Synthesis, anti-HCV, antioxidant, and peroxynitrite inhibitory activity of fused benzosuberone derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.10.033

Reaction of benzosuberone 1 with dimethylformamide-dimethylacetal (DMF-DMA) gives 2-dimethylamino-methylenebenozosuberone 2 which in turn reacts with heterocyclic amines to furnish new heterocyclic ring systems 6-9. Moreover, enaminone 2 reacts with hydrazine hydrate and hydroxylamine hydrochloride to afford the corresponding benzo[6,7]cyclohepta[1,2-c]pyrazole (10) and benzo[6,7]cyclohepta[2,1-d]isoxazole (12), respectively. In addition, the reactions of enaminone 2 with active methylene compounds afforded benzo[6,7]cyclohepta[1,2-b]pyridines (13-18). The X-ray crystallographic analysis of compounds 6 and 16, were recorded. We demonstrated the ability of nine new synthesized compounds to inhibit Hepatitis C Virus (HCV) and Subacute Sclerosing Panencephalitis (SSPE) due to structural similarity between ribavirin and some of the newly synthesized compounds were they contain triazoles and its bioisosters. In addition, the ability of ten synthesized compounds to react with the biologically relevant reactive nitrogen species, peroxynitrite was investigated indirectly by measurement of their ability to inhibit ONOO^--induced tyrosine nitration. The antioxidant activity of these ten compounds was also studied using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay.

Full text of DOI:10.1016/j.ejmech.2009.10.033

European Journal of Medicinal Chemistry 45 (2010) 492–500
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, anti-HCV, antioxidant, and peroxynitrite inhibitory activity of fused
benzosuberone derivatives
,
b
a
c
Thoraya A. Farghaly a , Naglaa A. Abdel Hafez , Eman A. Ragab , Hanem M. Awad ,
*
Mohamed M. Abdalla d
a Department of Chemistry, Faculty of Science, University of Cairo, Giza, Egypt
b Department of Applied Organic Chemistry, National Research Center, Dokki, Cairo, Egypt
c Department of Tanning Material and leather Technology, National Research Center, Dokki, Cairo, Egypt
d Uni-vet Pharmaceuticals Ltd. Balteem, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Reaction of benzosuberone 1 with dimethylformamide–dimethylacetal (DMF–DMA) gives 2-dimethy-
lamino-methylenebenozosuberone 2 which in turn reacts with heterocyclic amines to furnish new
heterocyclic ring systems 6–9. Moreover, enaminone 2 reacts with hydrazine hydrate and hydroxylamine
hydrochloride to afford the corresponding benzo[6,7]cyclohepta[1,2–c]pyrazole (10) and benzo[6,7]cy-
clohepta[2,1–d]isoxazole (12), respectively. In addition, the reactions of enaminone 2 with active
methylene compounds afforded benzo[6,7]cyclohepta[1,2–b]pyridines (13–18). The X-ray crystallo-
graphic analysis of compounds 6 and 16, were recorded. We demonstrated the ability of nine new
synthesized compounds to inhibit Hepatitis C Virus (HCV) and Subacute Sclerosing Panencephalitis
(SSPE) due to structural similarity between ribavirin and some of the newly synthesized compounds
were they contain triazoles and its bioisosters. In addition, the ability of ten synthesized compounds to
react with the biologically relevant reactive nitrogen species, peroxynitrite was investigated indirectly by
measurement of their ability to inhibit ONOOꢀ-induced tyrosine nitration. The antioxidant activity of
these ten compounds was also studied using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay.
Received 21 February 2009
Received in revised form
15 October 2009
Accepted 22 October 2009
Available online 29 October 2009
Keywords:
Benzosuberone
X-ray crystal structure
Hepatitis C virus
1,1-Diphenyl-2-picrylhydrazyl
Antioxidant
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
2. Chemistry
Enaminones are versatile reagents and their utility in hetero-
cyclic synthesis has recently earned considerable attention [1–4].
Benzosuberone derivatives are known as cytotoxic and anticancer
agents [5–8]. In this paper, we continue with our interest in the
development of new and simple methods for the synthesis of
substituted heterocycles from enaminone with multiplet functional
groups with anticipated biological activity [2]. Thus we used the
enaminone 2 namely, 2-dimethylaminomethylene benzosuberone
in the synthesis of a variety of novel fused benzosuberone and
investigated their anti-HCV activities. In addition, the 1,1-diphenyl-
2-picrylhydrazyl (DPPH) free radical scavenging method has been
used to measure the antioxidant property of the newly synthetic
compounds. These compounds were also examined for their ability
to protect against peroxynitrite-dependent nitration reactions.
Benzosuberone 1 reacts with dimethylformamide–dimethylace-
tal (DMF–DMA) and furnished a single product (examined by TLC)
that was identified as 2-dimethylamino-methylenebenzosuberone 2
(Scheme 1). Elemental analyses and spectral data were in complete
accordance with the assigned structure 2. For example, the 1H NMR
spectrum of compound 2 revealed two singlet signals at
d 3.07 and
7.58 ppm characteristic for N,N-dimethylamino and the exocyclic
C]CH protons, respectively [9]. The enaminone 2 was assigned the
E-configuration 2E based on their 1H NMR spectra which revealed
that the exocyclic C]CH proton signal at
E-isomers. On the other hand, Z-isomer 2Z of analogous structures
was reported to appear at 6.9 ppm [10].
d 7.58 ppm correlated for
d
As previously reported enominones can be used as potential
precursors for fused heterocyclic systems when reacting with
heterocyclic amines [11]. Thus, treatment of enaminone 2 with
3-aminotriazole 3, 4-phenyl-3-aminopyrazole 4, and 2-amino-
benzimidazole 5 in refluxing acetic acid gave three new ring systems
namely 9,10,11-trihydrobenzo[60,70]cyclohepta[20,10–e]triazolo[2,3–
a] pyrimidine (6), 9,10,11-trihydrobenzo[60,70]cyclohepta[20,10–
* Corresponding author.
E-mail address: thoraya-f@hotmail.com (T.A. Farghaly).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.033

T.A. Farghaly et al. / European Journal of Medicinal Chemistry 45 (2010) 492–500
493
H
N(Me)
H
O
2
O
O
N(Me)
2
DMF-DMA
reflux, 10h
1
2E
2Z
Scheme 1. Synthesis of enaminone 2.
e]pyrazolo[2,3–a]pyrimidine (8) and 9,10,11-trihydrobenzo[60,70]-
cyclohepta[20,10-e]pyrimido [1,2-a]benzimidazole (9), respectively
(Scheme 2). Condensation of 3-amino-[1,2,4]triazole (3) with enam-
inone 2 can proceed in two possible ways, as shown in Scheme 2. In
route 1, the nucleophilic amino group attacks the double bond via
a Michael addition reaction, followed by elimination of the dime-
thylamino group. Cyclization of intermediate 6a with concurrent
dehydration of 6bgave compound 6. On the otherhand, inroute 2, the
exocyclic amino group of triazole attacks the carbonyl group to
generate isomeric structure 7 via intermediate 7a and 7b. This
N
N
N
N
H
N
H
H
N
N
N
N
N
N
route 1
N
O
HO
N
N
N
H
NH
2
3
6
6b
6a
N
N
N
N
N
N
N
H
N
N
N
N
N
route 2
N(Me)
N(Me)
2
2
H
7
7a
7b
2
Ph
NH
2
Ph
N
Ph
Ph
N
H
N
H
N
N
N
4
N
H
N
N
H
N
N
O
HO
8
8b
8a
N
N
NH
2
N
N
N
H
5
H
N
N
H
N
H
N
N
N
O
HO
9
9b
9a
Scheme 2. Reaction of enaminone 2 with heterocyclic amines.

494
T.A. Farghaly et al. / European Journal of Medicinal Chemistry 45 (2010) 492–500
H
N
N
NH NH .H O
2
2
2
-HNMe
2
-H O
2
O
CH-N(Me)
10
2
O
N
2
NH OH
2
11
N
O
12
Scheme 3. Reaction of enaminone 2 with hydrazine hydrate and hydroxyl amine.
respectively (Scheme 4). The structure of the products were
assigned based on the 1H NMR which shows a characteristic singlet
signals at
d 8.14 and 8.05 ppm characteristic for the pyridine-H of
Fig. 1. Crystal structure of compound 6.
compounds 13 and 14, respectively [17].
Two possible structures 2-iminobenzo[6,7]cyclohepta[1,2–
b]pyran-3-carbonitrile (15) or 1,2-dihydro-2-oxo-benzo[6,7]cyclo-
hepta[1,2–b]pyridine-3-carbonitrile (16) have been proposed for
the reaction of enaminone 2 with malononitrile in refluxing etha-
nolic sodium ethoxide (Scheme 5). Structure 15 was ruled out based
on 1H NMR and IR spectral data. For example, 1H NMR spectrum of
regioselectivity has been unambiguously substantiated through the
X-ray crystallographic analysis of the product 6 rather than product 7
as shown in Fig.1. Typical bond lengths and bond angles are shown in
Table 1.
Reaction of enaminone 2 with hydrazine hydrate in absolute
ethanol resulted in the formation of a single product 10 as exam-
ined by TLC (Scheme 3). Mass spectrum and elemental analysis
showed that the reaction product 10 has the molecular formula
C12H12N2 with molecular ion peak at 184. 1H NMR spectrum,
the product, showed the absence of imine signal at
d 3.07 ppm.
Also, IR spectrum revealed the amidic carbonyl absorption band at
y
¼ 1650 cmꢀ1. Furthermore, the product 16 was substantiated by
independent synthesis from cyanoacetamide and enaminone 2.
The two reaction products were identical in all spectral data
(see Experimental). Also, the X-ray crystal structure indicated that
the reaction of enaminone 2 with each of malononitrile and
cyanoacetamide gave the same structure 16 as shown in Fig. 2. In
Table 2, selected bond angles and bond lengths are reported. In
showed two characteristic singlet signals at
d 7.47 and 12.40 ppm
for pyrazole-H and NH [12–14]. It is assumed that compound 10 is
formed via initial addition of the amino group in hydrazine to the
enamine double bond followed by elimination of dimethylamine
and water to give the final isolable product 10. Similarly, enami-
none 2 reacts with hydroxylamine hydrochloride in absolute
ethanol in the presence of anhydrous sodium acetate to give one
isolable product, namely, benzo[6,7]cyclohepta[2,1–d]isoxazole 12
rather than isomeric form benzo[6,7]cyclohepta[1,2–c]isoxazole 11
(Scheme 3). Structure 12 was assigned as the correct structure on
the basis of its 1H NMR spectrum where a resonance for H-3 of
a similar manner, the enaminone 2 was reacted with
u-cyanoace-
tophenone in acetic acid in the presence of ammonium acetate to
H C
3
COCH
3
COCH
3
CH
N
isoxazole appeared typically at
was ruled out as H-5 of isoxazole would be expected to resonate at
lower field around at 9.20 ppm [15,16].
Reaction of enaminone 2 with acetylacetone and ethyl acetoa-
cetate in glacial acetic acid in the presence of ammonium acetate
gave benzo[6,7]cyclohepta [1,2–b]pyridine derivatives 13 and 14,
d
¼ 8.50 ppm. The isomeric product
2
COCH
3
d
O
CH-N(Me)
2
13
H C
3
COCH
3
COOEt
CH
2
2
Table 1
N
COOEt
Selected bond angles and bond lengths of compound 6.
Bond
Angle
Bond
Lenghth (Å)
N2–N1–C4
N1–N2–C18
C9–N11–C18
C9–N6–C17
C4–C8–C16
127.5
100.5
102.4
116.0
120.3
N1–N2
N2–C18
C9–N11
N6–C17
C4–C8
1.372
1.331
1.329
1.310
1.3
14
Scheme 4. Reaction of enaminone 2 with active methylene compounds.

T.A. Farghaly et al. / European Journal of Medicinal Chemistry 45 (2010) 492–500
495
HN
CN
O
15
CN
CN
CH
2
O
CN
HN
16
CN
CH
2
CONH
2
H N
2
O
CN
CH-N(Me)
2
O
17
2
O
COPh
HN
18
CN
CH
2
HN
O
COPh
COPh
19
Scheme 5. Reaction of enaminone 2 with active methylene nitrile compounds.
give compound 18 (Scheme 5). 3-Benzoyl-5,6,7-trihydrobenzo[6,7]
cyclohepta[1,2-b]pyridine-2(1H)-one (18) was assigned as a reac-
tion product based on correct spectral and analytical data (see
Experimental).
3.1. Results and discussion
3.1.1. Hepatitis C virus (HCV) NS3-4A protease inhibitory activities
in both HCV replicon cells and in hamster brains
In continuation of our work, benzosuberone was allowed to
react with different enaminones 20a–c to give 5,6,7-trihydro-
benzo[6,7]cyclohepta[1,2-b]pyridine derivatives 21a–c (Scheme 6).
The spectral data were found to be in complete agreement with the
proposed structures (see Experimental).
In order to monitor the potential of nine new synthesized
compounds to inhibit HCV we used two methods for determination
of minimum inhibitory concentration (MIC) of these compounds.
Firstly, their antiviral activities in HCV Replicon Cells was measured
by their Hepatitis C Virus (HCV) NS3-4A Protease Inhibitory activ-
ities. Secondly, their antiviral chemotherapy for Subacute Scle-
rosing Panencephalitis (SSPE) were determined in Hamster Brains.
Ribaverin was used in both methods as positive control.
Table 3 showed the MIC values for the tested compounds and
ribaverin against HCV and SSPE. The results showed that all the
nine tested compounds are highly effective at very low concen-
tration compared to ribaverin in the following order:
21a > 6 > 14 >16 > 9 > 21b > 10 > 13 > 12 These results indicate
that these compounds are also very potent inhibitors of HCV in both
cases.
3. Pharmacology
Benzosuberone derivatives have some cytotoxic,antioxidant and
anti angiogenic activities [18] so depending on this strong biolog-
ical rational we aiming at test the antioxidant activities, this
together with testing the anti –HCV activities based on the struc-
tural similarity between our end products and ribavirin wer the
triazole and its bioisosters presents.The obtained results prompted
us to investigate the anti SS PE activities where it need special
structural features in drugs to be act on this highly resistant viral
category ,we though this due to the prescence of triazole or two
heteroatom five membed or one heteroatom six membered ring
containg electron deficient groups fused to the bezosuberone.
3.1.2. DPPH radical scavenging activity
Radical scavenging activities of compounds 2, 6, 9, 10, 12–14, 16,
21a,b were measured using model colourimetric test: DPPH radical

496
T.A. Farghaly et al. / European Journal of Medicinal Chemistry 45 (2010) 492–500
R
N
O
RCOCH=CHN(Me)
2
20
1
21 a-c
O
N
N
R = a,
N
b,
;
c,
;
Ph
N
Br
Ph
Scheme 6. Reaction of benzosuberone with enaminone 2.
3.1.3. Inhibition of peroxynitrite-induced tyrosine nitration
The reactive nitrogen species peroxynitrite (ONOOꢀ) has been
implicated in numerous human disease pathologies and its role is
usually inferred from the measurement of 3-nitrotyrosine (3-NT).
Peroxynitrite and species derived from it can oxidise and nitrate
lipids [19], proteins [20], DNA [21,22] and carbohydrates [23]
leading to tissue damage in a number of pathological conditions in
humans and in experimental animals. Due to the cytotoxicity of
ONOOꢀ and its apparent formation at sites of tissue injury [24,25],
there has been considerable interest in the ability of natural and
synthetic antioxidants to diminish ONOOꢀ damage. In the present
work, the ability of ten synthetic compounds to prevent ONOOꢀ-
mediated damage was examined using the model system: ONOOꢀ-
mediated tyrosine nitration. When the amino acid tyrosine is
exposed to ONOOꢀ at pH 7.4, 3-NT is formed [26]. Fig. 3A and
B show that the addition of the ten synthesized compounds,
significantly inhibited tyrosine from ONOOꢀ-mediated nitration in
a dose dependent matter. The relative inhibitory activity of these
compounds is summarized in Table 5 as IC50 values. Under our
experimental conditions the relative potencies of the ten
compounds were 2 >12 >13 > 21b > 10 > 16 > 6 > 14 > 9 > 21a.
Fig. 2. Crystal structure of compound 16.
scavenging test. The results are summarized in Table 3. The stable
radical DPPH has been used widely for the determination of
primary antioxidant activity, that is, the free radical scavenging
activities of pure antioxidant compounds, plant and fruit extracts
and food materials. The assay is based on the reduction of DPPH
radicals in methanol which causes an absorbance drop at 515 nm.
Among the ten compounds tested for antioxidant activity using
DPPH method six of the tested compounds exhibited good scav-
enging activity against the DPPH radical in the following order:
2 >14 > 21b > 10 > 12 > 21a (Table 4). The values were found to be
less than that of standard ascorbic acid. The other four compounds
were considerably less effective radical scavengers in the order
13 > 9 > 6 > 16 (Table 4).
The mechanism of antiviral activity is due to enhance host
T-cell-mediated immunity against viral infection through helping
to switch the host T-cell phenotype from type 2 to type 1. Also the
mechanism of antiviral activity is due to transient increase in the
mutation rate of HCV lethal mutagenesis and error catastrophe is
unlikely to be the mechanism of SSPE during treatment of CHC.
Te antioxidant activities of the newly synthesize compounds is
mainly due to the free scavenging activities of the benzosuberone
ring and heterocyclic ring system
4. Conclusion
In conclusion, enaminone 2 was used as precursor for the
synthesis of a variety of heterocyclic ring systems formed upon
reaction with heterocyclic amines, hydrazine hydrate, hydroxyl
amine and active methylene to afford heterocyclic derivatives with
high biological activity. The structures of the newly synthesized
compounds were confirmed by spectral data and elemental
Table 3
MIC of Ribaverin and the nine tested compounds against HCV and SSPE.
Tested Compound%
MIC
HCV
mg/ml
Subacute Sclerosing
Panencephalitis (SSPE)
Table 2
Selected bond angles and bond lengths of compound 16.
Ribaverin
16.15
7.66
7.09
6.22
5.98
6.00
4.45
3.58
2.21
1.15
77.89
51.11
41.12
40.15
38.40
34.89
23.01
12.55
11.12
7.56
12
13
10
21b
9
16
14
6
Bond
Angle
Bond
Length (Å)
C4–N2–C6
C6–C3–C9
C6–C3–C15
C9–C3–C15
N2–C4–C5
C3–C15–N18
O1–C6–N2
125.3
121.1
117.6
121.3
119.7
179.8
121.0
N2–C4
N2–C6
C3–C6
C3–C9
C4–C5
C3–C15
O1–C6
1.371
1.374
1.340
1.372
1.376
1.429
1.246
21a

T.A. Farghaly et al. / European Journal of Medicinal Chemistry 45 (2010) 492–500
497
Table 4
Decrease of DPPH absorbance (%) by ten synthesized compounds.
120
A
100
80
60
40
20
0
Compound No.
Decrease of DPPH absorbance%
mean ꢂ SD (n ¼ 3)
2
2
6
9
10
12
13
14
16
57.883 ꢂ 3.961
27.701 ꢂ 3.094
28.175 ꢂ 5.105
45.369 ꢂ 2.017
44.119 ꢂ 6.12
37.209 ꢂ 4.094
54.436 ꢂ 5.598
27.390 ꢂ 7.013
39.994 ꢂ 7.91
54.199 ꢂ 6.537
79.042 ꢂ 2.928
6
9
10
12
21a
21b
0
50
100
150
200
250
300
Concentration (µM)
Ascorbic acid (standard)
120
100
80
60
40
20
0
B
analyses and also, the structure of two compounds were confermed
by X-ray analyses. The studies described here demonstrate the
ability of nine new synthesized compounds to inhibit HCV at very
low concentration. In addition, ten of the newly synthesized
compounds were tested for antioxidant activity using DPPH
method, six of the tested compounds exhibited good scavenging
activity against the DPPH radical in the following order:
2 >14 > 21b > 10 > 12 > 21a. The values were found to be less than
that of standard ascorbic acid. The other four compounds were
considerably less effective radical scavengers in the order
13 > 9 > 6 > 16. Also, the ability of ten synthetic compounds to
prevent ONOOꢀ-mediated damage was examined using the model
system: ONOOꢀ-mediated tyrosine nitration which indicated
that the relative potencies of the ten compounds were
2 >12 >13 > 21b > 10 > 16 > 6 > 14 > 9 > 21a.
13
14
16
21a
21b
0
50
100
150
200
250
300
Concentration (µM)
Fig. 3. Inhibition of tyrosine nitration by ten compounds. Tyrosine (100
ONOOꢀ (100
15 min ꢂ compounds (0–300
m
M) and
C
m
M) incubated in phosphate buffer (100 mM), pH 7.4 at 37 ꢁ
for
m
M). 3-NT formed was measured by HPLC as described
under Materials and Methods. Data are mean ꢂ SD of 3 independent experiments.
Control ¼ ONOOꢀ þ tyrosine, (A) for compounds 2, 6, 9, 10 and 12, (B) for compounds
13, 14, 16, 21a and 21b.
5. Experimental protocol
reflection 2069 measured, qmax ¼ 21.50ꢁ. Fig. 1 illustrates the
structure as determined.
5.1. Chemistry
For compound 16: C15H12N2O, M ¼ 236.27, monoclinic,
All melting points were determined on an electrothermal Gal-
lenkamp apparatus. Solvents were distilled and dried by standard
literature procedures prior to use. The IR spectra were measured on
a Pye-Unicam SP300 instrument in potassium bromide discs. The
1H NMR spectra were recorded on a Varian Mercury VXR-300 MHz
a ¼ 8.1328 (4), b ¼ 9.2799 (4), c ¼ 16.2253 (8) Å, v ¼ 1220.75 (10),
a
¼
g
¼ 90.00ꢁ,
b
¼ 94.512 (2), space group: P21/c, Z ¼ 4,
Dx ¼ 1.286 Mg mꢀ3 reflection 2968 measured, qmax ¼ 22.98ꢁ. Fig. 2
illustrates the structure as determined. Full data can be obtained on
request from the CCDC [27].
spectrometer and the chemical shifts
d (ppm) down field from
tetramethylsilane (TMS) as an internal standard. The mass spectra
were recorded on a GCMS-Q1000-EX Shimadzu and GCMS 5988-A
HP spectrometers, the ionizing voltage was 70 eV. Elemental
analyses were carried out by the Microanalytical Center of Cairo
University, Giza, Egypt. Tyrosine, 3-nitrotyrosine (3-NT) and
1,1-Diphenyl-2-picryl hydrazyl (DPPH) were obtained from Sigma–
Aldrich (St. Louis, U.S.A).
5.3. Preparation of 2-dimethylaminomethylene-1-
benzosuberone (2)
A mixture of compound 1 (0.01 mol, 1.6 g) and DMF–DMF (2 ml)
was heated under reflux for 10 h. The reaction mixture was tritu-
rated with ethanol to give a solid product that was collected by
5.2. Crystallographic analysis
Table 5
IC50 values of the compounds tested for the inhibition of ONOOꢀ-mediated
3-nitrotyrosine formation.
The crystals were mounted on a glass fiber. All measurements
were performed on an ENRAF NONIUS FR 590. The data were
collected at a temperature of 25 ꢁC using the
to a maximum of a 20 of 22.986ꢁ. The structure was solved by direct
method using SIR 92 and refined by full-matrix least squares. Non-
hydrogen atoms were refined anisotropically. Hydrogen atoms
were located geometrically and were refined isotropically.
Compound No.
IC50 (
m
M) mean ꢂ SD (n ¼ 3)
u scanning technique
2
6
9
10
12
13
14
16
146.316 ꢂ 9.998
315.052 ꢂ 19.709
341.659 ꢂ 20.864
277.021 ꢂ 17.836
148.771 ꢂ 10.197
157.659 ꢂ 10.986
328.935 ꢂ 20.575
290.664 ꢂ 18.886
349.175 ꢂ 21.264
169.929 ꢂ 11.771
58.430 ꢂ 5.9
5.2.1. Crystal data
21a
21b
Trolox
For compound 6: C14H12N4, M ¼ 236.28, monoclinic, a ¼ 7.8943
(7), b ¼ 17.640 (2), c ¼ 8.2084 (9) Ao, v ¼ 1134.5 (2),
a
¼
g
¼ 90.00ꢁ,
b
¼ 97.020 (4), space group: P21/c, Z ¼ 4, Dx ¼ 1.383 Mg mꢀ3

498
T.A. Farghaly et al. / European Journal of Medicinal Chemistry 45 (2010) 492–500
filtration and crystallized from ethanol to give compound 2 as deep
orange crystals, yield (95%) m.p 85–86 ꢁC [28], 1H NMR (DMSO-d6)
reaction mixture was then heated under reflux for 5 h. The solid
formed was collected by filtration and crystallized from Dioxan/
Ethanol to give white solid, yield (70%), m.p > 300 ꢁC, 1H NMR
(DMSO-d6) 1.91–2.91 (m, 6H, 3 CH2) 7.30–7.37 (m, 4H, ArH), 8.50 (s,
1H, isoxazole-H), MS m/z(%) 186 (Mþ þ 1, 91), 185 (Mþ, 100), 158
(21), 157 (65), 156 (82), 126 (32), 125 (94), 116 (47), 77(29). Anal.
Calcd. For C12H11NO (185.23) C, 77.81; H, 5.99; N, 7.56. Formed C,
77.63; H, 5.69; N, 7.42%.
at
d 1.74–2.66 (m, 6H, 3CH2), 3.07 (s, 6H, 2CH3), 7.13–7.44 (m, 4H,
ArH), 7.58 (s, 1H, ]CH); IR 1720 cmꢀ1 MS m/z (%) 217 (Mþ þ 2, 15),
216 (Mþ þ 1100), 215 (Mþ, 53), 198 (26), 84(5), 77(2). Anal. Calcd.
For C14H17NO (215.30) C, 78.10; H, 7.96; N, 6.51. Found C, 77.93; H,
7.82; N, 6.30%.
5.4. Reaction of enaminone 2 with heterocyclic amines
5.7. Preparation of compounds 13 & 14
To a solution of 2 (0.005 mol, 1.08 g) in acetic acid (20 ml) was
added (0.005 mol) of the appropriate heterocyclic amines (3–5).
The mixture was heated under reflux for 6 h. The solid deposited
after cooling was then collected by filtration and crystallized from
the appropriate solvent. The physical constants together with the
spectral data of products 6, 8, 9 are depicted below.
To a solution of 2 (0.005 mol, 1.08 g) in acetic glacial in the
presence of ammonium acetate (0.5 g), was added acetylacetone or
ethyl acetoacetate (0.005 mol). The reaction mixture was heated
under reflux for several hours. The reaction was followed by TLC.
The solvent was evaporated under reduced pressure and the oil
residue was triturated with ethanol to give the solid products 13
and 14, respectively.
5.4.1. 9,10,11-Trihydrobenzo[60,70]cyclohepta[20,10-e]triazolo[2,3-a]
pyrimidine (6)
Golden yellow crystals yield (69%), m.p > 300 ꢁC. (Ethanol) 1H
NMR (DMSO-d6) 1.70–2.66 (m, 6H, 3CH2), 7.47–8.08 (m, 4H, ArH),
8.62 (s, 1H, pyrimidine-H), 8.88 (s, H, triazole-H). MS m/z (%) 237
(Mþ þ 1, 16), 236 (Mþ, 100), 235 (59), 128 (11), 115 (21), 89 (14), 77
(15). Anal. Calcd. for C14H12N4 (236.28) C, 71.17; H, 5.12; N, 23.71.
Found C, 71.20; H, 4.99; N, 23.54%.
5.7.1. 3-Acetyl-2-methyl-5,6,7- trihydrobenzo[6,7]cyclohepta[1,2–
b] pyridine (13)
Yellow solid, yield (60%); m.p 126–127 ꢁC (Ethanol) 1H NMR
(DMSO-d6) 1.71–2.45 (m, 6H, 3CH2), 2.62 (s, 3H, CH3), 2.67 (s, 3H,
CH3), 7.30–7.65 (m, 4H, ArH), 8.14 (s, 1H, pyridine-H); IR 1678
(C]O) cmꢀ1 MS m/z (%) 252 (Mþ þ 1, 16), 251 (Mþ, 52), 250 (25),
236 (100), 235 (20), 206 (18), 189 (15), 166 (30), 103 (36), 77 (41).
Anal. Calcd. For C17H17NO (251.33) C, 81.24; H, 6.82; N, 5.57. Found.
C, 81.21; H, 6.53; N, 5.30%.
5.4.2. 1-Phenyl-9,10,11-trihydrobenzo[60,70]cyclohepta[20,10-
e]pyrazolo[2,3-a] pyrimidine (8)
Golden yellow crystals, yield (72%) m.p > 300 ꢁC. (Dioxan/
Ethanol). 1H NMR (DMSO-d6) at
d
1.60–2.73 (m, 6H, 3 CH2); 7.15–
5.7.2. Ethyl-2-methyl-5,6,7–trihydrobenzo[6,7]cyclohepta[1,2–
b]pyridine-3-carboxylate (14)
8.23 (m, 9H, ArH); 8.54 (s, 1H, pyrimidine-H); 8.74 (s, 1H, pyrazole-
H). MS m/z (%) 312 (Mþ þ 1,36), 311 (Mþ, 100), 310(88). 285(14), 283
(14), 255(20), 238(17), 180 (10), 142 (21), 141 (25), 124 (11), 115 (13),
77 (18). Anal. Calcd. For C21H17N3 (311.39) C, 81.00; H, 5.50; N,13.49.
Found C, 80.90; H, 5.31; N, 13.41%.
Pale yellow solid, yield (62%); m.p. 154–156 ꢁC (Ethanol). 1H
NMR (DMSO-d6) 1.25 (t, J ¼ 7 Hz, 3H, CH3), 1.87–2.76 (m, 6H, 3CH2),
2.42 (s, 3H, CH3), 4.39 (q, J ¼ 7 Hz, 2H, CH2), 7.26–7.63 (m, 4H, ArH),
8.05 (s, 1H, Pyridine H). IR 1710 cmꢀ1 MS m/z (%) 282 (Mþ þ 1, 1),
281 (Mþ, 5), 200 (12), 187 (15), 186 (24), 170 (29), 144 (26), 141 (43),
131 (51),128 (100),118 (17),115 (83),103 (67), 91 (92), 77 (49). Anal.
Calcd. For C18H19NO2 (281.36) C, 76.84; H, 6.81; N, 4.98. Found C,
76.62; H, 6.65; N, 4.69%.
5.4.3. 9,10,11-Trihydrobenzo[60,70]cyclohepta[20,10-e]pyrimido[1,2-
a] benzimidazole (9)
Yellow crystals, yield (60%), m.p 226 ꢁC (Dioxan/Ethanol). 1H
NMR (DMSO-d6) at
d 1.95–2.84 (m, 6H, 3CH2); 7.09–7.88 (m, 8H,
ArH); 8.78 (s, 1H, pyrimidine-H). MS m/z (%) 287 (Mþ þ 2, 3), 286
(Mþ þ 1, 24), 285 (Mþ, 100), 284 (43), 270 (8), 257 (10), 135 (3), 115
(4), 102 (3), 90 (3), 77 (4). Anal. Calcd. For C19H15N3 (285.35) C,
79.98; H, 5.30; N, 14.72. Found C, 79.90; H, 5.08; N, 14.52%.
5.8. 2-Oxo-2,5,6,7-tetrahydro-1H-benzo[6,7]cyclohepta[1,2-
b]pyridine-3-carbonitrile (16)
To a solution of 2 (0.005 mol, 1.08 g) in ethanolic sodium eth-
oxide solution (0.12 g of sodium metal in 20 ml absolute ethanol)
was added (0.005 mol) malononitrile or cyanoacetamide. The
reaction mixture was heated under reflux for 5 h, and then poured
into ice-cold water. The solution was acidified with diluted HCl and
the solid deposited was collected by filtration and crystallized from
dioxan/ethanol, yield (60%), m.p > 300 ꢁC, 1H NMR(DMSO-d6) 2.04–
2.56 (m, 6H, 3CH2), 7.36–7.53 (m, 4H, ArH), 8.12 (s, 1H, pyridine-H),
12.66 (s, 1H, NH). IR 3386, 2229, 1647 cmꢀ1 MS m/z (%) 237 (Mþ þ 1,
13), 236 (Mþ, 100), 235 (23), 221 (28), 208 (10), 115 (2), 104 (5), 89
(4), 76 (5). Anal. Calcd. for C15H12N2O (236.28) C, 76.25; H, 5.12; N,
11.86. Found C, 76.05; H, 5.10; N, 11.62%
5.5. Synthesis of 4,5,6-trihydro-2H-benzo[6,7] cyclohepta[1,2-
c]pyrazole (10)
To a solution of 2 (0.005 mol, 1.08 g) in absolute ethanol (20 ml)
was added (0.005 mol, 0.25 g) of hydrazine hydrate. The mixture
was then heated under reflux for 2 h. The precipitate formed was
collected by filtration and crystallized from ethanol as pale yellow
crystals yield (78%) m.p 76 ꢁC [29], 1H NMR (DMSO-d6) at
d 1.74–
2.85(m, 6H, 3CH2), 7.17–7.27 (m, 4H, ArH), 7.47 (s, 1H, pyrazole-H),
12.60 (s,1H, NH). IR 3143 cmꢀ1 MS m/z (%) 185 (Mþ þ 1,17) 184 (Mþ,
100), 169 (27), 156 (36), 128 (26), 115 (28), 102 (12), 91 (13), 77
(20).Anal. Calcd. For C12H12N2 (184.24) C, 78.23; H, 6.57; N, 15.20.
Found C, 78.48; H, 6.40; N, 15.00%.
5.9. Synthesis of 3-benzoyl-5,6,7-
trihydrobenzo[60,70]cyclohepta[1,2-b] pyridine-2(1H)-one (18)
5.6. Synthesis of 4,5,6-trihydro-2H-benzo[6,7]cyclohepta[2,1-
To a solution of 2 (0.005 mol, 1.08 g) in ethanol (30 ml) con-
d]isoxazole (12)
taining piperidine (0.5 ml) as a catalyst, was added u-cyanoaceto-
phenone (0.005 mol, 0.71 g). The reaction mixture was heated
under reflux for several hours and the progress of the reaction was
monitored by TLC. The solvent was evaporated under reduced
pressure, the oil residue was treated with methanol to give
To a solution of 2 (0.005 mol, 1.08 g) in absolute ethanol was
added (0.005 mol, 0.35 g) of hydroxylamine hydrochloride in the
presence of anhydrous sodium acetate (0.005 mol, 0.41 g). The

T.A. Farghaly et al. / European Journal of Medicinal Chemistry 45 (2010) 492–500
499
compound 18 as buff crystals, yield (72%) m.p 105 ꢁC (ethanol) 1H
NMR 1.73–2.66 (m, 6H, 3CH2), 7.05–8.00 (m, 9H, ArH), 8.24 (s, 1H,
pyridine-H), 11.98 (s, 1H, NH). IR 3215, 1686, 1674 cmꢀ1 MS m/z (%)
316 (Mþ þ 1, 20), 315 (Mþ, 46), 314 (32), 147 (34), 104 (65), 89 (20),
77 (100). Anal. Calcd. for C21H17NO2 (315.38) C, 79.98; H, 5.43; N,
4.44. Found C, 79.75; H, 5.24; N, 4.12%
ether anesthesia, 50 ml of ribavirin or tested compound solutions at
dosages of 5, 10, and 20 mg/kg/day was injected for 10 days intra-
cranially to a depth of 2 mm by using a 27-gauge needle and was
placed within the subarachnoid space. At 1, 2, 3, 5, 7, 10, 12, 15, and
20 days after the initial injection, four hamsters from each group
were sacrificed. The brains were aseptically removed, washed
twice with phosphate-buffered saline (PBS), homogenized, and
suspended in PBS. The suspension was centrifuged at 1600 3g for
10 min. The supernatant was collected, ethanol was added to
remove proteins, and the mixture was heated at 90 ꢁC to evaporate
the ethanol. The protein-free samples were used to evaluate the
MIC in brain tissue by HPLC and bioassay.
5.10. Preparation of compounds 21a–c
To a solution of enaminones 20a–c (0.005 mol) in acetic acid
(20 ml) in the presence of ammonium acetate (0.5 g) was added
benzosuberone 1 (0.005 mol, o.80 g). The reaction mixure was
refluxed for 5 h., the solid formed was collected by filtration and
crystallized from acetic acid to yield compounds 21a–c.
5.11.2. Radical scavenging assays
5.11.2.1. DPPH radical scavenging assay. The antioxidant activity of
the ten compounds 2, 6, 9,10,12–14,16, 21a,b and the standard were
assessed on the basis of the radical scavenging effect of the stable
DPPHꢄ free radical [30]. Weighed quantities of the ten compounds
were dissolved in distilled DMSO and used. Compound 21a was not
fully soluble in DMSO (even after treating its solution for 5 min in an
ultrasonic bath). Solution of ascorbic acid used as standard for this
study was prepared in distilled H2O. All these solutions were serially
diluted with respective solvents to get lower dilutions.
5.10.1. 3-(6,7-Dihydro-5H-benzo[6,7]cyclohepta[1,2-b]pyridine-2-
yl)-1,7-diphenyl-1H-[1,2,4]triazolo[4,3-a]pyrimidin-5-one (21a)
Yellow solid, yield (85%), m.p 200 ꢁC, 1H NMR (DMSO-d6)
d 1.90–
2.70 (m, 6H, 3CH2); 6.70 (s, 1H, Pyrimidine-H), 7.49–8.30 (m, 14H,
ArH); 8.71 (d, J ¼ 8 Hz, 2H, pyridine-H), 9.44 (d, J ¼ 8 Hz, 2H, pyri-
dine-H). MS m/z (%) 482 (Mþ þ 1, 5), 481 (Mþ, 11), 456 (19), 376
(22), 178 (34), 128 (37), 89 (65), 77 (100). IR 1685 cmꢀ1. Anal. calcd.
For C31H23N5O (481.56) C, 77.32; H, 4.81; N,14.54. Found C, 77.15; H,
4.50; N, 14.31%.
10
m
l of each compound or standard (from 0.0
m
M/ml to 100
mM/
ml) was added to 90
m
l of DPPHꢄ in methanol solution (100
mM) in
a 96 well microtitre plate. After incubation in the dark at 37 ꢁC for
30 min, the decrease in absorbance of each solution was measured
at 515 nm using ELISA micro plate reader (Bio Rad Laboratories
Inc., California, U.S.A., Model 550). Absorbance of blank sample
containing the same amount of DMSO and DPPHꢄ solution was
prepared and measured as well. The experiment was carried out in
triplicate. The scavenging potential was compared with a solvent
control (0% radical scavenging) and ascorbic acid. Radical scav-
enging activity was calculated by the following formula:
5.10.2. 2-(2-Naphthyl)-6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-
b]pyridine (21b)
Pale yellow sheets, yield (85%), m.p 178–180 ꢁC, 1H NMR (DMSO-
d6) at
d 1.70–2.61 (m, 6H, 3CH2), 7.59–8.47 (m, 10H, ArH), 8.47 (d,
J ¼ 8 Hz, 2H, pyridine-H), 8.84 (s, 1H, naphthyl-H) 9.11 (d, J ¼ 8 Hz,
2H, pyridine-H). MS m/z (%) 321 (Mþ, 2), 316 (40), 202 (80), 157(66),
105 (100), 95 (87), 80 (67), 69 (80). Anal. Calcd. For C24H19N (321.43)
C, 89.68; H, 5.96; N, 4.36. Found C, 89.55; H,5.68; N, 4.21%.
5.10.3. 2-(4-Bromophenyl)-6,7-dihydro-5H-
%Reduction of absorbance [ ½ðAB L AAÞ=ABꢅ 3 100
benzo[6,7]cyclohepta[1,2-b] pyridine (21c)
Golden yellow crystals, yield (82%), m.p 212 ꢁC. 1H NMR (DMSO-
where: AB – absorbance of blank sample; AA – absorbance of tested
extract solution (t ¼ 30 min).
d6) at
d 1.90–2.4 (m, 6H, 3CH2); 7.74–7.83 (m, 8H, ArH), 8.15 (d,
J ¼ 8 Hz, 2H, pyridine-H), 8.20 (d, J ¼ 8 Hz, 2H, pyridine-H). MS m/z
(%) 352 (Mþ þ 2, 10), 351 (Mþ þ 1, 15), 350 (Mþ, 33), 270 (14), 193
(22), 157(80), 105 (64), 95 (87), 77 (100). Anal. Calcd. For C20H16BrN
(350.26) C,68.58; H,4.60; N,4.00. Found C, 68.32; H, 4.28; N,3.91%.
5.11.3. Reaction with reactive nitrogen species
5.11.3.1. Synthesis of peroxynitrite. Peroxynitrite (ONOOꢀ) was
synthesized as previously described [26]. Briefly, an acidic solution
(0.6 M HCl) of H2O2 (0.7 M) was mixed with KNO2 (0.6 M) on ice for
1 s and the reaction quenched with ice-cold NaOH (1.2 M). Residual
H2O2 was removed by mixing with granular MnO2 prewashed with
NaOH (1.2 M). The stock solution was filtered and then frozen
overnight (ꢀ20 ꢁC) and the top layer of the solution collected
for the experiment. Concentrations of stock ONOOꢀ were rede-
5.11. Pharmacology
5.11.1. Hepatitis C virus (HCV) NS3-4A protease inhibitory activities
in HCV replicon cells
5.11.1.1. Determination of minimum inhibitory concentration (MIC),
of ribavirin and different tested compounds in HCV replicon cells was
performed as follow. Briefly, 1 ꢃ104 replicon cells per well were
plated in 96-well plates. On the following day, replicon cells were
incubated at 37 ꢁC for the indicated period of time with antiviral
agents serially diluted in DMEM plus 2% FBS and 0.5% dimethyl
sulfoxide (DMSO). Total cellular RNA was extracted using an
RNeasy-96 kit (QIAGEN, Valencia, CA), and the copy number of HCV
RNA was determined using a quantitative RTPCR (QRT-PCR) assay.
Each datum point represents the average of five replicates in cell
culture. The cytotoxicity of tested compound was measured under
the same experimental settings using a tetrazolium (MTS)-based
cell viability assay (Promega, Madison, WI). For the cytotoxicity
assay with human hepatocyte cell lines, 1 ꢃ104 parental Huh-7
cells per well or 4 ꢃ104 HepG2 cells per well were used.
termined before each experiment at 302 nm using
a molar
absorption coefficient of 1670 cmꢀ1 Mꢀ1. Concentrations of 200–
250 mM were usually obtained. Once thawed, ONOOꢀ solutions
were kept in ice for no longer than 30 min before use.
5.11.3.2. Reaction of compounds with peroxynitrite. The ability of
tested compounds to inhibit peroxynitrite-induced tyrosine nitra-
tion was investigated via reaction of equimolar concentrations
(100
m
M) of tyrosine and peroxynitrite in the presence of increasing
M) in 100 mM
concentrations of each tested compound (0–300
m
phosphate buffer, pH 7.4 at 37 ꢁC for 15 min. Snap-freezing reaction
mixtures prior to HPLC analysis successfully terminated reactions.
The formation of 3-nitrotyrosine (3-NT) was monitored by HPLC
analyzed with photodiode array detection (see below). 3-NT
formed was characterised and quantified by use of an authentic
standard (elution time and unique spectral characteristics).
5.11.1.2. MIC of the tested compounds in Hamster Brains for antiviral
chemotherapy for Subacute Sclerosing Panencephalitis (SSPE). Under

500
T.A. Farghaly et al. / European Journal of Medicinal Chemistry 45 (2010) 492–500
[9] K.M. Dawood, J. Heterocycl. Chem. 42 (2005) 221–225.
[10] P. Bennett, J.A. Donnelly, D.C. Meaney, P.O. Boyle, J. Chem. Soc. Perkin Trans. I
(1972) 1554–1559.
[11] K.M. Dawood, A.M. Farag, E.A. Ragab, J. Chin. Chem. Soc. 51 (2004) 853–857.
[12] C.E. Grothaus, F.B. Dains, J. Am. Chem. Soc. 58 (1936) 1334.
[13] D.J. Anderson, C.R. Muchmore, J. Heterocycl. Chem. 32 (1995) 1189–1196.
[14] S.O. Abdallah, N.H. Metwally, H.F. Anwar, M.H. Elnagdi, J. Heterocycl. Chem. 42
(2005) 781–786.
[15] A.Z.A. Hassanien, S.A.S. Ghozlan, M.H. Elnagdi, J. Chin. Chem. Soc. 51 (2004)
575–579.
[16] E. Dominguez, E. Ibeas, E.M. Demarigorta, J.K. Palacios, R. San Martin, J. Org.
Chem. (1996) 5435–5439.
5.11.3.3. HPLC analysis. Reaction mixtures were analyzed using
reverse-phase HPLC. Analysis was performed on an Agilent 1100
system with a Zorbax ODS C18 column (150 ꢃ 4.6 mm i.d., 4
mm) and
guard column (15 ꢃ 4.6 mm i.d., 4 m). Mobile phase A consisted of
m
methanol/water/5 N HCl (5/94.9/0.1 v/v/v) and mobile phase B of
acetonitrile/water/5 N HCl (50/49.9/0.1 v/v/v). The following gradient
systemwas used (min/% acetonitrile): 0/0, 5/0, 40/50, 60/100, 65/100,
and 65.1/0 with a flow rate of 0.7 ml/min. The eluent was monitored
by photodiode array detection at 280 nm for 3-NT measurements
with spectra of products obtained over the 220–600 nm range.
[17] B. Al-Saleh, H. Behbehani, M.A. El-Apasery, M.H. Elnagdi, J. Chem. Res. (2004)
575–577.
[18] C.J. Grubbs, R.A. Lubet, V.R. Atigadda, K. Christov, A.M. Deshpande, V. Tirmal1,
G. Xia, K.I. Blandl, I. Eto, W.J. Brouillette, D.D. Muccio, Carcinogenesis 27 (2006)
1232–1239.
Appendix. Supplementary data
[19] R. Radi, J.S. Beckman, K.M. Bush, B.A. Freeman, Arch. Biochem. Biophys. 288
(1991) 481–487.
[20] H. Ischiropoulos, A.B. Al-Mehdi, FEBS Lett. 364 (1995) 279–282.
[21] V. Yermilov, J. Rubo, H. Oshima, FEBS Lett. 376 (1996) 207–210.
[22] J.P.E. Spencer, J. Wong, A. Jenner, O.A. Aruoma, C.E. Cross, B. Halliwell, Chem.
Res. Toxicol., 91(996)1152–1158.
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2009.10.033.
References
[23] J.S. Beckman, T.W. Beckman, J. Chen, P.M. Marshall, B.A. freeman, Proc. Natl.
Acad. Sci. U.S.A. 87 (1990) 1620–1624.
[24] B. Halliwell, K. Zhao, M. Whiteman, Free Radic. Res. 31 (1999) 651–669.
[25] H. Ischiropoulos, Arch. Biochem. Biophys. 356 (1998) 1–11.
[26] J.S. Beckman, J. Chen, H. Ischiropoulos, J.P. Crow, Meth. Enzymol. 223 (1994)
229–240.
[27] Crystal data for compounds 6 and 16 (ref. CCDC 703976 and CCDC 703975)
can be obtained on request from director, Cambridge Crystallographic Data
Center, 12 Union Road, Cambridge CB2 1EW, UK
[28] F. Bondavalli, O. Bruno, E.L. Presti, G. Menozzi, L. Mosti, Synthesis (1999)
1169–1174.
[1] S.M. Riyadh, I.A. Abdelhamid, H.M. Al-Matar, N.M. Hilmy, M.H. Elnagdi,
Heterocycles 75 (2008) 1849–1904.
[2] T.A. Farghaly, J. Chem. Res. (2008) 152–156.
[3] G. Negri, C. kascheres, A.J. Kascheres, J. Heterocycl. Chem. (2004) 461–491.
[4] A.A. Elassar, A.A. El-Khair, Tetrahedron 59 (2003) 8463–8480.
[5] J.R. Dimmock, G.A. Zello, E.O. Oloo, J.W. Quail, H.B. Kraatz, P. Perjesi, F. Aradi,
K. Takacs-Novak, T.M. Allen, C.L. Santos, J. Batzarini, E. Declerq, J.P. Stables, J.
Med. Chem. (2002) 3103–3111.
[6] A.E.G. Hammam, N.A. Abdel-Hafez, W.H. Midura, M. Mikolajczyk, Z. Natur-
forsch 55b (2000) 417–421.
[7] A.G.E. Amr, A.M. Mohamed, S.F. Mohamed, N.A. Abdel-Hafez, A.E.F. Hammam,
Bioorg. Med. Chem. 14 (2006) 5481–5488.
[8] N.A. Abdel-Hafez, O.I. Abdel Salam, A.G. Hammam, Egypt. J. Chem. 49 (1)
(2006) 63–66.
[29] J.M. Khanna, N. Anand, Indian J. Chem. (1968) 6–10.
[30] E.J. Gamez, L. Luyengi, S.K. Lee, L.F. Zhou, H. Frog, A.D. Kinghorn, J. Nat. Prod. 61
(1998) 706–708.