Synthesis, structural and biological characterization of 5-phenylhydantoin derivatives as potential anticonvulsant agents

Article abstract of DOI:10.1007/s00706-012-0791-8

Considering the importance of hydantoin derivatives in treatment of status epilepticus, four 5-phenylhydantoins, whose lipophilicities were estimated to be similar to that of phenytoin, were synthesized. Evaluation of their anticonvulsant activities was performed on rats by subcutaneous pentylenetetrazol seizure test and intravenous pentylenetetrazol threshold test, and spontaneous locomotor activity test was used to assess possible sedative effects. X-ray analysis of three compounds suggested that certain analogies might be drawn between interactions in crystal packing and biological interactions responsible for their anticonvulsant activity. It was found that 5-ethyl-5-phenyl- 3-propylhydantoin exhibits the most favorable pharmacological properties among the synthesized compounds, i.e., anticonvulsant activity comparable to phenytoin with lower liability for induction of sedation in rats.

Full text of DOI:10.1007/s00706-012-0791-8

Monatsh Chem (2012) 143:1451–1457
DOI 10.1007/s00706-012-0791-8
ORIGINAL PAPER
Synthesis, structural and biological characterization
of 5-phenylhydantoin derivatives as potential anticonvulsant
agents
•
Nemanja Trisovic Tamara Timic Jovana Divljakovic Jelena Rogan
•
•
•
ˇ
´
Dejan Poleti Miroslav M. Savic Gordana Uscumlic
´
´
•
•
´
ˇ´
´
Received: 24 November 2011 / Accepted: 8 May 2012 / Published online: 26 June 2012
Ó Springer-Verlag 2012
Abstract Considering the importance of hydantoin
derivatives in treatment of status epilepticus, four 5-phen-
ylhydantoins, whose lipophilicities were estimated to be
similar to that of phenytoin, were synthesized. Evaluation
of their anticonvulsant activities was performed on rats by
subcutaneous pentylenetetrazol seizure test and intravenous
pentylenetetrazol threshold test, and spontaneous locomo-
tor activity test was used to assess possible sedative effects.
X-ray analysis of three compounds suggested that certain
analogies might be drawn between interactions in crystal
packing and biological interactions responsible for their
anticonvulsant activity. It was found that 5-ethyl-5-phenyl-
3-propylhydantoin exhibits the most favorable pharmaco-
logical properties among the synthesized compounds, i.e.,
anticonvulsant activity comparable to phenytoin with lower
liability for induction of sedation in rats.
arrhythmias many years ago. Phenytoin (5,5-diphenylhy-
dantoin, PHE) is one of the oldest reportedly nonsedative
anticonvulsant drugs, being employed in the control of
tonic–clonic seizures, some forms of partial seizures, or
a combination of these [1, 2].
The therapeutic effects of PHE result from selective
inhibition of the neuronal voltage-sensitive sodium channel
(NVSC) [3, 4]. It binds to the inactivated state of NVSC in
a voltage- and frequency-dependent manner [5], and neu-
rons, which undergo prolonged depolarization or repetitive
firing, are more completely inhibited [1]. Regarding the
molecular mode of action of PHE, Smythies [6] suggested
that it forms an array of molecules stacked on a b-turn
segment of the proteic part of the putative receptor through
the formation of hydrogen bonds with the carbonyl group
at position 2 and the NH at position 3. The hypothesis that
the ability to form hydrogen bonds plays an important role
in the anticonvulsant action of these compounds was
additionally confirmed in our previous study [7], in
which the solvatochromic comparison method was applied
to quantify and correlate multiple solvent effects on their
transport properties and interactions with biological
receptors. The amount and position of the hydrophobic
elements of the molecular structure strongly affect the
binding to NVSC [8], mostly as a result of the necessity for
hydantoins to enter or cross the cell membrane prior to
interaction with the channel protein. A comparative study
of binding affinities of hydantoins and related nonhydan-
toins (hydroxyamides, oxazolidinedione, hydroxyacids,
and amino acids) revealed that the hydantoin ring is
important for derivatives bearing short alkyl side-chains,
but it is much less important for derivatives with long
hydrophobic chains, which can effectively occupy an
additional hydrophobic site of the receptor [9]. In general,
structure–activity relationship studies of anticonvulsants
Keywords Hydantoin derivatives Á
Anticonvulsant activity Á Sedative effects Á
Crystal structure
Introduction
Derivatives of hydantoin (imidazolidine-2,4-dione) are
well known for their pleiotropic biological activity and
were introduced for therapy of epilepsy and cardiac
ˇ
´
ˇ´
´
N. Trisovic Á J. Rogan Á D. Poleti Á G. Uscumlic (&)
Faculty of Technology and Metallurgy, University of Belgrade,
Karnegijeva 4, 11000 Belgrade, Serbia
e-mail: goca@tmf.bg.ac.rs
´
´
´
T. Timic Á J. Divljakovic Á M. M. Savic
Faculty of Pharmacy, University of Belgrade,
Vojvode Stepe 450, 11000 Belgrade, Serbia
123

ˇ ´
N. Trisovic et al.
1452
have revealed the need for retention of at least one
hydrophobic site and hydrogen bond donors/acceptors, in
certain relative orientations, for the activity to be exhibited
[10]. In this context, it was suggested that binding of a
ligand to NVSC is governed by the correct conformational
arrangement of the phenyl ring at position 5 with respect to
the hydantoin ring [11].
Results and discussion
Evaluation of anticonvulsant activity was performed in rats
using the subcutaneous pentylenetetrazol (s.c. PTZ) seizure
test and intravenous pentylenetetrazol (i.v. PTZ) threshold
test. Seizures were classified using the procedures of
Lamberty and Klitgaard [15] as: facial and ear twitching
(I), convulsive waves through the body (II), rearing,
myoclonic jerks (III), turn over onto side position (IV),
generalized tonic–clonic seizures (V). The spontaneous
locomotor activity (SLA) assay was used to demonstrate
possible sedative effects of synthesized compounds. All
compounds were dissolved in vehicle (VEH; 85 % distilled
water, 14 % propylene glycol, 1 % Tween 80), and were
administered intraperitoneally 20 min before testing. PTZ
was dissolved in the same vehicle for s.c. PTZ seizure test
and in saline for i.v. PTZ threshold test.
In this paper, we present structural as well as biological
examination, in comparison with PHE, of four 3,5-disub-
stituted 5-phenylhydantoin ligands 1–4 (Table 1), whose
lipophilicities were estimated to be similar to that of PHE.
Such a choice of compounds gains support from the work
of Brouillette et al. [12], who demonstrated that 5-pentyl-
5-phenylhydantoin exhibited the same binding affinity for
NVSC as that of PHE. The structural characteristic of the
investigated compounds is that two hydrophobic regions
are separated by a polar hydantoin region, while one polar
and one hydrophobic region exist in the PHE molecule.
Our aim was to assess the anticonvulsant potential of these
ligands using the pentylenetetrazol (PTZ) test [13], as well
as their sedative liability. PHE has a significant role in the
therapy of generalized convulsive status epilepticus [14],
but a number of potentially serious drawbacks hamper its
use [2]. PTZ is widely employed for experimental induc-
tion of convulsive status epilepticus [13], and the potential
protective action of some novel ligands, in conjunction
with low incapacitating/sedative action in rats, would offer
the prospect of more detailed experimental analysis of
treatment of status epilepticus.
The s.c. PTZ test was a preliminary screening assay to
evaluate the anticonvulsant potential of the investigated
5-phenylhydantoins. As a result of this screening, com-
pounds 3 and 4 were subjected to quantification of their
anticonvulsant activity in i.v. PTZ threshold test. The effect
of PHE and 3,5-disubstituted 5-phenylhydantoins on gen-
eralized clonic seizure threshold determined 20 min after
i.p. injection is given in Table 2. Data are presented as
mean standard error (SE, n = 6). Statistical analysis
using one-way analysis of variance (ANOVA) indicated a
significant overall effect (p \ 0.05), and Tukey’s post hoc
analysis revealed that PHE and compound 3 at dose of
80 mg kg-1 significantly lowered the clonic seizure
threshold in rats (p \ 0.05). This means that compound 3
possesses anticonvulsant activity comparable to that of
PHE.
Table 1 Structures and estimated lipophilicities of the investigated
compounds
Locomotor activity was measured in the SLA test; total
distance traveled and distance traveled in 5-min intervals
are shown in Table 2 and Fig. 1, respectively. It can be
observed in Fig. 1 and Table 2 that compound 4 and PHE
showed significant decrease (p \ 0.05) in total distance
traveled and in distance traveled in the intervals 0–5 min
and 5–10 min, compared with VEH. Sedative effect of
PHE and compound 4 was also manifested in comparison
with compound 3. On the other hand, compound 3 exerted
a significant decrease (p \ 0.05) in locomotor activity in
the period 10–15 min (Fig. 1). These results indicate that
compound 3 possesses low sedative potential and sub-
stantial activity against seizures induced by i.v. PTZ,
despite the fact that it was not so effective in the s.c. PTZ
screening test.
No.
R1
R2
log Pa
PHE
C6H5
C6H5
i-C3H7
C2H5
CH3
H
2.52
2.55
2.35
2.46
2.46
1
2
3
4
a
CH2=CHCH2
CH2=CHCH2
n-C3H7
Furthermore, an X-ray study of 5-phenylhydantoin
derivatives 2–4 was performed to explore potential quali-
tative correlations between the situations of hydantoins in
crystals and their anticonvulsant properties. Examination
of the conformational characteristics of molecules under
n-C4H9
Estimated using Advanced Chemistry Development (ACD/Labs)
software ACD version 11.02
123

Synthesis and structural and biological characterization
1453
Table 2 Anticonvulsant and locomotor activity
No.
Dose/mg kg-1 i.p.
PTZ (70 mg kg-1, s.c.)
PTZ (100 mg cm-3, i.v.
infusion at rate of 0.2 cm3 min-1
Threshold PTZ/mg kg-1
SLA
)
Protected/total
Degree of convulsions
Total distance
traveled/m
PHE
40
3/8
4/6
1/8
3/8
0/8
0/8
0/8
–
III, III, III, IV, V
II, IV
49.91 2.86
–
80
59.68 8.95*
5.08 0.97*
120
80
II, III, III, III, III, V, V
III, III, III, III, V
III, III, IV, V, V, V, V, V
II, II, III, III, III, III, IV, V
II, III, III, IV, V, V, V, V
–
45.68 4.29
–
1
2
3
–
–
120
80
–
–
–
–
120
40
–
–
44.20 3.35
59.43 3.04*
–
–
80
2/8
2/8
–
II, II, II, III, V, V
II, III, III, III, V, V
–
9.08 0.99
120
40
–
4
45.33 4.81
43.01 4.14
–
–
80
3/7
4/8
2/6
II, II, II, V
5.44 0.83*
–
120
–
II, II, V, V
VEH
V, V, V, V
36.90 1.50
9.35 1.07
Each value is the mean SE of 6 animals (for PTZ threshold test) or 6–8 animals for SLA
* p \ 0.05 versus VEH
p \ 0.05 versus corresponding dose of compound 3
provide a useful representation of the actual biological
target site.
The molecular structures of 2–4 are shown in Fig. 2.
Bond distances and angles of hydantoin rings are very
similar in all three compounds. The value of torsion angle
N1–C5–C6–C7 in PHE is only 2° [10], while correspond-
ing torsion angles in 2, 3, and 4 are -12.8(3)°, 6.4(3)°, and
-22.6(4)°, respectively. It was shown that, besides
log p value, 5-phenyl ring orientation strongly affects
anticonvulsant activity. On the basis of the observation of a
large number of hydantoin derivatives, it was demonstrated
that the minimum energy conformations of the most potent
compounds contain phenyl ring torsion angles between -
11° and 11° [12]. Only the value of the torsion angle in 3,
i.e., 6.4(3)°, is in this range, and even though this is a
‘‘soft’’ parameter, it supports its favorable pharmacological
properties.
Fig. 1 Effects of PHE and 3,5-disubstituted 5-phenylhydantoins on
distance traveled in the activity chamber during six consecutive 5-min
intervals. *p \ 0.05 compared with VEH; p \ 0.05 compared with
compound 3
The crystal packings of 2–4 are governed by hydrogen
N–HÁÁÁO bonds (Table 3). As a consequence, chains par-
allel to b-axis in 2 and parallel to c-axis in 4 are formed
(Fig. 3a, c, respectively). Furthermore, the chains in 4 are
stacked in a three-dimensional framework connected by
weak face-to-face p–p interactions (centroid–centroid dis-
nonbiological conditions does not necessarily reflect
in vivo situations. Nevertheless, X-ray crystallographic
data may afford insight into their probable conformations.
It is recognized that the structural features required for an
ideal receptor, which hydantoins target, are all present
within the hydantoins themselves, viz. a combination of
hydrogen-bond acceptors and donors to bind the hydantoin
ring, and hydrophobic groups to bind the hydrocarbon side-
chains [16, 17]. The environment in the crystal may thus
˚
tance of about 3.80 A) between every second pair of
phenyl rings (Fig. 3c). In this way, hydrophilic and
hydrophobic layers parallel to the bc-plane are formed. The
hydrogen bonds are present in hydrophilic layers, but there
are two kinds of alternative hydrophobic layers where p–p
123

ˇ ´
N. Trisovic et al.
1454
Fig. 2 Molecular structures of
2 (a), 3 (b), and 4 (c) with atom
labeling schemes (40 %
probability displacement
ellipsoids). H atoms are shown
as small spheres of arbitrary
radii. In 2, there are two pairs of
disordered atoms C14A/C14B
and C15A/C15B with individual
site occupancies of 0.5
Table 3 Geometry of hydrogen bonds for 2–4
Experimental
˚
˚
˚
No. D–HÁÁÁA
d(D–H)/A d(HÁÁÁA)/A d(DÁÁÁA)/A D–HÁÁÁA/°
Phenytoin was commercially obtained (Fluka), while
5-methyl-, 5-ethyl-, and 5-isopropyl-5-phenylhydantoin
were synthesized by a modification of the method of
Bucherer [18] (Scheme 1). These compounds were further
alkylated at position 3 using the corresponding alkyl halide
in K2CO3/N,N-dimethylformamide [19]. The molecular
structures and the purities of the synthesized 3,5-disubsti-
tuted 5-phenylhydantoins were confirmed by melting
points, elemental analysis, Fourier-transform infrared
(FT-IR), 1H and 13C nuclear magnetic resonance
(NMR) spectroscopy, and X-ray crystal structure analysis.
Elemental analysis was realized using an Elemental Vario
EL III microanalyzer. FT-IR spectra were recorded on a
Bomem MB 100 spectrophotometer. 1H and 13C NMR
spectral measurements were performed on a Bruker AC 200
spectrometerat200 MHzforthe1HNMRand50 MHzforthe
13C NMR spectra or on a Bruker 300 spectrometer at
2
3
4
N1–HN1ÁÁÁO2a 0.86
2.093
2.018
2.034
2.902(2)
2.858(3)
2.842(3)
157
165
156
N1–H1ÁÁÁO1b 0.86
N1–H1ÁÁÁO2c 0.86
Symmetry codes: (a) x, y – 1, z; (b) –x ? 2, –y, –z; (c) x, –y ? 1/2,
z ? 1/2
interactions or van der Waals forces exist. Compound 3
builds completely different supramolecular architecture:
hydrogen N–HÁÁÁO bonds (Table 3; Fig. 3b) between
neighboring molecules make R22 (8) centrosymmetric
dimers along b-axis. The existence of centrosymmetric
dimers in the crystal packing of 3 may well be responsible
for the differences in its biological activity, when com-
pared with 2 and 4.
1
300 MHz for the H NMR and 75 MHz for the 13C NMR
Conclusions
spectra. Spectra were recorded at room temperature in dime-
thyl sulfoxide (DMSO-d6). Chemical shifts are expressed in
ppm values referred to tetramethylsilane (TMS, dH = 0 ppm)
in the 1H NMR spectra and the residual solvent signal
(dC = 39.5 ppm) in the 13C NMR spectra.
The present study reveals that different hydantoin deriva-
tives with similar log
p
value exhibit different
anticonvulsant effects, suggesting that they likely bind to a
specific site on the NVSC. Furthermore, the structural
findings described herein suggest that the correct confor-
mational arrangement of the phenyl ring at position 5 with
respect to the hydantoin ring and binding modes in crystal
packing might well be related to their biological activity.
The obtained results indicate that ligand 3 exerts anticon-
vulsant activity comparable to the standard medicine used
in the treatment of status epilepticus, PHE, with lower
liability for induction of sedation in rats. Further studies
should examine the potential of 3 to induce the other
adverse effects hampering routine clinical use of PHE.
General procedure for preparation
of 5-alkyl-5-phenylhydantoins
The appropriate ketone (0.020 mol) was dissolved in
50 cm3 50 % ethanol, and 7.70 g (NH4)2CO3 (0.080 mol)
and 2.60 g KCN (0.040 mol) were then added. This mix-
ture was warmed under a condenser at temperature of
60 °C for 15 h, after which the solution was concentrated
to approximately two-thirds of the initial volume, chilled in
123

Synthesis and structural and biological characterization
1455
Fig. 3 Crystal packings of 2 along a-axis (a), 3 along a-axis showing
centrosymmetric dimers (b), and 4 along b-axis (c) (hydrogen bonds
represented by dashed lines). In a only one-half of the partially
disordered allyl group is shown, and one row of the molecules
connected by hydrogen bonds is emphasized in upper part
Scheme 1
O
R2X, K2CO3
DMF
R1 KCN, (NH4)2CO3
50% EtOH
R1
NH
R1
NH
O
O
O
O
N
N
R2
H
R1 = i-C3H7, C2H5, CH3
1-4
Table 4 Synthesis of compounds 1–4
K2CO3 (0.041 mol)wasadded.Alkylhalide(allylbromide,n-
propyl bromide, or n-butyl chloride, 0.011 mol) was then
added, and the reaction mixture was stirred overnight. The
reaction mixture was poured into three volumes of water and
extracted with ethyl acetate. The ethyl acetate extracts were
washed with 5 % NaOH and water, and then dried over
MgSO4. The solvent was removed, and the resulting solid was
No. R1
R2
Yield/% M.p./°C
Found
Reported
1
2
3
4
C6H5
CH2=CHCH2 68
114–117 114–117 [20]
i-C3H7 CH2=CHCH2 72
112–114
83–85
–
C2H5
CH3
n-C3H7
n-C4H9
65
74
84–86 [21]
89.8 [22]
88–90
1
recrystallized from ethanol. The melting points, FT-IR, H
and 13C NMR spectra of compounds 1, 3, and 4 are in
agreement with literature data (Table 4) and undoubtedly
corroborate their structures.
an ice-bath, and filtered on a Bu¨chner funnel. The product
was dissolved in 5 % NaOH solution, filtered from unre-
acted ketone, and reprecipitated by acidification with
hydrochloric acid. Recrystallization from 60 % ethanol
yielded a crystalline product.
3-Allyl-5-isopropyl-5-phenylhydantoin (2, C15H18N2O2)
White crystalline solid; yield: 72 %; m.p.: 112–114 °C; IR
-1
ꢀ
(KBr): m = 3,308 (NH), 1,771 (C=O), 1,711 (C=O) cm
;
1H NMR (300 MHz, DMSO-d6): d = 9.08 (s, 1H, NH),
7.55–7.29 (m, 5H, Ph-H), 5.78–5.66 (m, 1H,
CH2CH=CH2), 5.04 (dd, J = 1.5 Hz, J = 10.35 Hz, 1H,
CH2CH=CH2), 4.96 (dd, J = 1.5 Hz, J = 17.10 Hz, 1H,
CH2CH=CH2), 3.93 (d, J = 5.4 Hz, 2H, CH2CH=CH2),
General procedure for preparation of 3,5-disubstituted
5-phenylhydantoins
The appropriate 5-substituted 5-phenylhydantoin (0.010 mol)
was dissolved in 60 cm3 N,N-dimethylformamide, and 5.60 g
123

ˇ ´
N. Trisovic et al.
1456
Table 5 Crystal data, data collection, and refinement details for 2, 3, and 4
Compound
2
3
4
Chemical formula
Formula weight
Crystal habit, color
Crystal system
Space group
C15H18N2O2
258.31
C14H18N2O2
246.30
C14H18N2O2
246.30
Plate-like, colorless
Orthorhombic
Pbca
Plate-like, colorless
Monoclinic
P21/c
Block, colorless
Monoclinic
P21/c
˚
a/A
16.3379 (6)
6.2509 (2)
28.3984 (18)
–
7.7724 (10)
20.220 (3)
8.6004 (11)
90.045 (12)
1,351.6 (3)
4
14.748 (2)
8.4420 (11)
11.0540 (13)
94.583 (14)
1,371.9 (3)
4
˚
b/A
˚
c/A
b/8
3
˚
Volume/A
2,900.2 (2)
8
Z
q
calc/g cm–3
1.183
1.210
1.193
F(000)
l(Mo Ka)/mm-1
1,104
528
528
0.079
0.082
0.081
Limiting Miller indices
-19 B h B 17
-7 B k B 4
-34 B l B 32
6,686/2,749 (Rint = 0.0418)
-9 B h B 6
-23 B k B 24
-9 B l B 10
5,943/2,571
(Rint = 0.0860)
1,698/0/166
1.037
-18 B h B 17
-9 B k B 10
-13 B l B 10
5,723/2,806
(Rint = 0.0625)
1,598/1/167
1.019
Reflections collected/unique
Data/restraints/parameters
Goodness of fit on F2
R indices [I [ 2r(I)]
2,056/4/181
1.170
R1 = 0.0726, wR2 = 0.1507
R1 = 0.0636,
wR2 = 0.0996
R1 = 0.1097,
wR2 = 0.0854
0.118/-0.135
R1 = 0.0727,
wR2 = 0.1948
R1 = 0.1276,
wR2 = 0.1623
0.430/-0.269
R indices (all data)
R1 = 0.0992, wR2 = 0.1393
0.205/-0.175
-3
˚
Dqmax/Dqmin/e A
2.59–2.45 (m, 1H, CH(CH3)2), 0.84 (d, J = 6.6 Hz, 3H,
CH(CH3)2), 0.64 (d, J = 6.9 Hz, 3H, CH(CH3)2) ppm; 13
and refined using riding model with fixed C–H and N–H
distances. Crystal data, data collection, and refinement
details are listed in Table 5.
C
NMR (75 MHz, DMSO-d6): d = 172.1 (C4), 153.8 (C2),
136.1 (i-Ph-C), 129.8 (CH2CH=CH2), 126.2 (m-Ph-C),
125.6 (p-Ph-C), 123.2 (o-Ph-C), 114.3 (CH2CH=CH2),
68.1 (C5), 37.5 (CH2CH=CH2), 33.0 (CH(CH3)2), 14.4
(CH(CH3)2),14.0 (CH(CH3)2) ppm.
Crystallographic data for the structures in this paper
were deposited with the Cambridge Crystallographic Data
Centre as supplementary publication numbers CCDC
830412–830414. These data can be obtained free of
charge from http://www.ccdc.cam.ac.uk/data_request/cif,
by emailing data_request@ccdc.cam.ac.uk, or by contact-
ing The Cambridge Crystallographic Data Centre, 12,
Union Road, Cambridge CB2 1EZ, UK; fax: ?44-1223-
336033.
Crystal structure determination
X-ray single-crystal diffraction data were collected using
an Oxford Gemini S diffractometer equipped with a
charge-coupled device (CCD) detector at 293 K. In all
˚
cases, Mo Ka radiation (k = 0.71073 A) was used and a
Biological methods
multiscan correction for absorption was applied. The
structures were solved by direct methods (SIR92 [23]) and
refined on F2 by full-matrix least-squares (SHELXL97 [24]
and WinGX [25]). For all three compounds (2–4), nonhy-
drogen atoms were refined anisotropically. Hydrogen
atoms were placed at geometrically calculated positions
All biological assays were performed on male Wistar rats
weighing 220–250 g (Military Farm, Belgrade, Serbia),
housed in a temperature-controlled room with light/dark
cycles and free access to food and water. Animals were
randomly assigned to different drug treatment regimes.
123

Synthesis and structural and biological characterization
1457
Acknowledgments This work was performed within the framework
of research project no. 172013 supported by the Ministry of Education
and Science of the Republic of Serbia.
The s.c. PTZ seizure test [26]
Pentylenetetrazol (70 mg kg-1 for rats) was used as a
standard for chemically induced seizures. It was injected
s.c. 20 min after pretreatment with PHE or investigated
compounds (80 and 120 mg kg-1). Animals that did not
experience stage II or higher within the 30 min observation
period were considered as protected. Vehicle treatment did
not affect PTZ-induced seizures.
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0.2 cm3 min-1
.
The clonic seizure threshold was determined by insert-
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Measurement of locomotor activity [27]
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Twenty minutes after receiving the appropriate treatment
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without any habituation period. Besides the total distance
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by dividing the locomotor activity data into 5-min bins.
Data are presented as mean SE (n = 6–8) and were
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