5-(4-Chlorophenyl)-5,6-dihydro-1,3-oxazepin-7(4H)-one derivatives as lipophilic cyclic analogues of baclofen: Design, synthesis, and neuropharmacological evaluation

Article abstract of DOI:10.1016/j.bmc.2008.07.064

In trials to preserve the pharmacological profile and improve the bioavailability via lipophilicity increment of baclofen 1 and searching for more potent and less toxic muscle relaxants and analgesics, nine substituted cyclic analogues of 1 were designed and synthesized. The target derivatives 5-(4-chlorophenyl)-5,6-dihydro-1,3-oxazepin-7(4H)-one (11-19) were obtained through amide formation to the corresponding intermediates (2-10) followed by cyclization using acetic anhydride. The structures of the target compounds (11-19) were confirmed by IR, ¹H NMR, MS, and elemental analyses. The neuropharmacological activities of these lipophilic cyclic analogues (11-19) were assessed for their effects on motor activity, muscle relaxation, pain relief and impaired cognition, by intraperitoneal administration at a dose of 3 mg/kg with reference to those of baclofen 1. Our results showed that compounds 11-14 are devoid of all of the tested pharmacological effects associated with 1. In all paradigms tested, undecyl, tridecyl, heptdec-8-enyl and benzyl substituted analogue derivatives (15, 16, 18, and 19) revealed a significant neurological activity being vividly favorable comparable with baclofen 1. 2-Benzyl-5-(4-chlorophenyl)-5,6-dihydro-1,3-oxazepin-7(4H)-one derivative 19 is the most active candidate with high significant neurological potencies, while 5-(4-chlorophenyl)-2-(dec-8-enyl)-5,6-dihydro-1,3-oxazepin-7(4H)-one derivative 17 displayed activity at relatively higher time interval. These results probe a new structurally distinct class incorporating 1,3-oxazepine nucleus as promising candidates as GABA_B agonists for further investigations.

Full text of DOI:10.1016/j.bmc.2008.07.064

Bioorganic & Medicinal Chemistry 16 (2008) 7983–7991
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
5-(4-Chlorophenyl)-5,6-dihydro-1,3-oxazepin-7(4H)-one derivatives
as lipophilic cyclic analogues of baclofen: Design, synthesis,
and neuropharmacological evaluation
b
Atef A. Abdel-Hafez a, , Basel A. Abdel-Wahab
*
a Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
b Department of Pharmacology, Faculty of Medicine, Assiut University, Assiut 71526, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
In trials to preserve the pharmacological profile and improve the bioavailability via lipophilicity incre-
ment of baclofen 1 and searching for more potent and less toxic muscle relaxants and analgesics, nine
substituted cyclic analogues of 1 were designed and synthesized. The target derivatives 5-(4-chloro-
phenyl)-5,6-dihydro-1,3-oxazepin-7(4H)-one (11–19) were obtained through amide formation to the
corresponding intermediates (2–10) followed by cyclization using acetic anhydride. The structures of
the target compounds (11–19) were confirmed by IR, 1H NMR, MS, and elemental analyses. The neuro-
pharmacological activities of these lipophilic cyclic analogues (11–19) were assessed for their effects
on motor activity, muscle relaxation, pain relief and impaired cognition, by intraperitoneal administra-
tion at a dose of 3 mg/kg with reference to those of baclofen 1. Our results showed that compounds
11–14 are devoid of all of the tested pharmacological effects associated with 1. In all paradigms tested,
undecyl, tridecyl, heptdec-8-enyl and benzyl substituted analogue derivatives (15, 16, 18, and 19)
revealed a significant neurological activity being vividly favorable comparable with baclofen 1. 2-Ben-
zyl-5-(4-chlorophenyl)-5,6-dihydro-1,3-oxazepin-7(4H)-one derivative 19 is the most active candidate
with high significant neurological potencies, while 5-(4-chlorophenyl)-2-(dec-8-enyl)-5,6-dihydro-1,3-
oxazepin-7(4H)-one derivative 17 displayed activity at relatively higher time interval. These results
probe a new structurally distinct class incorporating 1,3-oxazepine nucleus as promising candidates as
GABAB agonists for further investigations.
Received 14 June 2008
Revised 22 July 2008
Accepted 23 July 2008
Available online 29 July 2008
Keywords:
1,3-Oxazepinone
Baclofen
Lipophilicity
Neuropharmacological activity
GABA agonist
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
with information derived from ‘knockout’ mice, has provided the
basis for much of the speculation about the potential therapeutic
c
-Amino butyric acid (GABA), the most abundant inhibitory
benefits of both agonists and antagonists for the GABAB receptor.
Pharmacological studies with 4-amino-3-(4-chlorophenyl)butyric
acid (baclofen 1), the highly selective GABAB receptor agonist, have
pointed to a role for GABAB receptors in a number of central ner-
vous system disorders such as epilepsy, cognition, pain, muscle
relaxation gastroesophageal reflux, and addiction.6 The most pre-
dominant pharmacological effects of baclofen are the muscle relax-
ant, anti-nociceptive and anti-drug craving effects, as well as the
reduction in cognitive behavior.7 Baclofen 1 has been in clinical
use for the treatment of spasticity associated with brain and spinal
cord injuries, as well as dystonia for over 30 years.8 The centrally
mediated muscle-relaxant properties of baclofen 1 are well estab-
lished clinically, making it the drug of choice in spasticity associ-
ated with cerebral palsy, multiple sclerosis, stiff-man syndrome,
and tetanus. However, the side effects produced by this drug,
including drowsiness, insomnia, dizziness, weakness, ataxia, ortho-
static hypotension, and impairment of cognitive functions are fre-
quently not tolerated by patients.9–13 Various clinical studies
demonstrated that baclofen 1 does not penetrate the blood–brain
neurotransmitter in the central nervous system, is implicated in
the regulation of several physiological and psychological pro-
cesses.1 Ionotropic GABA receptors, including GABAA and GABAC
receptors, and metabotropic GABAB receptors are the two subtypes
of GABA receptors.2 The GABAB receptor is a heterodimer made up
of two subunits, GABAB(1) and GABAB(2), both necessary for GABAB
receptors to be functionally active.3 The former contains the GABA-
binding domain, whereas GABAB(2) provides the G-protein-cou-
pling mechanism and also incorporates an allosteric modulatory
site within its heptahelical structure.4 The receptors are located
in the brain both pre- and post-synaptically where they are cou-
pled to Ca2+ and K+ channels. Therefore, when receptor activation
occurs, a variety of effects might be expected to occur as a conse-
quence of inhibition of transmitter release and/or neuronal hyper-
polarization.5 Having a selective agonist for the receptor, together
* Corresponding author. Tel.: +20 88 230 3006; fax: +20 88 233 2776.
E-mail address: atef@aun.edu.eg (A.A. Abdel-Hafez).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.064

7984
A. A. Abdel-Hafez, B. A. Abdel-Wahab / Bioorg. Med. Chem. 16 (2008) 7983–7991
barrier (BBB) in a sufficient extent. This has been largely overcome
by the introduction of intrathecal administration using an indwell-
ing pump.14,15 But there are several potential complications of
chronic intrathecal baclofen infusion, including surgical side ef-
fects and malfunction of the system.16 Serious surgical complica-
tions include epidural hematoma and abscess formation of the
surgical site or in the epidural space. Seroma formation at the
pump pocket is fairly common and can be minimized by careful
homeostasis and a small size of subcutaneous pocket. Severe prob-
lems associated with persistent cerebrospinal fluid leak, including
dural puncture headache, cranial nerve palsy, and intracranial sub-
dural hematoma, have also been reported.17 In an effort to improve
the bioavailability of baclofen 1, in the present study we design
and synthesize a series of 5-(4-chlorophenyl)-5,6-dihydro-1,3-oxa-
zepin-7(4H)-one derivatives (11–19) as lipophilic cyclic analogues
of baclofen 1 in a trial to enhance the permeation of 1 through
blood–brain barrier (BBB) to improve its therapeutic potency,
and to reduce the possible adverse effects. The central neurophar-
macological properties of target compounds (11–19) were studied
in mice and compared with the neuropharmacological effects and
structure of the reference drug baclofen 1.
(11–19). Acylation of the amino group of baclofen 1 with acyl chlo-
ride afforded the intermediates N-acylamino-3-(4-chloro-
phenyl)butyric acid derivatives (2–10). Intramolecular cyclization
through dehydration of 2–10 afforded the lactone type structures
(11–19). Acetic anhydride as mild and rapid dehydrating agent
was used successfully for the synthesis of the target lactones 11–
19. The structures of baclofen derivatives (11–19) were confirmed
by spectroscopic means including IR, 1H NMR, and EI-MS (Tables 1
and 2). Evidence of lactone formation in 11–19 emerged from the
presence of absorption bands in their IR spectra at 1758–
1770 cmꢀ1, assigned to ester C@O. 1H NMR spectra of compounds
(11–19) exhibited a multiplet signal at d 3.37–3.47 assigned to the
methine proton (H-5). A characteristic pair of doublets at d 2.62–
2.74, 2.77–2.87, 3.47–3.56, and 3.71–3.84 recognized to be H-6
and H-4, respectively. Two doublets appeared at d 7.12–7.18 and
7.30–7.36 reflected the p-substituted pattern of aromatic protons
of all derivatives (11–19) except compound 19 showed a multiplet
signal at d 7.16–8.12 assigned for nine aromatic protons. The pres-
ence of 1H NMR spectral data for residues in position 2 is also as-
signed. The EI-MS spectra of the target derivatives (11–19)
revealed molecular ion peaks consistent with their respective
molecular formulas. Based on the above findings, with the aid of
elemental analyses that showed satisfactory results within the ac-
cepted experimental errors, the structures of 11–19 were
established.
2. Results and discussion
2.1. Chemistry
2.2. Neuropharmacological activity
Scheme 1 illustrates the procedures used for the synthesis of 5-
(4-chlorophenyl)-5,6-dihydro-1,3-oxazepin-7(4H)-one derivatives
The aim of the present studies was to broadly characterize the
behavioral neuropharmacological effects of 5-(4-chlorophenyl)-
5,6-dihydro-1,3-oxazepin-7(4H)-one derivatives (11–19) as lipo-
philic cyclic analogues of baclofen 1. Loss of righting reflex, loco-
motor activity, rotarod test, traction test, hot-plate test, and
effect on passive avoidance behavior are paradigms employed. Bac-
lofen 1, as a positive control, and the tested derivatives (11–19)
were administered in mice intraperitoneally at a dose of 3 mg/
kg; 30, 45, 60, and 90 min before experimental testing. Our results
demonstrated that the methyl, heptyl, octyl, and decyl substituted
analogues (11–14) are devoid of all the tested neuropharmacolog-
ical effects associated with baclofen 1. While undecyl, tridecyl, dec-
9-enyl, heptadec-8-enyl, and benzyl substituted derivatives (15–
19) showed a comparable activity in the selected neuropharmaco-
logical effects tested (Tables 3–5).
R
O
O
1
2
O
7
N3
6
R
N
H
COOH
H2N
COOH
RCOCl
4
5
(Ac)2O
15 min.
100-150 °C
reflux
5~10 hr
Cl
Cl
1
Cl
11-19
2-10
Compound No.
R
Results obtained from the righting reflex test (Fig. 1) showed a
highly significant increase (p < 0.01) in the onset of the loss of
righting reflex after administration of 15, 18, and 19 derivatives
compared with baclofen 1 (Fig. 1A). While the tridecyl and dec-
8-enyl substituted analogues (16 and 17) displayed a significant
increase (p < 0.05) in the onset of the loss of righting reflex com-
pared with baclofen 1 (Fig. 1A). Also there was significant in-
crease (p < 0.05) in the duration of loss of righting reflex after
administration of compounds 15 and 17, and highly significant
increase (p < 0.01) after administration of compounds 16, 18,
and 19 relative to baclofen-treated group (Fig. 1B). Measurement
of locomotor activity of mice showed significant (p < 0.05) de-
crease in locomotor activity record after administration of unde-
cyl derivative (15) compared with baclofen-treated group after
30 and 45 min, which became highly significant (p < 0.01) after
60 and 90 min. On the other hand, the tridecyl, heptadec-8-enyl,
and benzyl substituted analogues (16, 18, and 19) showed highly
significant (p < 0.01) decrease in locomotor activity after all time
intervals tested (Fig. 2A–D) relative to that of baclofen 1. While
administration of compound 17 resulted in significant decrease
(p < 0.05) in locomotor activity only after 60 min and highly sig-
nificant decrease (p < 0.01) after 90 min of its administration
(Fig. 2C and D).
2, 11
3, 12
4, 13
5, 14
6, 15
7, 16
8, 17
9, 18
10, 19
Scheme 1. Synthesis of the target derivatives (11–19).

A. A. Abdel-Hafez, B. A. Abdel-Wahab / Bioorg. Med. Chem. 16 (2008) 7983–7991
7985
Table 1
Physical and IR data of substituted 5-(4-chlorophenyl)-5,6-dihydro-1,3-oxazepin-7(4H)-one derivatives (11–19)
Compounda
ClogPb
Mp (°C)
Yield (%)
Solventc
Molecular formulad (Molecular weight)
IR KBr (cmꢀ1
)
11
12
13
14
15
16
17
18
19
4.58
7.76
8.29
9.35
9.87
10.93
8.86
12.56
6.15
184–186
154–155
116–118
101–102
112–114
94–95
107–109
77–78
219–220
74
79
69
71
81
73
64
72
84
A
B
B
B
B
B
B
B
A
C12 H12ClNO2 (237.68)
C18 H24ClNO2 (321.84)
C19 H26ClNO2 (335.87)
C21 H30ClNO2 (363.92)
C22 H32ClNO2 (377.95)
C24 H36ClNO2 (406.00)
C21 H28ClNO2 (361.91)
C28 H42ClNO2 (460.09)
C18 H16ClNO2 (313.78)
1768 (C@O, ester), 1580, 1090
1765 (C@O, ester), 1575, 1085
1765 (C@O, ester), 1575, 1085
1762 (C@O, ester), 1570, 1085
1760 (C@O, ester), 1570, 1085
1760 (C@O, ester), 1570, 1085
1762 (C@O, ester), 1575, 1085
1758 (C@O, ester), 1570, 1085
1770 (C@O, ester), 1580, 1090
a
The mass spectrum revealed a proper molecular ion [M]+ for all compounds.
Calculated partition coefficient and ClogP for baclofen = ꢀ1.332.18
Solvent of crystallization: A, ethanol; B, ethanol–CHCl3.
b
c
d
Satisfactory microanalysis was obtained ( 0.04%) for C, H and N.
Table 2
1H NMR data of substituted 5-(4-chlorophenyl)-5,6-dihydro-1,3-oxazepin-7(4H)-one derivatives (11–19)
Compounda
–CH2–CO–
Methine
H
–CH2–N@
Ar-H
Others
11
2.72 (1H, dd, J = 16.2, 6.9 Hz,
H-6b), 2.84 (1H, dd, J = 16.2,
7.2 Hz, H-6a)
3.43
(1H, m,
H-5)
3.52 (1H, dd, J = 16.1, 6.8 Hz,
H-4b), 3.79 (1H, dd, J = 16.1,
7.2 Hz, H-44a)
7.18 (2H, d, J = 8.6, H-
30, 50), 7.36 (2H, d,
J = 8.6, H-20, 60)
1.76 (3H, s, CH3)
12
13
14
15
16
17
18
19
2.69 (1H, dd, J = 16.1, 6.8 Hz,
H-6b), 2.80 (1H, dd, J = 16.1,
7.0 Hz, H-6a)
2.67 (1H, dd, J = 16.0, 6.8 Hz,
H-6b), 2.79 (1H, dd, J = 16.1,
6.9 Hz, H-6a)
2.66 (1H, dd, J = 16.1, 6.9 Hz,
H-6b), 2.78 (1H, dd, J = 16.1,
7.1 Hz, H-6a)
2.64 (1H, dd, J = 16.1, 6.9 Hz,
H-6b), 2.80 (1H, dd, J = 16.0,
7.1 Hz, H-6a)
2.62 (1H, dd, J = 16.1, 7.0 Hz,
H-6b), 2.77 (1H, dd, J = 16.0,
7.1 Hz, H-6a)
2.69 (1H, dd, J = 16.0, 6.9 Hz,
H-6b), 2.81 (1H, dd, J = 16.2,
7.2 Hz, H-6a)
2.70 (1H, dd, J = 16.1, 6.9 Hz,
H-6b), 2.80 (1H, dd, J = 16.1,
7.1 Hz, H-6a)
2.74 (1H, dd, J = 16.2, 6.9 Hz,
H-6b), 2.87 (1H, dd, J = 16.2,
7.2 Hz, H-6a)
3.41
(1H, m,
H-5)
3.39
(1H, m,
H-5)
3.37
(1H, m,
H-5)
3.38
(1H, m,
H-5)
3.39
(1H, m,
H-5)
3.42
(1H, m,
H-5)
3.41
(1H, m,
H-5)
3.47
(1H, m,
H-5)
3.49 (1H, dd, J = 16.1, 6.9 Hz,
H-4b), 3.73 (1H, dd, J = 16.1,
7.3 Hz, H-4a)
3.50 (1H, dd, J = 16.2, 6.8 Hz,
H-4b), 3.75 (1H, dd, J = 16.2,
7.3 Hz, H-4a)
3.48 (1H, dd, J = 16.2, 6.9 Hz,
H-4b), 3.72 (1H, dd, J = 16.2,
7.0 Hz, H-4a)
3.47 (1H, dd, J = 16.1, 6.8 Hz,
H-4b), 3.71 (1H, dd, J = 16.1,
7.0 Hz, H-4a)
3.48 (1H, dd, J = 16.1, 6.9 Hz,
H-4b), 3.73 (1H, dd, J = 16.1,
7.1 Hz, H-4a)
3.51 (1H, dd, J = 16.2, 6.8 Hz,
H-4b), 3.80 (1H, dd, J = 16.1,
7.2 Hz, H-4a)
3.50 (1H, dd, J = 16.0, 6.8 Hz,
H-4b), 3.79 (1H, dd, J = 16.0,
7.2 Hz, H-4a)
3.56 (1H, dd, J = 16.1, 7.1 Hz,
H-4b), 3.84 (1H, dd, J = 16.1,
6.9 Hz, H-4a)
7.13 (2H, d, J = 8.5, H-
30, 50), 7.31 (2H, d,
J = 8.5, H-20, 60)
7.12 (2H, d, J = 8.5, H-
30, 50), 7.32 (2H, d,
J = 8.5, H-20, 60)
7.13 (2H, d, J = 8.5, H-
30, 50), 7.31 (2H, d,
J = 8.5, H-20, 60)
7.14 (2H, d, J = 8.5, H-
30, 50), 7.31 (2H, d,
J = 8.5, H-20, 60)
7.14 (2H, d, J = 8.6, H-
30, 50), 7.30 (2H, d,
J = 8.6, H-20, 60)
7.17 (2H, d, J = 8.6, H-
30, 50), 7.33 (2H, d,
J = 8.6, H-20, 60)
7.16 (2H, d, J = 8.5, H-
30, 50), 7.32 (2H, d,
J = 8.5, H-20, 60)
2.12 (2H, t, –CH2–), 1.34 (2H, p, –CH2–), 1.21 (8H, m,
4ꢁ –CH2–), 0.80 (3H, t, –CH3)
2.16 (2H, t, –CH2–), 1.36 (2H, p, –CH2–), 1.22 (10H,
m, 5ꢁ –CH2–), 0.81 (3H, t, –CH3)
2.18 (2H, t, –CH2–), 1.37 (2H, p, –CH2–), 1.24 (14H,
m, 7ꢁ –CH2–), 0.82 (3H, t, –CH3)
2.23 (2H, t, –CH2–), 1.40 (2H, p, –CH2–), 1.29 (16H,
m, 8ꢁ –CH2–), 0.84 (3H, t, –CH3)
2.24 (2H, t, –CH2–), 1.40 (2H, p, –CH2–), 1.30 (20H, m,
10ꢁ –CH2–), 0.85 (3H, t, –CH3)
5.27 (1H, m, –CH@CH2–), 4.23 (2H, m, –CH@CH2–),
2.14 (2H, q, –CH2–), 1.74 (2H, t, –CH2–), 1.35 (2H, p, –
CH2–), 1.01 (10H, m, 5ꢁ –CH2–)
5.11 (2H, m, 2ꢁ –CH–), 2.13(4H, m, 2ꢁ –CH2–), 1.81
(2H, t, –CH2–), 1.70 (2H, p, –CH2–), 1.11 (20H, m, 10ꢁ
–CH2–), 0.83 (3H, t, –CH3)
7.16–8.12 (9H, m)
3.52 (2H, s, –CH2–)
a
All compounds measured in DMSO.
Table 3
Effect of baclofen 1 and 5-(4-chlorophenyl)-5,6-dihydro-1,3-oxazepin-7(4H)-one derivatives (11–19) in dose 3 mg/kg, ip on the righting reflex and locomotor activity in mice
Compound
Righting reflex
Duration (min)
Locomotor activity (m)
Onset (min)
After 30 min
After 45 min
After 60 min
After 90 min
Control
Baclofen
11
12
13
14
15
16
17
—
—
45.24 5.46
30. 21 4.32*
45.24 5.46
45.24 5.46
45.24 5.46
45.24 5.46
20.12 4.73**
15.24 3.42**
25.36 5.62*
11.45 3.54**
0.00 0.0**
43.46 6.71
28.48 6.47**
43.46 6.71
43.46 6.71
43.46 6.71
43.46 6.71
16.21 4.22**
13.73 3.56**
20. 56 5.94**
9.33 3.14**
0.00 0.0**
42.79 5.63
30.76 4.15*
42.79 5.63
42.79 5.63
42.79 5.63
42.79 5.63
14.11 4.47**
14.76 3.22**
19.43 5.97**
10.61 3.46**
0.00 0.0**
40.13 5.18
35.64 4.64
40.13 5.18
40.13 5.18
40.13 5.18
40.13 5.18
15.66 4.63**
13.11 3.54**
18.56 5.73**
9. 25 3.44**
0.00 0.0**
20.24 5.46**
35.23 4.36**
—
—
—
—
—
—
—
—
38.23 6.38**
32.16 6.84**
36.63 4.23**
38.65 7.72**
42.12 5.32**
60.25 5.67**
75.93 6.38**
55. 33 7.39**
93.37 5.32**
112.36 5.61**
18
19
Results represent the mean (n = 5) SEM.
*
p < 0.05 vs control group.
p < 0.01 vs control group.
**
Results of rotarod test (Fig. 3) reflected that no significant
change in rotarod endurance after 30 min of administration of
compounds 15–19 and after 45 min of administration of com-
pounds 15 and 17 relative to baclofen 1 (Fig. 3A and B, respec-
tively). Also, administration of compounds 16 and 18 caused
significant (p < 0.05) reduction in the rotarod endurance relative

7986
A. A. Abdel-Hafez, B. A. Abdel-Wahab / Bioorg. Med. Chem. 16 (2008) 7983–7991
Table 4
Effect of baclofen 1 and 5-(4-chlorophenyl)-5,6-dihydro-1,3-oxazepin-7(4H)-one derivatives (11–19) in a dose 3 mg/kg, ip on the rotarod and traction tests in mice
Compound
After 30 min
After 45 min
After 60 min
After 90 min
Rotarod
endurance
Traction test
% successful
Rotarod
endurance
Traction test
% successful
Rotarod
endurance
Traction test
% successful
Rotarod
endurance
Traction test
% successful
Control
Baclofen
11
12
13
14
15
16
17
300
0
100
0
300
0
100
0
300
0
100
0
300
0
100
0
214 16**
50 3.1**
225 16*
52 5.3**
234 16*
53 5.4**
254 13*
53 4.5**
300
300
300
300
0
0
0
0
100
0
0
0
0
300
300
300
300
0
0
0
0
100
0
0
0
0
300
300
300
300
0
0
0
0
100
0
0
0
0
300
300
300
300
0
0
0
0
100
0
0
0
0
100
100
100
100
100
100
100
100
100
100
100
100
232 14*
219 9**
222 15*
198 12**
234 16*
45 4.3**
40 3.5**
55 3.2**
35 4.1**
27 4.3**
188 14**
174 9**
43 3.2**
40 3.1**
50 2.1**
35 4.2**
25 3.7**
178 14**
169 9**
185 15**
155 12**
0.00**
40 3.8**
35 2.4**
50 4.3**
33 3.3**
25 2.5**
145 14*
133 9**
155 13**
128 12**
0.00**
40 3.6**
35 5.7**
53 4.4**
31 2.4**
22 2.8**
198 15**
175 12**
185 16**
18
19
Results represent the mean (n = 5) SEM.
*
p < 0.05 vs control group.
p < 0.01 vs control group.
**
Table 5
Effect of baclofen 1 and 5-(4-chlorophenyl)-5,6-dihydro-1,3-oxazepin-7(4H)-one derivatives (11–19) in a dose 3 mg/kg (ip) on the hot plate and passive avoidance reflex (PAR)
tests in mice
Compound
Retention latency (s)
After 30 min
After 45 min
Hot-plate test PAR test
After 60 min
After 90 min
Hot-plate test
PAR test
Hot-plate test
PAR test
Hot-plate test
PAR test
Control
Baclofen
11
12
13
14
15
16
17
8.12 2.14
13.42 2.46*
8.12 2.24
8.12 2.13
8.12 2.14
8.12 2.34
7.82 3.51
8.33 2.21
8.32 3.22
9.34 2.88
9.53 2.36
163.23 6.26
125.34 4.54*
163.23 6.36
163.23 6.26
163.33 6.47
163.53 6.22
152.34 7.32
147.24 7.63
148.82 5.23
146.23 5.52*
125.64 6.57*
8.22 2.57
15.35 2.23*
8.22 2.58
8.22 2.57
8.22 2.67
163.23 6.24
121.46 4.83*
163.23 6.24
163.23 6.44
163.23 6.34
163.23 6.27
114.73 7.33**
109.34 7.54**
118.32 6.85**
110.36 6.54**
106.56 6.77**
9.23 2.35
14.36 2.13*
9.22 2.37
9.23 2.25
9.23 2.35
160.43 8.46
121.44 7.52*
160.43 7.43
160.43 8.46
160.43 8.26
160.43 8.38
88.14 13.4**
82.36 12.23**
95.35 9.32*
75.44 12.76**
70.34 9.55**
9.33 2.13
14.22 2.25*
9.33 2.13
9.33 2.13
9.33 2.13
164.56 8.66
128.56 7.64*
164.56 8.66
164.56 8.66
164.56 8.66
164.56 8.66
72.45 13.66**
67.56 12.56**
95.45 11.23**
68.44 12.38**
65.34 9.27**
8.22 2.55
9.23 2.45
9.33 2.13
13.21 3.11*
15.32 2.36*
14.34 3.41*
15.77 2.47*
15.38 3.32*
18.56 1.52*
18.45 2.35*
19.73 3.11*
23.61 2.17**
25.63 3.44**
24.43 1.52**
26.13 2.47**
21.42 3.21**
28.43 2.51**
28.73 3.25**
18
19
Results represent the mean (n = 5) SEM.
*
p < 0.05 vs control group.
p < 0.01 vs control group.
**
B
A
150
50
**
Baclofen
**
**
*
Compound 15
Compound 16
Compound 17
Compound 18
Compound 19
**
**
40
*
100
50
0
30
20
10
0
**
*
*
Figure 1. Effect of baclofen 1 and compounds (15–19) in dose 3 mg/kg, ip on the righting reflex test in mice. (A) onset and (B) duration of the loss of righting reflex. Results
represent mean (n = 5) SEM. *p < 0.05 significant increase vs baclofen-treated group. **p < 0.01 significant increase vs baclofen-treated group.
to baclofen 1 after 45 min (Fig. 3B) and compounds 15–18 after 60
and 90 min (Fig. 3C and D). While compound 19 showed a signifi-
cant decrease (p < 0.05) in rotarod endurance after 45 min (Fig. 3B)
and highly significant (p < 0.01) reduction in the rotarod endurance
relative to baclofen 1 after 60 and 90 min of its administration (Fig.
3C and D). In traction test, the dec-8-enyl substituted derivative
(17) revealed no significant decrease in the percent of success of
the traction test in measurements carried out at all time intervals
relative to baclofen-treated group (Fig. 4A–D). While compound
15, showed significant (p < 0.05) decrease in the percent of success
of the traction test only at 90 min after its administration (Fig. 4D).
Also significant activity (p < 0.05) showed by compounds 16 and 18
at 30 and 45 min time intervals compared with baclofen-treated
group (Fig. 4A and B). Highly significant (p < 0.01) decrease in the
percent of success of the traction test at all time intervals relative
to that of baclofen was displayed by the benzyl substituted deriv-
ative 19 (Fig. 4A–D). Many studies indicated the role of GABAB
receptors in sedation and muscle-relaxant effect, suggesting that
activation of GABAB receptors may decrease anxiety.25 Baclofen 1
has demonstrated anxiolytic-like effects in a number of tests and
reversed the anxiogenic response induced by withdrawal from
chronic diazepam or alcohol treatment.26–29 Clinically, baclofen 1
reversed the anxiety associated with alcohol withdrawal,30 post-
traumatic stress,31 and panic disorder.32

A. A. Abdel-Hafez, B. A. Abdel-Wahab / Bioorg. Med. Chem. 16 (2008) 7983–7991
7987
A
B
40
30
20
10
0
40
Baclofen
30
Compound 15
Compound 16
Compound 17
Compound 18
Compound 19
*
*
20
**
**
**
**
10
**
**
0
C
D
40
30
20
10
0
50
40
*
30
**
**
**
**
20
10
0
**
**
**
**
**
Figure 2. Effect of baclofen 1 and compounds (15–19) in dose 3 mg/kg, ip on the locomotor activity of mice after (A) 30 (B) 45 (C) 60, and (D) 90 min of administration.
Results represent mean (n = 5) SEM. *p < 0.05 significant decrease vs baclofen-treated group. **p < 0.01 significant decrease vs baclofen-treated group.
A
B
400
300
200
100
0
300
200
100
Baclofen
Compound 15
Compound 16
Compound 17
Compound 18
Compound 19
*
*
*
0
C
D
300
300
200
100
0
200
100
0
*
*
*
*
*
*
*
*
**
**
Figure 3. Effect of baclofen 1 and compounds (15–19) in dose 3 mg/kg, ip on the rotarod test endurance in mice after (A) 30 (B) 45 (C) 60, and (D) 90 min of drug
administration. Results represent mean (n = 5) SEM. *p < 0.05 significant decrease vs baclofen-treated group. **p < 0.01 significant decrease vs baclofen-treated group.
Results obtained from measurement of hot-plate retention la-
tency shown in Table 5 indicted that baclofen 1 showed signifi-
cant increase (p < 0.05) in hot-plate retention latency at all
time intervals compared with control group. While administra-
tion of undecyl, tridecyl, and dec-9-enyl, hepatdec-8-enyl, and
benzyl substituted analogues 15–19 showed no significant
change in hot-plate latency compared with baclofen (Fig. 5A
and B). Administration of compounds 15 and 16 resulted in sig-
nificant increase (p < 0.05) in hot-plate retention latency mea-
sured at 60 min, and highly significant increase in hot-plate
latency at 90 min following administration of these compounds
(Fig. 5C and D, respectively). On the other hand, relative to bac-
lofen-treated group, the heptadec-8-enyl and benzyl substituted
derivatives 18 and 19 revealed highly significant (p < 0.01) in-
crease in hot-plate retention latency after 60 and 90 min, and
compound 17 showed significant increase (p < 0.05) in hot-plate
latency measured 60 and 90 min after its administration (Fig.
5C and D). Our results indicated that the cyclic baclofen ana-
logues 15–19 exert analgesic activity, but with relatively delayed
onset and longer duration than baclofen 1, which is known to ex-
ert analgesic activity.33 These results are in accordance with the
study shown by Cutting and Jordan who reported that baclofen 1
prolonged the time to foot withdrawal in the mouse hot-plate
test.34 Also, study of Balerio and Rubio demonstrated that baclo-

7988
A. A. Abdel-Hafez, B. A. Abdel-Wahab / Bioorg. Med. Chem. 16 (2008) 7983–7991
A
B
100
80
60
40
20
0
100
Baclofen
Compound 15
Compound 16
Compound 17
Compound 18
Compound 19
80
60
*
*
*
*
40
20
0
**
**
C
D
100
80
60
40
20
0
100
80
60
40
20
0
*
*
*
**
**
**
**
Figure 4. Effect of baclofen 1 and compounds (15–19) in dose 3 mg/kg, ip on the %. Success of traction in mice after (A) 30 (B) 45 (C) 60, and (D) 90 min of drug
administration. Results represent mean (n = 5) SEM. *p < 0.05 significant decrease vs baclofen-treated group. **p < 0.01 significant decrease vs baclofen-treated group.
A
B
20
15
10
5
20
15
10
5
Baclofen
Compound 15
Compound 16
Compound 17
Compound 18
Compound 19
**
0
**
0
C
D
30
20
10
0
**
**
**
**
30
20
10
0
**
*
**
*
*
*
**
**
Figure 5. Effect of baclofen 1 and compounds (15–19) in dose 3 mg/kg, ip on the retention latency of hot-plate test in mice after (A) 30 (B) 45 (C) 60, and (D) 90 min of
administration. Results represent mean (n = 5) SEM. *p < 0.05 significant increase vs baclofen-treated group. **p < 0.01 significant increase vs baclofen-treated group.
fen 1 elicits a dose-dependent anti-nociceptive effect in mice
using the hot-plate test.35
45 min of their administration (Fig. 6B). Compound 16 showed sig-
nificant decrease in the retention latency of the PAR test at level
p < 0.05 after 60 min of its administration (Fig. 6C) that became
highly significant at level p < 0.01 after 90 min of its administration
(Fig. 6D). Compounds 18 and 19 showed highly significant
(p < 0.01) decrease in the retention latency of the PAR test after
60 and 90 min of its administration (Fig. 6C and D). Compound
15 showed no significant change in PAR test latency compared
with baclofen after 30 and 45 min of its administration, while it
started to show significant decrease (p < 0.05) in PAR latency after
60 min and highly significant decrease (p < 0.01) after 90 min of its
As shown in Table 5, compounds (11–19) also differed in their
influence on cognitive function compared with the control group.
Baclofen 1 and its derivatives (15–19) showed significant decrease
in the retention latency of passive avoidance reflex (PAR) test com-
pared with control (Table 5). While compared with baclofen 1, re-
sults showed that there was no significant change in the retention
latency of PAR test after 30 min of their administration of these
compounds 15–19 (Fig. 6A). Compounds 16, 18, and 19 showed
significant decrease in the retention latency of the PAR test after

A. A. Abdel-Hafez, B. A. Abdel-Wahab / Bioorg. Med. Chem. 16 (2008) 7983–7991
7989
B
A
C
150
200
150
100
50
Baclofen
**
*
Compound 15
Compound 16
Compound 17
Compound 18
Compound 19
100
50
*
**
**
0
0
D
150
150
100
50
*
*
100
50
0
*
*
**
**
**
**
**
**
**
**
0
Figure 6. Effect of baclofen 1 and compounds (15–19) in dose 3 mg/kg, ip on retention latency of the passive avoidance reflex (PAR) test in mice after (A) 30 (B) 45 (C) 60, and
(D) 90 min of drug administration. Results represent mean (n = 5) SEM. *p < 0.05 significant decrease vs baclofen-treated group. **p < 0.01 significant decrease vs baclofen-
treated group.
administration (Fig. 6C and D). Compound 17 showed significant
decrease (p < 0.05) in PAR test latency only after 60 and 90 min
of its administration (Fig. 6C and D). These data are consistent with
previous reports demonstrating cognitive impairing effects of bac-
lofen 1 on passive avoidance behavior.36–38 Baclofen 1, an agonist
of GABAB receptors, impaired cognition and passive avoidance re-
flex in a dose-related manner.39–42 Zarrindast et al. showed that
low doses of baclofen (0.125 mg/kg and 0.25 mg/kg) showed only
a tendency (without significance) to impair acquisition of passive
avoidance in mice but diminished the improvement produced by
physostigmine.43 This is in accordance with our results, where a
significant impairment in cognition observed after administration
of baclofen 1 and its cyclic derivatives 15–19, but these compounds
showed delayed impairment in cognitive function but with more
prolonged duration than that of baclofen 1.
During the last decade, however, innumerable attempts to de-
velop specific agonists for the GABAB receptors site, baclofen 1 re-
mains the clinical drug of choice in the treatment of spasticity.44–46
Up to now, baclofen 1 is the only clinically useful selective GABAB
receptors agonist. Therefore, additional efficient GABAB receptor
agonists are eagerly awaited. In ongoing efforts to search for more
potent and less toxic GABAB receptor agonist, nine new lipophilic
cyclic analogues of baclofen (11–19) are designed and synthesized.
The main target of our design of 5-(4-chlorophenyl)-5,6-dihydro-
1,3-oxazepin-7(4H)-one derivatives (11–19) is to preserve the
pharmacological profile and improve the bioavailability through
lipophilicity increment of baclofen 1. Structural modifications of
baclofen 1 are designed in a manner where baclofen back-forma-
tion via metabolic hydrolysis from the target molecules (11–19)
can be achieved.
and 19) to baclofen 1 may gives rise to longer duration of action.
2-Benzyl-5-(4-chlorophenyl)-5,6-dihydro-1,3-oxazepin-7(4H)-one
derivative 19 is the most active candidate highly affecting the
potencies at all time intervals tested in addition to its 10-times
reduction in locomotor activity, 6-times reduction in the rotarod
endurance, 3-times decrease in the retention latency of PAR, and
2-times increase in hot-plate retention latency and decrease in
the percent of success of retention test compared with baclofen 1
after 90 min of its administration. The neurological activity of 5-
(4-chlorophenyl)-2-(dec-8-enyl)-5,6-dihydro-1,3-oxazepin-7(4H)-
one derivative 17 which appeared at relatively higher time interval
in most terms may prove that metabolic activation of 17 to baclo-
fen 1 is a necessary after crossing BBB to reach the site of action at
a higher concentration.
Although no clear correlation between the structure and the neu-
ropharmacological activity emerged from the present results, it is
obvious that the R residue at C-2 of baclofen cyclic analogues (15–
19) effectively enhances the neuropharmacological potency. On
the other hand, the number of carbon atoms and their nature (satu-
rated, unsaturated, or aromatic) of the R residue at C-2 possibly af-
fected the neuropharmacological activity of these compounds.
Partition coefficient, logP values, is an important parameter for
determining the biological properties of compounds affecting the
central nervous system (CNS). Since the cross of BBB by CNS agents
is highly affected by lipophilicity, calculated lipophilicity (ClogP)
of the target derivatives (11–19) was estimated by using the ClogP
program based on the fragment method developed by Leo.18 As it
is clear from our results that it is difficult to correlate all logP values
with the neuropharmacological potency of the target derivatives
(11–19), which may reflect that the intrinsic activity in addition to
the bioavailability plays an important role in their activities.
The most interesting data from this study are that simple cycli-
zation of baclofen 1 to 1,3-oxazepinone with addition of lipophilic
moieties may lead to a dramatic increase in the neuropharmaco-
logical potency, which may reflect a new era of research on GABAB
agonists. Generally, it can be concluded that 5-(4-chlorophenyl)-
5,6-dihydro-1,3-oxazepin-7(4H)-one derivatives represent a new
From the results obtained, a significant neuropharmacological
activity was displayed by undecyl, tridecyl, heptdec-8-enyl, and
benzyl substituted analogues derivatives (15, 16, 18, and 19) in
all paradigms tested. In terms of potency, the neuropharmacologi-
cal activity of these derivatives (15, 16, 18 and 19) is vividly
favorable comparable with baclofen 1. On the other hand, vulner-
ability to metabolic activation of these compounds (15, 16, 18,

7990
A. A. Abdel-Hafez, B. A. Abdel-Wahab / Bioorg. Med. Chem. 16 (2008) 7983–7991
distinct class of structures with therapeutic strategy for the treat-
ment of various neurological diseases. Although initial evidence
obtained from the present experiments about the neuropharmaco-
logical potencies of the synthesized derivatives (11–19) is very
encouraging, further experiments are being actively investigated
in our laboratory and will be published in due course.
allel to those carried on control group. Animals were divided into
groups five mice for each. Each group is placed in a separate cage.
3.3.3. Drug treatment
L-Baclofen and tested compounds (11–19) were dissolved di-
rectly before experiments in DMSO to prepare solutions for giving
all drugs in a dose 3 mg/kg intrapretoneally, 30, 45, 60, and 90 min
before experimental testing. Control group was treated with DMSO
free from drugs.
3. Materials and methods
3.1. Chemistry
3.3.4. In vivo experiments
3.3.4.1. Loss of righting reflex.
The test was carried out as
Melting points were determined on a micromelting point appa-
ratus (L-272, Yanaco, Kyoto, Japan) and are uncorrected. IR spectra
were performed with a JASCO FT/IR-230 infrared spectrophotome-
ter. Thin layer chromatography was carried out on pre-coated Sil-
ica gel 60 F254 plates (0.25 mm thickness, Merck, Darmastat,
Germany) and spots were detected under UV light. Silica gel 60
(60–230 mesh, Merck, Darmastat, Germany) was used for column
chromatography. 1H NMR spectra were measured with a JEOL-
JNM-GX 400 (400 MHz) spectrophotometer and chemical shifts
were given in d ppm relative to tetramethylsilane (TMS). Electron
impact (EI) mass spectra were measured with a JEOL GC-mate
spectrometer at an ionization voltage of 70 eV. Elemental analyses
were performed at the microanalytical center of Toyama Medical
and Pharmaceutical University, Toyama, Japan. All chemicals used
were of analytical grade.
described before by Boehm et al.19 Animals were injected with bac-
lofen or tested compounds (3 mg/kg, ip), and the length of loss of
righting reflex (sleep-time) was measured. Upon loss of the right-
ing reflex, mice were placed on their backs in a sleep trough (90°
angle), and the time to regain the righting reflex was measured.
Loss of righting reflex was defined as the inability of a mouse to
right itself within 30 s. Return of the righting response was defined
as the ability of a mouse to right itself twice in 1 min sleep-time, or
duration of loss of righting reflex was defined as the time between
loss and return of the righting response.
3.3.4.2. Locomotor activity
3.3.4.2.1. Recording device. Horizontal locomotor activity was
assessed
in
transparent
Plexiglas
boxes
(dimensions:
19 ꢁ 31 ꢁ 16 cm), and activity was detected and registered using
the TSE Moti system (TSE, Bad Homburg, Germany), which is based
on the registration of infrared light beam interruptions along the
x-, y-, and z-axes, as caused by an animal’s movements data were
directly stored into the computer.20
3.3.4.2.2. Experimental procedures. Mice received injections
with L-baclofen 1 and the tested derivatives (11–19) (3 mg/kg ip),
or vehicle (DMSO) and also were immediately returned to their
home cages for 30, 45, 60, and 90 min. Subsequently, they were
individually placed in Plexiglas boxes, and their spontaneous loco-
motor activity was registered.
3.1.1. Synthesis of 4-acylamino-3-(4-chlorophenyl)butanoic
acid derivatives (2–10)
To a solution of 1 (1 equiv mol) in dioxane, a solution of the
appropriate acid chloride (1 equiv mol) in dioxane was added.
The reaction mixture was heated at 100 ꢂ 150 °C for 5 ꢂ 10 h.
The residue after evaporation of dioxane was purified by SiO2 gel
column chromatography using CHCl3/MeOH as eluent, and the res-
idues after solvent evaporation were used for further reaction.
3.1.2. Synthesis of 5-(4-chlorophenyl)-5,6-dihydro-1,3-
oxazepin-7(4H)-one derivatives (11–19)
3.3.4.3. Rotarod test.
The rotarod apparatus consisted of a
A solution of the appropriate derivative (2–10) (0.3 g) in acetic
anhydride (5 mL) was refluxed for 15 min. After cooling to room
temperature, the product precipitated was filtered and crystallized
from the appropriate solvent. Yields and physical constants are
listed in Table 1, and 1H NMR data are presented in Table 2.
cylinder subdivided into five available mice positions, each 6 cm
in diameter, which was positioned 30 cm above the table and ro-
tated at a speed of 12 rpm.21 The mice were placed singly on the
cylinder. On the day before the start of the experiment, animals
were trained to stay on the rotarod for 300 s. Mice that failed to
learn the test or did not reach the criterion (300-s endurance) were
excluded from the study. During the test day, the length of time
each mouse remained on the cylinder (‘endurance time,’ maximal
score 300 s) was measured 30, 45, 60, and 90 min after application
of a test compound or vehicle.
3.2. Calculation of logP values
The logP values of the synthesized derivatives (11–19) were
computed with a routine method called calculated logP (ClogP)
contained in a PC-software package (Mac log P 2.0, BioByte Corp.,
CA, USA). A representation of the molecular structure where hydro-
gens are omitted, or suppressed (SMILES notation), is entered into
the program, which computes the logP based on the fragment
method developed by Leo.18 Results are given in Table 1.
3.3.4.4. Traction test.
The effect of drugs on muscle strength
was examined in the traction test. The animal was suspended by
the front paws from a horizontal wire. The untreated mice grasped
the stick with both forepaws and, when allowed to hang free,
placed at least one hind foot on the stick. The test was successfully
completed when the animal was able to touch the wire with at
least one hind paw within 5 s. Inability to perform was scored as
a failure of traction.22 The animals were tested 30, 45, 60, and
90 min after application immediately prior to the rotarod test.
3.3. Neuropharmacological evaluation
3.3.1. Chemicals
L-Baclofen is purchased from (Sigma co., USA) through the office
of International co. of chemicals, Jeddah, Saudi Arabia.
3.3.2. Animals
3.3.4.5. Hot-plate test.
The test was carried out as described
by Dambrova et al.23 Mice were placed into a 19 cm wide glass cyl-
inder on a heated (54 °C) metal plate (Model DS35, Ugo Basile,
Italy). The latency to lick one of the hind paws or to jump off the
plate was determined. Mice were removed from the hot plate
immediately after the response. The cut-off time was set to 30 s
Male albino mice (Laboratory Animal Breeding Facility, Jazan,
KSA) weighing 20–25 g were housed under standard conditions
(21–25 °C, 12 h light–dark cycle) with unlimited access to food
and water. All experimental procedures were carried out in the
morning and procedures for the tested drugs were carried out par-

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3.3.4.6. Effect on passive avoidance behavior.
Starting on
the day before the training trial and continuing throughout the
experiment, they were housed singly in the experimental room.
The apparatus and procedure were as previously described in de-
tail.24 Briefly, for the training trial, mice were gently placed into
the light side of the two-compartment trough-shaped apparatus.
The door to the dark compartment was opened and a button
pressed to initiate timing by the computer. When the mouse broke
a photocell beam located 10.5 cm into the dark compartment, the
latency from opening the door to the animal breaking the beam
(step-through latency) was automatically recorded, and a Camp-
den Instruments 521 C Shock Source (Campden Instruments Ltd,
Leicester, UK) was automatically activated. This resulted in the
application of a foot shock (0.5-mA rectangular current waves) be-
tween the stainless steel plates, which comprised the dark com-
partment. This ended when the mouse escaped back to the light
compartment or after 5 s elapsed, whichever came first. If an ani-
mal did not enter the dark compartment within 150 s on the train-
ing trial, it was removed from the apparatus without receiving any
shock and was excluded from the retention test. The retention test
was carried out on the following day, and the same procedure was
followed except that the shock generator was switched off.
3.3.4.6.1. Drugs.
L-Baclofen 1 and the tested derivatives (11–
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90 min before the training trial.
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3.3.5. Statistical analysis
All results are expressed as means SEM. For testing the signif-
icance of the differences between groups Student’s t-test was used.
p-values less than 0.05 were considered to be significant and less
than 0.01 to be highly significant. All statistical analyses of data
were carried out with the help of computer system using the com-
mercially available software (Graph pad Prism V5, USA).
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