Synthesis and antihepatotoxic activity of 2-(substituted-phenyl)-5-(2,3- dihydro-1,4-benzodioxane-2-yl)-1,3,4-oxadiazole derivatives

Article abstract of DOI:10.3109/14756366.2010.489899

Novel 1,3,4-oxadizole derivatives containing the 1,4-dioxane ring system were synthesised starting from 2,3-dihydro-1,4-benzodioxane-2-carbohydrazide. The synthesised compounds were evaluated for antihepatotoxic activity against CCl4-induced hepatotoxicity in rats. Some compounds demonstrated a significant antihepatotoxic activity comparable to the standard drug Silymarin.

Full text of DOI:10.3109/14756366.2010.489899

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(2): 216–221
© 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2010.489899
rESEarCH artICLE
Synthesis and antihepatotoxic activity of 2-(substituted-
phenyl)-5-(2,3-dihydro-1,4-benzodioxane-2-yl)-1,3,4-
oxadiazole derivatives
Bahar Ahmed*, Habibullah, and Shamshir Khan
Antihepatotoxic Research Laboratory, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard
(Hamdard University), New Delhi, India
abstract
Novel 1,3,4-oxadizole derivatives containing the 1,4-dioxane ring system were synthesised starting from 2,3-dihydro-
1,4-benzodioxane-2-carbohydrazide. The synthesised compounds were evaluated for antihepatotoxic activity
against CCl4-induced hepatotoxicity in rats. Some compounds demonstrated a significant antihepatotoxic activity
comparable to the standard drug Silymarin.
Keywords: 1;4-benzodioxane, 1;3;4-oxadiazole, antihepatotoxic activity
Introduction
three isomers of flavonolignan i.e. silybin, silychristin and
silydianin. Silybin is the most potent component contain-
ing the 1,4-dioxan ring system, whereas the other isomers
namely silychristin and silydianin do not possess the 1,4-
dioxan ring system and hence do not display any signifi-
cant antihepatotoxic activity. We have therefore concluded
that the 1,4-dioxane ring system plays an important role in
exhibiting antihepatotoxic activity and if compounds are
prepared containing the 1,4-dioxane ring, they will exhibit
the antihepatotoxic activity. us we have prepared
some new 1,3,4-oxadizole derivatives containing the
1,4-dioxane ring system namely, 2-(substituted-phenyl)-
5-(2,3-dihydro-1,4-benzodioxane-2-yl)-1,3,4-oxadiazole
derivatives and evaluated them for antihepatotoxic activity
against CCl4 induced hepatotoxicity in rats. Among them,
compounds 3Ai, 3Avii, 3Axiv and 3Axvii were found to
show significant antihepatotoxic activity as comparable to
standard drug silymarin.
e liver is an organ of paramount importance as it
plays an essential role in maintaining the biological
equilibrium of vertebrates. Traditional drugs used in the
treatment of liver diseases are sometimes inadequate
to cater for the needs of a large population. In spite of
tremendous strides in modern medicine, there are few
drugs available for the treatment of liver disorders. Many
natural products of herbal origin are in use for the treat-
ment of liver ailments [1–4]. e drugs available in the
modern systems of medicine are mainly corticosteroids
and immunosuppressive agents, these bring about only
symptomatic relief and in most cases have no influence
on the disease process. Further, their use is associated
with the risk of relapses and the danger of side effects.
Benzodioxane represents a series of synthetic and nat-
ural compounds of considerable medicinal importance.
Compounds containing dioxane ring systems exhibit a
variety of biological activities such as antihepatotoxic
[5,6], α-adrenergicblockingagents[7], anti-inflammatory
[8], and D2 antagonist/5-HT1A partial agonist activity [9].
ecompoundsilymarinisolatedfromseedsofSilybum
marianum commonly known as “milk thistle” has been
found to be a potent antihepatotoxic agent against a variety
of toxicants. Silymarin has been found to be a mixture of
Materials and methods
Chemistry
e IR spectra were recorded on a Brucker spectrom-
eter (Central Instrumental Facility (CIF), Hamdard
University, New Delhi). e mass spectra were
Address for Correspondence: Bahar Ahmed, Antihepatotoxic Research Laboratory, Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Jamia Hamdard (Hamdard University), New Delhi, India. Tel. (+91)-011-260-59688; E-mail: drbahmed@rediffmail.com
(Received 06 February 2010; revised 21 April 2010; accepted 23 April 2010)
216

Synthesis and antihepatotoxic activity 217
recorded on a Bruker daltronics high resolution mass
spectrometer, the H NMR (300 MHz) was recorded on
mixture was cooled and poured over crushed ice. e
precipitate thus obtained was filtered, washed with
sodium bicarbonate, dried and recrystallised with ben-
1
a Bruker DPX 300 spectrometer in CD3OD and DMSO-
d6 using TMS as the internal standard reference and the
chemical shifts were in δ (ppm). Elemental analyses
were performed on Elementar Vario EL III, Carlo Erba
1108 (Central Instrumental Facility (CIF), Hamdard
University, New Delhi). e melting points were deter-
mined by capillary method.
1
zene: methanol. H NMR (300 MHz, DMSO-d6): δ 4.33
(2H, m, unresolved doublet, CH2-3), 5.02 (1H, brs, unre-
solved doublet, CH-2), 6.88–7.67 (4H, m, Ar-H, ring A),
7.87 (5H, m, Ar-H, ring B); FTIR (KBr) cm−1: 3162 (=C-H,
aromatic), 1678(C=C), 1492(C=N), 1078(C-O-C). HR-MS
(m/z): 281.197 [MH]+ (Calcd. for C16H12N2O3, 280.2782);
Anal Calcd. fo C16H12N2O3: C, 68.56; H, 4.32; N, 9.99; O,
17.13; Found: C, 68.46; H, 4.42; N, 10.05; O, 17.12.
Synthesis of ethyl-l,4-benzodioxane-2-carboxylate (1)
Anhydrous potassium carbonate (50 g) was added
in portions to a stirred solution of 55 g of catechol in
200 ml of dry acetone followed by the dropwise addition
of 34.5 g of ethyl 2,3-dibromopropionate. Another 50 g
of potassium carbonate and 34.5 g of the dibromoester
were added similarly and this was repeated twice
more using a total of 200 g of potassium carbonate and
137.5 g of ester. Stirring and refluxing was continued for
another 15 h. e reaction mixture was then filtered and
the solid was washed several times with acetone. e
filtrate was concentrated to about 75 ml and the residue
was diluted with 50 ml of cold water. e oily layer was
separated from the aqueous layer and the latter was
extracted repeatedly with ether. e combined oily layer
and ether extracts were washed with water, dried over
magnesium sulphate and evaporated. e dark residue
was distilled at 96–97°C (0.1 mm) to yield 38 g of ester 1
2-(2-Bromo-phenyl)-5-(2,3-dihydro-1,4-benzodioxane-2-yl)-
1,3,4-oxadiazole (3Aii)
1H NMR (300 MHz, DMSO-d6): δ 4.24 (2H, m, unresolved
doublet, CH2-3), 5.15 (1H, brs, unresolved doublet,
CH2-2), 6.67–7.91 (4H, m, Ar-H, ring A), 7.65 (5H, m,
Ar-H, ring B); FTIR (KBr) cm−1: 3069 (=C-H, aromatic),
1670 (C=C), 1485 (C=N), 1067 (C-O-C), 756 (C-Br); Anal
Calcd. for C16H11BrN2O3: C, 53.5; H, 3.09; N, 7.8; O, 13.36;
Found: C, 53.43; H, 3.19; N, 7.67; O, 13.43.
2-(3-Bromo-phenyl)-5-(2,3-dihydro-1,4-benzodioxane-2-yl)-
1,3,4-oxadiazole (3Aiii)
1H NMR (300 MHz, DMSO-d6): δ 4.26 (2H, m, unresolved
doublet, CH2-3), 5.41 ( 1H, brs, unresolved doublet,
CH2-2), 6.58–7.23 (4H, m, Ar-H, ring A), 7.56 (5H, m,
Ar-H, ring B); FTIR (KBr) cm−1: 3106 (=C-H, aromatic),
1654 (C=C), 1498 (C=N), 1053 (C-O-C), 768 (C-Br); Anal
Calcd. for C16H11BrN2O3:C, 53.5; H, 3.09; N, 7.8; O, 13.36:
Found: C, 53.45; H, 3.08; N, 7.84; O, 13.43.
1
as colourless semisolid. H NMR (300 MHz, DMSO-d6):
δ 1.23 ( 3H, t, J= 7.1 Hz, CH3-12), 4.2 (2H, q, J = 7.1, 5.7
Hz, CH2-12), 4.3 (2H, d, J = 2.7, CH2-3), 4.77 (1H, t, J = 2.7,
CH-2), 6.84 (4H, m, Ar-H); FTIR cm−1: 3052 (=C-H, aro-
matic), 1772 (C=O), 1653 (C=C), 1292 (C-O, ester)
2-(4-Bromo-phenyl)-5-(2,3-dihydro-1,4-benzodioxane-2-yl)
-1,3,4-oxadiazole (3Aiv)
Synthesis of 2,3-dihydro-1,4-benzodioxane-2-
carbohydrazide (2)
1H NMR (300 MHz, DMSO-d6): δ 4.35 (1H, dd, J = 5.4, 9.9
Hz, CH2-3, H-α), 4.62 (1H, dd, J= 3.3, 3.2 Hz, CH2-3, H-β),
5.97 (1H, brs, unresolved doublet CH-2), 6.87–7.19 (4H,
m, Ar-H, ring A), 7.47–8.02 (4H, m, Ar-H, ring B); FTIR
(KBr) cm−1: 3156 (=C-H, aromatic), 1687 (C=C), 1493
(C=N), 1043 (C-O-C), 746 (C-Br); HRMS (m/z): 359.1955
[M]+ (Calcd for C16H11BrN2O3, 359.1742). Anal Calcd. for
C16H11BrN2O3: C; 53.5; H, 3.09; Br, 22.25; N, 7.8; O, 13.36.
Found: C; 53.48; H, 3.15; N, 7.78; O, 13.26.
To a solution of ethyl-1,4-benzodioxane-2-carboxylate
(0.01 mol)inethanol(20 ml),hydrazinehydrate(0.01 mol)
was added and the reaction mixture was refluxed. e
progress of the reaction was monitored by TLC. After
the completion of the reaction (usually 16 h), the excess
solvent was removed under reduced pressure. e reac-
tion mixture was poured over crushed ice. e solid thus
separated was filtered, dried and crystallised with metha-
nol to give a white powder; mp: 110–112°C; Yield: 80%; 1H
NMR (300 MHz, DMSO-d6): δ3.91 (2H, br-s, NH2-13), 4.24
(1H, dd, J= 6, 11.4 Hz, Ha-3), 4.46 ( 1H, dd, J= 6, 11.4 Hz,
Hb-3), 4.78(1H, d, J= 6, CH2), 6.91 (4H, m, Ar-H), 7.78 (1H,
s, NH-12); FTIR (KBr) cm−1: 3052 (=C-H, aromatic), 1772
(C=O), 1673 (C=C), 1259 (-NH2), 1195 (-NH), 758 (C=C);
Anal Calcd. for C9H10N2O3: C, 55.67; H, 5.19; N, 14.43; O,
24.72. Found: C: 55.37; H, 5.02; N, 14.67; O, 24.73.
2-(2-Chloro-phenyl)-5-(2,3-dihydro-1,4-benzodioxane-2-yl)-
1,3,4-oxadiazole (3Av)
1H NMR (300 MHz, DMSO-d6): δ 4.92 (2H, m (unresolved
doublet), CH2-3), 5.62 (1H, brs, unresolved doublet,
CH-2), 6.74–7.82 (4H, m, Ar-H, ring A), 7.02–7.39 (4H,
m, Ar-H, ring B); FTIR (KBr) cm−1: 3197(=C-H, aromatic),
1648 (C=C), 1489 (C=N), 1028 (C-O-C), 745 (C-Cl). Anal
Calcd. for C16H11ClN2O3:C, 61.06; H, 3.52; N, 8.9; O, 15.25;
Found C, 61.12; H, 3.45; N, 8.87; O, 15.29.
Synthesis of 2-(phenyl)-5-(2,3-dihydro-1,4-
benzodioxane-2-yl)-1,3,4-oxadiazole (3Ai)
2-(3-Chloro-phenyl)-5-(2,3-dihydro-1,4-benzodioxane-2-yl)-
1,3,4-oxadiazole (3Avi)
A solution of 0.01 mole of 2,3-dihydro-1,4-benzodiox-
ane-2-carbohydrazide, 0.01 mole benzoic acid and 5 ml
of POCl3 was refluxed with stirring for 6–7 h. e reaction
1H NMR (300 MHz, DMSO-d6): δ 4.54 (2H, m, unresolved
doublet, CH-3), 5.22 ( 1H, brs, unresolved doublet, CH-2),
© 2011 Informa UK, Ltd.

218 Bahar Ahmed et al.
6.88–7.57 (4H, m, Ar-H, ring A), 7.23–7.45 (5H, m, Ar-H,
ring B); FTIR (KBr) cm−1: 3057 (=C-H, aromatic), 1643
(C=C), 1468 (C=N), 1023 (C-O-C), 768 (C-Cl). Anal Calcd.
for C16H11ClN2O3: C, 61.06; H, 3.52; Cl, 11.26; N, 8.9; O,
15.25; Found C, 61.03; H, 3.48;N, 8.78: O, 15.3.
H, 4.79; N, 9.52; O, 16.31; Found: C: 69.42; H, 4.81; N, 9.48;
O, 16.29.
2-(4-Hydroxy-phenyl)-5-(2,3-dihydro-1,4-benzodioxane-2-yl)-
1,3,4-oxadiazole(3Axii)
1H NMR (300 MHz, DMSO-d6): δ 10.24 (1H, s, ArOH),
4.37 (2H, m, unresolved doublet, CH2-3), 5.26 ( 1H, brs,
unresolved doublet, CH-2), 6.88–7.67 (4H, m, Ar-H,
ring A), 7.26–7.34 (4H, m, Ar-H, ring B); FTIR (KBr)
cm−1: 3145 (=C-H, aromatic), 1646 (C=C), 1479 (C=N),
1023 (C-O-C); Anal Calcd. for C16H12N2O4: C, 64.86; H,
4.08; N, 9.46; O, 21.6; Found: C, 64.82; H, 4.25; N, 9.45;
O, 21.56.
2-(4-Chloro-phenyl)-5-(2, 3-dihydro-1,4-
benzodioxane-2-yl)-1,3,4-oxadiazole (3Avii)
1H NMR (300 MHz, DMSO-d6): δ 4.25 (2H, m, unresolved
doublet, CH-3), 5.02 ( 1H, brs, unresolved doublet,
CH2-2), 6.88–7.67 (4H, m, Ar-H, ring A), 7.87 (5H, m,
Ar-H, ring A); FTIR (KBr) cm−1: 3158 (=C-H, aromatic),
1642 (C=C), 1475 (C=N), 1016 (C-O-C), 743 (C-Cl) Anal
Calcd. for C16H11ClN2O3: C, 61.06; H, 3.52; Cl, 11.26; N, 8.9;
O, 15.25. Found: C, 60.98; H, 3.48; N, 8.85; O, 15.3.
2- (3,4-Dihydroxy-phenyl)-5-(2,3-dihydro-1,4-benzodioxane-
2-yl)-1,3,4-oxadiazole (3Axiii)
1H NMR (300MHz, DMSO-d6): δ 4.39 (2H, m (unresolved
doublet), CH2-3), 5.02 (1H, brs, unresolved doublet,
CH-2), 6.88–7.05 (4H, m, Ar-H, ring A), 6.26–7.12(3H, m,
Ar-H, ring B), 10.36 (2H, s, Ar-OH); FTIR (KBr) cm−1: 3042
(=C-H, aromatic), 1648 (C=C), 1469 (C=N), 1048 (C-O-C);
Anal Calcd. for C16H11N2O5: C, 61.54; H, 3.87; N, 8.97; O,
25.62; Found: C, 61.58; H, 3.85; N, 8.89; O, 25.59.
2-(2, 4-Dichloro-phenyl)-5-(2,3-dihydro-1,4-benzodioxane-2-
yl)-1,3,4-oxadiazole (3Aviii)
1H NMR (300 MHz, DMSO-d6): δ 4.35 (2H, m, unre-
solved doublet, CH2-3), 5.91 ( 1H, brs, unresolved dou-
blet, CH-2), 6.88–7.07 (4H, m, Ar-H, ring A), 7.73–7.92
(3H, m, Ar-H, ring B); FTIR (KBr) cm−1: 3050 (=C-H, aro-
matic), 1693 (C=C), 1478 (C=N), 1070 (C-O-C), 827, 734
(C-Cl). Anal Calcd. for C16H10Cl2N2O3: C, 55.04; H, 2.89;
Cl, 20.31; N, 8.02; O, 13.75. Found: C, 54.94; H, 2.75; Cl,
20.28; N, 8.53; O, 13.65.
2-(4-Methoxy-phenyl)-5-(2,3-dihydro-1,4-benzodioxane-2-yl)-
1,3,4-oxadiazole (3Axiv)
1H NMR (300 MHz, DMSO-d6): δ3.84 (3H, s, Ar-OCH3),
4.62 (2H, m, unresolved doublet, CH2-3), 5.87 (1H, brs,
unresolved doublet, CH-2), 6.91–7.16 (4H, m, Ar-H, ring
A), 7.92–7.94 (4H, m, Ar-H, ring B); FTIR (KBr) cm−1:
3062 (=C-H, aromatic), 1611 (C=C), 1494 (C=N), 1180,
1017 (C-O-C); Anal Calcd. for C17H14N2O4: C, 65.80; H,
4.55; N, 9.03; O, 20.62; Found: C, 65.78; H, 4.58; N, 9.13;
O, 20.69.
2-(2-Methyl-phenyl)–5-(2,3-dihydro-1,4benzodioxane-2-yl)-
1,3,4-oxadiazole (3Aix)
1H NMR (300 MHz, DMSO-d6): δ 2.35 (3H, s, Ar-CH3),
4.52 (2H, m, unresolved doublet, CH2-3), 5.17 ( 1H, brs,
unresolved doublet, CH-2), 6.78–7.57 (4H, m, Ar-H,
ring-A), 7.12–7.46 (4H, m, Ar-H, ring-B); FTIR (KBr) cm−1:
3048 (=C-H, aromatic), 2970 (Ar-CH3), 1638 (C=C), 1474
(C=N), 1025 (C-O-C); Anal Calcd. for C17H14N2O3:C, 69.38;
H, 4.79; N, 9.52; O, 16.31. Found: C, 69.25; H, 4.72; N, 9.54;
O, 16.34.
2-(3, 4-dimethoxy-phenyl)-5-(2, 3-dihydro- 1,4-benzodioxane-
2-yl)-1, 3, 4-oxadiazole (3Axv)
1H NMR (300 MHz, DMSO-d6): δ 3.76 (2H, s, Ar-OCH3),
4.52 (2H, m, unresolved doublet, CH2-3), 5.35 ( 1H, brs,
unresolved doublet, CH-2), 6.88–7.67 (4H, m, Ar-H,
ring-A), 7.01–7.32 (3H, m, Ar-H, ring-B); FTIR (KBr) cm−1:
3067 (=C-H, aromatic), 1664 (C=C), 1469 (C=N), 1245,
1030, 1024 (C-O-C); Anal Calcd. for C18H16N2O5: C, 63.52;
H, 4.74; N, 8.23; O, 23.51; Found: C, 63.48; H, 4.79; N, 8.26;
O, 23.49.
2-(3-Methyl-phenyl)–5-(2,3-dihydro-1,4-
benzodioxane-2-yl)-1,3 4-oxadiazole (3Ax)
1H NMR (300 MHz, DMSO-d6): δ 2.42 (3H, s, Ar-CH3),
4.41 (1H, dd, J = 5.4, 12.3 Hz, CH2-3, H-α), 4.62 (1H, dd,
J= 2.1, Hz, CH2-3, H-β), 5.18 (1H, brs, unresolved doublet
CH-2) 6.88–7.01 (4H, m, Ar-H, ring A), 7.25–7.97 (4H, m,
Ar-H, ring B); FTIR (KBr) cm−1: 3197 (=C-H, aromatic),
2950 (Ar-CH3), 1687 (C=C), 1490 (C=N), 1076 (C-O-C);
Anal Calcd. for C17H14N2O3: C, 69.38; H, 4.79; N, 9.52; O,
16.31; Found: C, 69.46; H, 4.78; N, 9.49; O, 16.27.
2-(4-amino-phenyl)-5-(2,3-dihydro-1,4-benzodioxane-2-yl)-
1,3,4-oxadiazole (3Axvi)
2-(4-Methyl-phenyl)-5-(2,3-dihydro-1,4-benzodioxane-2-yl)-
1,3,4-oxadiazole (3Axi)
1H NMR (300 MHz, DMSO-d6): δ 4.35 (2H, s, Ar-NH2),
4.61 (2H, m (unresolved doublet), CH2-3), 5.25 (1H,
brs, unresolved doublet, CH-2), 6.73–7.21 (4H, m, Ar-H,
ring-A), 7.66–8.15 (4H, m, Ar-H, ring-B); FTIR (KBr)
cm−1: 3072 (=C-H, aromatic), 1648 (C=C), 1449 (C=N),
1320 (C-N), 1036 (C-O-C); Anal Calcd. for C16H13N3O3:
C, 65.08; H, 4.44; N, 14.23; O, 16.25; Found: C, 65.1; H,
4.45; N, 14.24; O, 16.21.
1H NMR (300 MHz, DMSO-d6): δ 2.26 (3H, s, Ar-CH3),
4.27 (2H, m, unresolved doublet, CH2-3), 5.43 ( 1H, brs,
unresolved doublet, CH-2), 6.68–7.37 (4H, m, Ar-H, ring
A), 7.34–7.87 (4H, m, Ar-H, ring B); FTIR (KBr) cm−1:
3142 (=C-H, aromatic), 2850 (Ar-CH3), 1668 (C=C), 1475
(C=N), 1038 (C-O-C); Anal Calcd. for C17H14N2O3:C: 69.38;
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and antihepatotoxic activity 219
1. e starting material ethyl-l,4-benzodioxane-2-car-
boxylate (1) was prepared by a reaction between cat-
echol and ethyl-2,3-dibromopropionate in dry acetone
in the presence of anhydrous potassium carbonate,
which on treatment with hydrazine hydrate afforded
the corresponding hydrazide (2). e reaction of the
hydrazide (2) with substituted aryl carboxylic acids in
phosphorus oxychloride (POCl3) afforded the cyclised
products; 2-(substituted-phenyl)-5 -(2,3-dihydro-1-
,4-benzodioxane-2-yl)-1,3,4-oxadiazoles (3Ai–3Axvii).
e synthesised compounds were characterised by
IR, 1H-NMR, mass spectroscopic data and elemental
analysis.
Testing the antihepatotoxic activity of the synthesised
compounds
Animals
Male albino rats weighing 150–200 g were used for the
study. e animals were housed in clean metabolic cages
and maintained at a controlled temperature (23 2°C).
ey were fed with a standard pellet diet and had water
ad libitum. e animals were maintained at 25°C to 28°C
with40–70%RHand12 hlight/darkcyclesandwerefasted
for 12 hours prior to the experiment. e protocol was
approved by the Institutional Animal Ethical Committee
constituted by Jamia Hamdard for such a purpose.
Adult rats of either sex weighing 150–200 g were
divided into eight groups each consisting of six animals
(Table 1). Group I received liquid paraffin only (1.5 ml/
kg, orally) and served as control. Rats of the remaining
seven groups received suspension of carbon tetrachlo-
ride (CCl4) in liquid paraffin (1:1, v/v, 1.5ml of CCl4/kg,
per oral.) to induce hepatic damage 24 h before the start
of treatment. Group III received the CCl4 suspension,
in addition to silymarin (10 mg/kg, po) daily. Groups
IV-VIII received the synthesised compounds 3Ai, 3Avii,
3Axii, 3Axiv, 3Axvii (10 mg/kg, po, for each compound)
orally every day in addition to the CCl4 suspension for
8 days. Blood was withdrawn through the retro-orbital
plexus of the rats on the 8th day. Serum was separated
from the blood of each rat by centrifugation for estima-
tion of glutamate oxaloacetate transaminase (GOT) and
glutamate pyruvate transaminase (GPT) [10], alkaline
phosphatase (ALP) [11], and total protein [12]. e rats
were sacrificed and the livers rapidly exercised immedi-
ately after sacrifice. e liver was fixed in formalin (10%),
serially sectioned and microscopically examined after
staining with hematoxylin and eosin.
Table 1. e chemical structures, melting points and percentage
yields for the synthesised compounds in scheme 1.
R
O
N
O
N
(3A)
O
Molecular
Compound R
Formula
mp (°C) Yield (%)
3Ai
3Aii
3Aiii
3Aiv
3Av
H
C16H12N2O3
160–62
175–77
156–58
135–37
189–91
201–203
182–84
148–150
139–41
134–36
142–44
128–30
165–67
87–89
76
68
72
75
69
82
73
79
70
81
77
64
65
71
74
67
58
2-bromo
3-bromo
4-bromo
2-chloro
3-chloro
4-chloro
C16H11BrN2O3
C16H11BrN2O3
C16H11BrN2O3
C16H11ClN2O3
C16H11ClN2O3
C16H11ClN2O3
3Avi
3Avii
3Aviii
3Aix
3Ax
3Axi
3Axii
3Axiii
3Axiv
3Axv
3Axvi
3Axvii
2,4–dichloro C16H10Cl2N2O3
Statistical analysis
2–methyl
3–methyl
4–methyl
4–hydroxy
C17H18N2O4
C17H14N2O3
C17H14N2O3
C16H12N2O4
e data obtained were analysed by one-way ANOVA fol-
lowed by Dunnett’s test. e level of significance was set
at P< 0.05.
3,4–dihydroxy C16H11N2O5
4– methoxy C17H14N2O4
3,4–dimethoxy C18H16N2O5
4–amino C16H13N3O3
Benzodioxino C18H14N2O5
results and discussion
141–43
55–57
Chemistry
e synthetic route used to prepare the starting materials
and the title compounds have been outlined in Scheme
160–62
O
O
OH Br
+
COOC2H5
COOC2H5
(b)
O
CONHNH2
+
COOH
(a)
H
CH2
Br
OH
O
(2)
(1)
R
(c)
R
O
O
N
N
O
(3A)
Scheme 1. Reagents and conditions (a) K2CO3, acetone, reflux with stirring; (b) NH2.NH2.H20, ethanol, reflux; (c) POCl3, stirring.
© 2011 Informa UK, Ltd.

220 Bahar Ahmed et al.
released in serum. It has been shown that protective
agents exert their action against CCl4 mediated lipid
peroxidation, either through a decreased production of
free radical derivatives or due to the antioxidant activ-
ity of the protective agent itself.
Antihepatotoxic activity of the synthesised
compounds
e CCl4-induced hepatotoxicity is mediated by the
primary and secondary bond formation of the reactive
species to critical cellular molecules such as DNA, lipid,
proteins or carbohydrates. It has been well established
that the hepatotoxicity by CCl4 is due to the enzymatic
activation to release the CCl3. radical in the free state,
which in turn disrupts the structure and function of the
lipid and protein macromolecules in the membrane
of the cell organelles. Hence, elevated levels of serum
enzymes are indicative of cellular leakage and the loss
of the functional integrity of the cell membrane due to
the toxicity produced by CCl4. A significant rise in the
serum enzymatic concentration, namely SGOT and
SGPT, could be taken as an index of liver damage. It
generally induces the deposition of fat in the liver and
plays a significant role in inducing triacyl glycerol accu-
mulation, depletion of GSH, increased lipid oxidation,
membrane damage, depression of protein synthesis
and the loss of enzyme activity. Being cytoplasmic in
location, the damage marker enzymes SGOT, SGPT are
As shown in Table 2, the activities of the liver enzymes
serum glutamate oxaloacetate transaminase (SGOT),
serum glutamate pyruvate oxaloacetate transaminase
(SGPT), alkaline phosphatase (ALP) were markedly
increased, whereas total proteins (TP) were decreased
in the CCl4-treated rats in comparison with the normal
values. Administration of silymarin (standard drug) and
the synthesised compounds at a dose level of 10mg/kg
body weight, prevented the CCl4-induced elevation of
SGOT, SGPT, ALP, as well as preventing the decrease
in total protein. Silymarin (10mg/kg) significantly
decreased the level of SGOT, SGPT, ALP and increased
the level in total protein. e histopathological studies
also showed a significant recovery of the hepatocytes
of the liver in both the standard drug and compound
treated animals (Table 3), which again correlated with
the results of the biochemical parameters. e results of
Table 2. e effect of the synthesised compound on serum enzymatic activity in CCl4 induced liver damage in rats.
SGOT
(IU/L)
SGPT
(IU/L)
ALP
(KA units)
Albumin
(g/dl)
Total protein
(g/dl)
Groups n=5 Treatment
Dose
–––
I
Normal control
34.82 0.697
74.52 0.695
45.6 1.18
42.08 3.57
3.67 0.123
2.41 0.148
6.46 0.53
3.54 0.57
II
III
Toxic control
1.5ml/kg (po)
10mg/kg (po)
85.27 2.05
66.157 2.886**
Silymarin
56.19 0.808** 61.29 1.78** 48.62 3.385**
3.15 0.181** 4.2 0.23**
(standard drug)
IV
Compd. 3Ai
Compd. 3Avii
Compd. 3Axii
10mg/kg (po)
10mg/kg (po)
10mg/kg (po)
64.03 0.995** 69.45 2.35*
62.84 0.662** 68.5 1.40** 37.57 2.548**
62.57 0.699** 62.35 2.27*** 54.42 2.432**
53.03 2.462*
4.14 0.125** 3.67 0.37**
4.76 0.165** 4.14 0.56*
4.59 0.112* 3.43 0.23**
4.69 0.109** 3.87 0.19**
V
VI
VII
VIII
Compd. 3Axiv 10mg/kg (po)
Compd. 3Axvii 10mg/kg (po)
63.58 9.061
65.87 2.39
63.15 3.67
42.83 2.046 **
42.022 4**
4.32 1. 28
4.71 0.378**
65.87 0.965**
SGOT, Serum glutamate oxaloacetate transaminase; SGPT, Serum glutamate pyruvate transaminase; ALP, alkaline phosphatase: TP, total
protein; p.o., per oral.
*** P<0.0001; ** P < 0.01; *P < 0.05 vs CCl4; P > 0.05 ns.
Values are mean SEM (n=5). ANOVA followed by dunnett’s test was performed.
Table 3. Histopathological changes in the livers of the Wistar rats.
GroupsN=5 Treatment
Microscopic observations
I
Normal control
Normal control liver samples showed normal architecture without any degeneration, necrosis, or
inflammation.
II
III
Toxic control
Toxic control liver samples showed prominent centrilobular necrosis with prominent and enlarged
central vein. ere is significant periportal inflammation reflecting liver damage.
Silymarin (standard
drug)
Standard control liver samples showed hepatocytes with uniformly staining cytoplasm and mild
dilatation of sinusoidal spaces. e central vein was clearly visible. Liver samples also showed good
recovery with the absence of necrosis.
IV
V
3Ai
3Avii
Liver samples showed hepatocytes with few sinusoidal spaces and no portal triad inflammation.
Liver histology of compound 3Avii was almost normal with only very little sinusoidal dilatation seen
in some hepatic lobules. e portal vein appeared clearly with the disappearance of necrosis.us
indicating a potent antihepatotoxic activity.
VI
3Axii
3Axiv
3Axvii
Liver hepatocytes were found almost normal with some sinusoidal dilatation. Portal vein appeared
clearly with the disappearance of necrosis thus indicating a potent antihepatotoxic activity.
VII
VIII
Pretreatment with compound 3Axiv significantly prevented the CCl4-induced hepatotoxicity as
revealed by the hepatic cells with well reserved cytoplasm, and marked decrease in inflammatory cells.
Some of the 1–2 hepatocyte rows around the central vein demonstrated hepatic cell degeneration,
necrosis (loss of nucleus), less injury of endothelial cells around central vein and less fat vacuoles in
hepatocytes.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and antihepatotoxic activity 221
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normal control rats. Administration of the synthesised
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of the tissues as neither fatty accumulation nor necrosis
was observed. e central vein appeared clearly indi-
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acknowledgements
e authors would like to thank the Head, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia
Hamdard, New Delhi, India for providing necessary
research facilities. We would also like to thank Showkat
Ahmad Shah, In charge, Animal House facility, Jamia
Hamdard, New Delhi for providing experimental animals
for carrying out the antihepatotoxic activity.
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declaration of interest
e authors would like to thank University Grant
Commission, New Delhi, for approving the major
research project for financial assistance.
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© 2011 Informa UK, Ltd.