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.ROCEEDINGS OF THE NEUROSCIENCE SOCIETY
OF NIGERIA (NSN) CONFERENCE, JOS, 2006.
Anticonvulsant Activity of Some N, N'-(2,3,5,6- Tetramethyl-l,4-Phenylene)
Bis (4-Substituted Benzamides)
By
Afolabi, E. 0*. and Ebuga, Solomon
Dept. of Pharmaceutical Chemistry, University of Jos, Jos. NIGERIA.
Abstract
The anticonvulsant
activity
of four analogues
of N,N'-(2,3,5,6-tetramethyl-I,4-phenylene)
bis (4substitutedbenzamides) were determined at three dose levels in accordance with the Antiepleptic Drug Development
program (ADD) of National Institute of Health (NIH), USA. These compounds are analogues of 4-amino-N-(2,6dimethylphenyl)benzamide (Ameltolide®, or LY20 1116) which is the most potent benzamide anticonvulsant studied to
date. The result of our anticonvulsant screening confirmed the superiority of 4-aminobenzamides over other parasubstituted analogues.
Key Words: Anticonvulsant,
Benzamide, Epilepsy and Seizures.
Introduction
Epilepsy is a neurological disorder
associated with excessive temporary neuronal
discharge, characterized by discrete recurrent
episodes which results in disturbance of
movement sensation, behaviour, perception
and consciousness (Guelen and van der Kleijn,
1978). Epilepsy affects about 0.5-1 % of the
world's population (Daniels and Jorgensen,
1982).
There are many drugs used in the
management
of epilepsy. Among these
anticonvulsants
armamentarium,
the
benzamides have shown some promising
evidence
in anticonvulsant
properties.
Structurally,
compounds
possessing
anticonvulsant activities, e.g. Valproate, are
carboxylic acids and their amides (Murray and
Kier, 1977). However the basic event
responsible for epilepsy should be approached
rationally when developing any anticonvulsant
drug.
The substituted-benzamides
have
generated a lot of interest, as neuroleptics, in
recent years (Blaney et al., 1983). The
*Address
benzamides have a unique behavioral profile
.md they appear to exert their pharmacological
activity selectively
at the D2 dopamine
receptors.
This D2 receptor
subtype is
responsible for their therapeutic use as antischizophrenic.
Clark
et al., (1984)
demonstrated
that N-substituted-4aminobenzamides
show' a high level of
protection
against maximal electrostatic
induced convulsions in animal models. In this
study, four N, N'- (2,3,5,6-tetramethyl-l,4pheny lene) bis (4-substi tuted benzamides)
were prepared, screened and evaluated for their
anticonvulsant activity (see Table 1). These
compounds are analogues of 4-amino-N-(2,6dimethylphenyl) benzamide (Ameltolide" or
LY20 1116) which is the most potent benzamide
anticonvulsant studied to date.
Materials and Method
Animals
Mice (25-40 g) were obtained from National
Veterinary Research Institute, Vom, Nigeria.
The animals were kept in the Animal house of
the Department of Pharmacology, University of
correspondence to this author at the Department of Pharmaceutical Chemistry,
Faculty of Pharmaceutical Sciences, Univeristy of Jos, PMB 2048, Jos.Nigeria.
Tel. +234-73-6 \05514 Ex!. 211 GSM: 08035889579. E-mail: a/olabie@unijos.edu.ng
8
Afolabi £.0. / Proceeding
0/ the
Neuroscience
SOc'
Jos, Nigeria for 2 days to acclimatize to
laboratory condition before the commencement
of experiment. They were fed with standard
feed and water ad libitum.
Synthesis of compounds
A typical procedure for the synthesis of the
compounds, is as follow: A mixture of 2.623g
(O.OIM) triphenylphosphine, 9.6mls (O.1M)
carbon
tetra-chloride
and 30mls
of
tetrahydrofuran (THF) was refluxed for thirty
(30) minutes. The solution was cooled in an ice
bath to 5°C and a mixture of 1.565g (O.OIM) 4p~p
+ CC~ -
[P~p+-ccl.JCr
,I
RCOOH/EtN
[P~P+-OOCR]Ct + CHCI3
t RN~
P~P=O
+ HCI + RCONHR
of Nig. (NSN) Conference,
Jos, 2006
chlorobenzoic
acid and 1.4mls (O.OIM)
triethylamine was added and allowed to stand at
5°C for ten (10) minutes. One equivalent of
2,3,5 ,6-tetramethyl-l
,4-phenyldiamine
(0.005M) was added and the mixture was
heated and refluxed for another forty-five (45)
minutes. The precipitated triphenylphosphite
was removed by filtration while the solvents
were removed under vacuum. The product was
purified with preparative TLC on silica, while
its CHN analysis agreed within ± 0.04% range
from the expected formula. Its melting point
was determined and recorded uncorrected.
AH
Y
R
= OH. CN, Cl,
NI:!
para-substituted benzoic acid
H'N*~
H:JC
CH3
H:JC
CH:J
2 3 5 6-tetramethylphenyldiamine
(TMA)
Wittig Reaction with Ester-Exchange
Anticonvulsant test
Animals were pretreated with the test
compounds
for 30 minutes
and later
pentylenetetrazol (85mg/Kg) was administered
intraperi-toneally, The animals were observed
for 30 minutes. Failure to observe a single
episode of clonic spasms for at least 5 seconds
duration was considered as protection, and the
results expressed as number of animals
protected/number of animals tested.
Results
The results of this study are shown on
Table 1. All the four test compounds in this
series have some anticonvulsant
activity
against pentylenetetrazole induced tonic clonic
seizures at doses lower than 300mglKg. The 4hydroxyl, 4-chloro and 4-cyano substituted
analogues appear to be quite toxic at the high
dose level of3 OOmg/Kg.
All the t est
compounds delayed the onset of seizures and
reduced the number of seizures as compared
with the negative control group; (ipPTZ only).
Considering prolongation of protection against
death due to seizure, only 100mgIKg of the 4amino offered a longer protection than with the
negative control group (ipPTZ only) while the
standard drug (cabamazepine) offered a more
prolonged
protection
than all the test
compounds even though all the animals in the
control group eventually died after 646 seconds
following administration of pentylenetetrazole.
Afolabi E. 0. ! Proceeding of the Neuroscience
Discussion
The observed actrvity of the test
compounds
(see Table 1) may not be
phenytoin- or carbamazepine-like. This is
because convulsant activity of maximal
electro-shock
is blocked,
by the
pentylenetetrazole-induced seizure. Swinyard
et al. (1989) demonstrated
that some
compounds are termed "preventing seizure
spread" rather than "raising seizure threshold".
In this experiment, the compounds are seen to
increase seizure threshold, probably through
GABA pathway.
Previous studies (Parli et al., 1987)
have shown that the metabolic product of
benzamides (N-acetyl, analogue) produced
anticonvulsant activity in laboratory animals.
Pentylenetetrazole(PTZ)
blocks Gamma
amino butyric acid (GAB A) receptors, thus,
interfering with the function of this inhibitory
neurotransmitter
(Kupferberg, 1989). The
anticonvulsant
activity of the compound
suggests that it is mediated, at least in part,
through a GABAagonist effect.
Since
convulsion
induced
by
pentylenetetrazole occurs in the brain and not
in the spinal cord, then for the 4- amino
analogue to have delayed onset of seizure,
proves that the compounds possibly crossed the
blood brain barrier (BBB) to antagonize the
action of pentylenetetrazole which is a non
competitive
gamma amino butyric acid
(GABA) antagonist. Moreover, because a dose
as low as 10mg/Kg of the 4- amino analogues
could offer a better delayed onset of seizure, it
means that this compound
has high
lipophilicity and significant percentage of the
amount administered can cross the BBB to
have a substantial concentration at the site of
action in the brain.
References
Blaney, F. E.; Michael S. G Clark, D. V.
Gardner, M. S. Hadley, * David Middleton,
and White T. 1. (1983): Anilides Related to
Substituted
Benzamides.
Potential
Antipsychotic Activity ofN-( 4-Amino-5-
Soc. of Nig. (NSN) Conference, Jos, 2006
9
chloro-2-meht oxyp heny 1) - 1- (ph
enylmet hy 1) -4-piperidinecarboxamide' J
Med. Chem. 1983,26, 1747-1752
Clark, C. R., M. 1. M. Wells, R. T. Sansom, G. N.
Norris, R. C. Dockens, And W. R. Ravis.
(1984). "Anticonvulsant
activity of 4aminobenzamide." Journal of Medicinal
Chemistry. 27: pp. 779-782.
Daniels, T. C.; Jorgensen, E. C. (1982): In
"Wilson and Gisvold's Textbook of Organic
Medicinal and Pharmaceutical Chemistry",
8th ed.; Doerge, R. F. Ed.; 1. B. Lippincott:
Philadelphia, p 375.
Guelen, P. 1. M.; van der Kleijn, E. (1978):
"Rational Anti-Epileptic Drug Therapy";
Elsevier: Amsterdam, p 1.
Kupferberg, H.1. (1989): Antiepileptic drug
development program: A cooperative effort
of government and industry. Epilepsia 30
(suppi.l), S51-S56.
Murray, W. 1.; and Kier, L. B. (1977):
"Structure-Activity
Studies
on
Hallucinogenic
Amphetamines
Using
Molecular Connectivity" J. Med. Chem.
(Academic Press) 1977, 15, 591.
Parli, C. 1.; Evenson, E.; Potts, B. D.; Beedle, E.;
R Lawson, R.; Robertson, D. W; and
Leander, 1. D. (1987) "Metabolism of the
prodrug DEGA (N-(2,6-dimethylphenyl)4[[(Diethylamino )acetyl]amino ]benzamid
e) ...." Drug Metab Dispos.: 16: 707-711
Swinyard, E.A., Woodhead, 1.H., White, H.S. &
Franklin, M.R. (1989): General principles:
experimental selection, quantification, and
evaluation
of antic on vu Is ants.
In:
Antiepileptic Drugs, Third Edition R.H.
Levy, R.H. Mattson, B. Melrum, 1.K. Penry
and F.E. Dreifuss eds, pp. 85-102. New
York: Raven Press.
10
Afolabi £.0. / Proceeding of tire Neuroscience
Soc. of Nig. (NSN) Conference, Jos. 2006
Table 1: Anticonvulsant Activity of N,N' -(2,3,S,6-tetramethyl-l,4-phenylene )bis
(4-substitutedbenzamides)
TMA bis (4-Substituted-benzamid~
-c\
L'
HO
_.
n 'rd
N-
~?
~ ,-N-.
,
/=.
'L.!;'
-OH
/
4-OH-TMA
(TMA~is-4-Hydrozyl-benzamide (mp 12(fC)
'"*"'
'"*"'u.o-~2
'"*"' -
t..c-o-r-~ ~-/i ~VCN
4-CN-TMA
(TMA)bis-4·<:yano-benzamide(mp 9l1'C)
Cl-o-r-~ ~-/i ~CI
(TMA)bis-4-chloro-benzamide
H~~
4-CI-TMA
84°C)
Dose
30mg/Kg
100mgl~
300mg/~
Onset of
Seizure (s)
64.0:!: 4.2
93.33:!: 13.7
70.0:!: 1.0
Episode
of Seizure
4
3
2
Time of Death
(s)
115.0 + 17.0
146.0:!: 14.6
114:!:1.0
Number
Survived
0/4
1/4
0/4
30mg/~
100mg'Kg
300mg/~
59.75:!: 3.2
178.5:t7.4
Lethal dose
4
2
210.5:!:13.3
197.25:!: 7.1
0/4
1/4
0/4
30mg/Kg
100mgl~
300mg/~
60.5:!: 7.1
68.75 ± 14.9
Lethal dose
5
6
163.0:!: 6.5
170.0 ± 12.2
0/4
0/4
0/4
10mg/~
30mg/Kg
100mg/Kg
95.25 ± 7.6
93.25 ± 4.9
89.7 + 15.4
4
4
2
218.3± 15.7
260.75 ± 12.7
184.5± 9.1
0/4
0/4
0/4
300mg/Kg
90mg/Kg
10mg/Kg
59.25+4.5
64.0 ± 7.0
33.75:!: 5.9
5
7
4
219.25 +8.9
222.5 ± 56.6
646.25:!: 50.3
0/4
"0/4
0/4
(mp
~-/i
4-NHz-TMA
(TMA)bis-4-amino-benzamide(mp 133"C)
..
(-)C {Negative Control (ipPTZ only)}
(+)C {positive Control (Carbamazepine + ipPTZ)}
Onset of Seizure(s)
200.00 180.00
160.00
140.00
120.00
en
li'
E 100.00
i=
80.00
60.00
,'"
"~
-.
40.00
20.00
"*
0.00
Cl
Cl
~
~
E
8
C')
4-OH-TMA
E
8
••••
4-CN-TMA
Cl
~
E
8
C')
i088
Cl
Cl
~
E
~
C')
4-C1-TMA
~
Cl
1!b
~
0
4-NH2-TMA
I
0
C')
(-)C : (+)C
Figure 1: Onset of Seizure(s) of TMA bis (4 -substituted -benzamides )
11
Afo/abi E. a. i Proceeding of the Neuroscience Soc. of Nig. (NSN) Conference, Jos, 2006
Episode of Seizure
8
7
~
5 ~
Cl)
.0
E 4
:l
Z
3
~
2
I
~Ir . --'1 11
1
,.
Cl
Cl
~
~
E
lij
F
Cl
~
tII
.5
•..
.5
E
lij
..,
0
0
4-OH-TMA
.5
E
Cl
F
~
~
tII
~
..,
0
E
lij
..,
S
•..
Cl
0
.5
E
S
•..
4-CN-TMA
•..E
0
4-CI-TMA
~Cl
~
0
~
tII
..,.5
lij
4-NH2-TMA
(-)C
(+)C
Figure 2: Episode of Seizure of TMA bis (4 -Substituted -benzamides )
TIme of Death (s)
700.00
600.00
500.00
'iiI 400.00
Q)
E
i= 300.00
200.00
I
~
100.00
0.00
I
I~~~:~,"~~
i ~.~
.5
.5e
•.. ..,
o
..,
0
4-0H-TMA
SE
.'
~>'
I
Cl
Cl
Cl
Cl
~
~
~
~ i ~
S
..,
lij
E:
E
0,
0
E
..,
JI
S
•..
4-CN-TMA
E
E
~!lij
4-CI-TMA
Cl
F
i .5
~~
.5
Cl
<:)
..,
<:)
•••.
4-NH2-TMA
Cl
<:)
M
(-)C
Figure 3: Time of Death (s) with TMA bis (4 -Substituted -benzamides)