Synthesis and evaluation of hepatoprotective activity of some new mannich bases bearing benztriazole moiety

Article abstract of DOI:10.4067/S0717-97072010000300021

A series of benztriazoles bearing Mannich bases (2 -10) were synthesized from benztriazole by aminomethylation with formaldehyde and various substituted secondary amines. Titled compounds were synthesized by Mannich reaction and they were characterized by IR and 'HNMR spectroscopy. All these Mannich bases were screened for hepatoprotective activity on carbon tetrachloride induced liver damage in rats. Only compounds 4 (250 mg/kg) protected significantly the animals from carbon tetrachloride induced hepatotoxicity. Compound 4 N-morpholinyl methyl benztriazole exhibited significant activity comparable to that of standard drug silymarin.

Full text of DOI:10.4067/S0717-97072010000300021

J. Chil. Chem. Soc., 55, Nº 3 (2010)
SyntheSiS and evaluation of hepatoprotective activity of Some new mannich baSeS
bearing benztriazole moiety
1AiyAlu RAjAsekARAn* And 2MuthusAMy PeRiyAsAMy , 1kMCh College of PhARMACy,
CoiMbAtoRe indiA., 2eRode College of PhARMACy, eRode, indiA
dr. Aiyalu Rajasekaran.M.Pharm, Ph.d., Professor and Principal, kMCh College of Pharmacy, kovai estate, kalapatti Road, Coimbatore – 641 048
(Received: December 29, 2009 - Accepted: July 22, 2010)
abStract
A series of benztriazoles bearing Mannich bases (2 -10) were synthesized from benztriazole by aminomethylation with formaldehyde and various substituted
1
secondary amines. Titled compounds were synthesized by Mannich reaction and they were characterized by IR and HNMR spectroscopy. All these Mannich
bases were screened for hepatoprotective activity on carbon tetrachloride induced liver damage in rats. Only compounds 4 (250 mg/kg) protected significantly the
animals from carbon tetrachloride induced hepatotoxicity. Compound 4 N-morpholinyl methyl benztriazole exhibited significant activity comparable to that of
standard drug silymarin.
Keywords: Mannich bases, benztriazoles, hepatoprotective, Serum Glutamate Oxaloacetate Transaminase, Serum Glutamate Pyruvate Transaminase,
Alkaline Phosphatase .
internal standard. Elemental analysis was performed on Heraceus Carlo Erba
introduction
1108 and the analysis indicated by the symbols of the elements was within ±
0.4% of theoretical values.
Liver is playing important role in metabolism, secretion and storage.
Any injury to liver can result in many disorders ranging from elevation of
liver enzymes to liver failure. Commonly, liver injuries are caused by toxic
chemicals like carbon tetrachloride (CCl4), aflatoxin and therapeutic agents
like antibiotic, anti-tubercular drugs as well as alcohol, microbial agents [1].
Administration of CCl4 is biotransformed by cytochrome P-450 system to
produce the trichloromethyl free radical which causes lipid peroxidation and
membrane damage. Also, CCl4 increases the serum level of marker enzymes,
serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate
transaminase (SGOT) and alkaline phosphatase (ALP) indicating the induction
of hepatotoxicity [2]. Silymarin has proved to possess multiple actions as a
hepatoprotective against various hepatotoxic substances in its preclinical
studies. The antioxidant property and cell-regenerating functions as a result of
increased protein synthesis are considered as most important. It can prevent
the absorption of toxins into the hepatocytes by occupying the binding sites
as well as inhibiting many transport proteins at the membrane. These actions
along with antiperoxidative property make silymarin a suitable candidate for
the treatment of iatrogenic and toxic liver diseases [3].
general procedure for the synthesis of mannich bases of benztriazole
Benztriazole (0.01 mol) was dissolved in methanol (50 mL) in a 250 mL
beaker under ice cold condition. Various secondary amines (0.01 mol) were
added in small quantities with constant stirring under ice cold condition. A
measured quantity of formaldehyde (0.01 mol) was added, slowly with
constant stirring for 4 hrs. The contents of the beaker were kept over night
in the freezer. Crystallized product was filtered and dried. The product was
recrystallized using methanol. The reaction sequence is outlined in Scheme 1.
The yield, melting point and Rf value were reported in Table 1.
Mannich reaction is an important tool for synthesis of novel compounds.
Mannich bases are physiologically reactive because of the basic function
rendering the molecule soluble in aqueous solvents when it is transformed into
aminium salt. Mannich bases have been reported to possess antibacterial [4-
6], antitubercular [7-9], antimalarial [10-11], anti-HIV [12] and analgesic and
antinflammatory [13-14]activities.1,2,4-triazole nucleus has been incorporated
into wide variety of therapeutically interesting molecules to transform them
into better drugs. Various 1,2,4-triazole derivatives have been reported to
possess anticancer [15], antioxidant [16], antibacterial [17], antifungal [18],
analgesic [19], anti-inflammatory [20] and anti-emetic activity [21]. Mannich
bases of 1,2,4-triazole were reported for anticancer [22] activity, protozocidal
and antibacterial activity [23]. Some of the modern-day drugs with triazole
nucleus are the antiviral agent Ribavirin, antimigrane agent Rizatriptan,
anxiolytic agent Alprazolam, antifungal agents Flucanozole and Itraconazole.
Amino azoles are reported for potential hepatoprotectant activity [24]. There is
no report on the synthesis and hepatoprotective evaluation of Mannich bases of
1,2,4-benztriazoles. Hence it was contemplated to synthesize some congeners
of benztriazole containing Mannich bases with a view to explore their potency
as hepatoprotective agents.
materialS and methodS
All melting points were taken by open capillary tubes and were uncorrected.
Thin layer chromatography was performed on precoated Silica Gel 60 F254
plates from E.Merck using methanol and benzene as mobile phase (50:50) and
visualized by exposure to iodine vapors. IR spectra recorded on a Perkin Elmer
IR spectrophotometer, using KBr pellets. NMR spectra were recorded on a
Bruker DRX 300 (300MHZ) NMR spectrophotometer in CDCl3 using TMS as
Sheme 1: Synthetic protocol of benztriazole Mannich bases.
e-mail: rsekaran2001in@yahoo.co.in
366

J. Chil. Chem. Soc., 55, Nº 3 (2010)
table 1: Physical and analytical data of newly synthesized compounds.
Compound
R
M.P. (°C)
Yield (%)
Rf value*
0.2
Molecular formula
Mol Weight
204.27
C2H5
2
100_102
48
C11H16 N4
N
N
C2H5
C6H5
3
4
-------
80-82
62
54
0.67
0.4
C19H16 N4
300.35
218.25
C6H5
C11H14 N4O
N
O
5
6
172-174
110-112
70
60
0.91
0.70
C11H15 N5
N
NH
217.27
248.28
N
C15H12 N4
C6H5
N
7
8
82-84
59
64
0.78
0.73
C15H14 N4O
266.29
328.36
CH3
O
C6H5
N
C6H5
165-168
C20H16 N4O
O
C6H4OH
CH3
N
9
120-122
72
0.34
C15H14 N4O2
282.29
O
NaOOCH2C
220-222
68
0.45
C21H15Cl2N4NaO2
N
Cl
10
449.26
Cl
*chloroform and benzene as mobile phase, spot detection-Iodine vapour
367

J. Chil. Chem. Soc., 55, Nº 3 (2010)
Infrared spectra of all compounds showed strong absorptions at 2860
and 2846 cm-1 for the CH2 group (Mannich methylene). 1HNMR spectra all
compounds showed a singlet at d 4.2 ppm for methylene protons of –NCH2N-
group.
histopathological Studies
One animal from the treated groups showing maximal activity as thick
sections and stained, using haematoxylin-eosin dye, and finally observed under
indicated by improved biochemical parameters from each test, positive control,
hepatotoxin and control groups were utilized for this purpose. The animals
were sacrificed and the abdomen was cut open to remove the liver. Then 5
mm thick piece of the liver were fixed in Bouin’s solution (mixture of 75 ml
of saturated picric acid, 25 ml of 40% formaldehyde and 5ml of glacial acetic
acid) for 12 h and then embedded in paraffin, using conventional methods and
cut into 5 m microscope for histopathological changes in liver architecture, and
their photomicrographs were taken.
Compound 2
IR (KBr) n/ cm-1: 3099 (Ar-H), 2975(C-H), 1640 (C-N), 1600 (C=C).
1HNMR (d, CDCl3); 2.9 (t, 6H, (CH2-CH3)2), 3.2 (q, 4H, (CH2-CH3)2), 7.4-7.9
(m, 4H, Ar-H).
Compound 3
IR (KBr) n/ cm-1: 3078 (Ar-H), 2998(C-H), 1656 (C-N), 1500 (C=C).
1HNMR (d, CDCl ); 6.7-7.9 (m, 14H, Ar-H).
Compound 4 3
Statistical analysis
IR (KBr) n/ cm-1: 3102 (Ar-H), 2984(C-H), 1644 (C-N), 1540 (C=C).
1HNMR (d, CDCl3); 2.5 (t, 4H, (-N-CH2-CH2-O-), 3.7 (t, 4H, (-N-CH2-
CH -O-), 7.4-7.9 (m, 4H, Ar-H).
The mean values ± SEM was calculated for each parameter. Percentage
reduction against the hepatotoxins by the test sample was calculated by
considering enzyme level difference between the hepatotoxin treated. For
determining the significant inter-group difference, each parameter was
analyzed separately, and one way analysis of variance (ANOVA) was carried
out. Then the individual comparison of the group means values were done using
Dunnett’s ‘t’ test procedure [29]. P values <0.05 were considered significant.
2 Compound 5
IR (KBr) n/ cm-1: 3325 (NH), 3098 (Ar-H), 2974(C-H), 1654 (C-N), 1500
(C=C). 1HNMR (d, CDCl3); 2.4 (t, 4H, (-N-CH2-CH2-N-), 2.7 (t, 4H, (-N-CH2-
CH2-N-), multiplet at d 2.46-3.45 ppm for piperazine proton, 7.4-7.9 (m, 4H,
Ar-H).
Compound 6
reSultS
IR (KBr) n/ cm-1: 3094 (Ar-H), 2978(C-H), 1650 (C-N), 1570 (C=C).
1HNMR (d, CDCl ) 6.4-7.6 (m, 10H, Ar-H).
Benztriazole 1 on treatment with substituted secondary aromatic amines in
presence of formaldehyde gave Mannich bases in good yields. The experimental
animals after treatment with CCl the SGOT, SGPT and ALP levels have been
increased significantly (Table 2).4S, ignificant reduction was observed for SGOT
and SGPT levels in silymarin and compound 4 treated groups when compared
with CCl -treated rats. The histological studies have shown a recovery of
the hepat4ocytes after the administration of compound 4 (N-Morpholinyl
methylbenztriazole) which clearly indicated the partial protection of liver cells
compared to silymarin. The liver section treated with compound 4 showed
partial disappearance of fatty deposit and necrosis comparable to standard
drug silymarin (Fig 1- 4). Compounds 5 and 6 showed lesser hepatoprotective
activity than compound 4 and failed to show the statistical significance when
compared to CCl4 treated groups.
Compound 7 3
IR (KBr) n/ cm-1: 3089 (Ar-H), 2987(C-H), 1694 (C=O), 1642 (C-N),
1570 (C=C). 1HNMR (d, CDCl3) 2.0 (s, 3H, CH3), 6.4-7.6 (m, 9H, Ar-H).
Compound 8
IR (KBr) n/ cm-1: 3078 (Ar-H), 2983(C-H), 1680 (C=O), 1624 (C-N),
1590 (C=C). 1HNMR (d, CDCl3) 6.4-7.9 (m, 14H, Ar-H).
Compound 9
IR (KBr) n/ cm-1: 3556 (OH), 3088 (Ar-H), 2992(C-H), 1676 (C=O), 1641
(C-N), 1590 (C=C). 1HNMR (d, CDCl3) 2.0 (s, 3H, CH3), 6.4-7.9 (m, 8H, Ar-
H).
Compound 10
IR (KBr) n/ cm-1: 3102 (Ar-H), 2990(C-H), 1710 (C=O), 1630 (C-N),
1590 (C=C), 721 (C-Cl). 1H NMR (d, CDCl3) 6.4-7.9 (m, 11H, Ar-H).
table 2: Effect of synthesized compounds on CCl4 induced hepatotoxicity.
evaluation of hepatoprotective activity
SGOT U/
mL
SGPT U/
mL
ALP U/
mL
Total Protein
(mg/mL)
Groups
Control
Hepatoprotective studies for compounds 4, 5 and 6 were carried out on
healthy wistar rats (190-210 g). They were divided into six groups of four
animals each. The study protocol was approved by the institutional animal
ethics committee for the purpose of control and supervision on animals
(CPCSEA), New Delhi, India. Registration Approval No:509/01/C/CPCSEA/
dated 10th July 2002. Carbon tetrachloride mixed with liquid paraffin (1:1) was
used as hepatotoxic agent. The compounds 4, 5 and 6 were administered in the
form of aqueous suspension in 1% v/v of carboxymethyl cellulose for seven
day after carbon tetrachloride administration. On the seventh day of the start
of respective treatment the rats were anesthetized by light ether anesthesia and
the blood was withdrawn from retro orbital plexus. It was allowed to coagulate
for 30 min and serum was separated by centrifugation at 2500 rpm. The serum
SGPT, SGOT, ALP and total protein were estimated in semi-autoanalyser using
specific enzymatic kits (Nicolas Primal diagnostic Division, Mumbai, India).
The standard drug silymarin procured from Sigma-Aldrich, USA was used as a
positive control, since it protects CCl4-induced hepatotoxicity [25-26].
17.0 ±
2.56
23.0 ±
1.86
6.0 ± 1.12
12.29 ± 1.10
13.50 ± 1.08ns
12.85 ± 1.12ns
13.10 ± 1.18ns
13.40 ± 1.21ns
13.45 ±1.06ns
55.0 ±
2.78a
57.0 ±
1.67a
11.0 ±
1.98b
CCl4 treated
CCl4 +
Silymarin
33.0 ±
2.76*
36.0 ±
1.45*
9.10 ±
1.32ns
CCl4 +
Compound 4
37.0 ±
2.11**
40.0 ±
2.56**
10.10 ±
1.29ns
CCl4 +
Compound 5
41.0 ±
1.42ns
43.0 ±
2.32ns
10.25 ±
1.45ns
CCl4 +
Compound 6
44.0 ±
1.73ns
45.0 ±
1.78ns
10.30 ±
1.89ns
All data were expressed as mean ± SD (n=6)
treatment Schedule
group i (normal control): These group of rats were not given neither carbon
tetrachloride nor the synthesized compounds.
group ii (ccl -treated rats): These rats were given carbon tetrachloride (1
ml/kg) for the fi4rst day of study to produce toxicity in the liver.
group iii (Silymarin treated rats): These rats were given a single dose of
carbon tetrachloride (1 ml/kg) on first day and then silymarin (10 mg/kg) was
given for 6 days.
aP<0.001 indicates significance when compared to control.
bP<0.01 indicates significance when compared to control.
*P<0.001 indicates significance when compared to CCl treated
**P<0.05 indicates significance when compared to CCl44 treated
ns – Not significant when compared to CCl4 treated
group iv (compound 4): These rats received were given a single dose of
carbon tetrachloride (1 ml/kg) on first day and then compound 4 (250 mg/kg)
was given for six days.
group v (compound 5): These rats were given a single dose of carbon tet-
rachloride (1 ml/kg) on first day and then compound 5 (250 mg/kg) was given
for six days.
group vi (compound 6): These rats were given a single dose of carbon tet-
rachloride (1 ml/kg) on first day and then compound 6 (250 mg/kg) was given
for six days.
368

J. Chil. Chem. Soc., 55, Nº 3 (2010)
figure 1:Normal hepatocytes without necrosis.
figure 2: Liver treated with Carbon tetrachloride.
The liver section showing necrotic normal hepatocytes without any
Necrosis abundant fatty depositions.
The liver section showing the development with and degeneration of liver
or fatty deposition
figure 3: Liver treated with silymarin.
figure 4: Liver treated with compound 4.
Sections showing good recovery with complete disappearance of fatty
necrosis and fatty deposition
Section showing the partial recovery from necrosis and fatty deposition
activity of this compound 4 bearing the morpholine moiety may be due to its
antioxidant activity.
diScuSSion
Mannich bases 2 -10 obtained by synthesis were in correlation with the
IR and NMR study. Since Mannich bases are reported for hepatoprotective
activity, the titled compounds were screened for hepatoprotective activity.
Carbon tetrachloride is a hepatotoxin commonly used for the production of
experimental liver toxicity [1]. The serum transaminase level is most widely
used as a measure of hepatic injury, due to its ease of measurement and high
degree of sensitivity [2]. It is useful for the detection of early damage of hepatic
tissue and requires less effort than that required for a histologic analysis,
moreover without sacrifice of the animals. In the present study, AST levels
in normal control group were in conformity with the findings of Kapoor et al
[30]. Seven days pretreatment with the compounds 4 (250 mg/kg) protected
the animals significantly from carbon tetrachloride induced hepatotoxicity.
Hepatoprotective activity of compound 4 could probably due to the presence
of morpholine ring since morpholine derivatives are reported to possess
hepatoprotective [27, 28] and antioxidant activities [31].
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