-MS [M+H]+ m/z: 328.36, 100%.
This article is protected by copyright. All rights reserved.

4-{2-[N-(2-hydroxyethyl)-N-(methyl)amino]ethoxy}-9H-xanthen-9-one (14a)
C18H20NO4Cl; M=349.81; mp 210-212 °C (14 – base mp 79-81 °C); Rf=0.26 (CH3OH/ethyl
4.00
61.80
5.76
acetate 1/1); Ncalc/found
=
/
4.03, Ccalc/found
=
/
61.71, Hcalc/found
=
/
5.80; 1H NMR (DMSO-d6): δ
(ppm) 10.92 (bs, 1H, NH+), 8.19 (ddd, J=8.0, J=1.8, J=0.5, H-8), 7.90 (ddd, J=8.5, J=7.1,
J=1.8, H-6), 7.78 (dd, J=8.0, J=1.5, H-1), 7.70 (ddd, J=8.5, J=7.1, J=1.8, H-5), 7.61 (dd,
J=8.0, J=1.5, H-3), 7.50 (ddd, J=8.0, J=7.1, J=1.0, H-7), 7.41 (dd, J=8.0, J=8.0, H-2), 5.46
(bs, 1H, OH), 4.68 (t, J=5.0, 2H, Ar-O-CH2), 3.92 (t, J=5.5, 2H, CH2-OH), 3.74 (bs, 2H, N-
CH2-CH2-O-Ar), 3.45 (bs, 2H, N-CH2-CH2-OH), 3.04 (s, 3H, CH3-N); (base C18H19NO4;
M=313.35, LC-MS [M+H]+ m/z: 314.08, 99.02%).
R,S-4-{2-[(1-hydroxypropan-2-yl)amino]ethoxy}-9H-xanthen-9-one hydrochloride (15a)
C18H20NO4Cl; M=349.81; mp 221-223 °C (15 – base mp 136-138 °C); Rf=0.20
4.00
61.80
5.76
(CH3OH/ethyl acetate 1/1); Ncalc/found
=
/
3.89, Ccalc/found
=
/
61.66, Hcalc/found
=
/5.42; (base IR
(KBr, cm-1): 3276, 3144, 3099, 2971, 2947, 2929, 2870, 1653, 1496, 1346, 1285, 1230, 1076,
1
755); H NMR (DMSO-d6): δ (ppm) 9.20, 8.95, 8.89 (bs, 2H, NH+, NH), 8.21 (dd, J=8.0,
J=1.7, 1H, H-8), 7.92 (ddd, J=8.5, J=7.1, J=1.7, 1H, H-6), 7.81 (dd, J=8.1, J=1.5, 1H, H-1),
7.71 (dd, J=8.5, J=1.1, 1H, H-5), 7.61 (dd, J=8.0, J=1.5, 1H, H-3), 7.52 (ddd, J=8.0, J=7.1,
J=1.1, 1H, H-7), 7.43 (dd, J=8.1, J=8.0, 1H, H-2), 5.45 (t, J=4.9, 1H, OH), 4.56 (t, J=5.4, 2H,
CH2-O-Ar), 3.79-3.72 (m, 1H, CHH-OH), 3.66-3.59 (m, CHH-OH), 3.57-3.42 (m, 3H, CH2-
13
N, CH), 1.33 (d, J=4.4, 3H, CH3); C NMR: 175.79 (C=O), 155.21 (C-4b), 146.92 (C-4),
146.81 (C-4a), 146.01 (C-6), 135.53 (C-8), 125.90 (C-7), 124.51 (C-2), 123.93 (C-8b),
122.03 (C-8a), 120.86 (C-5), 118.27 (C-3), 117.51 (C-1), 65.31 (Ar-O-CH2-), 61.01 (-
CH2OH), 55.03 (>CH-), 42.88 (-CH2-N), 13.21 (-CH3); LC-MS [M+H]+ m/z: 314.08, 100%;
(base C18H19NO4; M=313.35; LC-MS [M+H]+ m/z: 314.08, 97.99%).
4-{2-[(1-hydroxy-2-methylpropan-2-yl)amino]ethoxy}-9H-xanthen-9-one hydrochloride
(16a)
C19H22NO4Cl; M=363.84; mp 252-254 °C (16 – base mp 156-158 °C); Rf=0.22
3.85
62.72
6.09
(CH3OH/ethyl acetate 1/1); Ncalc/found
=
/
3.75, Ccalc/found
=
/
62.14, Hcalc/found
=
/6.34; (base
4,28
69,71
6,47
C19H21NO4; M=327.38; Ncalc/found
=
/
4,23, Ccalc/found
=
/
69,52, Hcalc/found
=
/6,42); IR (KBr,
cm-1): 3404, 3276, 3138, 3100, 2971, 2947, 2929, 2872, 2706, 1651, 1496, 1285, 1076, 756;
IR base (KBr, cm-1): 3276, 3139, 2972, 2947, 2930, 2871, 1654, 1496, 1285, 1076, 755; base
1H NMR (DMSO-d6): δ (ppm) 8.18 (dd, J=7.9, 1.5 Hz, 1H, Ar-H6), 7.87 (ddd, J=8.6, 7.2, 1.8
This article is protected by copyright. All rights reserved.

Hz, 1H, Ar-H8), 7.64 - 7.74 (m, 2H, Ar-H5, Ar-H1), 7.44 - 7.55 (m, 2H, Ar-H2, Ar-H7), 7.36
(t, J=7.9 Hz, 1H, Ar-H3), 4.54 (s, 1H, OH), 4.19 (t, J=5.9 Hz, 2H, Ar-O-CH2-), 3.20 (d, J=5.3
Hz, 2H, -CH2-OH), 2.93 (t, J=5.9 Hz, 2H, -CH2-NH-), 1.77 - 1.92 (m, 1H, NH), 0.98 (s, 6H,
1
+
>C-(CH3)2); H NMR (DMSO-d6): δ (ppm) 8.96 (bs, 2H, NH2 ), 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), 7.81 (dd, J=8.0, J=1.5, 1H, H-1),
7.69 (ddd, J=8.7, J=1.0, J=0.4, 1H, H-5), 7.61 (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), 7.43 (dd, J=8.1, J=8.0, 1H, H-2), 5.65 (t, J=5.2, 1H, OH),
+
4.53 (t, J=5.6, 2H, Ar-O-CH2-), 3.55 (d, J=5.2, 2H, CH2-OH), 3.45 (bs, 2H, CH2-NH2 ), 1.34
13
(s, 6H, 2x -CH3); C NMR: 175.82 (C=O), 155.20 (C-4b), 146.92 (C-4), 146.01 (C-4a),
135.54 (C-6), 125.90 (C-8), 124.50 (C-7), 123.94 (C-2), 122.00 (C-8b), 120.84 (C-8a),
118.28 (C-5), 118.17 (C-3), 117.39 (C-1), 65.13 (Ar-O-CH2-), 64.58 (-CH2OH), 59.75 (Cq),
40.10 (-CH2-N), 20.30 (2x -CH3); LC-MS [M+H]+ m/z: 328.36, 97.49%; (base C19H21NO4;
M=327.38, LC-MS [M+H]+ m/z: 328.36, 97.49%).
R,S-4-{2-[(1-hydroxybutan-2-yl)amino]ethoxy}-9H-xanthen-9-one hydrochloride (17a)
C19H22NO4Cl; M=363.84; mp 183-185 °C (17 – base mp 125-127 °C); Rf=0.27
4,28
(CH3OH/ethyl
acetate
1/1);
base
C19H21NO4;
M=327.38,
Ncalc/found
=
/
,
4,16
69,71
6,47
Ccalc/found
=
/
69,62, H=calc/found
/
6,27; IR (KBr, cm-1): 3381, 3166, 2975, 2811, 2466, 1649,
1
1594, 1496, 1278, 753; base H NMR (DMSO-d6): δ (ppm) 8.20 (ddd, J=8.0, J=1.8, J=0.5,
1H, H-8), 7.89 (ddd, J=8.5, J=7.1, J=1.7, 1H, H6), 7.74 (dd, J=8.0, J=1.4, 1H, H-1), 7.67
(ddd, J=8.5, J=1.1, J=0.6, 1H, H-5), 7.54 (dd, J=8.0, J=1.4, 1H, H-3), 7.49 (ddd, J=8.0,
J=7.1, J=1.1, 1H, H-7), 7.38 (dd, J=8.0, J=8.0, 1H, H-2), 4.46 (t, J=5.3, 1H, OH), 4.29-4.20
(m, 2H, Ar-O-CH2), 3.46-3.40 (m, 1H, CHH-OH), 3.35-3.29 (m, 1H, CHH-OH), 3.03 (t,
J=5.9, 2H, CH2-N), 2.56-2.51 (m, 1H, CH), 1.92 (bs, 1H, NH), 1.46-1.35 (m, 2H, CH2-R),
0.89 (t, J=7.4, 3H, CH3); LC-MS [M+H]+ m/z: 328.36, 98.29%.
Pharmacology
Antiepileptic activity and neurotoxicity assays were carried out within the Antiepileptic Drug
Development Program, Epilepsy Branch, National Institute of Neurological and
Communicative Disorders and Stroke, National Institute of Health in Rockville, USA (27).
Compounds were injected as suspensions in 0.5% methylcellulose at doses 30, 100 and 300
mg/kg b.w. intraperitoneally (i.p.) into mice. The preliminary evaluation was a qualitative
assay which used small groups of animals (1-8) and included three tests: maximal
electroshock seizure (MES), subcutaneous pentylenetetrazol (ScMet), and neurotoxicity
This article is protected by copyright. All rights reserved.

(rotarod), noted at 30 min and 4 h after administration (28).
The MES were elicited by 60 Hz alternating current at 50 mA (mice) or 150 mA (rats)
delivered for 0.2 s via corneal electrodes. A drop of 0.9% NaCl solution was placed into each
eye prior to applying the electrodes. Protection in the MES test was defined as the abolition
of the hindlimb tonic extension component of the seizure.
The ScMet test was performed by administration 85 mg/kg of pentylenetetrazole in 0.9%
NaCl solution into the posterior midline of mice. Seizures were awaited at minimal time of 30
min subsequent to subcutaneous administration of pentylenetetrazole. A failure to observe
even a threshold seizure (a single episode of clonic spasm of at least 5 s in duration) was
regarded as protection.
TOX defined as neurological deficit was measured in mice (i.p.) or rats (p.o.) by the rotarod
test. The animal was placed on a 1 inch diameter knurled plastic rod rotating at 6 rpm.
Neurotoxicity was indicated by the inability of the animal to maintain equilibrium on the rod
for at least 1 min in each of the three trials. In rats, neurological deficit was indicated by
ataxia and loss of placing response and muscle tone.
The pilocarpine test involves determination whether the investigated compound can halt
acute pilocarpine-induced status. A challenge dose of pilocarpine (50 mg/kg) is administered
i.p. and the rats are observed until the first convulsive (e.g. stage 3, 4, or 5) seizure (time
zero). The seizure severity is determined using the Racine scale (29) At this point a
minimally toxic dose of the candidate drug is administered to a group of 8 male albino
Sprague Dawley rats (150-180 g) i.p. Efficacy is defined by the ability of an investigational
drug to halt the further expression of pilocarpine induced convulsive seizures (e.g. stage 3, 4,
or 5).
Receptor binding studies
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 test compound, 50 μL of
radioligand and 150 μL of diluted membranes or the tissue suspension. Specific assay
conditions for each receptors are shown in Table 2. Recombinant human proteins were used
for 5-HT1A, 5-HT6, and 5-HT7 receptors. The radioactivity was measured in MicroBeta2
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.
This article is protected by copyright. All rights reserved.

Metabolic stability
Metabolism prediction was performed with use of MetaSite 5.1.1. Mass 3.2.1 by Molecular
Discovery Ltd. (Germany) (30).
Sprague-Dawley rat liver microsomes (RLMs), glucose-6-phosphate, NADP, glucose-6-
phosphate dehydrogenase and levallorphan were procured from Sigma Aldrich.
In vitro metabolism of compounds 9 and 12 using rat liver microsomes
Reaction samples consisted of RLMs (0.2 mg microsomal protein/mL), substrate (20 µM),
and NADPH-regenerating system (NADP, glucose-6-phosphate, glucose-6-phosphate
dehydrogenase and 100 mM potassium phosphate buffer) in the presence of potassium
phosphate buffer (100 mM, pH 7.4). At first, the mixtures containing microsomes, test
compound and buffer were preincubated at 37 °C for 15 min before NADPH-regenerating
system was added to commence the metabolic reaction. The mixture was then incubated at
various time points ranging from 5 to 120 min. Subsequently, an internal standard
(levallorphan, 20 µM) was added, and the samples were quenched by addition of perchloric
acid. The reaction mixtures were centrifuged and supernatants were collected for LC-MS/MS
analysis (UPLC/MS, Waters Corporation, Milford, MA, USA). In control samples NADPH-
regenerating system was replaced with phosphate buffer (31-33). Microsomal incubations
were conducted in duplicate. The in vitro half times (t1/2) and intrinsic clearance (Clint) of
compounds 9 and 12 in liver microsomes were determined according to procedures
previously described (34).
Results
Chemistry
Calculated parameters for 8-17 such as LogP, topological polar surface area TPSA, molecular
volume have been performed by means of Molinspiration online toolkit (14) and are
presented in Table 1.
Compounds 8-17 were obtained by N-alkylation of appropriate aminoalkanols using (2-
xanthonoxy)ethyl-, (2-xanthonoxy)propyl-, (3-xanthonoxy)ethyl-, (4-xanthonoxy)ethyl-, or 4-
(xanthonoxy)propyl chloride, or (3-xanthonoxy)ethyl bromide (respectively). The reaction
was performed in the presence of K2CO3 as a proton acceptor in toluene solution. The yield
of alkylation was in the range 45-70%. Appropriate xanthonoxyalkyl chlorides were achieved
This article is protected by copyright. All rights reserved.

by chlorination of appropriate xanthonoxyethanol or appropriate (xanthonoxy)propanol with
use of SOCl2 or by reaction of 3-chloro-1-bromopropan or 2-chloro-1-bromoethan with
appropriate hydroxyxanthone in acetone in presence of K2CO3 as a proton acceptor as well as
TEBA, KI. 3-(2-Bromoethoxy)-9H-xanthen-9-one 5 was achieved for the reason of
confirmation of method since its precursor 3-(2-hydroxyethoxy)-9H-xanthen-9-one 3 was
achieved with mp different from reference (26).
The amine products 8-9 and 14-17 were converted to hydrochlorides with EtOH saturated
with HCl in EtOH. The synthesis is shown in Scheme 1.
Pharmacology
Compounds 8a-17a were evaluated in preliminary anticonvulsant screening in MES, ScMet
and TOX tests in mice, i.p. Compounds 8a-9 and 12-13 are classified as class 1 ASP due to
their activity at 100 mg/kg b.w. or less. Simultaneously, the active compounds 8a-9 and 12
exhibit neurotoxicity at the dose 100 mg/kg b.w. 0.5 h after administration. Moreover, in case
of these compounds some activity lasted until 4 h after administration without neurotoxicity,
which may lead to presumption that metabolism may produce active and non-toxic
compounds. The results are presented in Table 3.
Compounds 9, 12-13 were advanced within the screening program to the next phase for oral
administration (p.o.) to rats and the results of activity in MES and neurotoxicity were
observed until 4 h after administration. It is interesting that the compounds are inactive at the
dose 30 mg/kg b.w. until 2, 4, or 1 h after administration, respectively. The results of 9 and
12 suggest activity rather not of the compound itself, but of a metabolite. These results vary
significantly from phenoxyalkylaminoalkanols, which exhibit activity immediately after
administration and time of peak effect is usually 0.25 h or 0.5 h after administration (6). The
results are presented in Table 4. None of the compounds are neurotoxic at the used dose.
Compound 12 was also evaluated in the next stage of ASP – the pilocarpine test for
verification of activity in a rodent model of status epilepticus. The activity was observed at
300 mg/kg b.w. The results are presented in Table 5. It is coherent with the results of MES
(rats, p.o.) (Table 4) that in spite of lack of activity for 2 h after administration, some activity
appears after 4 h. Death observed at 200 mg/kg of experiment might probably be related to
the risk of status epilepticus itself.
This article is protected by copyright. All rights reserved.

Due to our former results of receptor binding of xanthone derivatives, compounds 8a, 9 and 12 were
subject to preliminary serotonergic receptor evaluation. They did not exhibit satisfying binding. The
results are presented in Table 6.
The results of activity of compounds 9 and 12 in rats, i.p. and p.o. drew our attention towards
metabolism of these compounds by rat liver microsomes. For the purpose of this study,
prediction of metabolism was performed with use of MetaSite 5.1.1. Mass 3.2.1. The results
of proposed three most probable metabolites with their molecular mass and calculated LogP
are presented in Figure 2. Then the compounds were subject to microsomal metabolism and
the results were compared.
Three metabolites of compound 9 (M1-M3) and five metabolites of compound 12 (M1-M5)
were identified in rat liver microsomes (Table 7, Figure 3). In case of both compounds the
major and most abundant metabolite, M1 was the product of oxidation of alcohol moiety in
the parent compounds, whereas M2 metabolites of both compounds were the products of
dealkylation (Figure 4). MetaSite prediction of oxidation of alcohol groups of both xanthone
derivatives as the most probable pathway of biotransformation was confirmed by in vitro
testing and MS/MS spectra. Both compounds demonstrated Clint values lying in the medium
category range (26 and 32 µL/mg/min) (Table 8).
Discussion
For preliminary estimation whether the relatively large xanthonoxy moiety improves activity
in the synthesized group of compounds, a coherent group of 2-aminoalkan-1-ols was chosen,
among so far the optimal 2-aminopropan-1-ol, 2-aminobutan-1-ol, as well as their simple
modifications.
Calculation of several physicochemical parameters by means of Molinspiration online toolkit
was an important step in the research (14). Such calculations are useful at early stage of drug
discovery process in terms of predicting essential parameters and facilitating rational drug
design. The parameters include LogP, topological polar surface area TPSA, molecular
volume, as well as violations from the Lipinski rule of five (Table 1). The first calculated
parameter, LogP (with use of ChemBioDraw) was in the range 2.76-3.68, and miLogP
This article is protected by copyright. All rights reserved.

(Molinspiration toolkit), was found in the range 2.98-4.34, while in the literature optimum
value for CNS active compounds is about 2-3 (37). The most beneficial value of TPSA
should be <120 Å2 for orally administered drugs and <60-70 Å2 for compounds designed to
penetrate blood-brain barrier. The proposed structures are consistent with the rule for oral
drugs and they are on the edge of the rule for CNS drugs (calculated values are in the range
62.91-91.93 Å2). The volumes range 285.78-340.62. None of the compounds exhibited any
violations from the Lipinski rule of five (38), making them potentially promising drug-like
agents.
As it was stated in the Introduction, the pharmacological evaluation of anticonvulsant activity
of the achieved compounds verified that the optimal position of substituents in the xanthone
ring was expected to be C2 and on the basis of twelve presented compounds the assumptions
were proved. Three out of four most active compounds are 2-xanthonoxy derivatives, i.e. 8a-
9, and 12. Compound 13 is the derivative of xanthone substituted in position C3.
Substitutions in position 4 did not provide activity below 300 mg/kg b.w. (mice, i.p.). The
observed most probable metabolites M1 look rather hydrophilic and are larger than the parent
compound, therefore, they may not be likely to cross blood-brain barrier.
The aminoalkanol moiety optimal in the studied group of compounds was proved to be
aminobutanol – either 2-aminobutan-1-ol (as in 9, 12-13, 17a) which is coherent with
reference structures II and IV or 1-aminobutan-2-ol (8a).
As long as the linker between xanthonoxy and aminoalkanol moiety is concerned, among the
used ethylene and propylene – propylene is more beneficial, on the basis of comparison
between compounds 9 and 12. This result is another surprise contrary to our findings in other
aroxyalkylaminoalkanol derivatives, where ethylene is superior to propylene (structures I and
II) (6).
The results of activity of compounds 9 and 12 in rats, i.p. and p.o. where activity was
observed 2 and/or 4 h after administration, became premises for study of metabolism of these
compounds by rat liver microsomes. The achieved values of Clint (Table 8) were lower than
those reported previously for imipramine (36). Basing on the data for 9 and 12 it can be
concluded that both compounds are relatively stable metabolically. Considering the activity
of compounds 9 and 12 in rats (Tables 3-4) either it may be a very active metabolite (possibly
M1 in both cases) which appears late, or it may be other ADME factors other than
metabolism, i.e. absorption or distribution, that may influence time of onset of activity of
This article is protected by copyright. All rights reserved.

compounds 9 and 12.
Conclusions
As a conclusion, according to our former studies in terms of structure-activity relationship of
xanthone derivatives and phenoxyalkylaminoalkanols, synthesis of the chosen group of N-
[(xanthonoxy)alkyl]aminoalkanols where 2-, 3- or 4-hydroxyxanthone is used in place of
phenol, was a logical choice.
The assumptions were promising, especially with use of xanthone and expectations regarding
position C2 of substituents, as well as choice of aminoalkanols. Moreover, in spite of the
choice of very active reference structures, their modification and design with consistence of
the Lipinski rule described in the Introduction, calculations consistent with literature
premises, the experiments showed that the calculation of parameters did not fulfill
expectations for rational finding of an active molecule in vivo.
On the other hand, research regarding metabolism of active compounds provides wide view
regarding the metabolic pathway in the organism and its products.
Having completed screening documentation regarding the title group of compounds, one can
suggest further possible modifications in the other xanthone ring, which might enhance
anticonvulsant activity. Moreover, when a larger group of derivatives results from our
research, in order to compare derivatives in a full manner, it may be reasonable to synthesize
enantiomers of chiral active derivatives.
Acknowledgements
The authors thank Professor Jeff Jiang, Professor James Stables, Professor Steve White,
Professor Harvey Kupferberg, and dr. Tracy Chen from National Institute of Neurological
Disorders and Stroke (NIH, Rockville, USA) for providing the results of anticonvulsant
evaluation, as well as Professor Katarzyna Kieć-Kononowicz for coordination of the
cooperation with the Faculty of Pharmacy at Jagiellonian University Medical College.
The authors declare no conflict of interests.
The research was co-financed by European Regional Development Fund within the
Innovative Economy Program and by Jagiellonian University Medical College funding no.
This article is protected by copyright. All rights reserved.

K/ZDS/005487.
Bibliography
1.
2.
Sander J. (2003) The epidemiology of epilepsy revisited. Curr Opin Neurol. 16: 165-170.
Fischer W. (2002) Anticonvulsant Profile and Mechanism of Action of Propranolol and Its
Two Enantiomers. Seizure J Br Epilepsy Assoc 11: 285–302.
3.
4.
5.
Chew C.Y.C., Collett J., Singh B.N. (1979) Mexiletine: A Review of Its Pharmacological
Properties and Therapeutic Efficacy in Arrhythmias. Drugs 17: 161–181.
Alexander G.J., Kopeloff L.M., Alexander R.B., Chatterjie N. (1986) Mexiletine: biphasic
action on convulsive seizures in rodents. Neurobehav Toxico. Teratol 8: 231–235.
Marona H., Waszkielewicz A., Kieć-Kononowicz K. (2009) Derivatives of Aminoalkanols,
Method of Obtaining of Aminoalkanols and Their Use. PCT/PL2009/000004; WO
2009/093916A3.
6.
7.
Waszkielewicz A.M., Cegła M., Żesławska E., Nitek W., Słoczyńska K., Marona H. (2015) N-
[(2,6-Dimethylphenoxy)alkyl]aminoalkanols—their Physicochemical and Anticonvulsant
Properties. Bioorg Med Chem 23: 4197–4217.
Waszkielewicz A.M., Szneler E., Cegła M., Marona H. (2013) Synthesis and Evaluation of
Anticonvulsant Activity of Some N-[(4-Chlor-2-Methylphenoxy)ethyl]- and N-[(4-Chlor-2-
Methylphenoxy)acetyl]aminoalkanols. Lett Drug Des Discov 10: 34–42.
8.
9.
Marona H., Waszkielewicz A.M., Szneler E. (2005) Preliminary Evaluation of Anticonvulsant
Activity of Some Aroxyacetamides and Aroxyethylamines. Acta Pol Pharm 62: 345–353.
Waszkielewicz A.M., Gunia-Krzyżak A., Cegła M. (2015) Synthesis and Evaluation of
Anticonvulsant Activity of N-(2,5-Dimethylphenoxy)- and N-[(2,3,5-
Trimethylphenoxy)alkyl]aminoalkanols Acta Pol Pharm – Drug Res 72: 89–99.
10.
11.
12.
Marona H., Antkiewicz-Michaluk L. (1998) Synthesis and Anticonvulsant Activity of 1,2-
Aminoalkanol Derivatives. Acta Pol Pharm 55: 487–498.
Marona H. (1998) Evaluation of Some 2-Substituted Derivatives of Xanthone for
Anticonvulsant Properties. Pharmazie 53: 405–409.
Szkaradek N., Gunia A., Waszkielewicz A.M., Antkiewicz-Michaluk L., Cegła M., Szneler E.,
Marona H. (2013) Anticonvulsant Evaluation of Aminoalkanol Derivatives of 2- and 4-
Methylxanthone. Bioorg Med Chem 21: 1190–1198.
13.
Waszkielewicz A.M., Gunia A., Szkaradek N, Pytka K., Siwek A., Satała G., Bojarski, A.J.,
Szneler E., Marona H (2013) Synthesis and Evaluation of Pharmacological Properties of Some
New Xanthone Derivatives with Piperazine Moiety. Bioorg Med Chem Lett 23: 4419–4423.
14.
15.
http://www.molinspiration.com/services/properties.html.
Marona H., Szkaradek N., Kubacka M., Bednarski M., Filipek B., Cegla M., Szneler E. (2008)
Synthesis and Evaluation of Some Xanthone Derivatives for Anti-Arrhythmic, Hypotensive
Properties and Their Affinity for Adrenergic Receptors. Arch Pharm (Weinheim) 341: 90–98.
16.
Valenti P., Rampa A., Bisi A., Fabbri G., Andrisano, V.; Cavrini, V. (1995) Cholinergic
Agents. Synthesis and Acetylcholinesterase Inhibitory Activity of Some ω -[N-Methyl-N-(3-
Alkylcarbamoyloxyphenyl)-Methyl]aminoalkoxyxanthen-9-Ones. Med Chem Res 5: 255–264.
This article is protected by copyright. All rights reserved.

17.
18.
19.
Marona H., Szkaradek N., Karczewska E., Trojanowska D., Budak A., Bober P., Przepiórka
W., Cegla M., Szneler E. (2009) Antifungal and Antibacterial Activity of the Newly
Synthesized 2-Xanthone Derivatives. Arch Pharm (Weinheim) 342: 9–18.
Marona H., Szkaradek N., Rapacz A., Filipek B., Dybała M., Siwek A., Cegła M., Szneler E.
(2009) Preliminary Evaluation of Pharmacological Properties of Some Xanthone Derivatives.
Bioorg Med Chem 17: 1345–1352.
Ullmann F., Zlokasoff M.(1905) Ueber Arylsalicylsäuren und deren Ueberführung in
Xanthone. Ber Dtsch Chem Gesellschaft 38: 2111-2119.
20.
21.
Davies J., Lamb F., Suschitzky H. (1956) Studies in the Xanthone Series. Part II.* Preparation
and Reactions of 1-Formyl-2-Hydroxyxanthone. Journal of the Chemical Society 1790–1793.
Gnerre C., Thull U., Gaillard P., Carrupt P.A., Testa B., Fernandes E., Silva F., Pinto M., Pinto
M.M.M., Wolfender J.L., Hostettmann K., Cruciani G. (2001) Natural and synthetic xanthones
as MAO inhibitors: biological assay and 3D-QSAR. Helv Chim Acta 84: 552-570
22.
23.
Finnegan RA, Bachman PL. (1965) Natural occurrence of 2-hydroxyxanthone. J Pharm Sci
54(4): 633-635.
Ullmann F., Denzler W. (1906) Ueber o-Aminobenzophenonderivate. Berichte der Dtsch
Chem Gesellschaft 39: 4332–4339.
24.
Monache F.D., Mac-Quhae M.M., Monache G.D., Bettolo G.B.M., De Lima R.A. (1983)
Xanthones, xanthonolignoids and other constituents of the roots of vismia guaramirangae.
Phytochemistry 22: 227-232.
25.
26.
Lin Ch.N., Liou S-S., Ko F., Teng C-M. (1993) γ-Pyrone Compounds. IV: Synthesis and
Antiplatelet Effects of Mono- and Dioxygenated Xanthones and Xanthonoxypropanolamine. J
Pharm Sci 82: 11-16.
Marona H. (1998) Synthesis and Anticonvulsant Effects of Some Aminoalkanolic Derivatives
of Xanthone. Pharmazie 53: 672–676.
27.
28.
Carvalho M.J., Carvalho L.M., Ferreira A.M., Silva A.M.S. (2003) A New Xanthone from
Hedychium Gardnerianum. Nat Prod Res 17: 445–449.
Stables J.P., Kupferberg H.J. The NIH Anticonvulsant Drug Development (ADD) Program:
Preclinical Anticonvulsant Screening Project. (1997) Molecular and Cellular Targets for Anti-
epileptic Drugs; Avanzini, G.; Regesta, P.; Tanganelli, A.; Avoli, M., Eds.; John Libbey &
Company Ltd: London, 23129: 191–198.
29.
White S.H., Woodhead J.H., Wilcox K.S., Kupferberg H.J., Wolf H.H. General Principles of
Discovery and Preclinical Development of Antiepileptic Drugs.( 2002) Antiepileptic Drugs,
fifth ed.; Levy, R. H.; Mattson, R. H.; Meldrum, B. S.; E., P., Eds.; Lippincott Williams &
Wilkins: Philadelphia, 36–48.
30.
31.
Racine R.J. (1972) Modification of Seizure Activity by Electrical Stimulation. II. Motor
Seizure. Electroencephalogr Clin Neurophysiol;32:281–294.
Cruciani G., Carosati E., De Boeck B., Ethirajulu K., Mackie C., Howe T., Vianello R. (2005)
MetaSite: Understanding Metabolism in Human Cytochromes from the Perspective of the
Chemist. J Med Chem 48: 6970–6979.
32.
Hamelin B. Bouayad A. Drolet B., Gravel A., Turgeon J. (1998) In Vitro Characterization of
This article is protected by copyright. All rights reserved.

Cytochrome P450 2D6 Inhibition by Classic Histamine H1 Receptor Antagonists. Drug Metab
Dispos 26: 536–539.
33.
34.
Huang J., Si L., Fan Z., Hu L., Qiu J., Li G. (2011) In Vitro Metabolic Stability and Metabolite
Profiling of TJ0711 Hydrochloride a Newly Developed Vasodilatory β-Blocker Using a Liquid
Chromatography–tandem Mass Spectrometry Method. J Chromatogr B 879: 3386–3392.
Sakai C., Iwano S., Yamazaki Y., Ando A., Nakane F., Kouno M., Yamazaki H., Miyamoto Y.
(2014) Species Differences in the Pharmacokinetic Parameters of Cytochrome P450 Probe
Substrates between Experimental Animals such as Mice Rats Dogs Monkeys and
Microminipigs and Humans. J Drug Metab Toxicol 5: 173-185.
35.
36.
37.
38.
Cheng Y., Prusoff W. H. (1973) Relationship between the Inhibition Constant (K1) and the
Concentration of Inhibitor Which Causes 50 per Cent Inhibition (I50) of an Enzymatic
Reaction. Biochem Pharmacol 22: 3099–3108.
Singh J. K., Solanki A., Shirsath V. S. (2012) Comparative in-Vitro Intrinsic Clearance of
Imipramine in Multiple Species Liver Microsomes: Human, Rat, Mouse and Dog. J Drug
Metab Toxicol 3: 1–6.
Hansch C., Bjorkroth J.P., Leo A. (1987) Hydrophobicity and Central Nervous System
Agentsꢀ: On the Principle of Minimal Hydrophobicity in Drug Design. J Pharm Sci 76: 663-
687.
Lipinski C.A., Lombardo F., Dominy B.W., Feeney P.J. (2001) Experimental and
Computational Approaches to Estimate Solubility and Permeability in Drug Discovery and
Development Settings. Adv Drug Deliv Rev 46: 3–26.
This article is protected by copyright. All rights reserved.

Scheme 1: Synthesis of the starting materials (1-7) and the title compounds (8-17).
This article is protected by copyright. All rights reserved.

Table 1: Structures and logP of the title compounds (8a-17a) (mice, i.p.).
a)
Compd. Position
n
2
Z
Config. LogPcalc miLogPb) TPSA Volume
[Å2]
[Å3]
C2
R,S
2.95
3.27
71.70 302.58
8a
x HCl
R,S
2.95
3.62
71.70 302.58
9
R,S
3.68
4.34
71.70 340.62
10
3
-
3.19
3.32
3.84
3.89
71.70 318.82
71.70 319.38
11
12
R,S
C3
C4
2
2
R,S
2.95
3.62
71.70 302.58
13
-
2.76
2.42
2.98
3.06
62.91 286.13
71.70 285.78
14a
15a
x HCl
R,S
x HCl
x HCl
-
2.82
2.95
3.54
3.60
71.70 302.01
71.70 302.58
16a
17a
R,S
x HCl
Calculations were performed with use of a) ChemBioDraw Ultra 12.0 (CambridgeSoft) b)
Molinspiration online toolkit (14) for base forms.
This article is protected by copyright. All rights reserved.

Table 2: Preliminary data on anticonvulsant activity and neurotoxicity of the title compounds
(mice, i.p.).
Compd
Dose
MES a)
ScMet a)
TOX b)
ASP
class c)
0.5 h 4 h 0.5 h 4 h 0.5 h
4 h
|
3
–
–
|
|
|
|
–
–
1
8a
10
|
|
|
30
1/1
–
–
–
|
–
–
|
–
–
–
|
100
300
3
3/3 1/3
7/8
4/4
–
|
–
|
|
|
|
|
1
9
10
–
|
|
|
–
|
30
1/1
–
–
–
–
–
–
–
–
–
|
–
–
–
–
–
–
–
–
|
–
–
–
–
–
–
–
–
–
–
–
–
|
100
300
30
3/3 3/3
1/1 1/1
7/8
4/4
–
–
–
–
–
–
|
–
–
–
–
–
|
3
|
10
100
300
30
–
–
–
11
100
300
30
5/8
4/4
–
–
–
–
–
|
–
–
|
1
1
3
2
2
4
12
100
300
30
3/3 2/3
6/8
4/4
–
|
–
|
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1/2
–
13
100
300
30
1/3
1/1
–
–
–
–
14a
15a
16a
17a
100
300
30
–
8/8
4/4
–
–
–
100
300
30
–
–
1/1
–
4/4
–
100
300
30
–
–
1/1
–
4/4
2/4
This article is protected by copyright. All rights reserved.

100
300
–
–
–
–
|
–
|
6/8
4/4
–
|
1/1
a) Number of animals protected / number of animals tested; b) number of animals exhibiting toxicity /
number of animals tested in the rotorod test; c) ASP classification: 1 – anticonvulsant activity at doses
100 mg/kg or less; 2 – anticonvulsant activity at doses greater than 100 mg/kg; 3 – compound inactive
at 300 mg/kg; 4 – compound either active or inactive but toxic at doses of 30 mg/kg; – indicates that
the compound was not active or toxic in the particular case; | indicates that the compound was not
tested in the particular case.
Table 3: Anticonvulsant activity and neurotoxicity of the tested compounds (rats, p.o.).
Compound Test
Dose
Time [h]
[mg/kg b.w.]
0.25 0.5 1.0 2.0 4.0
MESa)
TOXb)
30
30
30
30
30
30
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1/4
–
–
–
9
MES
TOX
MES
TOX
–
–
1/4
–
12
13
–
–
1/4
–
–
–
–
–
a) Number of animals protected / number of animals tested; b) number of animals exhibiting toxicity /
number of animals tested in the rotorod test; – indicates that the compound was not active or toxic in
the particular case.
Table 4: Pilocarpine induced status epilepticus results (rats, i.p.).
Compd.
Dose
[mg/kg b.w.]
30
Time [h] a)
No. of
deaths
0.25
–
0.5
–
1.0
–
2.0
–
4.0
–
7/7
12
100
–
–
–
–
1/2
2/2
at dose 200
300
2/2
2/2
2/2
2/2
a) Number of animals protected / number of animals tested.
This article is protected by copyright. All rights reserved.

Table 5: Results of binding to serotoninergic receptors of reference and tested compounds.
Compd.
Ki [nM]
5-HT1A 5-HT6 5-HT7b
2136 16240 8301
9a
buspiron
clozapine
12
-
-
143
4
18
Inhibition constants (Ki) were calculated according to the equation of Cheng and Prusoff (36).
Radioligand binding assays to rats brain tissues using [3H]8-OH-DPAT for 5-HT1A, [3H]-LSD for 5-
HT6, [3H]-5-CT for 5-HT7.
Table 6: Results of binding to serotoninergic receptors of reference and tested compounds.
Ki [nM]
Compound
5-HT1A
[3H]-8-OH-DPAT
320.0 11.3
659.0 21.4
> 5000
5-HT6
5-HT7
[3H]-LSD
[3H]-LSD
-
-
-
-
-
-
8a
9
12
-
-
serotonin
1.2 0.05
1.0 0.1
0.4 0.02
methiothepin
-
Inhibition constants (Ki) were calculated according to the equation of Cheng and Prusoff
(35).
Table 7: Half life (t1/2) and intrinsic clearance (Clint) data for compounds 9 and 12 in rat liver
microsomal system.
Compound
t1/2
[min]
136
108
5
Clint
[µL/mg/min]
26
32
302
9
12
Imipramine (37)
Table 8: Half life (t1/2) and intrinsic clearance (Clint) data for compounds 9 and 12 in rat liver
microsomal system.
Compound
t1/2
[min]
136
108
5
Clint
[µL/mg/min]
26
32
302
9
12
Imipramine (36)
This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.