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Synthesis and Pharmacological Activity of Two Derivatives of the Amide of Valproic Acid.

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Full Papers
Synthesis and Pharmacological Activity of Two Derivatives of the
Amide of Valproic Acid
Elly Bechara) and Henri Astrougb) *
a) Department of
b,
PharmaceuticalChemistry, Faculty of Pharmacy, 2 Dunav Street, Sofia, 1O00, Bulgaria
Department of Pharmacology and Toxicology, Faculty of Pharmacy, 2 Dunav Street, Sofia, IOOO,Bulgaria
Key Words: Valpramide derivatives; acute toxicity; antiseizure activity; hexobarbital sleeping time;
spontaneous locomotor activity
Summary
Results and Discussion
Synthesis
Valproic acid (VPA), a synthetic branched-chain fatty acid, and its
pro-drug the primary amide (VPD) are effective and widely used
anti-epileptic agents. Although the use of VPA has grown in recent
years, major side effects are still associated with this drug. We
presume that it is possible, without loosing the VPD pharmacological profile, to obtain new compounds by undertaking substitutions
in the CONH group. N,N'-bis-(2-propylpentanoyl)- 1,2-ethanediamine (3) and N,N-bis-(2-propylpentanoyl)-1,3-propanediamine (4) were obtained from VPA (1) using a method reported
in the literature. The chemical structures of the new compounds
were demonstrated by elemental analysis, IR, and 'HNMR spectroscopy. Both compounds are less toxic and more effective in
protecting the animals from death caused by PTZ than VPD after
intraperitoneal administration to mice.
Introduction
Valproic acid (VPA), a synthetic branched-chain fatty acid,
and its pro-drug the primary amide VPD) are effective and
widely used antiepileptic agents 11, I. Although the use of
VPA has grown in the recent years, major side effects are still
associated with this drug. Many patients suffer from medication toxicity under VPA and its existing analogues [31. There
are many reports of the synthesis and antiseizure activity of
series of VPD analogues but also of their teratogenicity and
hepatotoxicity
Some of the compounds exhibit a general
anticonvulsive activity similar to that of VPA. However,
modifications are made in the aliphatic part of VPD while the
amide group is left unchanged [71. We presume that it is
possible to obtain new compounds by making substitutions
in the CONH group without losing the VPD pharmacological
profile. By synthesizing these compounds it should become
feasible to introduce two linked VPD fragments into the
organism and possibly to delay their hepatic metabolism
compared to VPD.
We report here the synthesis of two VPD derivatives and
describe their acute toxicity after oral and intraperitoneal
administration in mice. The pharmacological activity on the
central nervous system (spontaneous locomotor activity and
effects on hexobarbital induced sleep) after intraperitoneal
administration in mice was assayed. The effects of the compounds on seizures provoked by subcutaneous pentylenetetrazole (PTZ) application were also investigated.
1
Arch. Phann. Phann. Med. Chem.
N,N'-bis-(2-propylpentanoyl)- 1,3-propanediamine (3) and
N,N'-bis-(2-propylpentanoyl)-1,3-propanediamine (4) were
obtained from VPA (1) using a method reported in the literature [8] (Scheme l).
Valproyl chloride (2) is obtained from valproic acid (1)
using thionyl chloride. The compounds are obtained by the
interaction of valproyl chloride (2) and diamine in a molar
ratio of 2: 1 in anhydrous dioxane in the presence of triethylamine used as an acceptor for the HCl at 0 "C and with
continuous stirring. Compounds 3 and 4 are recrystallized
from anhydrous dioxane. Their structure is proven by JR and
'H NMR spectral analysis, as well as by elemental analysis.
The compounds N,N'-bis-(2-propylpentanoyl)-l,2-ethanediamine (3) and N,N-bis-(2-propylpentanoyl)-l,3-propanediamine (4) were synthesized using the method described in
a Spanish patent (Scheme 1).
Pharmacology
The newly synthesized compounds were assayed for acute
toxicity, influence on locomotor activity, hexobarbital sleeping time, and anticonvulsive effects (PTZ-provoked seizures).
H7C3,
CHCOOH
H7Ci
,
i
H7C3,
0
CH-C-CI
H d
2
HzN(CHzhNH2
HzN(CHzbNH2
1,4-Doxane, N(CZHsh, ?= 0" c
0 WILEY-VCH Verlag GmbH, D-6945 1 Weinheim, 1997
0365-6233197109 1010273 $17.50 +.50/0
274
Bechar and Astroug
Table 1. Acute toxicity (LDso) of the compounds 3,4, and VPD in mice after
oral and intraperitoneal administration (n indicates the number of animals).
Table 2. Influence of compounds 3 and 4 on hexobarbital induced sleeping
time in mice (n indicates the number of animals per group).
Compd
Compound
n
Route of
administration
LDso
Range of values
(mmolkg) (mmolkg)
3
12
12
p.0.
i.p.
>16.00*
>16.00 *
4
12
12
p.0.
i.p.
>15.31*
25
p.0.
5.39
4.965.82
25
i.p.
2.72
2.5C2.97
Control
(VPD)
*p
>15.31*
n
Doses
( I n 0 LD,,)
mmoVkg i.p.
Sleeping time
min
(Xf SD)
Control
Hexobarbital Na
10
0.30
17.08 f 3.07
3
prior to
Hexobarbital Na
10
1.60
32.83 f 1.72*
4
10
prior to
Hexobarbital Na
0.30
I .53
23.16f 3.18*
0.30
I0.05 statistically significant compared to the corresponding route of
administration for the control (VPD).
* p 50.05 statistical significance in comparison to the control group.
Analysis of the obtained experimental data on the acute
toxicity (LD5o) of the compounds showed that both of them
are significantly (p < 0.05) less toxic (3 times) than the
reference drug VPD after oral as well as after intraperitoneal
administration to mice (see Table 1).
Analysis of the data from the experiments on the influence
on spontaneous locomotor activity in mice shows that there
exits a marked difference between the effects of the compounds after the different routs of administration.Compound
3 causes a statistically significant increase of the locomotor
activity only for the first 30 and 50 min after oral and
intraperitoneal administration respectively. After 60 min,
compound 3 orally administered does not affect the locomotor activity. Administered intraperitoneally, this compound
tends to decrease locomotion.The route of application modifies the effects of compound 4 in a more important way -after
intraperitoneal administration locomotion is reduced significantly with about 100% for the whole period of the experi-
ment, while after oral administration the locomotor activity
is increased nearly 4 times between 40 and 70 min (Fig. 1).
This could be due to a massive production of an active
metabolite following oral administration.
The effects of the compounds in doses 1/10 of LD5o on
hexobarbital induced sleeping time are presented in Table 2.
Both compoundsprolong the sleeping time induced by hexobarbital with 92.2% and 35.6% for compound 3 and compound 4 respectively after intraperitoneal administration.
These results correlate with observed reduced locomotion 30
to 40 min after the administration.
The effects of the compounds on seizures provoked by
pentylenetetrazole (PTZ) are shown in Table 3. The dose of
PTZ (0.65 mmolkg s.c.) causes death of all the animals. We
administered the compound 3 and 4 (dose 1/10 LD5o i.p.) at
different time intervals before PTZ. Compound 3 prolongs
the latency period both to the first (I) and to the last (VI)
degree of seizure. This effect is most prominent when the
Table 3. Antiseizure effect of compounds 3 and 4 . Influence of the period of time before the administration of FTZ and
the dose. (n indicates the number of animals)
Compd
Dose
mmoVkg
n
0.65
S.C.
1.60
i.p.
Time
prior
toPTZ
rnin
Latency to the
I degree of
seizure
min k SD
Latency to the
VI degree of
seizure
rnin k SD
Mortality
20
-
3.54 f 0.44
16.28 f0.47
100
10
10
10
10
10
30
45
90
120
45
6.17 f 1.58*
11.07 ? 2.57***
7.16f 1.51 **
4.95 ? 1.02*
4.27 f 0.73*
16.33 & 18.23'
26.79 f 8.46 *
18.27 f 8.45
19.50 k 4.33*
20
0
50
70
40
10
10
30
60
90
120
90
3.02k 1.15
5.67 f 0.9**
6.93 f 1.8**
3.93 f 0.50
4.90 k 0.65**
14.33 f 6.36
20.11 f 8.37
21.83##
20.32 f 2.86
20.75 f 4.54
80
60
10
80
60
%
~~
PTZ
(control)
3
prior to
PTZ
4
prior to
FTZ
0.80
i.p.
1.53
i.p
0.77
i.p.
10
10
10
* p 2 0.05; ** p I O . 0 1 ; *** p 2 0.001 statistical significance in comparison to the control group.
'
- only
two animals out of 10 reached this degree of seizure. ##-only one animal out of 10 reached this degree of seizure.
Arch. P h a m P h a m Med. Chem. 330,273-276 (1997)
275
Valproic Acid Amide Derivatives
Experimental
A
A
-0- Compound 3 (I p )
- 0- Cornpound 3 (p o )
Compound 4 (I p )
A Compound 4 (p 0 )
A
A
Chemistry
A
v
C
._
>
c
'5
.-
-
*
P
b
c
E
A
A
I
a\
100-
A
0
,o--0---n
.--.,o-o-o'
0-
0
8
-4
-I
20
0
40
60
80
100
120
Time [rnin]
Figure 1. Effects of compounds 3 and 4 administered p.0. and i.p. in doses
1/10 of LD5o on locomotor activity of mice recorded on 10 min intervals up
to the 120-th minute. Asterisks indicate statistically significant differences
(p s 0.05) from the corresponding control groups (n = 5 animals for each
group).
compound is applied 45 min before PTZ - no animal reaches
the VI degree and none of them dies. Compound 4 prolongs
the latency period corresponding both to the first (I) and to
the last (VI) degree of seizure, the effect being most prominent when the compound is applied 90 min before PTZ - only
one of the animals reached degree VI and died.
In order to establish the threshold for the antiseizureactivity
we administered the compounds at the time of their respective
maximum effect in doses 1/20 L D 5 0 i. p. The lower doses
exert significantlyweaker effects, both on the latencies to the
I and VI degrees of the scale and on the mortality rate.
The data show aslightly more favorable index ( L D 5 d E D 5 0 )
for the effect "protection from death caused by PTZ' for
compound 3 compared to compound 4, but both compounds
prove to be more effective than VPD (see
and 4).
The results of our work indicate that these comuounds aDDear
to be promising for further detailed pharmacological and
t o x ~ c o ~ o ginvestigations
~ca~
and
clinical application.
New modified derivatives
prove interesting in respect
of reduced toxicity and better antiseizure effects.
I .
Melting points were measured in "C in capillary tubes (Buchi 535 apparatus) and not corrected. Spectra were recorded using the following instruments: IR spectra (cm-'). - Shimadzu FTIR 8101 M; 'H NMR spectra (6
values in ppm relative to internal TMS) - Bruker WP 100 (100 MHz).
Elemental analysis (for C, H, N) for these compounds were within k 0.4%
of the theoretical values unless otherwise indicated.
The compounds N,N'-bis-(2-propylpentanoyl)-l,2-ethanediamineand
N,"-bis-(2-propylpentanoyl)-1,3-propanediamine are synthesized according to a method reported in the literature (Scheme 1) from valproyl chloride
and diamine (2/8 molar ratio) in dioxane in the presence of triethylamine as
an acceptor of HCI at 0 "C and continuous stirring. The structure is proven
by IR and 'H NMR spectroscopy as well as by elemental analysis.
Valproyl chloride ( 2 )
Valproic acid (1) 144.20 g (1 mol) was added dropwise to thionyl chloride
142.76 g (1.2 mol). The mixture was stirred continuously and the temperature was maintained at 10 "C. After that the temperature is raised to 80 "C
and is kept at that level until the SO2 and the HCI are completely evaporated.
The remaining mixture is distilled under low pressure using a water vacuum
pump. The fraction distilled between 176 and 180 "C is collected to yield
146 g (90%).
N,K-bis-(2-propylpentanoyl)1.2-ethanediamine (3)
1.2 (20 mol) of 1,2-diaminoethane
4.04 (40 mol) of triethylamine were mixed in 200 ml of anhydrous dioxane. While stirring continuously (mechanical stirrer) at a temperature of 0 "C (f2 "C), 6.48 g (40 mmol)
of freshly distilled valproyl chloride mixed with 50 ml anhydrous dioxane
was added slowly (dropwise). After that the temperature had risen to 80 "C
and triethylamine was filtered while hot. The filtrate was concentrated to
30 ml and then cooled down. The separated crystals were filtered and
re-crystallized from acetone to yield 5.9i g- (95%) of 3 mp 207-208 "C. IR
(fluorolube, nujol): v = 3100-2910 cm-' (CHLCH~)with max. at 2920,
2950, 1635.8 (c=o, amide I). 3289 (NH secondary amid@, v = 1541 cm-'
(NH, amide II), 1452.6 (CCH3); 'H NMR (RCCOOH): 6 (ppm) = 0.95 (t,
12H, CH3), 1.28-1.74 (m,m, 16H, CHrCH2CH3). 2.66 (q,2H, CH), 3.79 (t,
4H, N-CH~CHZN).
Anal. (CisH36N202).
Table 4. Mean effective doses (ED5o) of compounds 3 and 4 - protection against PTZcaused animal mortality
(n indicates the number of animals).
Compd
3
n
Administered
doses
mmolkg i.p.
Mean effective
dose (ED50)
mmol/kg
Confidence
limits
(p 5 0.05)
mmoykg
Index
LDsdEDso
10
1.60
1.20
0.80
0.48
0.95
0.73 f 1.23
>16.84
1.53
1.22
0.92
0.46
0.85
0.63 f 1.16
>18.00
10
10
10
4
10
10
10
10
VPD'21
Arch P h a m P h a m Med. Chem. 330,273-276(1997)
0.34
11.52
276
N,K-bis-(2-propylpenta~yl)-1,3-propanediamine
(4)
Compound 4 was synthesized using the procedure reported for compound
3 the only difference being the use of 1.48 g (20 mmol) 1.3-diaminopropane
in stead of 1.2 g (20 mmol) 1,2-diaminoethane. The separated crystals were
filtered and recrystallized from acetone to yield 6.31 g (96.8%) of 4 mp
213-214 "C. IR (fluorolube, nujol): n = 3100-2910 cm-' (CHz.CH3) with
max. at 2932,2955, 1637.8 (C=O, amide I), 3285.2 (NH secondary amide),
= 1558.7cm-' (NH, amide 11). 1454 (CCH3); 'H NMR (F3CCOOH):d (ppm)
=0.95 (t. 12H, CH3). 1.24-1.78 (m,m, 16H, CHZCHZCH~),
2.1 (q,2H, CH),
3.67 (t. 4H, NCHzCHzN), 2 . 7 4 (4. 2H. NCHzCHzCHzN). Anal.
(CwH38NzOz) .
Bechar and Astroug
Antiseizure activity. Seizures were induced by subcutaneous administration of pentylenetetrazole (PTZ)(dose 0.65 mmol/kg). Compound 3 was
applied intraperitoneally 30.45.90, and 120 min before PTZ. Compound 4
was applied intraperitoneally 30,60,90, and 120 min before PTZ. The onset
of the seizures, seizure intensity, and latency to the I and VI degree of seizure
was estimated by a 6-score scale proposed by Roussinov ( 1983f9'. The index
LD5dEDso wa5 estimated after calculating EDso by the Litchfield-Wilcoxon(Io)method.
Statistics. The results underwent statistical processing by Student-Fischer
t-test at p 20.05, p 20.01, and p S 0.001.
References
Pharmacology
Materials and Meihods
The experiments were performed on 348 male white mice weighing
between 18 and 22 g. For all of the experiments the compounds were
solubilized in saline (0.9% NaCI) by adding 1-2 drops of Tween 80. For the
evaluation of the acute toxicity two ascending doses (12.8 and 16 mmoVkg
b.w. and 12.2 and 15.3 mmovkg b.w. for compounds 3 and 4 respectively)
were administered to two 4 groups of 6 animals without reaching LDso.
Infruence on locomotor activity: Every group containing 5 mice was put
into an actometer (Activity Cage, Ugo Basile, Italy) immediately after the
intraperitoneal administration of the compounds. The locomotor activity was
measured in arbitrary units at 10-min intervals for 120 min starting from the
20th minute. The results are presented as % of the controls treated intraperitoneally with saline.
Influence on hexobarbital sleeping time: The compounds were administered intraperitoneally to male mice (dose 1/10 of LDso). The same volume
of saline (0.1 ml per 10 g b.w.) was administered to the controls. Hexobarbital sodium dissolved in saline was administered (dose 0.30 mmoVkg b.w.)
30 min after the compounds. Sleeping time was measured in minutes by
observing the righting reflex recovery.
[l] V. Rogiers, A. Callaerts, A. Vercmisse, M. Arkrawi, E. Shephard, I.
Phillips, Phrm. Weekbl. Sci. Ed. 1992,14(3A), 127-131.
[2] G . Carraz, M. Darbon, S. Lebreton, H. Beriel, Therapie, 1964, 19,
468475.
[3] A. Haj-Yehia, S. Hadad, M. Bialer, Pharm. Res. 1992.9, 10.58-1063.
[4] A. Haj-Yehia, M. Bialer, P h r m . Res. 1989, 6,682-689.
[S]
M. Bialer, A. Haj-Yehia, K.Badir, S. Hadad, Pharm. World Sci. 1994,
16,2-6.
[6] H. Nau, H. Siemes, Pharm. Weekbl. Sci. Ed. 1992,14(3A), 101-107.
[7] G. Tailandier, G. Benoit-Guyod, A. Boucherle, M. Broll, P. Eymard,
Eur. J. Med. Chem. 1975,10,453462.
[8] Span. pat. 386303.16.03.1973, C.A., 80, 145733~.
[9] K. Roussinov, Bull. Inst. Physiol. Bulg. Acad. Sci. 1972,16, 315-322.
[lo] J. T. Litchfield, F. Wilcoxon, J. Pharmacol. Exp. Ther. 1949, 96,
99- 102.
Received April 16, 1997 [ F E W ]
Arch. P h a m P h r m Med Chem 330,273-276 (1997)
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acid, two, synthesis, amid, activity, valproic, pharmacological, derivatives
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