Synthesis, tautomerism, and antimicrobial, anti-HCV, anti-SSPE, antioxidant, and antitumor activities of arylazobenzosuberones

Article abstract of DOI:10.1016/j.bmc.2009.10.012

2-Dimethylaminomethylene-1-benzosuberone 1 was coupled with diazotized aniline derivatives to afford a series of the hitherto unreported 2-arylazo-1-benzosuberones 3a-i. The tautomeric structure and the effect of substituents on the tautomeric form (s) of

Full text of DOI:10.1016/j.bmc.2009.10.012

Bioorganic & Medicinal Chemistry 17 (2009) 8012–8019
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, tautomerism, and antimicrobial, anti-HCV, anti-SSPE, antioxidant,
and antitumor activities of arylazobenzosuberones
b
Thoraya A. Farghaly a, , Mohamed M. Abdalla
*
a Department of Chemistry, Faculty of Science, University of Cairo, Giza, Egypt
b MAPCO 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:
2-Dimethylaminomethylene-1-benzosuberone 1 was coupled with diazotized aniline derivatives to
afford a series of the hitherto unreported 2-arylazo-1-benzosuberones 3a–i. The tautomeric structure
and the effect of substituents on the tautomeric form (s) of the products 3a–i were discussed. Similar cou-
pling of the enaminone 1 with diazonium salts of heterocyclic amines gave the respective fused azolo-
triazino-benzosuberones. Some of the newly synthesized compounds showed potent antimicrobial,
anti-HCV, antioxidant, antitumor (as topoisomerase I inhibitors), and antimicrobial activities.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 31 August 2009
Revised 4 October 2009
Accepted 6 October 2009
Available online 12 October 2009
Keywords:
Azo-hydrazone tautomerism
Japp–Klingemann reaction
Antimicrobial
Antitumor
Anti-HCV
Anti- SSPE
Antioxidant activities
1. Introduction
thesized benzosuberone derivatives in inhibiting Hepatitis C Virus
(HCV) and Subacute Sclerosing Panencephalitis (SSPE), besides
In our previous papers,1–10 we were interested in the azo-
hydrazone tautomerism of various arylazo heterocycles as many
of them are useful in the field of material sciences and theoretical
chemistry.11,12 In addition to these applications, azo compounds
are used as photosensitive species in photographic or electropho-
tographic systems and are the dominant organic photoconductive
materials in commercial copiers.13 Also, hydrazones are known
as one of the most important classes of organic compounds, some
of which show significant biological activities such as analgesic,
antitumor, anti-inflammatory, antibacterial, and antifungal, and
they also play the role of anticancer, and immunomodulating
agents.14–19 On the other hand, benzosuberone ring system and
its fused systems present interesting pharmacological and biologi-
cal activities as potent antitumor agents20,21 against L1210 murine
leukemia and HT29 cell lines, as potent inhibitors of tubulin poly-
merization,22 and as blood platelet aggregation inhibitors.23 Based
on these findings, we report herein the results of coupling of 2-
their proved potent antioxidant activities.
2. Results and discussion
2.1. Chemistry
The required starting 2-dimethylaminomethylene-1-benzosu-
berone 1 was prepared by reacting 1-benzosuberone with dimeth-
ylformamide–dimethylacetal as previously described.24 Coupling
reaction of the enaminone 1 with diazotized anilines gave the
respective arylazo derivatives 3 and/or 4 (Scheme 1). On the basis
of their elemental and spectral (IR and mass) analyses (see Sec-
tion 4), the isolated products were assigned structure 3 rather than
structure 4. For example, the mass spectra revealed, in each case,
that the molecular ion peak was compatible with structure 3 rather
than with structure 4. The formation of the products (3a–i) is as-
sumed to proceed via Japp–Klingemann-type cleavage of dimethyl
aminomethylene moiety25 as depicted in Scheme 1.
dimethylaminomethylene-1-benzosuberone
1 with diazonium
salts of aniline derivatives and heterocyclic amines and elucidation
of the tautomeric structure(s) of the resulting products. Also, we
were interested in investigating the biological activities of the syn-
The isolated products 3 can have one or more of the three tau-
tomeric structures, namely, the keto-hydrazone 3A, hydroxy-azo
3B, and CH-azo structure 3C (Scheme 1). To elucidate the actual
tautomeric form(s) of these compounds, their electronic absorp-
tion spectra were first studied. The spectra of the compounds in
dioxane showed in each case three absorption bands in the regions
* Corresponding author.
E-mail address: thoraya-f@hotmail.com (T.A. Farghaly).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.012

T. A. Farghaly, M. M. Abdalla / Bioorg. Med. Chem. 17 (2009) 8012–8019
8013
Me
O
+
N
O
N(Me)2
Me
Ar-N=N-Cl
AcONa / EtOH
N
N
Ar
2
1
Japp-Klingemann
Ar
Ar
O
H
H
CHO
N
N
N
O
O
N
N
N
Ar
4
3A
3B
Ar
N
O
N
Ar = C6H4X
H
X: a, 4-OCH3; b,4-CH3; c, 3-CH3; d, H; e, 4-Cl;
f, 3-Cl; g,3-NO2; h, 4-NO2; i, 4-COCH3
3C
Scheme 1.
423–404, 288–284, and 276–231 nm (Table 1). This absorption
pattern is analogous to that reported for the azo chromophore.26
Furthermore, the spectrum of 3d, taken as a typical example of
the series studied, in solvents of different polarities, showed little,
if any, shift (Table 1). The small shift in kmax of 3d in different sol-
vents is due to solute–solvent interaction. This finding is in agree-
ment with the observation that o- and p-arylazo derivatives of
phenols and naphthols, unlike those of arylhydrazones derived
from the reaction of quinones with N-alkyl-N-phenylhydrazine,
are largely independent of the solvent polarity.27 This finding ex-
cludes the hydrazone tautomeric form 3A (Scheme 1).
Furthermore, the 1H NMR spectra of compounds 3 in deuterated
chloroform are characterized, in each case, by a singlet signal
assignable to the OH of the hydroxy-azo form near d 14.0 ppm28
and revealed absence the singlet signal near d 5.2729 for the CH
proton of the CH-azo form 3C. This finding indicates that the prod-
ucts 3a–i prepared exist in CDCl3 as a single tautomeric form,
namely hydroxy-azo form 3B (Scheme 1).
However, the 1H NMR spectra of compounds 3 in deuterated
dimethylsulfoxide indicated that such compounds exist as an equi-
librium of two tautomeric forms namely, the keto-hydrazone form
3A and the hydroxy-azo form 3B. This is because their 1H NMR
spectra revealed, in each case, two characteristic singlet signals
near d 10.0 and 14.0 ppm assignable to NH and OH protons, respec-
tively. The data in Table 1 indicate that the ratio of these two sig-
nals which is a function of the equilibrium constants (KT = [3A]/
[3B]) was found to depend on the substituent on the aryl moiety.
This ratio revealed that the ratio of the keto-hydrazone form 3A in-
creases and that of the hydroxy-azo form 3B decreases as the elec-
tron-donating property of the substituent increases.
Next, we investigated the behavior of the enaminone 1 toward
some diazotized heterocyclic amines as potential precursors for
fused heterocyclic systems.25 Thus, coupling the reaction of enami-
none 1 with the diazonium salt of 3-amino-1,2,4-triazole 5 in pyr-
idine afforded the corresponding hydrazone 6 which undergoes
in situ intramolecular cyclization to give 7,8-Dihydro-6H-benzo
[60,70] cyclohepta [10,20-e] [1,2,4] triazolo [3,4,-c] [1,2,4] triazine 7
(Scheme 2). The structure of the isolated product 7 was established
by its spectral (IR, 1H NMR and MS) data and elemental analyses
(see Section 4). Its mass spectrum revealed molecular ion peak at
m/z 237(M+) and its 1H NMR spectrum showed a characteristic sig-
nal at d 8.95 of the triazole CH proton.30
Table 1
UV Spectral data in dioxane and KT of compounds 3a–i in (DMSO-d6)
Compound kmax (log
no.
e)
Ratio of 3A in
Ratio of 3B in
KT
1H NMR
1H NMR
3a
3b
3c
3da
3e
3f
404 (5.32), 288
(4.75), 233 (4.27)
423 (4.65), 284
(4.14), 234 (4.15)
410 (4.72), 286
(4.19), 233 (4.17)
415 (4.27), 285
(3.98), 231 (3.91)
415 (4.96), 287
(4.42), 232 (4.27)
406 (4.55), 286
(4.13), 233 (4.08)
412 (4.62), 287
(4.35), 233 (4.18)
409 (4.70), 276
(3.93), 234 (4.02)
415 (4.10), 285
(3.91), 232 (3.80)
0.2
0.8
0.25
0.64
0.67
0.79
1.13
1.17
1.78
2.03
2.33
0.39
0.4
0.61
0.6
0.44
0.53
0.54
0.55
0.67
0.70
0.56
0.47
0.46
0.45
0.33
0.30
3g
3h
3i
a
Solvent kmax (log e): acetic acid 415 (4.43), 274 (3.85), 232 (3.94); acetone 411
(4.40), 298 (4.00), 231 (3.91); chloroform 418 (4.55), 282 (4.07), 236 (4.04); DMF
410 (4.36), 277 (3.81), 238 (3.89); ethanol 413 (4.51), 279 (3.96), 233 (4.01).

8014
T. A. Farghaly, M. M. Abdalla / Bioorg. Med. Chem. 17 (2009) 8012–8019
N
N
Cl
N
N
N
O
Cl
N N
5
N
H
N(Me)2
NH
8
1
N=N-Cl
NH
N
N
N
H
N
H
Ph
N
H
N
H
N
N
N
O
O
11
N
Ph
N
9
H
6
N
H
N
- H2O
- H2O
O
N
N
N
N
N
N
N
N
N
N
12
- H2O
10
7
Ph
N
N
N
N
13
Scheme 2.
In a similar manner, enaminone 1 coupled readily with diazo-
nium salt of 2-aminobenzimidazole 8 and 5-amino-3-phenylpyra-
zole 11 under the same experimental conditions, to afford a single
product in each case according to TLC (Scheme 2). The structure as-
signed for the isolated products was based on the elemental anal-
yses and spectral data. For example, 1H NMR spectrum of
compound 13 revealed a singlet signal at d 8.54 which is character-
istic of the proton of pyrazole ring.31
2.2.2. Pharmacology
Benzosuberone derivatives have some cytotoxic, antioxidant,
and anti-angiogenic activities.32 Also, in our previous work,24
we demonstrated the ability of nine newly synthesized benzosu-
berone derivatives to inhibit Hepatitis C Virus (HCV) and Suba-
cute Sclerosing Panencephalitis (SSPE), besides their proved
potent antioxidant activities. Based on these strong biological
activities, we aimed to test the antioxidant, anti-HCV, and anti-
tumor activities.
2.2. Biological screening
2.2.2.1. Hepatitis C virus (HCV) NS3-4A protease inhibitory
2.2.1. Antimicrobial activity
activities in both HCV replicon cells and hamster brains.
In
In vitro antimicrobial screening of seven of the synthesized
hydrazones 3 was evaluated against four antibacterial species,
namely, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus
subtilis, and Escherichia coli, and four fungal strains, namely, Asper-
gillus fumigatus, Penicillium italicum, Syncephalastrum racemosum,
and Candida albicans. Activities of the compounds and the mini-
mum inhibitory concentration (MIC) of the tested compounds
were evaluated by agar diffusion method. Compounds 3b, 3c, 3g,
and 3i exhibited highly significant activity against bacterial species
as compared with the reference drugs and also, the tested com-
pounds had moderate activity against the fungi as compared with
the reference (Terbinafin) as shown in Table 2. In addition, the MIC
was considered to be the lowest concentration of the tested sub-
stance exhibiting no visible growth of the bacteria or fungi on
the plate as shown in Table 2I.
order to monitor the potential of eight newly synthesized com-
pounds 3 to inhibit HCV, we used two methods for the determina-
tion of minimum inhibitory concentration (MIC) of these
compounds. Firstly, their antiviral activities in HCV replicon cells
were measured by their Hepatitis C Virus (HCV) NS3-4A Protease
Inhibitory activities. Secondly, their antiviral chemotherapy for
Subacute Sclerosing Panencephalitis (SSPE) was determined in
hamster brains. Ribaverin was used in both methods as a positive
control.
Table 3 shows the MIC values for the tested compounds 3 and
ribaverin against HCV and SSPE. The results showed that all the
nine tested compounds are highly effective at very low concentra-
tions compared to ribaverin in the following descending order: 3f,
3a, 3c, 3h, 3g, 3d, 3i, and 3b. It is worth to mention that all the
tested compounds are more potent than ribaverin.

T. A. Farghaly, M. M. Abdalla / Bioorg. Med. Chem. 17 (2009) 8012–8019
8015
Table 2
Preliminary antimicrobial activity for tested compounds
Microorganism
3b (l
g/mL)
3c (l
g/mL)
3f (lg/mL)
5.0
2.5
1.25
5.0
2.5
1.25
5.0
2.5
1.25
Aspergillus fumigatus
Penicillium italicum
Syncephalastrum racemosum
Candida albicans
Staphylococcus aureus
Pseudomonas aeruginosa
Bacillus subtilis
22
0
18
14
12
0
13
0
12
6
8
0
9
0
7
2
5
0
4
0
22
14
19
9
12
0
14
6
12
6
8
0
9
3
8
2
5
0
8
0
14
0
16
15
12
0
8
0
9
11
9
0
6
0
6
7
5
0
7
0
16
7
6
0
15
12
10
6
17
9
10
0
Escherichia coli
3g
3h
3i
Aspergillus fumigatus
Penicillium italicum
Syncephalastrum racemosum
Candida albicans
Staphylococcus aureus
Pseudomonas aeruginosa
Bacillus subtilis
20
0
19
15
10
0
11
0
12
8
6
0
8
0
9
5
0
0
8
0
18
0
16
12
9
0
14
8
8
0
9
6
3
0
8
0
5
0
4
0
0
0
3
0
20
17
16
10
15
0
11
6
9
7
8
0
10
0
6
4
6
3
5
0
7
0
19
12
13
5
16
12
Escherichia coli
Microorganism
7 (
lg/mL)
St. (lg/mL)
5.0
2.5
1.25
5.0
2.5
1.25
Aspergillus fumigatus
Penicillium italicum
Syncephalastrum racemosum
Candida albicans
Staphylococcus aureus
Pseudomonas aeruginosa
Bacillus subtilis
10
8
18
9
14
0
16
9
0
0
9
4
8
0
7
0
0
0
0
0
5
0
4
0
24
19
21
19
15
11
22
27
18
9
13
10
6
5
18
20
11
4
9
6
4
0
11
13
Escherichia coli
St. = reference standard Terbinafin was used as a standard antifungal agent while chloramaphenicol was used as a standard antibacterial agent.
Table 2I
in methanol which causes an absorbance drop at 515 nm. Among
the 10 compounds tested for antioxidant activity using DPPH
method, six exhibited good scavenging activity against the DPPH
radical in the following descending order: 3f, 3a, 3c, 3h, 3g, 3d,
3i, and 3b. It is worth to mention that all the tested compounds
are more potent than ascorbic acid.
The minimum inhibitory concentration (MIC)
Tested microorganisms
3b
3c
3i
Aspergillus fumigatus
Penicillium italicum
Syncephalastrum racemosum
Candida albicans
Staphylococcus aureus
Bacillis subtilis
0.313
—
0.313
1.25
0.313
1.25
0.625
0.313
2.50
0.625
1.25
0.313
1.25
0.625
0.313
—
0.625
1.25
0.625
0.625
—
2.2.2.3. Inhibition of peroxynitrite-induced tyrosine nitra-
Escherichia coli
tion.
The reactive nitrogen species peroxynitrite (ONOOꢀ)
has been implicated in human pathology and its role is usually in-
ferred from the measurement of 3-nitrotyrosine (3-NT). Peroxyni-
trite and species derived from it can oxidize and nitrate lipids,33
proteins,34 DNA35,36, and carbohydrates37 leading to tissue damage
in a number of pathological conditions in humans and in experi-
mental animals. Due to the cytotoxicity of ONOOꢀ and its apparent
formation at sites of tissue injury,38,39 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.40
2.2.2.2. DPPH radical scavenging activity.
Radical scaveng-
ing activities of eight newly synthesized compounds 3 were mea-
sured using a model colorimetric test: DPPH radical scavenging
test. The results are summarized in Table 4. The stable radical
DPPH has been used widely for the determination of primary anti-
oxidant 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
Table 3
MIC of ribaverin and the eight tested compounds against HCV and SSPE
The relative inhibitory activity of these compounds is summa-
rized in Table 5 as IC50 values. Under our experimental conditions
the relative potencies are in descending order: 3f, 3a, 3c, 3h, 3g, 3d,
3i, and 3b.
It is worth to mention that all the tested compounds are more
potent than trolox (Table 5).
Tested compound
MIC (lg/ml)
HCV
Subacute sclerosing panencephalitis (SSPE)
Ribaverin
16.15
1.011
1.141
1.021
1.071
1.000
1.043
1.034
1.092
77.89
6.010
7.06
6.011
6.122
6.001
6.111
6.079
6.123
3a
3b
3c
3d
3f
3g
3h
3i
2.2.2.4. In vivo efficacy study.
The in vivo activity of the
tested compounds and etoposide against murine Colon 38 cancer
was tested (Table 6). Treatment of Colon 38 implanted into mice
with ER-37328 induced a clear dose-dependent inhibition of tumor

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T. A. Farghaly, M. M. Abdalla / Bioorg. Med. Chem. 17 (2009) 8012–8019
Table 4
Table 7
Decrease of DPPH absorbance (%) by tested compounds
The correlation coefficients of the newly synthesized compounds with topoisomerase
I inhibitors
Compound no.
Decrease of DPPH absorbance% mean SD (n = 3)
Compound no.
The correlation coefficients with etoposide, doxorubicin,
SN-38 (topoisomerase I inhibitor), and cisplatin, calculated
according to the NCI COMPARE analysis procedure using the
tested compound as the benchmark
Ascorbic acid (standard)
79.042 2.928
83.89 0.099
80.12 0.088
83.11 0.091
80.98 0.064
84.13 0.092
81.14 0.082
82.00 0.081
80.98 0.089
3a
3b
3c
3d
3f
3g
3h
3i
Etoposide
Doxorubicin
SN-38
Cisplatin
3a
3b
3c
3d
3f
3g
3h
3i
0.38
0.32
0.37
0.34
0.40
0.35
0.36
0.33
0.36
0.29
0.35
0.32
0.37
0.33
0.34
0.31
0.35
0.27
0.34
0.3
0.36
0.33
0.32
0.29
0.33
0.25
0.32
0.25
0.34
0.2
0.31
0.26
Table 5
IC50 values of the compounds tested for the inhibition of ONOOꢀ-mediated 3-
nitrotyrosine formation
Compound no.
IC50 (lM) mean SD (n = 3)
Table 8a
The relative potencies of the newly synthesized compounds to etoposide
Trolox
3a
3b
3c
3d
3f
3g
58.430 5.9
54.190 0.0576
56.345 0.0511
55.088 0.0555
56.007 0.0533
54.099 0.0587
56.00 0.0543
Compound no.
Relative potency to etoposide
3a
3b
3c
3d
3f
4.90
4.27
4.66
4.40
5
3h
3i
5.130
0.0555
56.123 0.0522
3g
3h
3i
4.53
4.60
4.33
Table 6
Antitumor activities
Table 8b
The relative potencies of the newly synthesized compounds to etoposide
Compound no
Average GI50
Relative potency to etoposide
Compound no.
Relative potency to etoposide
3a
3b
3c
3d
3f
3g
3h
3i
0.61
0.7
0.64
0.68
0.6
0.66
0.65
0.69
4.918032787
4.285714286
4.6875
4.411764706
5
4.545454545
4.615384615
4.347826087
3a
3b
3c
3d
3f
3g
3h
3i
4.80
4.20
4.46
4.30
4.90
4.50
4.40
4.30
growth. Moreover, marked tumor regression was observed at
3 mg/kg. No body weight was observed at 3 mg/kg. All the tested
compounds showed potent antitumor activities and the order of
activity is in descending order: 3f, 3a, 3c, 3h, 3g, 3d, 3i, and 3b.
In a progress to determine the mechanism of action of the anti-
tumor activities. The correlation coefficients with etoposide, doxo-
rubicin, SN-38 (topoisomerase I inhibitor), and cisplatin were
calculated according to the NCI COMPARE analysis procedure using
the tested compound as the benchmark. These calculations culmi-
nated on the newly synthesized compounds that act as topoiso-
merase inhibitors (Table 7).
3. Conclusion
In conclusion, we synthesized new hydrazones through the cou-
pling of enaminone 1 with diazotized aniline derivatives and the
tautomeric structures of the product were studied. Also, coupling
of enaminone 1 with the diazonium salt of heterocyclic amine
afforded tetra and penta heterocyclic ring systems. The newly syn-
thesized compounds seem to be interesting for biological activity
studies. These compounds were investigated for antimicrobial,
anti-HCV, antioxidant, and antitumor (as topoisomerase I inhibi-
tors) activities. All the tested compounds are highly effective at
very low concentrations compared to the reference in all biological
activity studies.
To confirm this mechanistic pathway of action the following as-
says are done.
2.2.2.5. Inhibition of topoisomerase II activity.
The effects
of the tested compounds and etoposide on topoisomerase II activ-
ity were analyzed by means of a relaxation assay. The tested com-
pounds and etoposide inhibited topoisomerase II activity, and the
relative potency of each compound was determined and confirmed
in the following descending order: 3f, 3a, 3c, 3h, 3g, 3d, 3i, and 3b
(see Table 8a).
All the previously mentioned measurements revealed that all
the tested compounds are more active than etoposide and act sim-
ilar to its mechanism of action as topoisomerase I inhibitors (see
Table 8b).
4. Experimental
4.1. Chemistry
Melting points were uncorrected. IR spectra were recorded
(KBr) on a Pye Unicam SP-1000 Spectrophotometer. 1H NMR spec-
tra were obtained on a Varian Gemini 300 MHz spectrometer using
TMS as an internal reference. Chemical shifts were expressed as d
(ppm). Mass spectra were recorded on a GCMS-QP 1000 EX mass

T. A. Farghaly, M. M. Abdalla / Bioorg. Med. Chem. 17 (2009) 8012–8019
8017
spectrometer operating at 70 eV. The elemental analyses were per-
formed at the Microanalytical Center at Cairo University, Giza,
Egypt. 2-Dimethylaminomethylene-1-benzosuberone (1) was pre-
pared by following the literature method.24
4.1.1.5. 2-[4-Chlorophenylhydrazono]-1-benzosuberone (3e).
Yellow needles, yield (1.27 g, 85%), mp 146–148 °C (Ethanol);
1H NMR (DMSO-d6) 1.97–2.01 (m, 2H, CH2), 2.27–2.80 (m, 4H,
2CH2), 7.24 (d, J = 9 Hz, 2H, ArH), 7.31 (d, J = 9 Hz, 2H, ArH), 7.36–
7.63 (m, 4H, ArH), 10.32 (s, 0.53H, NH), 13.86 (s, 0.47H, OH); 1H
NMR (CDCl3); 1.97–2.76 (m, 6H, 3CH2), 7.12–7.36 (m, 4H,
ArH),7.38 (d, J = 9 Hz, 2H, ArH), 7.51 (d, J = 9 Hz, 2H, ArH), 13.93
4.1.1. Preparation of 2-(arylhydrazono)-1-benzosuberone (3a–i)
To a stirred solution of the enaminone 1 (1.10 g, 5 mmol) in eth-
anol (20 mL) was added sodium acetate trihydrate (0.7 g, 5 mmol)
and the mixture was cooled in an ice bath to 0–5 °C. To the result-
ing solution, while being stirred, was added dropwise over a period
of 20 min a solution of the appropriate arenediazonium chloride,
prepared as usual by diazotizing the respective aniline (5 mmol)
in hydrochloric acid (6 M, 3 mL) with sodium nitrite solution
(5 mmol) in (5 mL) water. The whole mixture was then left in a
refrigerator overnight. The precipitated solid was collected,
washed with water, and finally crystallized from the appropriate
solvent to give the respective hydrazones (3a–i).
(s, 1H, OH); IR
m
(cmꢀ1): 3406 cmꢀ1; Ms m/z (%) 298 (M+, 83),
172(27), 119(13), 116(50), 115(30), 107(53), 106(20), 91(30),
89(30), 79(87), 77(87), 65(100). Anal. Calcd for C17H15ClN2O
(298.77): C, 68.34; H, 5.06; N, 9.38. Found: C, 68.51; H, 4.99; N,
9.24.
4.1.1.6. 2-(3-Chlorophenylhydrazono)-1-benzosuberone (3f).
Orange needles, yield (0.91 g, 61%), mp 158–160 °C (Ethanol);
1H NMR (DMSO-d6) 1.97–2.46 (m, 2H, CH2), 2.62–2.82 (m, 4H,
2CH2), 6.92–7.66 (m, 8H, ArH), 10.34 (s, 0.54H, NH), 13.52 (s,
0.46H, OH); 1H NMR (CDCl3); 1.97–2.46 (m, 2H, CH2), 2.62–2.82
(m, 4H, 2CH2), 6.92–7.66 (m, 8H, ArH), 13.62 (s, 1H, OH); IR
m
4.1.1.1. 2-[4-Methoxyphenylhydrazono]-1-benzosuberone (3a).
Red solid, yield (0.91 g, 62%), mp 214–216 °C (Ethanol/Diox-
ane); 1H NMR (DMSO-d6) 2.02–2.04 (m, 2H, CH2), 2.51–2.82 (m,
4H, 2CH2), 3.84 (s, 3H, OCH3), 7.33–7.61 (m, 4H, ArH), 7.64 (d,
J = 9 Hz, 2H, ArH), 8.09 (d, J = 9 Hz, 2H, ArH), 12.20 (s, 0.2H, NH),
14.87 (s, 0.8H, OH); 1H NMR (CDCl3) 2.01–2.06 (m, 2H, CH2),
2.50–2.82 (m, 4H, 2CH2), 3.82 (s, 3H, OCH3), 7.33–7.61 (m, 4H,
ArH), 7.64 (d, J = 9 Hz, 2H, ArH), 8.09 (d, J = 9 Hz, 2H, ArH), 14.87
(cmꢀ1): 3448 cmꢀ1; Ms m/z (%) 300 (M++2, 5), 299 (M++1, 10),
298 (M+, 19), 172(49), 127(27), 117(39), 116(38), 111(31),
91(100), 77(58), 63(98). Anal. Calcd for C17H15ClN2O (298.77): C,
68.34; H, 5.06; N, 9.38. Found: C, 68.19; H, 5.21; N, 9.16.
4.1.1.7. 2-(3-Nitrophenylhydrazono)-1-benzosuberone (3g).
Yellow needles, yield (1.30 g, 84%), mp 160–162 °C (Ethanol/
Dioxane); 1H NMR (DMSO-d6) 1.99–2.04 (m, 2H, CH2), 2.51–2.81
(m, 4H, 2CH2), 7.33–8.26 (m, 8H, ArH), 10.60 (s, 0.55H, NH),
13.51 (s, 0.45H, OH); 1H NMR (CDCl3); 1.99–2.04 (m, 2H, CH2),
2.51–2.81 (m, 4H, 2CH2), 7.33–8.26 (m, 8H, ArH), 13.50 (s, 1H,
(s, 1H, OH); IR
m
(cmꢀ1): 3421 cmꢀ1; Ms m/z (%) 294 (M+, 61),
133(6), 132(67), 117(50), 107(89), 91(33), 88(39), 77(39), 74(28),
63 (100). Anal. Calcd for C18H18N2O2 (294.36); C, 73.45; H, 6.16;
N, 9.52. Found: C, 73.33; H, 6.09; N, 9.41.
OH); IR
m
(cmꢀ1): 3433 cmꢀ1; Ms m/z (%) 310 (M++1, 14), 309
(M+, 77), 172(76), 144(19), 117(44), 115(47), 91(100), 89(31),
77(36), 64(68). Anal. Calcd for C17H15N3O3 (309.33): C, 66.01; H,
4.89; N, 13.58. Found: C, 65.95; H, 4.75; N, 13.49.
4.1.1.2. 2-[4-Methylphenylhydrazono]-1-benzosuberone (3b).
Yellow needles, yield (1.14 g, 82%), mp 146–148 °C (Ethanol);
1H NMR (DMSO-d6) 1.97–2.01 (m, 2H, CH2), 2.27 (s, 3H, CH3),
2.27–2.80 (m, 4H, 2CH2), 7.24 (d, J = 9 Hz, 2H, ArH), 7.31 (d,
J = 9 Hz, 2H, ArH), 7.36–7.63 (m, 4H, ArH), 10.18 (s, 0.39H, NH),
13.86 (s, 0.61H, OH); 1H NMR (CDCl3) 1.97–2.04 (m, 2H, 1CH2),
2.25 (s, 3H, CH3), 2.41–2.75 (m, 4H, 2CH2), 7.24 (d, J = 9 Hz, 2H,
ArH), 7.31 (d, J = 9 Hz, 2H, ArH), 7.36–7.63 (m, 4H, ArH), 14.04 (s,
4.1.1.8. 2-(4-Nitrophenylhydrazono)-1-benzosuberone (3h).
Yellow needles, yield (1.36 g, 88%), mp 210–212 °C (Ethanol/
Dioxane); 1H NMR (DMSO-d6) 1.97–2.06 (m, 2H, 1CH2), 2.68–
2.86 (m, 4H, 2CH2), 7.31–7.68 (m, 4H, ArH), 7.70 (d, J = 9 Hz, 2H,
ArH), 8.19 (d, J = 9 Hz, 2H, ArH), 10.82 (s, 0.67H, NH), 13.43 (s,
0.33H, OH); 1H NMR (CDCl3); 1.97–2.79 (m, 6H, 3CH2), 7.14(d,
J = 9 Hz, 2H, ArH), 7.18–7.56 (m, 4H, ArH), 8.14 (d, J = 9 Hz, 2H,
1H, OH); IR
m
(cmꢀ1): 3436 cmꢀ1; Ms m/z (%) 278 (M+, 83),
172(27), 119(13), 116(50), 115(30), 107(53), 106(20), 91(30),
89(30), 79(87), 77(87), 65(100). Anal. Calcd for C18H18N2O
(278.36): C, 77.67; H, 6.52; N, 10.06. Found: C, 77.45; H, 6.41; N,
10.23.
ArH), 13.85 (s, 1H, OH); IR
m
(cmꢀ1): 3413, 3278, 1658 cmꢀ1; Ms
m/z (%) 310 (M++1, 10), 309 (M+, 65), 172(66), 138(17), 122(9),
116(97), 115(69), 103(51), 91(100), 77(59), 64(90). Anal. Calcd
for C17H15N3O3 (309.33): C, 66.01; H, 4.89; N, 13.58. Found: C,
66.32; H, 4.77; N, 13.74.
4.1.1.3. 2-[3-Methylphenylhydrazono]-1-benzosuberone (3c).
Yellow solid, yield (0.71 g, 51%), mp 110–112 °C (ethanol); 1H
NMR (DMSO-d6) 1.99–2.04 (m, 2H, CH2), 2.51 (s, 3H, CH3), 2.51–
2.84 (m, 4H, 2CH2), 7.04–7.79 (m, 8H, ArH), 10.18 (s, 0.4H, NH),
14.02 (s, 0.6H, OH); 1H NMR (CDCl3); 1.98–2.04 (m, 2H, CH2),
2.50 (s, 3H, CH3), 2.51–2.84 (m, 4H, 2CH2), 7.04–7.79 (m, 8H,
4.1.1.9. 2-(4-Acetylphenylhydrazono)-1-benzosuberone (3i).
Yellow solid, yield (1.19 g, 78%), mp 200–202 °C (Dioxane); 1H
NMR (DMSO-d6) 1.96–2.05 (m, 2H, CH2), 2.52 (s, 3H, COCH3),
2.66–2.84 (m, 4H, 2CH2), 7.30–7.92 (m, 4H, ArH), 7.93 (d,
J = 8 Hz, 2H, ArH), 7.95 (d, J = 8 Hz, 2H, ArH), 10.55 (s, 0.7H, NH),
13.59 (s, 0.3H, OH); 1H NMR (CDCl3); 1.96–2.05 (m, 2H, CH2),
2.52 (s, 3H, COCH3), 2.66–2.84 (m, 4H, 2CH2), 7.30–7.92 (m, 4H,
ArH), 7.93 (d, J = 8 Hz, 2H, ArH), 7.95 (d, J = 8 Hz, 2H, ArH), 13.59
ArH), 14.02 (s, 1H, OH); IR
m
(cmꢀ1): 3420 cmꢀ1; Ms m/z (%) 278
(M+, 18), 172(60), 131(23), 118(26), 116(74), 104(21), 91(100),
77(58), 65(51). Anal. Calcd for (278.36): C,77.67; H, 6.52; N,
10.06. Found: C,77.39; H, 6.46; N, 9.88.
(s, 1H, OH); IR
m
(cmꢀ1): 3395, 1702 cmꢀ1; Ms m/z (%) 306 (M+,
4.1.1.4. 2-Phenylhydrazono-1-benzosuberone (3d).
Orange
63), 172(75), 115(36), 107(80), 106(31), 91(30), 89(43), 77(97),
65(100). Anal. Calcd for C19H18N2O2 (306.37): C, 74.49; H, 5.92;
N, 9.14. Found: C, 74.31; H, 5,87; N, 9.00.
needles, yield (0.95 g, 72%), mp 90 °C. (Ethanol/Dioxane); 1H NMR
(DMSO-d6) 1.97–2.01 (m, 2H, CH2), 2.51–2.81 (m, 4H, 2CH2), 6.90–
7.65 (m, 9H, ArH), 10.23 (s, 0.44H, NH), 13.77 (s, 0.56H, OH); 1H
NMR (CDCl3); 1.95–2.76 (m, 6H, 3CH2), 6.94–7.55 (m, 9H, ArH),
4.1.2. Coupling of enaminone 1 with diazonium salts of
heterocyclic amines
A solution of enaminone 1 (1.10 g, 5 mmol) in pyridine (20 ml)
was cooled in an ice bath at 0–5 °C while being stirred. To the
resulting cold solution was added portionwise a cold solution of
14.00 (s, 1H, OH) IR
m
(cmꢀ1): 3420 cmꢀ1; Ms m/z (%) 265 (M++1,
9), 264 (M+, 36), 172(39), 145(11), 118(18), 116(38), 91(80),
77(85), 65(100). Anal. Calcd for C17H16N2O (264.33): C, 77.25; H,
6.10; N, 10.60. Found: C, 77.02; H, 6.34; N, 10.52.

8018
T. A. Farghaly, M. M. Abdalla / Bioorg. Med. Chem. 17 (2009) 8012–8019
the appropriate diazonium salt of 3-amino-1,2,4-triazole 5, 2-ami-
nobenzimidazole 8, or 5-amino-3-phenylpyrazole 11, prepared as
usual by diazotized heterocyclic amine (5 mmol) in nitric acid
(6 M, 3 ml) with sodium nitrite (0.35 g, 5 mmol) in water (5 ml).
After all the diazonium salt was added, the mixture was stirred
for further 30 min while cooling in an ice bath. The reaction mix-
ture was then left in a refrigerator for three days. The solid that
precipitated was filtered off, washed with water, dried, and finally
crystallized from the appropriate solvent to give product 7, 10, or
13, respectively. The physical constants and the spectral data of
the isolated products 7, 10, and 13 are listed below.
fuged at 1600 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.
4.1.5. Radical scavenging assays
DPPH radical scavenging assay: The antioxidant activities of the
tested compounds and the standard were assessed on the basis
of the radical scavenging effect of the stable DPPHÅ free radical.41
Weighed quantities of the ten compounds were dissolved in dis-
tilled DMSO and used. Solution of ascorbic acid used as a standard
for this study was prepared in distilled H2O. All these solutions
were serially diluted with respective solvents to get lower
dilutions.
4.1.2.1. 7,8-Dihydro-6H-benzo[60,70]cyclohepta[10,20-e]
[1,2,4]triazolo[3,4-c] [1,2,4]triazine (7).
Pale yellow solid,
yield (1.01 g, 85%), mp196–198 °C (Dioxane); 1H NMR (DMSO-d6)
2.39–2.71 (m, 6H, 3CH2), 7.51–8.20 (m, 4H, ArH), 8.95 (s, 1H, tria-
zole-H); Ms m/z (%) 238 (M++1, 12), 237 (M+, 12), 127(19), 101(21),
89(27), 87 (27), 74(15), 63(23), 53(100). Anal. Calcd for C13H11N5
(237.27): C, 65.81; H, 4.67; N, 29.52. Found: C, 65.98; H, 4.80; N, 29.31.
Each compound (10
100 M/ml) was added to 90
(100 M) in a 96-well microtitre plate. After incubation in the dark
l
l) or standard (from 0.0
lM/ml to
l
l
l
l of DPPHÅ in methanol solution
at 37 °C for 30 min, the decrease in absorbance of each solution
was measured at 515 nm using ELISA microplate reader (Bio Rad
Laboratories Inc., California, USA, Model 550). Absorbance of blank
sample containing the same amount of DMSO and DPPHÅ solution
was done and measured as well. The experiment was carried out
in triplicate. The scavenging potential was compared with a sol-
vent control (0% radical scavenging) and ascorbic acid. Radical
scavenging activity was calculated by the following formula:
4.1.2.2. 9,10-Dihydro-8H-benzo[60,70]cyclohepta[10,20-e]benzim-
idazo[2,1-c] [1,2,4]triazine (10).
Dark yellow solid, yield
(1.29 g, 90%), mp 206 °C. (Dioxane); 1H NMR (DMSO-d6) 2.05–
2.81 (m, 6H, 3CH2), 7.40–8.16 (m, 8H, ArH); Ms m/z (%) 287
(M++1, 24), 286 (M+, 100), 257(39), 129 (29), 128(40), 127(17),
115(39), 102 (28), 91(12), 89(32), 77(40), 63 (58). Anal. Calcd for
C18H14N4 (286.34): C, 75.51; H, 4.93; N, 19.57. Found: C, 75.24;
H, 4.77; N, 19.29.
% Reduction of absorbance ¼ ½ðAB ꢀ AAÞ=ABꢂ ꢁ 100;
where AB indicates absorbance of blank sample; and AA indicates
absorbance of tested extract solution (t = 30 min).
4.1.2.3. 2-Phenyl-7,8-Dihydro-6H-benzo[60,70]cyclohepta[10,20-
4.1.6. Reaction with reactive nitrogen species
4.1.6.1. Synthesis of peroxynitrite.
e]pyrazolo[5,1-c] [1,2,4]triazine (13).
Yellow solid, yield
Peroxynitrite (ONOOꢀ)
(1.25 g, 80%), mp 172 °C. (Ethanol/Dioxane); 1H NMR (CDCl3)
2.06–2.90 (m, 6H, 3CH2), 6.32–7.53 and 7.56–7.80 (m, 9H, ArH),
8.54 (s, 1H, pyrazole-H); Ms m/z (%) 313 (M++1, 3), 312 (M+, 22),
160(43), 144(46), 131(100), 118 (59), 115(489, 91(54), 89(60),
77(85). Anal. Calcd for C20H16N4 (312.38): C, 76.90; H, 5.16; N,
17.94. Found: C, 76.72; H, 5.02; N, 18.14.
was synthesized as previously described.40 Briefly, an acidic solu-
tion (0.6 M HCl) of H2O2 (0.7 M) was mixed with KNO2 (0.6 M)
on ice for one second and the reaction was quenched with ice-cold
NaOH (1.2 M). Residual H2O2 was removed by mixing with granu-
lar MnO2 prewashed with NaOH (1.2 M). The stock solution was fil-
tered and then frozen overnight (ꢀ20 °C) and the top layer of the
solution was collected for the experiment. Concentrations of stock
ONOOꢀ were redetermined before each experiment at 302 nm
using a molar absorption coefficient of 1670 cmꢀ1 Mꢀ1. Concentra-
tions of 200–250 mM were usually obtained. Once thawed, ONOOꢀ
solutions were kept in ice for no longer than 30 min before use.
4.1.3. Determination of minimum inhibitory concentration
(MIC) of ribavirin and different tested compounds in HCV
replicon cells
Briefly, 1 ꢁ 104 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 were
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 RT-PCR (QRT-PCR) assay. Each da-
tum 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 ꢁ 104 parental Huh-7 cells
per well or 4 ꢁ 104 HepG2 cells per well were used.
4.1.7. Reaction of compounds with peroxynitrite
The ability of tested compounds to inhibit peroxynitrite-in-
duced tyrosine nitration was investigated via reaction of equimolar
concentrations (100
ence of increasing concentrations of each tested compound (0–
300 M) in 100 mM phosphate buffer, pH 7.4, at 37 °C for
lM) of tyrosine and peroxynitrite in the pres-
l
15 min. Snap-freezing reaction mixtures prior to HPLC analysis
successfully terminated the reactions. The formation of 3-nitroty-
rosine (3-NT) was monitored by HPLC analyzed with photodiode
array detection (see below). 3-NT formed was characterized and
quantified by use of an authentic standard (elution time and un-
ique spectral characteristics).
4.1.4. MIC of the tested compounds in hamster brains for
antiviral chemotherapy for subacute sclerosing panencephalitis
(SSPE)
4.1.8. HPLC analysis
Reactionmixtureswereanalyzedusingreverse-phase HPLCanal-
ysis performed on an Agilent 1100 system with a Zorbax ODS C18
Under 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 intracranially to a depth of 2 mm by using a 27-gauge nee-
dle and was placed within the subarachnoid space. On 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 re-
moved, washed twice with phosphate-buffered saline (PBS),
homogenized, and suspended in PBS. The suspension was centri-
column (150 ꢁ 4.6 mm i.d.,
4
l
m) and
a
guard column
(15 ꢁ 4.6 mm i.d., 4 m). Mobile phase A consisted of methanol/
l
water/ 5 N HCl (5:94.9:0.1 v/v/v) and mobile phase B consisted of
acetonitrile/water/5 N HCl (50:49.9:0.1 v/v/ v). The following gradi-
ent system was 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

T. A. Farghaly, M. M. Abdalla / Bioorg. Med. Chem. 17 (2009) 8012–8019
8019
monitored by photodiode array detection at 280 nm for 3-NT mea-
surements with spectra of products obtainedoverthe220–600 nmrange.
with TBE buffer [89 mM Tris, 89 mM boric acid, and 2 mM EDTA
(pH 8.0)]. The DNA was stained with ethidium bromide and photo-
graphed under UV light.
4.1.9. Antitumor activity of novel topoisomerase II inhibitors
DNA topoisomerase II has been shown to be an important ther-
apeutic target in cancer chemotherapy. Herein, we describe studies
on the antitumor activity of novel topoisomerase II inhibitors.
The tested compounds inhibited topoisomerase II activity at 10
times lower concentration than etoposide in relaxation assay and
cells. Murine Colon 38 cancer, murine leukemia P388, and human
lung cancer LX-1 were obtained from the Cancer Chemotherapy
Center, Japan Foundation for Cancer Research (Tokyo, Japan). Hu-
man lung cancer PC-1 and human gastric cancer MKN-1, MKN-7,
MKN-28, and MKN-74 were obtained from Immuno Biology Labo-
ratory (Gunma, Japan). Human lung cancer A549 and human colon
cancer WiDr were obtained from Dainippon Pharmaceutical Co.,
Ltd (Osaka, Japan). Human gastric cancer HGC-27 and GT3TKB
were obtained from Riken Cell Bank (Ibaraki, Japan).The other cell
lines were purchased from American Type Culture Collection
(Rockville, MD).
4.1.13. Growth-inhibitory effect
The growth-inhibitory effects of the tested compounds 28 on 21
human solid tumor cell lines (8 lung, 7 colon, and 6 gastric cancer
cell lines) were determined. The tested compounds inhibited the
growth of these cell lines with an average GI50 given in Table 1
and relative potency to etoposide were determined. The correla-
tion coefficients with etoposide, doxorubicin, SN-38 (topoisomer-
ase I inhibitor), and cisplatin, calculated according to the NCI
COMPARE analysis procedure using the tested compound as the
benchmark, are given in Table 2, respectively. That is, the pattern
of the growth-inhibitory effect of the tested compounds was sim-
ilar to that of etoposide, but less similar to that of doxorubicin.
References and notes
1. Shawali, A. S.; Farghaly, T. A. Tetrahedron 2009, 65, 644.
2. Farghaly, T. A.; Abdallah, Z. A. Arkivoc 2008, xvii, 295.
3. Shawali, A. S.; Mosselhi, M. A. N.; Farghaly, T. A.; Shehata, M. R.; Tawfik, N. M. J.
Chem. Res. 2008, 452.
4. Shawali, A. S.; Mosselhi, M. A. N.; Altablawy, F. M. A.; Farghaly, T. A.; Tawfik, N.
M. Tetrahedron 2008, 64, 5524.
5. Shawali, A. S.; Sherif, S. M.; Farghaly, T. A.; Darwish, M. A. A. Afinidad 2008, 65,
314.
6. Shawali, A. S.; Mosselhi, M. A. N.; Farghaly, T. A. J. Chem. Res. 2007, 479.
7. Shawali, A. S.; Sherif, S. M.; Farghaly, T. A.; Shehata, M. R.; Darwish, M. A. A. J.
Chem. Res. 2007, 44.
8. Shawali, A. S.; Farghaly, T. A.; Edrees, M. M. Int. J. Pure Appl. Chem. 2006, 1, 531.
9. Shawali, A. S.; Zayed, M. M.; Farghaly, T. A. J. Heterocycl. Chem. 2005, 42, 185.
10. Shawali, A. S.; Farghaly, T. A. Tetrahedron 2004, 60, 3051.
11. Bhaskar, M.; Gnanamani, A.; Ganeshjeevan, R. J.; Chandraskar, R.; Sadulla, S.;
Radhakrishnan, G. J. Chromatogr., A 2003, 1018, 117.
4.1.10. Growth-inhibitory effect
Exponentially growing solid tumor cells in 0.1 ml of medium
were seeded in 96-well plates on day 0. On day 1, 0.1 ml aliquots
of medium containing graded concentrations of test drugs were
added to the cell plates. After incubation at 37 °C for 72 h, the cell
number was estimated by sulforhodamine B (SRB) assay, as de-
scribed by Skehan et al. The cell number on day 1 was also mea-
sured separately. GI50 values are the drug concentrations causing
a 50% reduction in the net protein increase in control cells. The
antitumor spectrum of ER-37328 was compared with those of
other drugs by means of the NCI COMPARE analysis procedure.
Exponentially growing P388 cells were seeded in 96-well plates
on day 0. On day 1, 0.1 ml aliquots of medium containing graded
concentrations of test drugs were added to the wells. After incuba-
tion at 37 °C for 72 h, the cell number was determined by MTT as-
say. IC50 values were calculated as the drug concentrations that
reduced the number of cells to 50% of the control number.
12. Navarro, A.; Sanz, F. Dyes Pigments 1999, 40, 131.
13. Carpicz, R.; Gulbinas, V.; Undzenas, A. J. Chin. Chem. Soc. 2000, 47, 589.
14. El-Gazzar, A. B. A.; El-Enany, M. M.; Mahmoud, M. N. Bioorg. Med. Chem. 2008,
16, 3261.
15. Alagarsamy, V.; Soloma, V. R.; Dhanabal, K. Bioorg. Med. Chem. 2007, 15, 235.
16. Ganguly, A. K.; Doll, A. K.; Girijavallabhan, V. M. Curr. Med. Chem. 2001, 8, 1419.
17. Kandile, N. G.; Mohamed, M. I.; Zaky, H.; Mohamed, H. M. Eur. J. Med. Chem.
2009, 44, 1989.
18. Vicini, P.; Incerti, M.; La Colla, P.; Loddo, R. Eur. J. Med. Chem. 2009, 44, 1801.
19. Banachiewicz, B. M.; Kaminska, T. Eur. J. Med. Chem. 2001, 36, 93.
20. Afonso, A.; Kelly, J. M.; Weinstein, J.; Wolin, R. L.; Rosenblum, S. B. US Patent 6
218 401, 2001; Chem. Abstr. 2001, 134, 295746.
4.1.11. In vivo efficacy study
Female C57BL/6 mice (6 weeks of age; Charles River, Atsugi, Ja-
pan) were housed in barrier facilities on a 12 h light/dark cycle,
with food and water ad libitum. About 30 mg of Colon 38 tumor
tissue was inoculated s.c. into the flank of animals. Mice were ran-
domized and separated into control and treatment (n = 5 each)
groups when the tumor volume reached approximately 300 mm3
on day 1. The tested compound was dissolved in 5% glucose. Etopo-
side was diluted in saline and doxorubicin was dissolved in saline.
The tested compound was administered on day 1 (single dose) by
iv injection into the tail vein, and etoposide and doxorubicin were
given at the maximum tolerated dose on the reported administra-
tion schedule. Control animals received 5% glucose. Tumor volume
and body weight were measured on the days indicated in the fig-
ures. Tumor volume was calculated according to the following
equation: tumor volume (mm3) = (length ꢀ width2)/2.
21. Joseph, B.; Algille, D.; Mérour, J. Y.; Léonce, S. Chem. Pharm. Bull. 2000, 48, 1872.
22. Sriram, M.; Hall, J. J.; Grohmann, N. C.; Strecker, T. E.; Wootton, T.; Franken, A.;
Trawick, M. L.; Pinney, K. G. Bioorg. Med. Chem. 2008, 16, 8161.
23. Nakayama, T.; Takashi, H. Jpn Kokai. Tokkyo Koho JP 03167178 A2, 1991; Chem.
Abstr. 1991, 115, 256204.
24. Farghaly, T. A.; Abdel Hafez, N. A.; Ragab, E. A.; Awad H. M.; Abdalla, M. M. Eur.
J. Med. Chem., in press.
25. Al-Mousawi, S.; Abdelkhalik, M. M.; John, E.; Elnagdi, M. H. J. Heterocycl. Chem.
2003, 40, 689.
26. Jones, R.; Ryan, A. J.; Sternhell, S.; Wright, S. E. Tetrahedron 1963, 19, 1497.
27. Daunis, J.; Follet, M.; Marzin, C. Org. Magn. Reson. 1980, 13, 330.
28. Nawrot-Modranka, J.; Nawrot, E.; Graczyk, J. Eur. J. Med. Chem. 2006, 41, 1301.
29. Hegarty, A. F.; Cashman, M. P.; Scott, F. L. Chem. Commun. 1971, 13, 884.
30. Wagle, S.; Adhikari, A. V.; Kumari, N. S. Eur. J. Med. Chem. 2009, 44, 1135.
31. Konova, E. S.; Makarov, V. A.; Alekeeva, L. M.; Avramenko, G. V.; Granik, V. G.
Russ. Chem. Bull. Int. Ed. 2006, 55, 735.
32. Grubbs, C. J.; Lubet, R. A.; Atigadda, V. R.; Christov, K.; Deshpande, A. M.;
Tirmal, V.; Xia, G.; Bland, K. I.; Eto, I.; Brouillette, W. J.; Muccio, D. D.
Carcinogenesis 2006, 27, 1232.
33. Radi, R.; Beckman, J. S.; Bush, K. M.; Freeman, B. A. Arch. Biochem. Biophys. 1991,
288, 481.
34. Ischiropoulos, H.; Al-Mehdi, A. B. FEBS Lett. 1995, 364, 279.
35. Yermilov, V.; Rubo, J.; Oshima, H. FEBS Lett. 1996, 376, 207.
36. Spencer, J. P. E.; Wong, J.; Jenner, A.; Aruoma, O. I.; Cross, C. E.; Halliwell, B.
Chem. Res. Toxicol. 1996, 9, 1152.
4.1.12. Relaxation assay
Topoisomerase II was purified from P388 cells. One unit of the
enzyme was defined as the activity to relax completely 0.125
of supercoiled pBR-322 DNA at 30 °C for 1 h. For the assay,
0.125 g of supercoiled pBR-322 DNA (Takara Shuzo Co., Ltd, To-
kyo, Japan) was relaxed with one unit of topoisomerase II in
20 l of the assay buffer [50 mM Tris–HCl (pH8.0), 100 mM KCl,
10 mM MgCl2, 0.5 mM DTT, 0.5 M EDTA, 1 mM ATP, and 30 g/
lg
37. Beckman, J. S.; Beckman, T. W.; Chen, J.; Marshall, P. A.; Freeman, B. A. Proc.
Natl. Acad. Sci. U.S.A. 1990, 87, 1620.
38. Halliwell, B.; Zhao, K.; Whiteman, M. Free Radicals Res. 1999, 31, 651.
39. Ischiropoulos, H. Arch. Biochem. Biophys. 1998, 356, 1.
40. Beckman, J. S.; Chen, J.; Ischiropoulos, H.; Crow, J. P. Methods Enzymol. 1994,
233, 229.
l
l
l
l
41. Gamez, E. J. C.; Luyengi, L.; Lee, S. K.; Zhu, L. F.; Zhou, B. N.; Fong, H. H. S.;
Pezzuto, J. M.; Kinghorn, A. D. J. Nat. Prod. 1998, 61, 706.
ml BSA] in the presence or absence of an inhibitor at 30 °C for
1 h. Samples were subjected to electrophoresis in 0.7% agarose gels