Synthesis, characterisation, and biological activity of three new amide prodrugs of lamotrigine with reduced hepatotoxicity

Article abstract of DOI:10.2478/s11696-010-0094-7

Lamotrigine (LTG) is an antiepileptic drug used for the prevention of convulsions. Except several known side effects, hepatic dysfunction is also reported. Hepatotoxic side effects occur due to the dichlorophenyl moiety which develops an abnormally low level of glutathione. Depletion of glutathione causes oxidative stress and hepatic cell damage. The goal of the present study was to test the action and side effects of the three compounds synthesised and compared to LTG. Three amide prodrugs of LTG were synthesised by its reaction with N-acetylamino acids, viz, glycine, glutamic acid, and methionine. Purified synthesised prodrugs were subjected to thin layer chromatography, melting point, solubility and partition coefficients determination and characterised by UV, FTIR, ¹H and ¹³C NMR spectroscopy. The synthesised prodrugs were subjected to in vitro hydrolysis and to anticonvulsant and hepatotoxic activity studies. Significant reduction in hepatotoxicity and comparable anticonvulsant activities were obtained in all synthesised prodrugs as compared to LTG.

Full text of DOI:10.2478/s11696-010-0094-7

Chemical Papers 65 (1) 70–76 (2011)
DOI: 10.2478/s11696-010-0094-7
ORIGINAL PAPER
Synthesis, characterisation, and biological activity of three new
amide prodrugs of lamotrigine with reduced hepatotoxicity
Saurabh K. Sinha, Prabhat K. Shrivastava, Sushant K. Shrivastava*
Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi, 221005, India
Received 21 May 2010; Revised 27 September 2010; Accepted 18 October 2010
Lamotrigine (LTG) is an antiepileptic drug used for the prevention of convulsions. Except several
known side effects, hepatic dysfunction is also reported. Hepatotoxic side effects occur due to the
dichlorophenyl moiety which develops an abnormally low level of glutathione. Depletion of glu-
tathione causes oxidative stress and hepatic cell damage. The goal of the present study was to test
the action and side effects of the three compounds synthesised and compared to LTG. Three amide
prodrugs of LTG were synthesised by its reaction with N-acetylamino acids, viz, glycine, glutamic
acid, and methionine. Purified synthesised prodrugs were subjected to thin layer chromatography,
melting point, solubility and partition coefficients determination and characterised by UV, FTIR,
1H and 13C NMR spectroscopy. The synthesised prodrugs were subjected to in vitro hydrolysis and
to anticonvulsant and hepatotoxic activity studies. Significant reduction in hepatotoxicity and com-
parable anticonvulsant activities were obtained in all synthesised prodrugs as compared to LTG.
c
ꢀ 2011 Institute of Chemistry, Slovak Academy of Sciences
Keywords: prodrug, glutathione, LTG, hepatotoxicity, in vitro hydrolysis, anticonvulsant activity
Introduction
increase in the level of alkaline phosphatase (ALP), as-
partate aminotransferase (AST), alanine aminotrans-
ferase (ALT), bilirubin, and direct bilirubin at the
therapeutic dose of LTG (Serwetman et al., 2008;
Overstreet et al., 2002; Moeller et al., 2008; Makin
et al., 1995; Sauvé et al., 2000). The mechanism re-
sponsible for LTG-induced hepatotoxicity has been
attributed to the accumulation of arene oxides, gen-
erated by the dichlorophenyl moiety of LTG, which
are rearranged to dichlorophenols (Santos et al., 2008;
Maggs et al., 2000; Nedelcheva et al., 1998). These
metabolites generate reactive oxygen species which
cause oxidative stress and cell damage (Siritantikorn
et al., 2007). Arene oxide and dichlorophenol metabo-
lites are deactivated primarily by glutathione (GSH)
conjugation (Maggs et al., 2000). The glutathione
tripeptide macromolecule is produced by its precur-
sor amino acids methionine, glutamic acid, cysteine,
and glycine. It exists in its reduced form (GSH), it
is enzymatically or non-enzymatically oxidised to a
disulfide dimer (GSSG) and used to detoxify hydro-
LTG, 6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5-di-
amine (C9H7Cl2N5; alternative names: lamotrigine,
lamictal), is a newly developed antiepileptic drug de-
rived from pyrimethamine. It is effective in treat-
ing both partial and generalised seizure. LTG most
probably exerts its antiepileptic activity by blocking
voltage-sensitive sodium channels in order to inhibit
the release of excitatory amino acids, principally glu-
tamate and aspartate in the central nervous system
(Shorvon & Stefan, 1997). However, hepatotoxicity,
which can cause liver failure, is the major disadvan-
tage associated with the use of this drug. Patients re-
ceiving chronic treatment with LTG in the form of
single or polytherapy are at high risk of hepatic en-
zymes elevation without clinical signs or symptoms
of hepatic dysfunctions or even fatal hepatotoxicity
(May et al., 1996; Fayad et al., 2000; Meshkibaf et
al., 2006). Several studies with different doses of LTG
were done and it was found that there is a significant
*Corresponding author, e-mail: skshrivastava.phe@itbhu.ac.in

S. K. Sinha et al./Chemical Papers 65 (1) 70–76 (2011)
71
NH2
Cl
NH2
N
O
C
N
N
H
H
N
H
C
ii
CH3CONH
COOH
COOH
H2N
CH3CONH
C
+
N
N
N
R
R
Cl
Cl
Cl
lamotrigine
Compound
LTG-Gl
R
H
i
H
C
H2N
CH2CH2COOH
CH2CH2SCH3
LTG-Ga
LTG-Mt
R
Fig. 1. Synthesis of LTG-acetylamino acid conjugates. Reaction conditions: i) AcOH, Ac2O, heat; ii) HOBt, DCC, DMF, r.t.;
amino acids used: glycine (Gl), glutamic acid (Ga), methionine (Mt).
gen peroxide and lipid hydroperoxides. Therefore, the
elevation of GSSG in tissues has been used as a marker
for oxidative stress (Younis et al., 2000).
cant differences between the control and experimental
groups. Data are expressed as mean SD.
This current research work is designed to minimise
the hepatotoxicity of LTG to a great extend by its con-
jugation with amino acids inducing the biosynthesis
of glutathione inducing amino acids. The synthesised
LTG-amino acid conjugates showed similar activity as
their parent drug molecule with significantly reduced
hepatic cell damage.
Preparation of N-acetylamino acids and LTG-
amino acid conjugates
Accurately weighed amino acid (10 mM) was sus-
pended in glacial acetic acid (4 mL) followed by the
addition of acetic anhydride (2 mL). The mixture was
gently heated until the whole amount of amino acid
dissolved. The reaction progress was monitored by
TLC using ethyl acetate/methanol/ammonia (ϕr =
17 : 2 : 1) as the mobile phase. On cooling, crystals of
acetylated amino acid separated at once. The product
was separated, washed with a little water, and dried
(Fig. 1) (Dakin, 1929).
Accurately weighed HOBt· H2O (234 mg, 1.53
mmol) and DCC (247 mg, 1.2 mmol) were added to
a solution of LTG (256 mg, 1 mmol) in anhydrous
DMF (2 mL) at 0◦C. The reaction mixture was con-
tinuously stirred for 30 min. N-Acetylamino acid (1.2
mmol) was then added at once, and the reaction mix-
ture was stirred for additional 24 h (reaction progress
was monitored by TLC as given above) and filtered.
The solvent was then removed under reduced pressure.
The solid residue was dissolved in water and filtered;
the filtrate was lyophilised. (Fig. 1) (Nam et al., 2003).
Experimental
LTG standard drug was obtained as a gift sam-
ple from Lupin Ltd (Pune, India). Amino acids
(glycine (Gl), glutamic acid (Ga), methionine (Mt)),
1-hydroxybenzotriazole (HOBt), and dicyclohexylcar-
bodiimide
(DCC) were purchased from Sigma–Aldrich Chemical
Ltd, USA. Methanol, ethyl acetate, and dimethyl for-
mamide were purchased from CDH Laboratory, India.
Melting points were determined using a BI 9300 Bum-
stead/electrothermal Stuart (SMPIO) melting point
apparatus. Thin layer chromatography was performed
on TLC silica gel 60 F254 aluminium sheets (Merck,
India). UV spectra were obtained using a JASCO
(Model 7800) UV-VIS spectrophotometer. FTIR spec-
tra were recorded on a Shimadzu FTIR 8400S spec-
trophotometer at the scanning range of 400–4000
In vitro hydrolysis
1
cm−1. H and 13C NMR spectra were recorded on a
JEOL AL 300 FT-NMR spectrophotometer in DMSO-
d6. In vitro hydrolysis studies of LTG-amino acid con-
jugates were performed using an HPLC system (Cecil
liquid chromatography, Cecil Instruments Ltd, India)
consisting of an Adept CE-4100-dual piston pump and
Phenomenex Luna C18(2) (250 mm × 4.60 mm, 5
µm). A mixture of buffer (KH2PO4, adjusted to pH
= 7 with triethylamine), acetonitrile, and methanol
(ϕr = 6 : 2 : 2) was used as mobile phase for the in
vitro hydrolysis study. Flow rate was kept at 1.5 mL
min−1 and the wavelength was adjusted to 307 nm
(Barbosa & Mídio, 2000).
To study the fate of amino acid and drug conju-
gates in a biological system, in vitro hydrolysis was
carried out in different aqueous buffer systems (pH
1.2, 7.4, and 9.0) simulating biological fluids at (37
1)◦C. The rate of hydrolysis and half life of the syn-
thesised conjugates were calculated according to Eqs.
(1) and (2), respectively (where K = hydrolysis rate
constant, t = time in h, a = initial concentration of
the conjugate, x = amount of the conjugate hydrol-
ysed into free parent drug, a − x = amount of parent
drug remaining in the conjugate form, t1/2 = half-life
of the conjugate).
The Student’s paired t-test (two tailed) using the
GraphPad Prism version 5.0 for Windows (Graph-
Pad Software, Inc., 2009) was used to analyse signifi-
2.303
t
a
k =
×
(1)
a − x

72
S. K. Sinha et al./Chemical Papers 65 (1) 70–76 (2011)
Table 1. Characteristics of LTG-amino acid conjugates
Compound
Formula
Mr
M.p./◦C
Yield/%
Rf
Rm
log P
λmax/nm
LTG-Gl
LTG-Ga
LTG-Mt
C13H12Cl2N6O2
C17H18Cl2N6O4
C17H22Cl2N6O2S
355.18
441.27
445.37
100–102
110–112
106–108
55
52
56
0.45
0.52
0.56
0.087
−0.034
−0.104
1.10
1.60
1.93
265.0
270.0
281.2
ꢀ
ꢁ
0.693
k
After applying shock, the rats were observed for the
type of convulsion induced and the hind limb exten-
sor response was taken as the end point. The animals
showing positive hind limb extensor response were
used for testing drug substance. After 24 h, the rats
were administered the drug and its conjugates suspen-
sions in 1 % gum acacia perorally. Maximal seizure
was induced after 1 h and 4 h of drug administration.
Abolition of the hind limb tonic extensor spasm was
recorded as the measure of anticonvulsant activity of
the drug (Tanaka & Kawasaki, 1957).
t1/2
=
(2)
LTG-amino acid conjugate (25 mg) was added to
the hydrochloric acid buffer USP (50 mL, pH 1.2) and
the solution was stirred maintaining the temperature
at (37 1)◦C. The samples were withdrawn from the
buffer solution after 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h,
5 h, 6 h, and 7 h, respectively, and they were replaced
by an equivalent volume of fresh buffer solution (this
replacement was added as the correction factor). The
samples were analysed using HPLC associated with a
UV detector at λmax 307 nm. The drug release was
determined at different time intervals from the stan-
dard calibration curve. Similar procedure was carried
out in pH 7.4 and 9.0 buffer solutions. The synthesised
drug-amino acid conjugate was found to be soluble in
water and MeOH.
Hepatotoxicity study
Blood samples were collected after four weeks of
the experimental period by heart puncture within 6
h to 8 h of the last dose of the drug and its conju-
gate from the test groups. Liver function tests were
carried out immediately after the separation of serum
using Span diagnostic reagent kits for ALP, ALT,
AST, bilirubin, and direct bilirubin estimation (Over-
street et al., 2002; Meshkibaf et al., 2006). Animals
were sacrificed and the liver sections were subjected to
histopathological observation at the Institute of Med-
ical Sciences, BHU, Varanasi. Coloured microscopical
images of the liver sections were taken on a Nikon
ECLIPSE 50i optical microscope (Nikon Corporation,
Japan) with the resolution of 400X attached to a cam-
era.
Biological evaluation and anticonvulsant
activity
Wistar rats of either sex (weighing 120–150 g)
were obtained from Central Animal House, Institute of
Medical Sciences, Banaras Hindu University, Varanasi
(Registration No. 542/02/ab/CPCSEA). They had ad
libitum access to water and semi-synthetic balanced
diet, with occasional supply of green vegetables (salad
leaves). Rats were caged five per a Perspex experi-
mental cage at room temperature (22–25◦C). Twelve
hours of light and dark cycles were strictly followed in
a fully ventilated room. Animals were divided into five
groups: control-I and experimental II, III, IV, and V.
Group-I received 1 % gum acacia suspension whereas
Results and discussion
Prior to the conjugation reaction, the free amino
group of amino acids was protected by acetylation
to give the corresponding N-acetylamino acid. LTG-
acetylamino acid conjugates were obtained by the
raction of LTG and N-acetylamino acid using 1-
hydroxybenzotriazole (HOBt) and N,N-dicyclohexyl-
carbodimide (DCC) as activators to form an amide
linkage between the free amino group of LTG and
the free carboxylic acid group of N-acetylamino acid
(Fig. 1).
Melting points, yields, and solubility (log P) of the
synthesised conjugates in methanol are given in Ta-
ble 1. Some other characterisation data, like Rf, Rm
(chromatography), and λmax (UV-VIS) are also given.
FTIR spectra of LTG-Gl, LTG-Ga, and LTG-Mt
conjugates exhibited strong bands at 1640–1660 cm−1
and 3260–3320 cm−1 corresponding to stretching vi-
group-II received LTG drug suspension (5 mg kg−1
,
p.o.), group-III the LTG-Gl conjugate, group-IV the
LTG-Ga conjugate, and group-V the LTG-Mt conju-
gate (5 mg kg−1, p.o. drug equivalent) (Meshkibaf et
al., 2006).
Anticonvulsant activity of the conjugates was stud-
ied on laboratory animals by electrically induced
seizure followed by the determination of acute toxi-
city. This test has predictive value concerning drugs
in respect to their potential therapeutic effect in the
management of grand mal epilepsy.
Maximal electroshock seizure (MES) test
Maximal seizure was induced by application of
electrical current across the brain through ear elec-
trodes. The stimulus parameters were: 50 mA, 0.2 s.
—
brations of C O and N—H bonds, respectively, and
—

S. K. Sinha et al./Chemical Papers 65 (1) 70–76 (2011)
Table 2. Spectral data of LTG-amino acid conjugates
73
Compound
LTG-Gl
Spectral data
IR, ν˜/cm−1: 3352 (N—H), 3093 (C—H), 1643 (C O), 1593 (C N)
—
—
—
—
1H NMR (DMSO-d6), δ: 7.96–7.93 (m, 2H, NH2), 7.70–7.50 (m, 3H, Haryl), 6.80–6.60 (d, 2H, NH2), 3.43 (s, 2H,
CH2), 2.50 (s, 3H, CH3)
13
—
—
—
C NMR (DMSO-d6), δ: 170 (1C, C O of acetyl), 170, 169 (1C, C O of amide), 160, 155 (3C, Ctriazine), 136,
—
132, 131, 130, 128, 127 (6C, Caryl), 43 (1C, CH2), 24 (1C, CH3)
—
—
LTG-Ga
LTG-Mt
IR, ν˜/cm−1: 3327 (N—H), 2875 (C—H, CH2), 1658 (C O), 1579 (C N), 1458 (C C), 1384 (C—Hbending, CH2)
— —
— —
1H NMR (DMSO-d6), δ: 8.09–7.84 (m, 4H, NH), 7.81–7.69 (m, 3H, Haryl), 4.5 (s, 1H, CH), 2.55–2.45 (m, 4H, CH2),
1.84 (s, 3H, CH3CO)
13
—
—
—
—
C NMR (DMSO-d6), δ: 179 (1C, COOH), 172 (1C, C O of amide), 171 (1C, C O of acetyl), 170, 160, 155 (3C,
Ctriazine), 134, 132, 131, 130, 128, 127 (6C, Caryl), 51 (1C, CH), 30, 27 (2C, CH2), 23 (1C, CH3)
IR, ν˜/cm−1: 3263 (N—H), 3070 (C—H, CH2), 2924, 2843 (C—H, CH3), 1651 (C O), 1545 (C N), 1396 (C—
—
—
—
—
Hbending, CH2)
1H NMR (DMSO-d6), δ: 8.32–8.01 (m, 4H, NH), 7.96–7.30 (m, 3H, Haryl), 3.21 (s, 1H, CH), 3.0–2.5 (m, 4H, CH2),
2.03 (s, 3H, CH3S), 1.59 (s, 3H, CH3CO)
13
—
—
—
C NMR (DMSO-d6), δ: 172 (1C, C O of amide), 170, 170 (1C, C O of acetyl), 160, 155 (3C, Ctriazine), 134,
—
132, 131, 130, 128, 127 (6C, Caryl), 53 (1C, CH), 32, 30 (2C, CH2), 25 (1C, CH3 of acetyl), 16 (1C, CH3S)
16
14
12
10
8
30
25
20
15
10
5
6
4
2
0
0
0
20
40
60
80
0
2
4
6
8
Time/min
Time/h
Fig. 3. Release profile of LTG-amino acid conjugates in a phos-
phate buffer solution (pH 7.4): LTG-Gl ( ); LTG-Ga
(ꢀ); LTG-Mt (ꢁ).
Fig. 2. Release profile of LTG-amino acid conjugates in a phos-
phate buffer solution (pH 9): LTG-Gl (ꢀ); LTG-Ga ( );
LTG-Mt (ꢁ).
indicating the formation of amide linkage in all con-
jugates. Further strong bands at 1593 cm−1, 1458
cm−1, and 3263 cm−1 were attributed to the stretch-
Table 3. Anticonvulsant activity (MES screening) of LTG and
LTG-amino acid conjugates in rats
Rats protected/rats tested
—
—
ing vibrations of C N, C C (aromatic), and N—H
—
—
1
(amide) bonds, respectively. H NMR spectra of LTG
showed a characteristic peak at δ 4.0 corresponding
to the NH2 group. For conjugates, signals at δ 8.32–
7.84, characteristic of amide proton, were observed.
Other signals (protons of aromatic ring and amino
acid chain) were in accordance with the structure of
the respective conjugate (Table 2). 13C NMR spec-
tra of LTG and its conjugates showed a characteristic
peak in the range of δ 134–127 corresponding to the
dichlorophenyl ring. The characteristic δ values for
carbon atoms of the triazine ring (170–155) and for
the amide carbon (172–169) in all conjugates were in
conformity with their respective structures (Table 2).
The release rate of LTG from its conjugates was
studied by comparing the drug release profiles using
Compounda
Time/h
Protection/%
1
2
3
4
Control
LTG
LTG-Gl
LTG-Ga
LTG-Mt
0/5
5/5
5/5
5/5
5/5
0/5
5/5
5/5
5/5
5/5
0/5
5/5
5/5
4/5
4/5
0/5
5/5
4/5
4/5
4/5
0
100
95
90
90
a) 5 mg kg−1 was used.
in vitro hydrolysis. It was found that the rate of hy-
drolysis increased with the increase of pH. The re-
sults indicated that in vitro release of LTG from the

74
S. K. Sinha et al./Chemical Papers 65 (1) 70–76 (2011)
Table 4. Biochemical parameters of rat serum exposed to LTG and LTG-amino acid conjugates
Biochemical parameter
Compounda
ALP
ALT
AST
Bilirubin
mg dL−1
Direct bilirubin
mg dL−1
unit L−1
unit L−1
unit L−1
Control
LTG
LTG-Gl
LTG-Ga
LTG-Mt
342.20
56.76
78.40b
56.84
42.49
76.20
52.06
7.579
149.6
7.831
0.351
0.045
0.035b
0.052c
0.03c
0.132
0.330
0.158
0.166
0.139
0.015
500.20
384.20
389.20
368.58
101.95
61.06
63.12
60.66
6.093b
4.388c
7.572c
6.303c
292
192.2
187.6
163
7.831b
14.618c
4.878c
16.294c
0.44
0.36
0.38
0.36
0.026b
0.054c
0.068c
0.014c
0.032c
a) 5 mg kg−1 was used; b) P < 0.001 compared with control; c) P < 0.001 compared with standard (LTG).
a
c
e
b
d
Fig. 4. Histopathological examination of liver section in nor-
mal and experimental rats at the magnification of 400×.
Normal rat liver parenchyma in control group (a); rat
liver treated with LTG (b) (arrows show hepatocyte
necrosis); rat liver treated with: LTG-Gl conjugate (c),
LTG-Ga conjugate (d), and LTG-Mt conjugate (e).

S. K. Sinha et al./Chemical Papers 65 (1) 70–76 (2011)
75
fective than the other two amino acid conjugates due
to its transformation into cystine (du Vigneaud et al.,
1944). It is a well established fact that N-acetyl cystine
prevents liver damage by inhibiting apoptotic path-
way and oxidative stress (San-Miguel et al., 2006).
Histopathological examination of rat liver was also
performed in order to study pathological changes in
liver tissue. The histopathology report also confirmed
that LTG-amino acid conjugates have lower hepato-
toxicity than the parent drug, LTG (Fig. 4).
conjugate was catalysed by alkaline buffer solutions.
Under pH 1.2, negligible liberation of LTG from the
conjugate was found whereas at pH 7.4, the drug was
released in 34 h and at pH 9.0 within 45 min. The
release of LTG-Gl, LTG-Ga, and LTG-Mt conjugates
was determined at different pH values. The conjugates
showed 24.14 %, 23.34 %, and 26.05 % release at pH
9.0 for LTG-Gl, LTG-Ga, and LTG-Mt, respectively
(Fig. 2). The release percentage at pH 7.4 was found
to be 10.98 %, 13.49 %, and 12.17 % for LTG-Gl, LTG-
Ga, and LTG-Mt, respectively (Fig. 3). The half lives
of LTG-Gl, LTG-Ga, and LTG-Mt conjugates at pH
9.0 were 90.92 min, 95.77 min, and 88.48 min, respec-
tively, while at pH 7.4 they were 36.60 h, 31.60 h, and
34.42 h, respectively.
The LTG-amino acid conjugates were analysed for
anticonvulsant activity using the electroshock method
and they were found to be active as anticonvulsants
(Table 3). LTG-treated rats were protected from tonic
seizures (% protection = 100). All the three conjugates
were also able to prevent seizures in experimental rats
with a comparable activity as the parent drug, LTG.
Among the three conjugates, the glycine conjugate
(% protection = 95) showed better anticonvulasant
activity compared to glutamic acid (% protection =
90) and methionine conjugates (% protection = 90).
Better anticonvulsant profile of the glycine conjugate
is due to its antagonistic action of glycine itself on the
metabotropic glutamate receptor (Attwell et al., 1998)
(Table 3).
Conclusions
Anticonvulsant activity of the LTG-Mt conjugate
comparable to that of LTG and the significantly lower
damage to liver, shown by biochemical results, prove
this conjugate to be a most promising alternative for
the treatment of convulsions at clinical level.
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