Anticonvulsant evaluation of aminoalkanol derivatives of 2- and 4-methylxanthone

Article abstract of DOI:10.1016/j.bmc.2012.12.051

A series of 17 new aminoalkanol derivatives of 6-methoxy- or 7-chloro-2-methylxanthone as well as 6-methoxy-4-methylxanthone was synthesized and evaluated for anticonvulsant activity. All compounds were verified in mice after intraperitoneal (ip) administ

Full text of DOI:10.1016/j.bmc.2012.12.051

Bioorganic & Medicinal Chemistry 21 (2013) 1190–1198
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Anticonvulsant evaluation of aminoalkanol derivatives of 2- and
4-methylxanthone
a
a
b
Natalia Szkaradek a, , Agnieszka Gunia , Anna M. Waszkielewicz , Lucyna Antkiewicz-Michaluk ,
⇑
Marek Cegła c, Edward Szneler d, Henryk Marona a
a Department of Bioorganic Chemistry, Chair of Organic Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Krakow, Poland
b Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland
c Chair of Organic Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Krakow, Poland
d Faculty of Chemistry, Jagiellonian University, Krakow, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 17 new aminoalkanol derivatives of 6-methoxy- or 7-chloro-2-methylxanthone as well as 6-
methoxy-4-methylxanthone was synthesized and evaluated for anticonvulsant activity. All compounds
were verified in mice after intraperitoneal (ip) administration in maximal electroshock (MES) and subcu-
taneous pentetrazole (scMet) induced seizures as well as neurotoxicity assessment. Eleven of the tested
substances showed protection against electrically evoked seizures in the majority of the tested mice at
the dose of 100 mg/kg. Additionally, one was effective at the dose of 30 mg/kg. Five substances were
active at the dose of 300 mg/kg or at the dose of 100 mg/kg in the minority of the tested mice. The most
promising compound revealed ED50 value of 47.57 mg/kg in MES (mice, ip, 1 h after administration) and
Received 16 October 2012
Revised 2 December 2012
Accepted 21 December 2012
Available online 9 January 2013
Keywords:
Anticonvulsant
MES
Neurotoxicity
Xanthone
Synthesis
at the same time its TD50 was evaluated as above 400 mg/kg. Those values gave PI (calculated as TD50
/
ED50) of more than 8.41. Three other synthesized xanthone derivatives also proved to act as anticonvul-
sants and showed ED50 values in MES test (mice, ip) ranged 80–110 mg/kg. Results were quite encourag-
ing and suggested that in the group of xanthone derivatives new potential anticonvulsants might be
found.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
covery of AEDs includes mostly in vivo screening tests and is based
on seizure types rather than etiology.4
Epilepsy belongs to the most common neurological disorders
and constitutes an important cause of disability and mortality. Re-
cent findings indicate that it affects about 50 million people world-
wide. The main objective of pharmacotherapy of epilepsy is to
control the seizures, which could mean either total elimination
or at least limitation of their number or severity. Despite the avail-
ability of many various antiepileptic drugs (AEDs), there are still
approximately one third of epilepsy patients that are inadequately
treated. Patients suffering from drug resistant epilepsy are charac-
terized by increased risks of premature death, injuries, psychoso-
cial dysfunction, and reduced quality of life. Moreover, the use of
antiepileptic drugs is associated with multiple and often serious
adverse effects.1–3 AEDs have various mechanisms of action that
are still not fully understood. Many of the AEDs reveal several
mechanisms of action and new proposed mechanisms and evi-
dence for clinically important targets are still emerging. Thus, dis-
While searching for new substances with anticonvulsant activ-
ity we directed our attention to derivatives of xanthone. Xanthone
skeleton possesses several potential substitution positions and
respectively to the kind of substituents and the place of substitu-
tion in one of the xanthone rings, many pharmacological activities
of xanthone derivatives were described (such as analeptic,5 antial-
lergic,6 antilipidemic,7 diuretic,8 antitumor9 or antimicrobial10–12).
It is important that one specific xanthone derivative reveals rather
one profile of in vivo activity without influence on other biological
targets, that was mentioned among others in our former publica-
tion13 when xanthones showed circulatory but not anticonvulsant
activity. Nevertheless, our former research proved that some xan-
thone derivatives can also act as anticonvulsants. They provided
protection against seizures, mainly MES-induced seizures and at
the same time exhibited low neurotoxicity.14–16 The very promis-
ing compounds were (S)-2-aminobutan-1-ol, and (R,S)- or (R)-2-
N-methylaminobutan-1-ol derivatives of 7-chloro-2-methylxanth-
one (Fig. 1) with anti-MES ED50 (mg/kg, in mice ip) values of 76.5,
27.0, and 33.4, respectively.14 We have also previously reported
some derivatives of 6-methoxy-2-methylxanthone with anticon-
vulsant activity for example, derivatives of (R,S)- and (S)-1-amino-
⇑
Corresponding author. Address: 9 Medyczna Str., 30-688 Krakow, Poland. Tel.:
+48 12 620 55 76; fax: +48 12 620 54 05.
E-mail address: nszkarad@cm-uj.krakow.pl (N. Szkaradek).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.051

N. Szkaradek et al. / Bioorg. Med. Chem. 21 (2013) 1190–1198
1191
were synthesized in
procedures.14,15,20
a
similar way to previously published
2.2. Pharmacology
The anticonvulsant activity and neurotoxicity of all synthesized
compounds 1–17 were evaluated according to the standard proto-
cols within Antiepileptic Drug Development (ADD) Program at Na-
tional Institute of Neurological Disorders and Stroke, National
Institutes of Health, Rockville, USA.21,22 At present there are three
in vivo screens routinely used by most of antiepileptic drugs dis-
covery programs that include the maximal electroshock (MES)
and subcutaneous pentetrazole (scMet) evoked seizure tests as
well as neurotoxicity assay. MES and scMet are considered as the
‘gold standards’ at the early stages of anticonvulsant testing. In
the MES and scMet activity against generalized tonic–clonic sei-
zures and generalized absence seizures might be recognized,
respectively.23
The anticonvulsant screening showed that all evaluated com-
pounds revealed activity in the MES test (Table 2), which indicates
the ability of substances to prevent seizure spread. The most prom-
ising structure seems to be 3, which was effective at the dose of
30 mg/kg (that is why that compound was evaluated also in lower
doses). Compounds 1, 2, 4, 5, 8–10, 12, 13, 15 and 17 were effica-
cious in the majority of the tested mice at the dose of 100 mg/kg.
Compounds 6, 7, 11, 14 and 16 were active at the dose of
300 mg/kg or at the dose of 100 mg/kg in the minority of the tested
mice. The activity in MES screen observed at 0.5 h after com-
pound’s injection could be explained by the short lasting anticon-
vulsant activity or the lack of active metabolites. Some of the
tested structures revealed also long duration of anticonvulsant
activity observed mainly at the dose of 300 mg/kg, while com-
pound 15 was active only at 4 h after administration (both at 100
and 300 mg/kg). Considering scMet results only one compound
(11) showed some activity but only in minority of the tested ani-
mals (1 of 5). Those finding suggest that those substances have lit-
tle or no ability to increase seizures’ threshold. Taking into account
neurotoxicity assay, it can be stated that the most promising xan-
thone derivatives (1, 2, 3 and 17) were devoid of neurotoxic effects
(or revealed some balance deficiency in as few as in two from eight
of the tested animals) at the smallest effective doses. Other com-
pounds showed very diversified range of neurotoxicity (see
Table 2).
Figure 1. General structure of the most active derivatives of 7-chloro-2-
methylxanthone.14
propan-2-ol as well as (R,S)-1-aminobutan-2-ol which showed val-
ues of ED50 in MES (mice, ip) of 37.4, 33.89, and 41.2 mg/kg, respec-
tively (Fig. 2).15
Considering our former findings we decided to continue the re-
search in the group of methylxanthone derivatives. Structures
were designed to check influence of both place and kind of substi-
tution on anticonvulsant activity. So far, mainly 2-methylxanthone
derivatives have been evaluated and described, thus the present
paper describes synthesis, evaluation of anticonvulsant activity
and neurotoxicity of aminoalkanol derivatives of both 6-meth-
oxy-2-methyl- and 6-methoxy-4-methylxanthone in order to esti-
mate influence of place of substitution on the anticonvulsant
activity. Herein we also report on 7-chloro-2-methylxanthone
derivatives of some new aminoalkanols, which constitute continu-
ation of our former research,14,17 for more efficient SAR analysis.
New branched aminoalkanol was introduced to evaluate its ability
to serve as pharmacophore element when compared to linear
aminoalkanols (aminopropanol and aminobutanol). The tested
compounds were obtained in the form of racemic mixture and
some additionally in the form of pure enantiomers to determine
stereochemistry and activity relationship.
2. Results and discussion
2.1. Chemistry
Chemical structures of the tested compounds are shown in
Table 1. The synthesis of 1–17 was a multistage process. At
first, substituted benzoic acid reacted with 2- or 4-methylphenol
in two step reaction involving Ullmann condensation and
electrophylic addition.18,19 6-Chloro-2-methyl-9H-xanthen-9-one,
6-chloro-4-methyl-9H-xanthen-9-one or 7-chloro-2-methyl-9H-
xanthen-9-one were obtained as products. 6-Chloro-2-methyl-
9H-xanthen-9-one and 6-chloro-4-methyl-9H-xanthen-9-one were
used in the methanolysis in order to synthesize 6-methoxy-2-
methyl-9H-xanthen-9-one and 6-methoxy-4-methyl-9H-xanthen-
9-one.9,15 Intermediate methyl derivatives of substituted xanthone
were used in the reaction with N-bromosuccinimide giving appro-
priate bromide derivatives (as shown in Scheme 1). The last step
constituted an aminolysis by means of appropriate aminoalkanol
carried out in toluene in the presence of anhydrous K2CO3 as a
proton acceptor. Raw bases were recrystallized from a toluene/
n-hexane (5:1 v/v) mixture. Compound 5 was obtained as hydro-
chloride salt from the appropriate base, as it was found difficult
to crystallize and precipitate the pure base. The substances
On the basis of the data obtained from ip screen in mice and
according to the Anticonvulsant Screening Project (ASP) disposi-
tion, 14 compounds: 1–3, 5–10, 12–13 and 15–17 were examined
for their anti-MES activity and neurotoxicity after oral administra-
tion (po) in rats at the dose of 30 mg/kg (see Table 3). According to
the obtained results, compounds 1 and 3 seemed to be the most
potent. They exhibited activity after 15 min of administration,
which persisted until 4 h of observation with concomitant lack of
neurotoxicity. Curiously enough, compound 17 which displayed
long effectiveness after ip injection in mice was active only after
0.5 h of oral administration into rats. One of probable reasons
may be its poor absorption from alimentary track.
Some clinically useful AEDs are ineffective in the standard MES
and scMet tests but still have anticonvulsant activity in vivo. In or-
der to identify potential AEDs with this profile, some compounds
may be tested in 6 Hz psychomotor seizure model.21 Similar to
the maximal electroshock (MES) test, 6 Hz test is used to assess a
compound’s efficacy against electrically-induced seizures but uses
a lower frequency (6 Hz) and longer duration of stimulation. Com-
pound 5 underwent 6 Hz test at the dose of 100 mg/kg without any
significant activity during 4 h of observation.
The most promising compounds were selected for quantitative
evaluation in mice after intraperitoneal administration (Table 4).
Figure 2. General structure of the most active derivatives of 6-methoxy-2-
methylxanthone.16

1192
N. Szkaradek et al. / Bioorg. Med. Chem. 21 (2013) 1190–1198
Table 1
2.4. Structure–anticonvulsant activity relationship
General route of synthesis and structures of the tested compounds 1–17
Currently presented results together with former find-
ings14,15,17,20,24,25 enabled drawing general conclusions about the
relationship between structure and anticonvulsant activity in the
group of xanthone derivatives. So far, we have described only
one compound with side chain in the position 4 of the xanthone
ring.17 It proved to be less potent than its 2-substituted analogue.
In our opinion, synthesis and biological assay of other 4 substituted
xanthone derivatives reported in the present publication was ra-
tional for completing structure–activity relationship study. Other
factors potentially influencing anticonvulsant activity are type of
aminoalkanol and stereochemistry.
O
O
O
O
8
5
1
4
7
6
2
3
R
aminolysis
Br
N
R''
toluene/K2CO3
R''
R'
Compound Configuration Position R00
Position R0
R
1
2
3
R,S
R
S
6
OCH3
2
H
CH2OH
C2H5
OH
*
From the group of 6-methoxy substituted xanthone derivatives
the most potent were those with 2-aminobutan-1-ol moiety, both
substituted in position 2 (compounds 1–3) as well as in position 4
(compounds 8–10). 2-Substituted substances were slightly more
potent than 4-substituted. Moreover 6-methoxy-2-substituted
xanthones were less neurotoxic than their 6-methoxy-4-substi-
tuted analogues. More evident differences in activity’s profile of
2- and 4-substituted xanthones were observed for 2-aminopropan-
1-ol and 1-aminopropan-2-ol derivatives. (R,S)-2-((1-Hydroxypro-
pan-2-ylamino)methyl)-6-methoxy-9H-xanthen-9-one, (R,S)-2-((2-
hydroxypropan-1-ylamino)methyl)-6-methoxy-9H-xanthen-9-one
and their enantiomers, which were subject of former publication,15
revealed good anti-MES activity in mice at the dose of 100 mg/kg
and possessed PI values ranging 2.2–2.6. Herein we report on their
4-substituted analogues: (R,S)-, (R)-, (S)-4-((1-hydroxypropan-2-
ylamino)methyl)-6-methoxy-9H-xanthen-9-one (compounds 4–6)
4
R,S
6
6
OCH3
CH3
4
4
H
H
CH3
*
5
6
7
R,S⁄HCl
R
S
CH2OH
CH3
*
8
9
10
R,S
R
S
6
OCH3
4
H
CH2OH
C2H5
*
CH2OH
C2H5
11
R,S
6
6
OCH3
OCH3
4
4
CH3
H
*
CH2OH
12
13
14
—
—
—
CH3
CH3
CH2OH
and
(R,S)-4-((2-hydroxypropan-1-ylamino)methyl)-6-methoxy-
9H-xanthen-9-one (compound 6) which were significantly less
potent and were not even qualified for anticonvulsant quantifica-
tion. Considering aminoalkanol type, the anticonvulsant activity
of derivatives containing 2-aminobutan-1-ol and 2-aminopropan-
1-ol were similar and quite promising in investigated groups of
7-chloroxanthone and 6-methoxyxanthone. Introduction of
branched aminoalkanol (2-amino-2-methylpropan-1-ol) as well
as N-methylation did not give clear understanding of the influence
on anticonvulsant activity. As for the stereochemistry four sets of
recemates together with enantiomers were tested. Considering
anti-MES activity it is not possible to establish relationship be-
tween stereochemistry and anticonvulsant properties because all
sets of compound gave different results. Racemate and enentio-
mers showed either similar results or differed in duration of activ-
ity or lower effective doses.
7
Cl
2
CH3
CH3
CH3
CH2OH
7
7
Cl
Cl
2
2
H
H
CH3
CH3
15
16
17
R,S
R
S
CH2OH
CH3
*
Protection index (PI = TD50/ED50) values calculated for substances 3
and 17 were >8.408 and 3.926, and were similar to that of phenyt-
oin (PI = 6.6) and carbamazepine (PI = 4.9). Compound 2 which is
an R enantiomer of compound 3 and their racemate (compound
1) possessed PI values of 2.510 and 2.662, respectively, that were
comparable to that of valproate (PI = 1.7).22
Reasumming, the spectrum of activity depended on the kind of
the substituents and position of the substitution in one of the xan-
thone’s rings and conclusions formulated above should be taken
under consideration in future investigations.
2.3. In vitro study
AEDs exert their action by different mechanisms and, what is
more, some of them can act by more than one mechanism. The
drug can influence the inhibitory or excitatory neurotransmitters
systems or transmembrane transport of ions. Our former results
suggested that anticonvulsant activity of xanthone derivatives
may result from influence on voltage-dependent Ca2+ channels
(VDCC), for example, 7-chloro-2-methylxanthone possessing 2-
3. Conclusions
In conclusion, we have synthesized a series of 17 new aminoalk-
anol derivatives of 6-methoxy- or 7-chloro-2-methylxanthone as
well as 6-methoxy-4-methylxanthone. All compounds were tested
for anticonvulsant activity and all of them showed activity in the
MES screen which is recognized as one of the two most widely
used seizures models for early identification of candidate anticon-
vulsants. Four compounds were especially promising and they
were used in quantitative assays. The results of the tested sub-
stances are quite encouraging, therefore further modification of
their structures might lead to discovering new potential anticon-
vulsants. In addition, it seems that anticonvulsant activity was
associated with both the aminoalkanol type and its configuration
as well as place of substitution in the xanthone skeleton.
aminopropan-1-ol moiety proved affinity to VDCC at low lM level
(EC50 = 7.6 l
M).20 Herein we present further investigation in that
field. The radioligand 3H-nitrendipine was used for estimating
VDCC affinity of the selected most active compounds. The obtained
results have shown that tested substances displaced 3H-nitrendi-
pine from its binding sites in
compounds 1 and 3 to VDCC were in low
(EC50 = 1.28 and 0.96 M, respectively). Compound 2 did not prove
the affinity to voltage-dependent calcium channels.
lM concentrations. The affinities of
l
M concentrations
l

N. Szkaradek et al. / Bioorg. Med. Chem. 21 (2013) 1190–1198
1193
Scheme 1. General route for synthesis of the parent compounds.
The most promising compound was (S)-2-((1-hydroxybutan-2-
ylamino)methyl)-6-methoxy-9H-xanthen-9-one (3) which re-
vealed ED50 value of 47.57 mg/kg in MES (mice, ip, after 1 h of
administration) and at the same time its TD50 was evaluated as
more than 400 mg/kg. Those values gave PI more than 8.41. Other
synthesized xanthone derivatives that is, (R,S)- (1) and (R)-2-((1-
hydroxybutan-2-ylamino)methyl)-6-methoxy-9H-xanthen-9-one
(2), as well as (S)-2-chloro-7-(1-hydroxypropan-2-ylami-
no)methyl-9H-xanthen-9-one (17) also proved to act as anticon-
vulsants and showed ED50 values in MES test (mg/kg mice, ip) of
79.96, 107.26, and 79.74, respectively.
60 F-254 Merck, Darmstadt, Germany) using the solvents indicated
below. Spots were visualized by UV light. Specific rotation was
measured on Jasco P-2000 polarimeter (1% w/v solutions in chloro-
form, sodium light 589 nm). Theoretical values of the partition
coefficient (LogP) of the tested compounds were calculated by
means of ACDLABS 12.0 program.
4.2. General procedure for the preparation of the tested
compounds
Synthesis of compounds 1–17 was multistage. The first stages
and intermediates were previously described9,14–16 and are shown
in Scheme 1. Compounds 1–11 and 14–17 were obtained by ami-
nation of 2-(bromomethyl)-6-methoxy-9H-xanthen-9-one, 4-(bro-
momethyl)-6-methoxy-9H-xanthen-9-one or 2-(bromomethyl)-
7-chloro-9H-xanthen-9-one with appropriate aminoalkanols in
toluene in a presence of anhydrous K2CO3, according to the earlier
published procedures.14,15,20 Compounds 12 and 13, as a tertiary
amines, were obtained from compounds 8 and 14 by N-methyla-
tion using mixture of 30% formaldehyde and 95% formic acid as
described in procedures.14 The crude bases were recrystallized from
toluene/n-hexane (5:1). Compound 5 was evaluated in the form of
hydrochloride salt. Conversion was carried out in propanol/acetone
(4:1 v/v) with an excess of ethanol saturated with HCl. The crude
product was recrystallized from acetone/ethanol (1:3 v/v).
4. Experimental protocols
4.1. Chemistry
Reagents: (R,S)-, (R)-, (S)-2-aminobutan-1-ol, 2-amino-2-meth-
ylpropan-1-ol, (R,S)-, (R)-, (S)-2-aminopropan-1-ol, 2,4-, 2,5-
dichlorobenzoic acid, o- and p-cresol, N-bromosuccinimide, ben-
zoyl peroxide, formaldehyde, formic acid and K2CO3 were pur-
chased from Alfa Aesar (Karlsruhe, Germany). Solvents were
commercially available materials of reagent grade. Melting points
(mp) are uncorrected and were determined using a Büchi SMP-
20 apparatus (Büchi Labortechnik, Flawil, Switzerland). The infra-
red spectra were recorded on potassium bromide pellets using a
Jasco FT/IR 410 spectrometer (Jasco Inc., Easton, MD, USA). The
1H NMR spectra were obtained in CDCl3 or DMSO-d6 with a Varian
Mercury-VX 300 NMR spectrometer (Varian Inc., Palo Alto, CA,
USA), a Bruker Avance II spectrometer (Brucker, Karlsruhe, Ger-
many) with 500.13 MHz or a Bruker Avance III spectrometer (Bruc-
ker, Karlsruhe, Germany) with 600.2 MHz with TMS or DMSO as an
internal standard, respectively. Results are presented in the follow-
ing format; chemical shift d in ppm, multiplicity, coupling constant
J in Hertz (Hz), number of protons, proton’s position. Multiplicities
are showed as the abbreviations: s (singlet), br s (broad singlet), d
(doublet), dd (doublet of doublets), t (triplet), m (multiplet). Ele-
mental analyses were performed on a Elementar Vario EL III (Ele-
mentar Analysensysteme, Hanau, Germany). The purity of
obtained compounds was confirmed by the thin-layer chromatog-
raphy (TLC), carried out on precoated aluminum sheets (Silica Gel,
4.2.1. (R,S)-2-((1-Hydroxybutan-2-ylamino)methyl)-6-methoxy-
9H-xanthen-9-one (1)
White solid (yield 60%), mp 140–142 °C. Anal. Calcd for
C18H17NO3: C, 69.70; H, 6.47; N, 4.27. Found: C, 70.01; H, 6.13; N,
4.09. IR (KBr) in vivo (cmꢀ1): 3417, 1620, 1452, 1259, 1103, 827,
779. 1H NMR (CDCl3, 600 MHz) d (ppm): 0.86 (t, J = 7.4, 3H, –
CH3), 1.36–1.45 (m, 2H, –CH2), 2.05 (br s, 1H, –NH–), 2.40–2.45
(m, 1H, –CH–), 3.32–3.36 (m, 1H, –CHH-CH3), 3.39–3.45 (m, 1H,
–CHH-CH3), 3.85 (s, 2H, Ar-CH2-NH–), 3.94 (s, 3H, Ar-O-CH3),
4.44 (t, J = 5.4, 1H, –OH), 7.05 (dd, J = 8.9, J = 2.4, 1H, H-7), 7.16
(d, J = 2.4, 1H, H-5), 7.58 (dd, J = 8.5, J = 0.4, 1H, H-4), 7.82 (dd,
J = 8.5, J = 2.3, 1H, H-3), 8.11 (d, J = 8.9, 1H, H-8), 8.13 (dd, J = 2.3,
J = 0.4, 1H, H-1). Rf = 0.54 (methanol/ethyl acetate (3/1)).
LogP = 3.09 0.56.

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N. Szkaradek et al. / Bioorg. Med. Chem. 21 (2013) 1190–1198
Table 2
The results of anticonvulsant activity evaluation in mice (ip)
Compound
Dose (mg/kg)
MESa
ScMeta
Neurotoxicityb
0.25 h
0.5 h
1.0 h
4.0 h
0.5 h
4.0 h
0.25 h
0.5 h
1.0 h
4.0 h
1
30
100
300
—
—
—
0/1
3/3
1/1
—
—
—
0/1
0/3
1/1
0/1
0/1
0/1
0/1
0/1
0/1
—
—
—
0/4
0/8
3/4
—
—
—
0/2
0/4
1/2
2
3
30
100
300
—
—
—
0/1
3/3
1/1
—
—
—
0/1
1/3
1/1
0/1
0/1
0/1
0/1
0/1
0/1
—
—
—
0/4
2/8
1/4
—
—
—
0/2
0/4
0/2
3
10
30
100
300
—
—
—
—
—
0/4
1/4
1/1
3/3
1/1
—
—
—
—
—
—
—
0/1
3/3
1/1
—
—
0/1
0/1
0/1
—
—
0/1
0/1
0/1
—
—
—
—
—
0/4
0/4
0/1
1/8
3/4
—
—
—
—
—
—
—
0/2
0/4
2/2
4
5
30
100
300
—
—
—
0/1
3/3
—
—
—
—
0/1
2/3
—
0/1
0/1
—
0/1
0/1
—
—
—
—
0/4
8/8
4/4
—
—
—
0/2
1/4
—
30
100
300
—
—
—
0/1
2/3
—
—
—
—
0/1
0/3
—
0/1
0/1
—
0/1
—
—
—
—
—
0/4
6/8
4/4
—
—
—
0/2
3/3
—
6
30
100
300
—
—
—
0/1
0/3
1/1
—
—
—
0/1
0/3
1/1
0/1
0/1
0/1
0/1
0/1
—
—
—
—
0/4
2/8
4/4
—
—
—
0/2
0/4
2/2
7
30
100
300
—
—
—
0/1
1/3
0/1
—
—
—
0/1
0/3
1/1
0/1
0/1
0/1
0/1
0/1
0/1
—
—
—
0/4
4/8
2/4
—
—
—
0/2
0/4
0/2
8
30
100
300
—
—
—
0/1
3/3
1/1
—
—
—
0/1
0/3
—
0/1
0/1
Death
0/1
0/1
—
—
—
—
0/4
8/8
4/4
—
—
—
0/2
0/4
1/1
9
30
100
300
—
—
—
0/1
2/3
1/1
—
—
—
0/1
0/3
1/1
0/1
0/1
0/1
0/1
0/1
0/1
—
—
—
0/4
0/8
4/4
—
—
—
0/2
0/4
0/2
10
11
12
13
14
15
16
17
30
100
300
—
—
—
0/1
2/3
1/1
—
—
—
0/1
1/3
1/1
0/1
0/1
0/1
0/1
0/1
0/1
—
—
—
2/4
4/8
4/4
—
—
—
0/2
1/4
1/2
30
100
300
—
0/3
—
0/1
0/2
1/1
—
0/3
—
0/1
0/3
1/1
0/1
0/1
0/1
1/5
0/1
1/5
—
0/3
—
1/4
3/8
1/4
—
0/3
—
1/2
0/4
0/2
30
100
300
—
—
—
0/1
2/2
—
—
—
—
0/1
0/3
—
0/1
0/1
—
0/1
0/1
—
—
—
—
0/4
8/8
4/4
—
—
—
0/2
0/4
—
30
100
300
—
—
—
0/1
2/3
1/1
—
—
—
0/1
2/3
1/1
0/1
0/1
0/0
0/1
0/1
0/1
—
—
—
1/4
3/8
3/4
—
—
—
0/2
0/4
0/2
30
100
300
—
1/3
—
0/1
0/3
1/1
—
0/3
—
0/1
0/3
1/1
0/1
0/1
0/1
0/1
0/1
0/1
—
0/3
—
0/4
1/8
2/4
—
1/3
—
1/2
0/4
0/2
30
100
300
—
—
—
0/1
0/3
0/1
—
—
—
0/1
2/3
1/1
0/1
0/1
0/1
0/1
0/1
0/1
—
—
—
0/4
0/8
0/4
—
—
—
0/2
0/4
0/2
30
100
300
—
—
—
0/1
1/3
1/1
—
—
—
0/1
0/3
1/1
0/1
0/1
0/1
0/1
0/1
0/1
—
—
—
0/4
0/8
2/4
—
—
—
0/2
0/4
0/2
30
100
300
—
—
—
0/1
2/3
0/1
—
—
—
0/1
3/3
1/1
0/1
0/1
0/1
0/1
0/1
0/1
—
—
—
0/4
0/8
0/4
—
—
—
0/2
0/4
0/2
— The compound was not tested in the particular case.
a
Number of animals protected/number of animals tested in the MES and ScMet tests.
Number of animals displaying motor impairment/number of animals used in the rotarod test.
b
4.2.2. (R)-2-((1-Hydroxybutan-2-ylamino)methyl)-6-methoxy-
9H-xanthen-9-one (2)
4.2.3. (S)-2-((1-Hydroxybutan-2-ylamino)methyl)-6-methoxy-
9H-xanthen-9-one (3)
White solid (yield 70%), mp 152–153 °C. Anal. Calcd for
White solid (yield 50%), mp 152–154 °C. Anal. Calcd for
C
19H21NO4: C, 69.70; H, 6.47; N, 4.28. Found: C, 69.97; H, 6.21;
C19H21NO4: C, 69.70; H, 6.47; N, 4.28. Found: C, 69.57; H, 6.19;
N, 4.38. IR and 1H NMR spectra as well as Rf and LogP values are
N, 4.11. IR and 1H NMR spectra as well as Rf and LogP values are
analogous to that of racemate (compound 1). ½a D20
ꢁ
¼ ꢀ19:05.
analogous to that of racemate (compound 1). ½a D20
¼ 19:05.
ꢁ

N. Szkaradek et al. / Bioorg. Med. Chem. 21 (2013) 1190–1198
1195
Table 3
The results of anticonvulsant activity evaluation in rats (po)
Compound
Test
Dose (mg/kg)
Time/results
1.0 h
0.25 h
0.5 h
2.0 h
4.0 h
1
2
MESa
TOXb
MESa
TOXb
30
30
1/4
0/4
3/4
0/4
0/4
0/4
2/4
0/4
2/4
0/4
30
30
0/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
1/4
0/4
3
MESa
TOXb
30
30
2/4
0/4
1/4
0/4
1/4
0/4
3/4
0/4
1/4
0/4
5
MESa
TOXb
30
30
0/4
0/4
2/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
6
MESa
TOXb
30
30
0/4
0/4
1/4
1/4
0/4
1/4
3/4
0/4
0/4
1/4
7
MESa
TOXb
30
30
0/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
8
MESa
TOXb
30
30
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
9
MESa
TOXb
30
30
1/4
0/4
0/4
0/4
1/4
0/4
2/4
0/4
1/4
0/4
10
12
13
15
16
17
MESa
TOXb
MESa
TOXb
MESa
TOXb
MESa
TOXb
30
30
0/3
0/3
1/4
0/4
1/4
0/4
2/4
0/4
0/4
0/4
30
30
0/4
0/4
1/4
0/4
1/4
0/4
0/4
0/4
1/4
0/4
30
30
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
30
30
0/4
0/4
0/4
0/4
2/4
0/4
0/4
0/4
1/4
0/4
MESa
TOXb
30
30
1/4
0/4
0/4
0/4
2/4
0/4
0/4
0/4
1/4
0/4
MESa
TOXb
30
30
2/4
0/4
2/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
a
Number of animals protected/number of animals tested in the MES test.
Number of animals displaying motor impairment/number of animals used in neurotoxicity test.
b
Table 4
–CH–), 3.95 (s, 3H, –O-CH3), 4.16 (s, 2H, Ar-CH2-), 6.92–8.28
(m, 6H, Ar-H). Rf = 0,43 (methanol/ethyl acetate (1/1)). LogP =
2.56 0.56.
The results of anticonvulsant quantification in mice after intraperitoneal
administration
Compound ED50 MES1 (mg/kg)
ED50 ScMet1 (mg/kg) TD50 (mg/kg)
2
1
79.96a
>500a
PI <0.43
212.85b
(171.85–275.38)
4.2.5. (R,S)-5-((1-Hydroxypropan-2-ylamino)methyl)-3-
methoxy-9H-xanthen-9-one hydrochloride (5)
(66.65–92.61)
PI = 2.66
White solid (yield 50%), mp 206–208 °C. IR (KBr) m
(cmꢀ1): 3399,
2
107.26b
>175b
269.27b
1605, 1439, 1245, 1104, 848, 772. 1H NMR (DMSO-d6, 500.13 MHz)
d ppm): 1.01 (d, J = 6.3, –CHCH3), 2.25 (br s, 1H, –NH–), 2,68–2.71
(m, 1H, –CH–), 3.29–3.36 (m, 2H, –CH2OH), 3.95 (s, 3H, –OCH3),
4.08–4.15 (m, 2H, Ar-CH2), 4.53 (t, J = 5.4, 1H, –OH), 7.06 (dd,
J = 8.9, J = 2.4, 1H, H-7), 7.17 (d, J = 2.4, 1H, H-5), 7.41 (t, J = 7.6,
1H, H-2), 7.89 (dd, J = 7.3, J = 1.5, 1H, H-3), 8.05 (dd, J = 7.9,
J = 1.6, 1H, H-1), 8.10 (d, J = 8.9, 1H, H-8). Rf = 0.49 (methanol/ethyl
acetate (3/1)). LogP = 2.56 0.56. Base of compound 5: Anal. Calcd
for C18H19NO4: C, 68.99; H, 6.11; N, 4.47. Found: C, 68.67; H, 6.06;
N, 4.41.
(68.07–148.03)
PI = 2.51
PI <1.54
(235.76–301.79)
3
47.57b
>200b
>400c
(33.87–60.16)
PI >8.41
17
79.74b
>375d
PI <0.84
313.09d
(251.13–382.27)
(63.71–102.12)
PI = 3.93
1
Dose of the compound which gives protection against seizures in 50% of tested
animals; 95% confidence interval is shown in brackets.
2
Dose of the compound which produces neurotoxicity in 50% of tested animals;
PI (protection index) = TD50/ED50; results observed after 0.5 ha; 1.0 hb; 0.25 hc;
2.0 hd of administration of the compound.
4.2.6. (R)-5-((1-Hydroxypropan-2-ylamino)methyl)-3-methoxy-
9H-xanthen-9-one (6)
White solid (yield 65%), mp 126–128 °C. Anal. Calcd for
18H19NO4: C, 68.99; H, 6.11; N, 4.47. Found: C, 68.95; H, 5.90;
4.2.4. (R,S)-5-((2-Hydroxypropan-1-ylamino)methyl)-3-
methoxy-9H-xanthen-9-one (4)
C
N, 4.20. IR (KBr)
m
(cmꢀ1): 3434, 3070, 2850, 1619, 1438, 1287,
White solid (yield 55%), mp 115–117 °C. Anal. Calcd for
1063, 761. 1H NMR (CDCl3, 600 MHz) d ppm): 1.01 (d, J = 6.3, 3H,
–CH3), 2.26 (br s, 1H, –NH–), 2.65–2.72 (m, 1H, –CH–), 3.28–3.37
(m, 2H, –CH2-OH), 3.95 (s, 3H, Ar-O-CH3), 4.07 (d, J = 14.6, 1H,
Ar-CHH-NH-), 4.13 (d, J = 14.6, 1H, Ar-CHH-NH-), 4.55 (t, J = 5.5,
1H, –OH), 7.06 (dd, J = 8.8, J = 2.4, 1H, H-7), 7.18 (d, J = 2.4, 1H, H-
5), 7.42 (dd, J = 7.9, J = 7.3, 1H, H-2), 7.89 (dd, J = 7.3, J = 1.9, 1H,
C
18H19NO4: C, 68.99; H, 6.11; N, 4.47. Found: C, 69.35; H, 5.86;
N, 3.76. IR (KBr)
m
(cmꢀ1): 3430, 1628, 1447, 1255, 923, 778. 1H
NMR (DMSO-d6, 300 MHz) d ppm): 1.18 (d, J = 6.2, 3H, –CH3),
2.50 (dd, J = 12.2, J = 9.3, 1H, –NH-CHH-CH–), 2.80 (dd, J = 12.1,
J = 3.1, 1H, –NH-CHH-CH–), 3.82 (s, 1H, –OH), 3.83–3.90 (m, 1H,

1196
N. Szkaradek et al. / Bioorg. Med. Chem. 21 (2013) 1190–1198
H-3), 8.05 (dd, J = 7.9, J = 1.9, 1H, H-1), 8.09 (d, J = 8.8, 1H, H-8).
¼ ꢀ22:43. Rf = 0.47 (methanol/ethyl acetate (3/1)).
LogP = 2.56 0.56.
4.2.13. 2-Chloro-7-((N-(1-hydroxy-2-methylpropan-2-
yl)methylamino)methyl)-9H-xanthen-9-one (13)
White solid (yield 55%), mp 132–134 °C. IR (KBr)
½ ꢁ
a 2D0
m
(cmꢀ1):
3262, 1671, 1463, 1314, 1150, 1073, 790. Rf = 0,50 (methanol/ethyl
acetate (3/1)). LogP = 4.19 0.56. Hydrochloride of compound 13:
Anal. Calcd for C19H21NO3Cl2: C, 59.69; H, 5.53; N, 3.66. Found: C,
59.74; H, 5.50; N, 3.87. 1H NMR (DMSO-d6, 600 MHz) d ppm):
1.37 (s, 3H, –CH3), 1.43 (s, 3H, –CH3), 2.53 (d, J = 4.9, 3H, ˜N-
CH3), 3.65 (d, J = 12.1, 1H, –CHH-OH), 3.84 (d, J = 12.1, 1H, –CHH-
OH), 4.22 (dd, J = 12.7, J = 9.4, 1H, Ar-CHH-N—), 4.74 (d, J = 12.7,
1H, Ar-CHH-N—), 5.98 (br s, 1H, –OH), 7.77 (dd, J = 8.9, J = 0.6, 1H,
H-5), 7.78 (d, J = 8.6, 1H, H-4), 7.95 (dd, J = 8.9, J = 2.7, 1H, H-6),
8.13 (dd, J = 8.6, J = 2.2, 1H, H-3), 8.14 (dd, J = 2.7, J = 0.6, 1H, H-
8), 8.43 (d, J = 2.2, 1H, H-1), 9.51 (br s, 1H, NH+).
4.2.7. (S)-5-((1-Hydroxypropan-2-ylamino)methyl)-3-methoxy-
9H-xanthen-9-one (7)
White solid (yield 55%), mp 126–128 °C. Anal. Calcd for
C
18H19NO4: C, 68.99; H, 6.11; N, 4.47. Found: C, 68.63; H, 5.92;
N, 4.74. IR and 1H NMR spectra as well as Rf and LogP values are
analogous to that of R enantiomer (compound 6). ½a D20
¼ 22:40.
ꢁ
4.2.8. (R,S)-5-((1-Hydroxybutan-2-ylamino)methyl)-3-methoxy-
9H-xanthen-9-one (8)
White solid (yield 55%), mp 114–116 °C. Anal. Calcd for
C19H21NO4: C, 69.71; H, 6.47; N, 4.28. Found: C, 69.47; H, 6.33;
N, 4.56. 1H NMR (CDCl3, 500.13 MHz) d ppm): 0.96 (t, J = 7.5, 3H,
–CH2CH3), 1.48–1.64 (m, 2H, –CH2CH3), 2.69–2.76 (m, 1H, –CH–),
3.43 (dd, J = 6.1, J = 10.7, 1H, –CHHOH), 3.78 (dd, J = 4.0, J = 10.7,
1H, –CHHOH), 3.94 (s, 3H, –OCH3), 4.10 (d, 1H, J = 13.3, ArCHH-),
4.12 (d, 1H, J = 13.3, ArCHH-), 6.92–6.96 (m, 2H, H-7, H-5), 7.30–
7.33 (m, 1H, H-2), 7.68 (d, J = 7.3, 1H, H-3), 8.22–8.28 (m, 2H, H-
1, H-8). Rf = 0.56 (methanol/ethyl acetate (3/1)). LogP = 3.09 0.56.
4.2.14. 2-Chloro-7-(1-hydroxy-2-methylpropan-2-
ylamino)methyl-9H-xanthen-9-one (14)
White solid (yield 50%), mp 162–163 °C. Anal. Calcd for
C18H18NO3Cl: C, 65.15; H, 5.46; N, 4.22. Found: C, 65.35; H, 5.19;
N, 4.06. IR (KBr)
m
(cmꢀ1): 3297, 3060, 2889, 1658, 1466, 1214,
1065, 822. 1H NMR (DMSO-d6, 500.13 MHz) d ppm): 1.03 (s, 6H,
(-CH3)2), 1.86 (s, 1H, –NH–), 3.27 (d, J = 5.5, 2H, –CH2-O–), 3.75
(s, 2H, –N-CH2–), 4.63 (t, J = 5.5, 1H, –OH), 7.58 (d, J = 8.6, 1H, H-
5), 7.70 (d, J = 8.9, 1H, H-4), 7.84 (dd, J = 2.2, J = 8.6, 1H, H-6),
7.88 (dd, J = 2.7, J = 9.0, 1H, H-3), 8.08 (d, J = 2.7, 1H, H-1), 8.11
(d, J = 2.0, 1H, H-8). Rf = 0.38 (methanol/ethyl acetate (3/1)).
LogP = 3.59 0.57.
Hydrochloride of compound 8: IR (KBr) m
(cmꢀ1): 3337, 1621, 1437,
1284, 1122, 764.
4.2.9. (R)-5-((1-Hydroxybutan-2-ylamino)methyl)-3-methoxy-
9H-xanthen-9-one (9)
White solid (yield 60%), mp 143–144 °C. Anal. Calcd for
4.2.15. (R,S)-2-Chloro-7-(1-hydroxypropan-2-ylamino)methyl-
9H-xanthen-9-one (15)
C
19H21NO4: C, 69.70; H, 6.47; N, 4.28. Found: C, 68.84; H, 6.42;
N, 4.32. IR and 1H NMR spectra as well as Rf and LogP values are
White solid (yield 65%), mp 154–156 °C. Anal. Calcd for
analogous to that of racemate (compound 8). ½a D20
¼ 17:84.
ꢁ
C
17H16NO3Cl: C, 64.26; H, 5.08; N, 4.41. Found: C, 64.58; H, 4.96;
N, 4.14. IR (KBr)
m
(cmꢀ1): 3420, 2867, 1656, 1467, 1298, 1050,
4.2.10. (S)-5-((1-Hydroxybutan-2-ylamino)methyl)-3-methoxy-
9H-xanthen-9-one (10)
819. 1H NMR (CDCl3, 300 MHz) d ppm): 1.12 (d, J = 6.4, 3H, –
CH3), 2.84–2.92 (m, 1H, –CH–), 3.30 (dd, J = 10.7, J = 7.2, 1H, –
CHHOH), 3.62 (dd, J = 10.5, J = 4.1, 1H, –CHHOH), 3.86 (d, J = 13.2,
1H, Ar-CHH–), 4.01 (d, J = 13.2, 1H, Ar-CHH–), 7.46 (d, J = 9.0, 1H,
H-5), 7.48 (d, J = 8.7, 1H, H-4), 7.66 (dd, J = 8.9, J = 2.6, 1H, H-6),
7.77 (dd, J = 8.6, J = 2.1, 1H, H-3), 8.23 (d, J = 2.1, 1H, H-1), 8.29
(d, J = 2.6, 1H, H-8). Rf = 0.33 (methanol/ethyl acetate (1/1)). LogP
=3.24 0.56.
White solid (yield 65%), mp 142–144 °C. Anal. Calcd for
C
19H21NO4: C, 69.70; H, 6.46; N, 4.28. Found: C, 69.58; H, 6.41;
N, 4.10 IR and 1H NMR spectra as well as Rf and LogP values are
analogous to that of racemate (compound 8). ½a D20
¼ ꢀ17:28.
ꢁ
4.2.11. 5-((1-Hydroxy-2-methylpropan-2-ylamino)methyl)-3-
methoxy-9H-xanthen-9-one (11)
White solid (yield 55%), mp 174–176 °C. Anal. Calcd for
4.2.16. (R)-2-Chloro-7-(1-hydroxypropan-2-ylamino)methyl-
9H-xanthen-9-one (16)
C
19H21NO4: C, 67.70; H, 6.47; N, 4.28. Found: C, 69.40; H, 6.57;
N, 4.25. IR (KBr)
m
(cmꢀ1): 3494, 3300, 3068, 2959, 1622, 1432,
White solid (yield 60%), mp 138–140 °C. Anal. Calcd for
1272, 1101, 761. 1H NMR (CDCl3, 600 MHz) d ppm): 1.09 (s, 6H,
(–CH3)2), 1.95 (br s, 1H, –NH–), 3.32 (d, J = 5.5, 2H, –CH2-OH),
3.94 (s, 3H, Ar-O-CH3), 3.98 (s, 2H, Ar-CH2-NH–), 4.61 (t, J = 5.5,
1H, –OH), 7.04 (dd, J = 8.9, J = 2.4, 1H, H-7), 7.11 (d, J = 2.4, 1H, H-
5), 7.40 (dd, J = 7.8, J = 7.3, 1H, H-2), 7.91 (dd, J = 7.3, J = 1.9, 1H,
H-3), 8.03 (dd, J = 7.8, J = 1.8, 1H, H-1), 8.08 (d, J = 8.9, 1H, H-8).
Rf = 0.46 (methanol/ethyl acetate (3/1). LogP = 3.69 0.56.
C
17H16NO3Cl: C, 64.25; H, 5.07; N, 4.40. Found: C, 64.59; H, 5.21;
N, 4.16. IR and 1H NMR spectra as well as Rf and LogP values are
analogous to that of racemate (compound 15). ½a D20
¼ ꢀ34:13.
ꢁ
4.2.17. (S)-2-Chloro-7-(1-hydroxypropan-2-ylamino)methyl-
9H-xanthen-9-one (17)
White solid (yield 65%), mp 138–140 °C. Anal. Calcd for
C
17H16NO3Cl: C, 64.25; H, 5.07; N, 4.40. Found: C, 64.05; H, 4.48;
N, 4.36. IR and 1H NMR spectra as well as Rf and LogP values are
4.2.12. (R,S)-5-((N-(1-Hydroxybutan-2-yl)-
analogous to that of racemate (compound 15). ½a D20
¼ 35:61.
ꢁ
methylamino)methyl)-3-methoxy-9H-xanthen-9-one (12)
White solid (yield 60%), mp 86–88 °C. Anal. Calcd for
C20H23NO4: C, 70.36; H, 6.79; N, 4.25. Found: C, 69.67; H, 6.60; N,
4.3. Pharmacology
4.08. IR (KBr)
m
(cmꢀ1): 3465, 1619, 1435, 1245, 1102, 760. 1H
NMR (CDCl3, 600 MHz) d ppm): 0.94 (s, 3H, –CH3 (Et)), 1.41–1.59
(m, 2H, –CH2– (Et)), 2.25 (s, 3H, –N-CH3), 2.59–2,65 (m, 1H, –
CH–), 3.47–3.53 (m, 1H, –CHH-OH), 3.62–3.69 (m, 1H, –CHH-OH),
3.94 (s, 3H, Ar-O-CH3), 4.02 (s, 2H, Ar-CH2-NH-), 4.34 (t, J = 5.5,
1H, –OH), 7.04 (dd, J = 8.4, J = 2.4, 1H, H-7), 7.09 (d, J = 2.4, 1H, H-
5), 7.41 (dd, J = 7.8, J = 7.2, 1H, H-2), 7.86 (dd, J = 7.2, J = 1.8, 1H,
H-3), 8.05 (dd, J = 7.8, J = 1.8, 1H, H-1), 8.08 (d, J = 8.8, 1H, H-8).
Rf = 0.65 (methanol/ethyl acetate (3/1)). LogP = 2.91 0.56.
Pharmacological tests were performed at Epilepsy Branch, Na-
tional Institute of Neurological Disorders and Stroke, National
Institutes of Health (Rockville, USA).
All compounds (1–17) were tested using MES, scMet and neuro-
toxicity assays, after intraperitoneal (ip) injection into male albino
mice (Carworth Farms No. 1) as suspensions in 0.5% methylcellulose
at the doses of 30, 100, and 300 mg/kg (for compound 3 also at the
doses of 3 and 10 mg/kg). Observations were carried out after 0.5 h

N. Szkaradek et al. / Bioorg. Med. Chem. 21 (2013) 1190–1198
1197
and 4 h (sometimes additionally 0.25 and 1 h) of compound’s
administration. Compounds that were interesting in the context of
ADD program, underwent additional tests including: anticonvulsant
identification in male albino rats (Sprague-Dawley) after oral
administration, anticonvulsant quantification in mice and 6 Hz test.
Tests in rats after oral administration (po) are performed because
one of the valuable property of a candidate anticonvulsant is its abil-
ity to inhibit convulsions when administered po. That screen dis-
closes the time of onset, the approximate time of peak effect (TPE)
and the duration of anticonvulsant activity or neurotoxicity.21–23
sue was homogenized at 0 °C in 30 vol of 50 mmol/l Tris–HCl
buffer, pH 7.6. The homogenate was centrifuged at 1000ꢂg for
10 min. The supernatant was decanted and recentrifuged at
25,000ꢂg for 30 min, and the resulting pellet was resuspended in
the buffer and recentrifuged under the same conditions. The final
pellet (fraction P2) was stored in ꢀ20 °C for 24 h. For binding stud-
ies the pellet was reconstituted in the Tris–HCl buffer, pH 7.6. The
radioligand [3H]Nitrendipine (NEN, spec. act. 87.6 Ci/mmol), for
estimating voltage-dependent Ca2+ channel was used. The incuba-
tion mixture (final volume 550
suspension, 50
l of radioligand solution [3H]nitrendipine concen-
tration 0.8 nmol/l prepared in the dark and 50 l buffer containing
seven concentrations of nifedipine (0.1 nM–10 M), and investi-
gated compounds 1, 2, 3 (1 nM–100 M). For measuring unspecific
binding, nifedipine in a concentration of 10 mol/l was used. The
ll) consisted of 450 ll membrane
l
4.3.1. The maximal electroshock test (MES)
l
Seizures 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 is defined as the abolition
of the hindlimb tonic extension component of the seizure.21–23
l
l
l
incubation was carried out in duplicate, in a shaking water bath,
at 25 °C for 30 min. Addition of a ligand initiated the incubation,
which was terminated by rapid filtration through GF/C Whatman
fiberglass filters. The filters were then rinsed twice with 5 ml por-
tions of ice-cold incubation buffer and placed in plastic scintillation
minivials. Scintillation fluid (Akwascynt, BioCare) was added (3 ml)
and the samples were counted for radioactivity in Beckman LS
3801 scintillation counter.
4.3.2. The subcutaneous pentetrazole seizure test (scMet)
The scMet was conducted by subcutaneous administration of
pentetrazole dissolved in 0.9% NaCl solution at the dose of
85 mg/kg (mice) or 70 mg/kg (rats) into animals. A minimal time
of 30 min subsequent to sc administration of pentetrazole was
used for seizure detection. 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.21–23
Acknowledgements
The authors would like to acknowledge Professor James P. Sta-
bles and Tracy Chen Ph.D. from National Institute of Neurological
Disorders and Stroke (NIH, Rockville, USA) for providing the results
4.3.3. Neurotoxicity (TOX)
Neurological deficit was measured in mice by the rotorod test.
The mouse was placed on a 1 in. diameter knurled plastic rod rotat-
ing 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.21–23
´
of pharmacological assays and Professor Katarzyna Kiec-Kon-
onowicz for coordination of the cooperation between the National
Institute of Neurological Disorders and Stroke and the Faculty of
Pharmacy Jagiellonian University Medical College.
The publication is supported by Jagiellonian University Medical
College (Grant Nos. K/ZDS/000951).
4.3.4. Anticonvulsant quantification
Anticonvulsant quantification, that is, the dose of drug required
to produce the biological responses in 50% of animals (ED50), and
the respective 95% confidence intervals, were determined for se-
lected compounds displaying sufficient antiepileptic activity and
low neurotoxicity in the primary evaluations. The details of these
procedures have been published elsewhere.21–23
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.12.051.
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