Stereoselective synthesis and antioxidant activity of azabicycloadducts derived from 9, 10-Phenanthrenequinone

Article abstract of DOI:10.1002/hc.20562

A facile synthesis of sprio {1-azabicyclo-[3, 3, 0]-6-octene-8, 1′ -phenanthrene} -2′-ones has been accomplished by [3 + 2] cycloaddition of azomethine ylide (amy) generated from 9,10- phenanthrenequinone and different secondary cyclic amino acids, namely

Full text of DOI:10.1002/hc.20562

Heteroatom Chemistry
Volume 20, Number 7, 2009
Stereoselective Synthesis and Antioxidant
Activity of Azabicycloadducts Derived from
9,10-Phenanthrenequinone
Komal Arora,1 D. Jose,1 D. Singh,2 R. S. Gupta,2 P. Pardasani,1
and R. T. Pardasani1
1Department of Chemistry, University of Rajasthan, Jaipur 302 055, India
2Department of Zoology, University of Rajasthan, Jaipur 302 055, India
Received 18 December 2008; revised 18 April 2009
INTRODUCTION
ABSTRACT: A facile synthesis of sprio{1-aza-
bicyclo-[3,3,0]-6-octene-8,1ꢀ -phenanthrene}-2ꢀ -ones
The monumental work of Huisgen [1] has estab-
lished 1,3-dipolar cycloaddition reactions as the
has been accomplished by [3 + 2] cycloaddition
of azomethine ylide (amy) generated from 9,10-
phenanthrenequinone and different secondary cyclic
amino acids, namely, thiazolidine-4-carboxylic
acid, L-pyrrolidine-2-carboxylic acid (L-proline),
and piperidine-2-carboxylic acid (pipecolinic acid)
with electron-deficient dipolarophiles in 67%–79%
yield. AM1 calculations have been performed to
understand the stereochemical course of the cy-
cloaddition. The products have been characterized
by elemental analyses and spectroscopic techniques,
most powerful methodology for the construction
of five-membered heterocycles [2]. The biological
importance of pyrrolidines [3] has also inspired
the development of [3 + 2] azomethine ylide cy-
cloadditions. In this context, we have examined
[3 + 2] cycloaddition reactions of indole-2,3-dione
and benzo[β]thiophene-2,3-dione with various
secondary cyclic amino acids [4]. However, the
work on 9,10-phenanthrenequinone has remained
unexplored. The fact that phenanthrenequinone
derivatives have been reported to possess strong
antioxidant properties [5] including free radical
scavenging activity and can reduce lipid peroxi-
dation prompted us to investigate cycloaddition
reactions of azomethine ylides derived from
phenanthrenequinone and various pharmacologi-
cally active secondary cyclic amino acids, namely
thiazolidine-4-carboxylic acid (TCA), L-proline and
piperidine-2-carboxylic acid (PCA) [6]. Literature
survey revealed that TCA exhibits strong antioxidant
properties [7]. It has been reported as an intracellu-
lar sulfhydryl antioxidant and free radical scavenger
[8]. Dietary supplementation with thiaproline
improves immune response (leukocyte function)
[9] as well as stimulates the phagocytic process
of macrophages [10], and neutralization of the
superoxide radical is also observed [11]. Therefore,
1
namely IR, H NMR, and 13C NMR spectroscopies as
well as mass spectrometry. Some of the synthesized
cycloadducts showed moderate antioxidant activ-
ꢁ
C
ity.
2010 Wiley Periodicals, Inc. Heteroatom Chem
20:379–392, 2009; Published online in Wiley InterScience
(www.interscience.wiley.com). DOI 10.1002/hc.20562
Correspondence to: R. T. Pardasani; e-mail: rtpardasani@
gmail.com
Contract grant sponsor: Department of Atomic Energy/Board
of Research in Nuclear Sciences, Mumbai, India.
Contract grant sponsor: University Grants Commission,
New Delhi, India.
c
ꢀ
2010 Wiley Periodicals, Inc.
379

380 Arora et al.
any heterocyclic system incorporating these two
moieties might be expected to have significant
biological activities. The results are reported herein.
in the presence of various dipolarophiles, namely
phenyl acetylene, diphenyl acetylene, 1-phenyl-1-
propyne, ethyl phenyl propiolate thereby producing
azabicycloadducts 4a–7c in 67%–80% yields.
Repeating the reaction with methyl acrylate af-
forded 8a–c in 73%–78% yields, respectively. The
product distribution is given in Table 1.
RESULTS AND DISCUSSION
The reaction of phenanthrenequinone
1 with
(R)-thiazolidine-4-carboxylic acid (TCA) 2a, L-
pyrrolidine-2-carboxylic acid (L-proline) 2b, and
PCA 2c in equimolar ratio in refluxing acetoni-
trile for 22 h generated, in situ, azomethine ylide
(Scheme 1) 3a–c, which was trapped as cycloadduct
The mechanism for the formation of cy-
cloadducts involves the formation of intermediate
azomethine ylide (amy) 3a–c formed during the reac-
tion by the loss of CO2 via a stereospecific cyclorever-
sion [12], which subsequently undergoes 1,3-dipolar
O
O
X
n
H
COOH
N
H
2a; X = S, n = 1
2b; X = CH2, n = 1
2c; X = CH2, n = 2
1
CH3CN, 22 h
reflux
2
2
n
H
X
O
N
3a–c
Ph
C
C
R
CH2=CHCOOMe
R=H, Ph, Me, COOEt
R
H
H
n
n
MeOOC
O
Ph
O
X
X
N
N
8a–c
4a–7c
SCHEME 1 Reaction of 9,10-pheanthrenequinone with α-amino acids (2a–c) in the presence of different dipolarophiles.
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Stereoselective Synthesis and Antioxidant Activity of Azabicycloadducts Derived from 9,10-Phenanthrenequinone 381
TABLE 1 Product Distribution of Synthesized Azabicycloadducts
Cycloadduct Amino Acid Dipolarophile Ph C C R, R = Cycloadduct Amino Acid Dipolarophile Ph C C R, R =
4a
4b
4c
5a
5b
5c
2a
2b
2c
2a
2b
2c
H
H
H
Ph
Ph
Ph
6a
6b
6c
7a
7b
7c
2a
2b
2c
2a
2b
2c
Me
Me
Me
COOEt
COOEt
COOEt
cycloaddition reactions with various dipolarophiles,
giving regiospecific spiro compounds. The results
are in good harmony with the observation of Grigg
et al. [13, 14] for the reaction of carbonyl com-
pounds with amines as well as with the theoretical
calculations.
AM1 Hamiltonians to understand the stereochemi-
cal course of the cycloaddition reaction. Geometry
optimization of azomethine ylide 3a indicated that
it has an almost planar structure (Fig. 1).
Instead of having an envelope shape, the thiapro-
line ring is planar and lies in the same plane as that
of phenanthrenequinone ring. It may exist as two
conformers, one in which the >C O group and C H
of the dipole are syn to each other 3asyn and the other
one in which these two groups are anti 3aanti (Fig. 2).
Phenyl acetylene may approach either of the
azomethine ylide (amy) syn or anti with the forma-
tion of products having two chiral centers. There-
fore, a total of 4 + 4 = 8 stereoisomers could be pos-
sible (Fig. 3).
Attack of phenyl acetylene on anti-azomethine
ylide (anti amy) results in the inward movement
of thiazolidine ring toward the phenanthrene nu-
cleus, and the transition state (TS) could not be lo-
cated even in a single case (Fig. 2). It may be due
to the steric hindrance between the quinone nu-
cleus and the thiaproline ring that makes it unstable
and hence fails to produce transition state geome-
try, ruling out the possibility of formation of prod-
ucts [4a(v)–4a(viii)]. Thus it leaves the possibility
of attack on only the syn azomethine ylide (amy)
and hence only four isomers 4a(i)–4a(iv) are left for
consideration. Out of these four possibilities, only
The structure of compounds has been estab-
lished from their spectral data. The IR spectrum of
the typical phenyl acetylene cycloadduct 4a (where
R = H) showed characteristic bands at 1675, 1290,
and 650 cm−1 for >C O, C N, and C S stretching vi-
brations, respectively. Its 1H NMR spectrum showed
a quartet at δ 1.86 for 5-H, a doublet at δ 2.25 for 4-H,
a broad multiplet at δ 2.90 for 2-H + 6-H; aromatic
protons were seen as multiplet in the range δ 7.26–
8.21 ppm. Its 13C NMR spectrum displayed a signal
at 180.2 for carbonyl carbon, aromatic carbons ap-
peared in the range δ 136.0–128.3 ppm, the olefinic
carbons appeared at δ 128.2 and 123.9, and spiro car-
bon appeared at δ 89.5, C-5 at δ 66.6, C-2 at δ 59.0 and
C-4 at 49.0 ppm. Additional evidence was gathered
from its mass spectrum. The molecular ion peak and
base peaks were present at m/z 381 (35%) and m/z
279 (100%), respectively. The mass fragmentation
pattern has been depicted in Scheme 2. Physical and
spectral data have been given in the Experimental
section, respectively.
MOLECULAR ORBITAL ANALYSIS
Detailed semiempirical molecular orbital studies
were conducted employing MOPAC 6 program on
Ph–C
(–102)
CH
C25H19NOS
m/z= 381, 35%
C17H13NOS
m/ z= 279,100%
–C = O
–C12H11NS
–C6H4
C24H19NOS
C12H8
C6H4
m/z= 353, 43%
m/z= 152,40%
m/z= 76, 32%
SCHEME 2 Mass fragmentation pattern of phenyl acetylene
cycloadduct 4a.
FIGURE 1 AM1-optimized geometry of azomethine ylide 3a.
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382 Arora et al.
Inward movement
H
S
Ph-C
C-H
S
Ph–C C–H
Outward movement
N
O
N
O
H
Front-side attack
Back-side attack
3asyn
3aanti
FIGURE 2 Mode of attack of dipolarophile (phenyl acetylene) on azomethine ylide.
two have concerted mechanism 4a(i) and 4a(ii) and
we could optimize the transition state in the case
of 4a(i) only. This can be explained using the fron-
tier molecular orbitals (FMO) approach along with
the endo approach of the phenyl ring. The favored
The ꢀH , HOMO, LUMO energies, and HOMO–
LUMO energy gaps of azomethine ylides 3a–c
with dipolarophiles are given in Table 2. From
f
the table, it may be concluded that HOMOdipole
–
LUMOdipolarophile energy gap is lower than the
LUMOdipole–HOMOdipolarophile gap and therefore
path involves the HOMOdipole and LUMOdipolarophile
.
The transition state of the concerted 1,3-dipolar cy-
cloadditions is usually controlled by FMOs of dipo-
larophiles and dipole (azomethine ylide).
the dominant FMO approach is HOMOdipole
LUMOdipolarophile
–
.
Both the HOMO and the LUMO of the dipole
show uneven distribution of the electron density
along the C N C dipole. In the HOMO case, the
orbital coefficient is larger at C1 (0.229) than at C2
(−0.162). Similarly in the LUMO of phenyl acetylene,
the atomic orbital coefficient on the C-atom bearing
the phenyl group is larger (0.202) than that of the
atom away from it (−0.142). Thus, there is a better
orbital overlap between C1 of azomethine ylide and
the C-atom bearing the phenyl group. (Fig. 4)
H
Ph
H
H
H
H
Ph
O
H
O
Ph
O
S
S
S
N
N
N
This results in the formation of product 4a(i),
thus ruling out the possibility of 4a(ii), in which case
we could not optimize the transition state. Besides
a secondary interaction between the two phenyl
rings, the endo approach also favors the formation
of product 4a(i). The AM1-optimized geometry of
cycloadduct 4a and energy profile diagram for the
4a (i)
4a (ii)
4a (iii)
S
S
Ph
H
H
H
H
Ph
H
O
N
O
N
S
O
N
S
Ph
H
4a (iv)
4a (v)
4a (vi)
S
H
H
H
Ph
N
N
O
O
Ph
H
4a (vii)
FIGURE 3 Possible stereoisomers of cycloadduct 4a.
4a (viii)
FIGURE 4 Atomic orbital coefficients and overlapping of
dipole (3a) with dipolarophile (phac).
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Stereoselective Synthesis and Antioxidant Activity of Azabicycloadducts Derived from 9,10-Phenanthrenequinone 383
TABLE 2 ꢀHf, HOMO, LUMO Energies and H-L and L-H Energy Gaps
Energy Gaps (ev)
ꢀHf (Kcal/mol)
HOMO (ev)
LUMO (ev)
H-L
L-H
Dipole azomethine ylide (3a)
Dipolarophiles
74.85
−7.71
−0.97
–
–
phac
diph
phpr
etph
74.65
97.81
64.88
−10.80
−65.98
59.72
−9.39
−8.75
−9.08
−9.71
−11.06
−7.44
−0.07
−0.44
−0.03
−0.66
−0.06
−0.60
7.64
7.27
7.68
7.05
7.65
–
8.42
7.78
8.01
8.74
10.09
–
meac
Dipole azomethine ylide (3b)
Dipolarophiles
phac
diph
phpr
etph
74.65
97.81
64.88
−10.80
−65.98
48.14
−9.39
−8.75
−9.08
−9.71
−11.06
−7.38
−0.07
−0.44
−0.03
−0.66
−0.06
−0.58
7.37
7.00
7.41
6.78
7.38
–
8.79
8.15
8.48
9.11
10.46
–
meac
Dipole azomethine ylide (3c)
Dipolarophiles
phac
diph
phpr
etph
meac
74.65
97.81
64.88
−9.39
−8.75
−9.08
−9.71
−11.06
−0.07
−0.44
−0.03
−0.66
−0.06
7.31
6.94
7.35
6.64
7.32
8.80
8.16
8.50
9.13
10.48
−10.80
−65.98
phac = phenyl acetylene; diph = diphenyl acetylene; phpr = 1-phenyl-1-propyne; etph = ethylphenyl propiolate; meac = methyl acrylate.
cycloadducts 4a, 5a, 4b, 5b, 4c, and 5c have been
depicted in Figs. 5 and 6, respectively.
2. The dominant FMO approach is HOMOdipole
−
LUMOdipolarophile as this energy gap is lower than
ꢀH -R, ꢀH -TS, ꢀH -P, Ea, and stabilization
energy of azomethine ylide with different dipo-
larophiles are presented in Table 3.
Parallel calculations have been performed on
other cycloadducts, and the following conclusions
may be drawn:
the LUMOdipole − HOMOdipolarophile gap.
f
f
f
3. The endo approach is favored, and the phenyl
group lies toward the phenanthrene ring.
ANTIOXIDANT ACTIVITY
1. Geometry optimization of the azomethine ylide
3a indicated that it has an almost planar struc-
ture. The thiaproline ring, instead of having an
envelope shape, is planar and lies in the same
plane as that of the phenanthrene moiety.
Free radicals are highly reactive species ow-
ing to the presence of unpaired valence shell
electron and possess damaging activity toward
macromolecules such as protein, DNA, and lipids
[15].
Antioxidants and radical scavengers have been
employed to study the mechanism of CCl4 toxi-
city as well as to protect liver cells from CCl4-
induced damage by breaking the chain reac-
tion of lipid peroxidation [16]. Therefore, the
present study aimed to establish the antioxidant
potential of azabicycloadducts derived from 9,10-
phenanthrenequinone, using CCl4-induced perox-
idative damage in rats. Initially, a control experiment
was performed with known antioxidant silymarin to
facilitate standardization of the data with following
test compounds.
The two compounds selected for the present
study are compound I: (5S, 8R)-spiro-{6,7-
diphenyl-1-aza-3-thia-bicyclo-[3,3,0]-6-octene-8,1ꢀ-
phenanthrene}-2ꢀ-one (5a) and compound (II):
FIGURE 5 AM1-optimized geometry of phenyl acetylene cy-
cloadduct 4a.
Heteroatom Chemistry DOI 10.1002/hc

384 Arora et al.
30.33 Kcal/mol
(Activation energy)
44.19 Kcal/mol
(Activation energy)
Reactants
3a+ phac
Reactants
3a+ diph
65.26 Kcal/mol
(Stabilization energy)
57.99 Kcal/mol
(Stabilization energy)
4a
product
5a
product
35.43 Kcal/mol
(Activation energy)
40.47 Kcal/mol
(Activation energy)
Reactants
3b+ phac
Reactants
3b+ diph
40.88 Kcal/mol
(Stabilization energy)
69.31 Kcal/mol
(Stabilization energy)
4b
product
5b
product
27.21 Kcal/mol
(Activation energy)
30.45 Kcal/mol
(Activation energy)
Reactants
3c+ phac
Reactants
3c+ diph
29.86 Kcal/mol
(Stabilization energy)
25.50 Kcal/mol
(Stabilization energy)
4c
product
5c
product
FIGURE 6 Energy profile diagram of cycloadducts 4a, 5a, 4b, 5b, 4c, and 5c.
(5S, 8R)-spiro-{6-ethoxycarbonyl-7-phenyl-1-aza-
Results
3-thia -bicyclo-[3,3,0]-6-octene-8,1ꢀ-phenanthrene}-
The results of biochemical and histological param-
eters revealed that the administration of CCl4 to
rats caused significant (p ≤ 0.001) oxidative dam-
age as evidenced by marker enzymes, antioxidant
defense system, and histo-architectural examina-
tions through liver and serum contents (Table 4 and
Figs. 7–11).
Rats treated with CCl4 showed a significant
(p ≤ 0.001) elevation in serum enzymatic activities
of SGOT (serum glutamic oxaloacetic transam-
inase), SGPT (serum glutamic-pyruvic transami-
nase), ALP (alkaline phosphatase), and total biliru-
bin when compared with normal vehicle controls
(group 1). The oral administration of compound
5a and 7a attenuated the CCl4-induced rise in
the SGOT, SGPT, ALP, and total bilirubin levels
(groups IV and V) when compared with group II
(the CCl4-treated group) (Table 4). The attenuation
of enhanced SGOT, SGPT, ALP, and total biliru-
bin level by compounds 5a and 7a was statistically
similar in nature with the standard drug silymarin
(known antioxidant), thus suggesting that the test
compounds are as active as the standard and hence
may prove to be potential antioxidant agents.
2ꢀ-one (7a).
Ph
6
H
4
2
5
Ph
O
3
S
7
8
N
1
2'
3'
5'
10'
9'
4'
6'
7'
8'
5a
C2H5OOC
H
6
4
2
5
Ph
O
3
S
7
8
N
1
2'
3'
5'
10'
9'
4'
6'
7'
8'
7a
Heteroatom Chemistry DOI 10.1002/hc

Stereoselective Synthesis and Antioxidant Activity of Azabicycloadducts Derived from 9,10-Phenanthrenequinone 385
TABLE 3 ꢀHf-R, ꢀHf-TS, ꢀHf-P, Ea, and Stabilization Energy of Azomethine Ylide with Different Dipolarophiles
ꢀHf (kcal/mol)
Compound
Reactants
R
TS
P
Ea (kcal/mol)
Stabilization Energy (kcal/mol)
4a
5a
7a
4b
5b
6b
4c
5c
6c
7c
3a + phac
3a + diph
3a + etph
3b + phac
3b + diph
3b + phpr
3c + phac
3c + diph
3c + phpr
3c + etph
149.50
172.66
64.05
134.37
157.52
124.60
122.79
145.95
113.02
37.34
179.83
216.85
98.42
169.80
197.99
158.28
150.00
176.40
144.50
61.59
84.24
114.67
25.03
93.49
89.21
86.14
90.93
120.45
78.04
7.55
30.33
44.19
34.67
35.43
40.47
33.68
27.21
30.45
31.48
24.25
65.26
57.99
39.02
40.88
69.31
38.46
29.86
25.50
34.98
29.79
phac = phenyl acetylene; diph = diphenyl acetylene; etph = ethylphenyl propiolate phpr = 1-phenyl-1-propyne; Ea = activation energy.
Table 4 depicts that the activities of hepatic
antioxidants such as SOD (superoxide dismutase),
CAT (catalase), GSH (reduced glutathione), and lipid
peroxidation (LPO) were altered significantly (p ≤
0.001) upon CCl4- induction to rats (group II) when
compared with group I (vehicle control). In contrast,
treatment with compounds 5a and 7a (10 mg/kg)
showed a significant restoring effect on CCl4-induced
peroxidative damage (groups IV and V). The restora-
tion of hepatic peroxidative damage by compounds
5a and 7a at the dose level of 10 mg/kg statistically
showed equal protection vis-a`-vis as good as sily-
marin (10 mg/kg) (group III of known antioxidant).
Histology of the liver sections of normal con-
trol animals (Fig. 7) showed normal hepatic cells
FIGURE 8 Photomicrograph of rat liver section with CCl4
with well-preserved cytoplasm, prominent nucleus,
treatment showing marked steatosis of the hepatocytes with
ballooning degeneration and distended portal vein, mild peri-
nucleolus, and visible central veins. The liver
portal fibrosis, and necrosis at H & E × 100.
FIGURE 9 Photomicrograph of rat liver section of CCl4
+
FIGURE 7 Photomicrograph of control rat liver section show-
ing well brought central vein, hepatic cells with preserved cy-
toplasm, and prominent nucleus at hematoxylin and eosin (H
& E) × 100.
silymarin (10 mg/kg body weight), showing histological pat-
tern almost similar to liver of vehicle-treated rats with pre-
served cytoplasm and prominent nucleus at H & E × 100.
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386 Arora et al.
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Stereoselective Synthesis and Antioxidant Activity of Azabicycloadducts Derived from 9,10-Phenanthrenequinone 387
inhibiting the lipid peroxidation and by many other
mechanisms and thus preventing diseases [17]. CCl4-
induced hepatotoxicity begins with the changes in
endoplasmic reticulum, which result in the loss of
metabolic enzymes located in the intracellular struc-
tures [18]. The toxic metabolite CCl•3 radical is pro-
duced, which further reacts with oxygen to give
trichloromethyl peroxy radical. Cytochrome P 450
2E1 is the enzyme responsible for this conversion.
This radical binds covalently to the macromolecules
and causes peroxidative degradation of lipid mem-
brane of the hepatocytes. In this view, the reduction
in the levels of SGOT and SGPT by the standard drug
silymarin as well as with the tested compounds 5a
and 7a is an indication of stabilization of plasma
membrane as well as repairing of hepatic tissue
damage caused by CCl4. This effect is in agreement
with commonly accepted view that serum levels of
transaminases return to normal with the healing
of hepatic parenchyma and regeneration of hepa-
tocytes [19]. Alkaline phosphatase is the prototype
of these enzymes that reflects the pathological alter-
ation in biliary flow [20]. CCl4-induced elevation of
this enzymatic activity in the serum is in line with
high level of serum bilirubin content. Silymarin and
both tested compounds induced suppression of the
increased serum ALP activity with the concurrent
depletion of raised bilirubin suggests the possibility
of the compounds 5a and 7a to have ability to sta-
bilize biliary dysfunction in rat liver during hepatic
injury with CCl4. Thus, administration of silymarin
and both compounds revealed its hepatoprotective
activity against the toxic effect of CCl4, which was
also supported by histological studies.
The measurement of LPO is a convenient
method to monitor oxidative cell damage. Because
the lipid peroxidation is accelerated when free radi-
cals are formed and scavenging of free radicals is one
of the major antioxidation mechanisms to inhibit the
chain reaction of LPO. This indicated the antilipid
peroxidation and adaptive nature of the systems as
brought about by silymarin as well as both com-
pounds against the damaging effects of free radicals
produced by CCl4. During hepatic injury, superox-
ide radicals are generated at the site of damage and
modulate SOD and CAT, resulting in the loss of activ-
ity and accumulation of superoxide radicals, which
damage the liver. Decreased CAT activity is linked
to exhaustion of the enzyme as a result of oxidative
stress caused by CCl4.
FIGURE 10 Photomicrograph of rat liver section of CCl4
+
compound 5a (10 mg/kg body weight), showing considerable
reduction in necrosis and fatty changes with pyknotic nuclei
and cytoplasmic clearing at H & E × 100.
sections of CCl4-intoxicated rats showed massive
fatty changes, necrosis, ballooning, degeneration,
and broad infiltration of the lymphocytes and loss of
cellular boundaries (Fig. 8). The histological archi-
tecture of the liver sections of the rats treated with
silymarin and test compounds 5a and 7a showed
more or less normal lobular pattern with a mild de-
gree of fatty changes, necrosis, and lymphocyte in-
filtration almost comparable to the normal control
(Figs. 9–11).
Discussion
Antioxidants may offer resistance against the per-
oxidative damage by scavenging the free radicals,
Reduction in liver GSH in CCl4-treated rats as
observed in this study indicates the damage to the
hepatic cells. Administration of silymarin and cho-
sen compounds 5a and 7a promoted the conver-
sion of GSSG (oxidized glutathione) into GSH by
FIGURE 11 Photomicrograph of rat liver section of CCl4
compound 7a (10 mg/kg body weight), showing moderately
regeneration in hepatocellular architecture at H & E × 100.
+
Heteroatom Chemistry DOI 10.1002/hc

388 Arora et al.
are reported in wave numbers (cm−1). The
1H NMR spectra and 13C NMR spectra have
been recorded on a Bruker DRX-300 MHz and
75.47 MHz models, respectively in CDCl3 and
(DMSO, 300 MHz) using tetramethylsilane as an in-
ternal standard. The chemical shifts (δ) are given in
ppm. The mass spectra were recorded on a JEOL-
SX 102 (FAB). Most of the spectra were recorded at
Central Drug Research Institute, Lucknow, India. El-
emental analyses were performed on a Perkin Elmer
series C, H, N, S analyzer 2400. The solvents were
purified by standard procedures [24, 25]. The opti-
cal rotations 20[α]D were measured on a D line of Na
lamp at 5893 A wavelength, at 20 C in 1 g concentra-
tion using chloroform as solvent. Acetonitrile was
dried by heating at reflux temperature with anhy-
drous calcium chloride for 5–6 h and then distilled it.
Phenanthrenequinone, TCA, L-proline, and pipecol-
inic acid were purchased from Merck and Aldrich
and used as supplied. Column chromatography was
performed on silica gel 60 (Merck).
the reactivation of hepatic glutathione reductase en-
zyme, and SOD and CAT activities were brought to
near normal levels in CCl4-treated rats. The avail-
ability of sufficient amount of GSH thus increased
the detoxification of reactive metabolites of CCl4
through the involvement of glutathione peroxidase
[21]. Therefore, the restoration of SOD, CAT, and
GSH levels after the enduration of both compounds
to such CCl4-treated rats account for the antioxidant
efficacy against free radicals. It might be attributed
to the peculiar oxidizing power of both compounds,
which can be explained on the basis of their strained
structures [22]. The two benzene rings in phenan-
threnequinone moiety are coaxial and owing to the
normal spatial requirements of the carbonyl group;
the diphenyl skeleton suffers a distortion or twisting
which produces a strain within the molecule.
CCl4-treated rats may potentiate focal hepato-
cyte damage and degeneration (Fig. 8). It is provoked
by the increased production of a high reactive in-
termediate of CCl4 like trichloromethyl free radical
(CCl•3), which is normally detoxified by endogenous
glutathione, SOD, and CAT but in excess it may de-
plete these antioxidants, allowing the reactive inter-
mediate to react with and destroy the hepatic cells
and other cells [23].
◦
˚
Computational Details
All calculations were carried out at MOPAC 6 pro-
gram using AM1 Hamiltonians on PCL-Pentium 4,
PC model for Windows, version 5.13 Serena soft-
ware was used as a graphical interface for drawing
and visualizing all structures and for preparing in-
put files for MOPAC 6. The energy terms are given in
kcal/mol.
These changes were very much reduced
histopathologically in rats treated with CCl4 and sily-
marin (Fig. 9) as well as with CCl4 and compound 5a
(Fig. 10) and compound 7a (Fig. 11) treated rats. As
the alterations produced in the antioxidants level in-
dicate involvement of deleterious oxidative changes,
increased levels of these antioxidants would there-
fore be important in protection against toxicity.
Synthesis of Compounds 4a−8c
A
Representative Method for the Synthesis
of Cycloadduct (5S, 8R)-Spiro-{7-phenyl-1-aza-3-
thia-bicyclo[3,3,0]-6-octene-8,1ꢀ-phenanthrene-2ꢀ-one
(4a). A mixture of phenanthrenequinone 1 (0.416 g;
2.0 mmol), thiazolidine-4-carboxylic acid (0.266 g;
2.0 mmol), and phenyl acetylene (0.219 g, 2.0 mmol)
in equimolar amount was heated at reflux temper-
ature under a nitrogen atmosphere for 22 h in dry
acetonitrile. After completion of the reaction as
monitored by TLC, unreacted acid was removed by
filtration. The filtrate was evaporated in vacuo to
half of its volume. The crude product so obtained
was purified using column chromatography over
silica gel, whereby chloroform/ethyl acetate (3:1)
fraction afforded the cycloadduct 4a as bright
CONCLUSION
It may be concluded that biochemical alterations ob-
served in hepatic damage seems to be mainly due to
an oxy-radical-mediated mechanism, involving lipid
peroxidation, under conditions of reduced antioxi-
dant levels that scavenge superoxide, hydrogen per-
oxide, and lipid peroxides. The results yet available
are encouraging vis-a`-vis standard drug silymarin
and suggest that compounds 5a and compound 7a
may be helpful in quenching free radicals by oxi-
dizing them and induction of an in vivo antioxidant
defense system.
1
yellow crystalline solid in 70% yield; mp 200◦C; H
EXPERIMENTAL
NMR ((DMSO, 300 MHZ), 300 MHz) δ: 1.86 (d, 5-H,
1H), 2.25 (d, 4-H, 2H), 2.90 (br m 2-H + 6-H, 4H),
7.26–8.21 (m, 13H, 5 ArH + 3ꢀ-H to 10ꢀ-H). 13C NMR
(DMSO, 75.47 MHz) δ: 180.2 (>C O), 136.0–128.3
(6 ArC + C-3ꢀ–C-10ꢀ), 128.2, 123.9 (C C), 89.5 (C-8),
The uncorrected melting points were taken in
open glass capillaries. The IR spectra were
recorded on a Nicolet Magna IR spectrometer
model 550 in KBr pellets, and band positions
Heteroatom Chemistry DOI 10.1002/hc

Stereoselective Synthesis and Antioxidant Activity of Azabicycloadducts Derived from 9,10-Phenanthrenequinone 389
66.6 (C-5), 59.0 (C-2), 49.0 (C-4) ppm. IR (KBr)
(5S, 7S, 8R)-Spiro-{7-methoxycarbonyl-1-aza-3-
thia-bicyclo-[3,3,0]-octane-8,1ꢀ-phenanthrene}-2ꢀ one
(8a). The cycloadduct was obtained in 73% yield
as dark brown crystals; mp 182◦C; 1H NMR (DMSO,
300 MHz) δ: 0.85 ((d, 6-H, 2H), 1.25 (t, 4-H, 2H), 1.84
(t, 7-H, 1H), 2.10 (dd, 2-H, 2H), 2.62 (s, OCH3, 3H),
4.12 (m, 5-H, 1H), 7.39 (t, J = 7.23 Hz, 5ꢀ-H + 8ꢀ-H,
2H), 7.72 (t, J = 8.13 Hz, 4ꢀH + 9ꢀ H, 2H), 8.01 (d,
J = 8.23 Hz, 3ꢀ-H + 10ꢀ-H, 2H), 8.19 (d, J = 7.69 Hz,
6ꢀ-H + 7ꢀ-H, 2H) ppm.
(νmax) cm−1: 3050, 1675, 1290, 650. Mass m/z: 381
[M+] (35%), 353 [M+ − CO] (43%), 279 [M+
−
C8H6] (100%), 152 [M+ − C13H11NOS] (40%). 20[α]D
(CHCl3) = 50. Elemental anal. (%) Calcd: C, 78.74;
H, 4.98; N, 3.67; Found: C, 78.82; H, 4.72; N, 3.45.
(5S,8R)-Spiro-{6,7-diphenyl-1-aza-3-thia-bicy-
clo-[3,3,0]-6-octene-8,1ꢀ-phenanthrene}-2ꢀ-one (5a).
A mixture of phenanthrenequinone (1) (0.41 g,
2.0 mmol), thiazolidine-4-carboxylic acid (2a)
(0.26 g, 2.0 mmol), and diphenyl acetylene (0.35 g;
2.0 mmol) in the molar ratio 1:1:1 was heated at
reflux temperature under nitrogen atmosphere for
22 h in dry acetonitrile (50 mL). The completion of
the reaction was judged by TLC. The unreacted acid
was filtered off, and the filtrate was evaporated in
vacuo to half of its volume. After failing in several
attempts to crystallize the product from various
solvent mixtures, the product was purified using
column chromatography over silica gel whereby
cycloadduct 5a was obtained as pale yellow crystals
in 75% yield; mp 156◦C; 1H NMR ((DMSO, 300
MHz) δ: 1.86 (d, 4-H, 2H), 2.89 (s, 2-H, 2H), 4.97 (t,
5-H, 1H), 7.20–8.21 (m, 18ArH) ppm. IR (KBr), νmax
(cm−1): 3065, 1675, 1285, 930, 680. 20[α]D (CHCl3) =
47. Elemental anal. (%) Calcd: C, 81.40; H, 5.03; N,
3.06; Found: C, 80.92; H, 4.89; N, 2.83.
IR (KBr) νmax (cm−1): 3075, 1690, 1650, 1275,
1230, 930, 675. 20[α]D (CHCl3) = 51. Elemental anal.
(%) Calcd.: C, 69.04; H, 5.20; N, 3.83; Found: C, 68.42;
H, 4.99; N, 3.74.
(2R, 5S)-Spiro-{3-phenyl-1-aza-bicyclo-[3,3,0]-3-
octene-2,1ꢀ-phenanthrene}-2ꢀ-dione (4b). The com-
pound was obtained in 75% yield as shining brown
1
crystals; mp 120◦C; H NMR (DMSO, 300 MHz) δ:
1.25 (m, 6-H, 2H), 2.08 (m, 7-H, 2H), 2.72 (t, 8-
H, 2H), 3.07 (q, 5-H, 1H), 3.49 (d, 4-H, 1H), 7.25–
8.36 (m, 13 H, 5 ArH+ 3ꢀ-H to 10ꢀ-H) ppm. IR
(KBr) νmax (cm−1): 3030, 2920, 1700, 1300, 790. 20[α]D
(CHCl3) = 54. Elemental anal. (%) Calcd.: C, 85.95;
H, 5.78; N, 3.85; Found: C, 85.29; H, 5.62; N, 3.79.
(2R, 5S)-Spiro-{3,4-diphenyl-1-aza-bicyclo-[3, 3,
0]-3-octene-2,1ꢀ-phenanthrene}-2ꢀ-one (5b). The cy-
cloadduct was obtained as black crystals in 76%
yield; mp 178◦C; 1H NMR (DMSO, 300 MHz) δ: 1.64
(m, 7-H, 2H), 1.89 (t, 6-H, 2H), 2.05 (t, 5-H, 1H),
4.67 (t, 8-H, 2H), 7.33–8.09 (m, 18ArH) ppm. IR
(KBr) νmax (cm−1): 3020, 2910, 1680, 1400, 800. 20[α]D
(CHCl3) = 62. Elemental anal. (%) Calcd.: C, 87.47;
H, 5.69; N, 3.18; Found: C, 87.13; H, 5.56; N, 2.91.
(5S,8R)-Spiro-{6-methyl-7-phenyl-1-aza-3-thia-
bicyclo-[3,3,0]-6-octene-8,1ꢀ -phenanthrene}-2ꢀ -one
(6a). The cycloadduct was obtained in 76% yield
1
as brown solid; mp 144◦C; H NMR (DMSO, 300
MHz) δ: 1.76 (d, 4-H, 2H), 2.25 (s, CH3, 3H), 2.89
(br m, 2-H, 2H), 3.86 (d, 5-H, 1H), 7.29 (t, 5ꢀH +
8ꢀH), 7.50 (m, 5 ArH), 7.70 (t, 4ꢀH + 9ꢀH), 8.01 (d,
3ꢀH + 10ꢀH), 8.18 (d, 6ꢀH + 7ꢀH) ppm. 13C NMR
(DMSO, 75.47 MHz) δ: 180.1 (>C O), 135.9–129.4
(6 ArC + C-3ꢀ–C-10ꢀ), 128.5, 123.8 (C C), 84.1 (C-8),
62.1 (C-5), 52.0 (C-2), 39.8 (C-4), 14.0 (CH3). IR
(KBr) νmax (cm−1): 3055, 1675, 1280, 920, 660. 20[α]D
(CHCl3) = 42. Elemental anal. (%) Calcd.: C, 78.98;
H, 5.31; N, 3.54; Found: C, 78.13; H, 5.12; N, 3.24.
(2R, 5S)-Spiro-{4-methyl-3-phenyl-1-aza-bicyclo-
[3,3,0]-3-octene-2,1ꢀ-phenanthrene}-2ꢀ-one
(6b).
The cycloadduct was obtained in 77% yield as coffee
brown solid; mp 192◦C; 1H NMR (DMSO, 300 MHz)
δ: 0.88 (m, 7-H, 2H), 1.29 (m, 6-H, 2H), 2.05 (s, CH3,
3H), 2.20 (t, 8-H, 2H), 3.48 (t, 5-H, 1H), 7.14–8.08
(m, 13ArH). IR (KBr) νmax (cm−1): 3010, 1690, 1275,
820. 20[α]D (CHCl3) = 59. Elemental anal. (%) Calcd:
C, 85.94; H, 6.10; N, 3.71; Found: C, 84.79; H, 5.87;
N, 3.59.
(5S,8R)-Spiro-{6-ethoxycarbonyl-7-phenyl-1-aza-
3-thia-bicyclo-[3,3,0]-6-octene-8,1ꢀ-phenanthrene}-2ꢀ-
one (7a). The compound was obtained as black
solid in 70% yield; mp 154◦C; 1H NMR (DMSO,
300 MHz) δ: 1.36 (t, CH3, 3H), 2.00 (d, 4-H, 2H), 2.92
(br s, 2-H, 2H), 3.94 (d, J = 6.89 Hz, 5-H, 1H), 4.30
(q, J = 7.16 Hz, OCH2, 2H), 7.26–8.18 (m, 13ArH).
IR (KBr) νmax (cm−1): 3060, 1680, 1670, 1280, 930,
660. 20[α]D (CHCl3) = 40. Elemental Anal. (%) Calcd:
C, 74.17; H, 5.07 N, 3.09; Found: C, 75.22; H, 4.97;
N, 2.99.
(2R,5S)-Spiro-{4-ethoxycarbonyl-3-phenyl-1-aza-
bicyclo-[3,3,0]-3-octene-2,1ꢀ -phenanthrene}-2ꢀ-one
(7b). The cycloadduct was obtained as brown
1
powder in 74% yield; mp 188◦C; H NMR (DMSO,
300 MHz) δ: 1.24 (q, CH3, 3H), 1.43 (m, 6-H + 7-H,
4H), 1.69 (t, 8-H, 2H), 3.71 (q, J = 6.96 Hz, OCH2,
2H), 4.30 (t, J = 7.14 Hz, 5-H, 1H), 7.39 (m, 5 ArH),
7.47 (t, 5ꢀH + 8ꢀH, 2H), 7.73 (t, 4ꢀH + 9ꢀH, 2H),
Heteroatom Chemistry DOI 10.1002/hc

390 Arora et al.
8.02 (d, 3ꢀH + 10ꢀH, 2H), 8.19 (d, 6ꢀH + 7ꢀH, 2H).
13C NMR (DMSO, 75.47 MHz) δ: 190.1 (>C O),
180.2 (O C O), 135.9–123.1 (6 ArC + C-3’–C-10’),
122.2, 121.5 (C C), 86.5 (C-2), 66.0 (OCH2), 62.0
(C-5), 49.8 (C-8), 41.8 (C-6), 35.7 (C-7), 19.1 (CH3).
IR (KBr) νmax (cm−1): 3075, 2950, 1700, 1680, 1270,
(6S,9R)-Spiro-{7-methyl-8-phenyl-1-aza-bicyclo-
[4,3,0]-7-nonen-9,1ꢀ-phenanthrene}-2ꢀ-one
(6c).
The compound was obtained as camel yellow
1
crystals in 67% yield; mp 200◦C; H NMR (DMSO,
300 MHz) δ: 1.25 (s, 2-H, 2H), 1.35 (m, 3-H + 4-H,
4H), 1.64 (br m, 5-H, 2H), 2.01 (s, CH3, 3H), 4.29 (t,
6-H, 1H), 7.38 (m, 2 × m-ArH + 1 × p-ArH, 3H), δ:
7.49 (t, 5ꢀH + 8ꢀH, 2H), 7.58 (d, 2 × o-ArH, 2H), 7.71
(t, 4ꢀH + 9ꢀH, 2H), 8.03 (d, 3ꢀH + 10ꢀH, 2H), 8.21
(d, 6ꢀH + 7ꢀH, 2H) ppm IR (KBr) νmax (cm−1): 3050,
1655, 1240, 770. 20[α]D (CHCl3) = 48. Elemental
Anal. (%) Calcd: C, 85.93; H, 6.39; N, 3.58; Found:
C, 85.23; H, 6.19; N, 3.42.
1190, 800. Mass m/z: 435 [M+] (50%), 261 [M+
−
C11H10O2] (100%), 233 [M+ − C12H10O3] (55%), 180
[M+ − C16H17NO2] (65%), 152 [M+ − C17H17NO3]
(45%). 20[α]D (CHCl3) = 60. Elemental anal. (%)
Calcd: C, 80.01; H, 5.74; N, 3.21 Found: C, 79.75; H,
5.54; N, 3.18.
(2R, 3R, 5S)-Spiro-{3-methoxycarbonyl-1-aza-
bicyclo-[3,3,0]-octane-2,1ꢀ-phenanthrene}
-2ꢀ-one
(6S,
9R)-Spiro-{7-ethoxycarbonyl-8-phenyl-1-
(8b). The compound was obtained as dark brown
solid in 74% yield; mp 130◦C; 1H NMR (DMSO, 300
MHz) δ: 1.52 (s, 7-H + 3-H, 4H), 1.85 (m, 6-H, 2H),
2.02 (m, 5-H + 4-H, 3H), 2.64 (s, 8-H, 2H), 2.77 (s,
OCH3, 3H), 7.35 (t, J = 7.04 Hz, 5ꢀH + 8ꢀH, 2H),
7.59 (t, J = 8.26 Hz, 4ꢀH + 9ꢀH, 2H), 7.98 (d, J =
8.42 Hz, 3ꢀH + 10ꢀH, 2H), 8.08 (d, J = 7.55 Hz, 6ꢀH
+ 7ꢀH, 2H). IR (KBr) νmax (cm−1): 3040, 2950, 1720,
1700, 1375, 1230. 20[α]D (CHCl3) = 48. Elemental
anal. (%) Calcd: C, 76.08; H, 6.05; N, 4.03; Found: C,
75.94; H, 5.93; N, 3.91.
aza-bicyclo-[4,3,0]-7-nonen-9,1ꢀ -phenanthrene}-2ꢀ-
one (7c). The compound was obtained as dark
brown solid in 71% yield; mp 154◦C; 1H NMR
(DMSO, 300 MHz) δ: 1.24 (t, CH3, 3H), 1.36 (t,
3-H, 2H), 1.67 (br m, 4-H + 5-H, 4H), 2.02 (d, 2-H,
2H), 3.71 (q, J = 6.96 Hz, OCH2, 2H), 4.29 (t, J =
7.24 Hz, 6-H, 1H), 7.36 (m, 2 × mArH + 1 × p-ArH,
3H), δ: 7.47 (t, 5ꢀH + 8ꢀH, 2H), 7.58 (d, 2 × o-ArH,
2H), 7.75 (t, 4ꢀH + 9ꢀH, 2H), 8.02 (d, 3ꢀH + 10ꢀH, 2H),
8.19 (d, 6ꢀH + 7ꢀH, 2H) ppm. 13C NMR (DMSO, 75.47
MHz) δ: 180.1 (>C O), 174.0 (O C O), 135.9–128.4
(6 ArC + C-3ꢀ–C-10ꢀ), 123.8, 119.5 (C C), 85.9 (C-9),
80.6 (C-6), 70.1 (OCH2), 62.0 (C-2), 54.0 (C-5), 35.7
(C-4), 25.4 (C-3), 14.0 (CH3). IR(KBr) νmax(cm−1):
3055, 2855, 1690, 1670, 1290, 1235, 760. Mass m/z:
449 [M+] (38%), 275 [M+ − C11H10O2] (100%), 192
[M+ − C16H19NO2] (52%), 180 [M+ − C17H19NO2]
(47%), 152 [M+ − C18H19NO3] (34%). 20[α]D (CHCl3)
= 68. Elemental anal. (%) Calcd: C, 80.17; H, 6.01;
N, 3.11; Found: C, 79.92; H, 5.91; N, 2.98.
(6S, 9R)-Spiro-{8-phenyl-1-aza-bicyclo-[4,3,0]-7-
nonen-9,1ꢀ-phenanthrene}-2ꢀ-one (4c). The com-
pound was obtained as brown powder in 74% yield;
mp 182◦C; 1H NMR (DMSO, 300 MHz) δ: 0.79 (m, 3-
H, 2H), 1.80 (s, 4-H, 2H), 2.10 (q, 5-H, 2H), 2.77
(t, 2-H, 2H), 3.71 (q, 6-H, 1H), 4.30 (t, 7H, 1H),
7.26–8.21 (m, 13 H, 5 ArH + 3ꢀ-H to 10ꢀ-H). 13C
NMR (DMSO, 75.47 MHz) δ: 180.7 (>C O), 143.8–
130.1 (6 ArC + C-3ꢀ–C-10ꢀ), 128.8, 124.4 (C C), 85.9
(C-9), 80.6 (C-6), 66.3 (C-2), 47.4 (C-5), 35.8 (C-4),
28.4 (C-3) ppm. IR (KBr) νmax (cm−1): 3060, 2910,
1675, 1235, 775. 20[α]D (CHCl3) = 63. Elemental anal.
(%) Calcd: C, 85.94; H, 6.10; N, 3.71; Found: C, 85.41;
H, 5.88; N, 3.55.
(6S,8S,9R)-Spiro-{8-methoxycarbonyl-1-aza-bic-
yclo-[4,3,0]-nonan-9,1ꢀ-phenanthrene}-2ꢀ-one (8c).
The compound was obtained as orange needles in
72% yield; mp 166◦C; H NMR (DMSO, 300 MHz)
1
δ: 2.30 (d, 5-H, 2H), 2.32 (m, 3-H + 4-H, 4H), 2.38
(t, 2-H, 2H), 2.98 (br m, 7-H + 8-H, 3H), 3.49 (m,
6-H, 1H), 4.64 (s, OCH3, 3H), 7.47 (t, J = 7.34 Hz,
5ꢀH + 8ꢀH, 2H), 7.73 (t, J = 8.27 Hz, 4ꢀH + 9ꢀH, 2H),
8.02 (d, J = 8.23 Hz, 3ꢀH + 10ꢀH, 2H), 8.19 (d, J =
7.71 Hz, 6ꢀH + 7ꢀH, 2H) ppm IR (KBr) νmax (cm−1):
3060, 1680, 1650, 1290, 1220, 760. 20[α]D (CHCl3) =
62. Elemental anal. (%) Calcd: C, 76.45; H, 6.37; N,
3.87; Found C, 76.12; H, 5.96; N, 3.79.
(6S,9R)-Spiro-{7,8-diphenyl-1-aza-bicyclo-[4,3,
0]-7-nonen-9,1ꢀ-phenanthrene}-2ꢀ-one (5c). The co-
mpound was obtained as chocolate brown solid in
72% yield; mp 170◦C; 1H NMR (DMSO, 300 MHz) δ:
1.25 (q, 5-H, 2H), 1.35 (m, 4-H, 2H), 2.01 (m, 3-H,
2H), 3.34 (t, 2-H, 2H), 4.31 (t, 6-H, 1H), 7.21–8.62
(m, 18ArH) ppm.
IR (KBr) νmax (cm−1): 3060, 2860, 1670, 1235.
20[α]D (CHCl3) = 54. Elemental anal. (%) Calcd: C,
87.41; H, 5.96; N, 3.09; Found: C, 86.92; H, 5.89; N,
2.93.
The cycloadducts 4a–8c are optically active, and
their specific rotations 20[α]D were in the range 40◦–
68◦ using chloroform (CHCl3) as solvent.
Heteroatom Chemistry DOI 10.1002/hc

Stereoselective Synthesis and Antioxidant Activity of Azabicycloadducts Derived from 9,10-Phenanthrenequinone 391
The remaining part of liver was fixed in 10% for-
malin solution, dehydrated with different ethanol
solutions (50%–100%), embedded in paraffin wax
then cut into 5 μm sections and stained with
hematoxylin–eosin dye for photomicroscopic obser-
vations.
Antioxidant Activity Procedure
Methodology.
Animals. Adult male, Wistar strain, albino rats
weighing 150–170 g were used. The animals were
housed in standard laboratory conditions and were
maintained on rat diet (Lipton India Ltd.) and
tap water ad libitum under a natural light–dark
(12L:12D) cycle. The study was approved by the de-
partmental ethical committee.
Statistical Analysis. All results were expressed
as mean
SEM and were analyzed using the stu-
dent’s t-test. A probability value of p ≤ 0.05 was con-
sidered as significant.
Selection of Dose Level of Compounds 5a and 7a.
The compounds 5a and 7a were found to be a prac-
tically nontoxic when administered orally to rats,
and LD50 value of both compounds was found to
be higher than 40 mg/kg body weight. The double
of minimum dose level of both compounds, namely
10 mg/kg body weight, was used for experimentation.
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Experimental Design. After 15 days of acclima-
tization, the animals were divided into following
groups with six rats in each group.
Group I: Vehicle-treated rats were kept on normal
diet and served as control.
Group II: Rats were intoxicated with CCl4
(1.0 mL/kg body weight/once a week with olive
oil, intraperitoneally, 1:1) for 15 days.
Group III: Rats received 10 mg/kg body weight/
day, orally with olive oil of silymarin and CCl4 for
15 days.
Group IV: Rats received 10 mg/kg body
weight/day, orally with olive oil of compound 5a
and CCl4 for 15 days.
Group V: Rats received 10 mg/kg body weight/day,
orally with olive oil of compound 7a and CCl4 for
15 days.
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