New pyrazoles incorporating pyrazolylpyrazole moiety: Synthesis, anti-HCV and antitumor activity

Article abstract of DOI:10.1016/j.ejmech.2009.11.050

Three series of novel pyrazole derivatives 2b-d, 4a-d and 6a-d were synthesized via two step procedure that utilizes hydrazonoyl chlorides 1a-d and enaminones 3a-d and 5a-d, respectively as starting materials. The structures of all the newly synthesized p

Full text of DOI:10.1016/j.ejmech.2009.11.050

European Journal of Medicinal Chemistry 45 (2010) 1042–1050
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New pyrazoles incorporating pyrazolylpyrazole moiety: Synthesis, anti-HCV
and antitumor activity
,
b
Sayed M. Riyadh a, Thoraya A. Farghaly a, Magda A. Abdallah a , Mohamed M. Abdalla ,
*
Mohamed R. Abd El-Aziz a
a Department of Chemistry, Faculty of Science, University of Cairo, Giza 12613, 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:
Three series of novel pyrazole derivatives 2b–d, 4a–d and 6a–d were synthesized via two step procedure
that utilizes hydrazonoyl chlorides 1a–d and enaminones 3a–d and 5a–d, respectively as starting
materials. The structures of all the newly synthesized products have been established on the basis of
analytical and spectral data. Moreover, some of the products 2–6 were tested against HCV and Subacute
Sclerosing Panencephalitis (SSPE). In addition, compounds 2–6 were also tested for the inhibition of
peroxynitrite-induced tyrosine nitration and antioxidant activity. The tested compounds are highly
effective at very low concentration as anti-HCV, SSPE antioxidant and anticancer in the following
ascending order 2d, 4c, 6b, 3b, 6c, 4d, 2b, 2c, 2a, 6a, 5b, 5a, 3a, 4b and 5c. It is worth to mention that all
tested compounds are more potent than the reference standards used for comparing activity. All the
measurements revealed that the mechanism of action of the anti cancer activities of all the tested
compounds is topoisomerase I inhibitor.
Received 14 September 2009
Received in revised form
20 November 2009
Accepted 27 November 2009
Available online 5 December 2009
Keywords:
Pyrazolylpyrazole derivatives
Enaminones
Hydrazonoyl chlorides
Anti-HCV
Subacute Sclerosing Panencephalitis (SSPE)
Antitumor activity
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
2. Chemistry
A survey of literature revealed that pyrazoles belong among
the most representative five-membered heterocyclic systems
[1–3]. Numerous synthetic pyrazole derivatives found use in
various pharmaceutical, agrochemicals, photographic and other
applications. Such examples of important pyrazole derivatives are
natural products (S)-pyrazolylalanine [4], pyrazomycin [5] and
synthetic compounds sildenafil, ionazolac [6–8], difenamizole [9–
11], mepirizole [12–14], phenidone [15–17] and bicyclic pyr-
azolidinone LY 186826 [18–26]. On the other hand, synthesis of
heterocyclic compounds represents the major topic of our
research interest [27–37]. So, based on these findings, we prepare
in this context, a series of novel pyrazole derivatives containing
one and two pyrazole rings as substituents, using enaminones and
hydrazonoyl chloride as starting materials. Our objective from
synthesis of these pyrazole derivatives is to study the effect of
introducing pyrazole moiety in position-4 on the biological
activity of target compounds.
The starting materials 1-(1-aryl-4-benzoyl-1H-pyrazol-3-yl)
ethanones 2a–d were prepared in one step by reaction of 1-phenyl-
3-(N,N-dimethylamino)-propenone with N-aryl 2-oxo-propane-
hydrazonoyl chlorides 1a–d in dioxane and in the presence of
triethylamine under reflux as previously described [38]. Reactions
of compounds 2a–d with dimethylformamide–dimethylacetal
(DMF–DMA) in dry toluene under reflux for 10 h, afforded products
that were identified as 1-(1-aryl-4-benzoyl-1H-pyrazol-3-yl)-3-
dimethylamino-propenones 3a–d (Scheme 1). The structures of the
latter products were established from their microanalyses, mass, IR
and 1H NMR spectra (see Experimental). When each of 3a–d was
reacted with N-phenyl 3-oxo-propane-hydrazonoyl chloride 1a in
dioxane in the presence of triethylamine under reflux, a single
product was obtained in each case as evidenced by TLC analysis of
the crude product. The microanalyses and mass spectral data of the
isolated products are in consistent with the assigned structure 4 (cf.
Scheme 1). Besides, the IR spectra of products 4 revealed in each
´
case three absorption bands for the carbonyl groups near y 1699–
1685, 1681–1673 and 1653–1642 cmꢀ1 (see Experimental). 1H NMR
spectra exhibited one singlet signal near
the acetyl protons, in addition to the signals at
8.94–8.86 ppm assigned for the proton at position-5 for each
d
2.69–2.63 ppm due to
d
9.18–9.05 and
* Corresponding author.
E-mail address: thoraya-f@hotmail.com (M.A. Abdallah).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.11.050

S.M. Riyadh et al. / European Journal of Medicinal Chemistry 45 (2010) 1042–1050
1043
PhOC
COCH3
DMF-DMA
Cl
Ph-CO-CH=CH-N(Me)2
Dioxane / Et3N /
NHAr
N
H3COC
N
N
Toluene /
1a-d
Ar
2a-d
O
Cl
NHPh
PhOC
N(CH3)2
H3COC
N
1a
N
Dioxane / Et3N /
N
Ar
3a-d
O
COCH3
PhOC
O
O
PhOC
N(CH3)2
N
N
DMF-DMA
Toluene /
N
N
N
N
N
N
Ph
Ar
4a-d
5a-d
Cl
Ph
Ar
Dioxane / Et3N /
NHPh
1a
H3COC
N
O
O
COCH3
PhOC
N
N
N
N
N
N
Ar
Ph
Ph
6a-d
Ar : a, C6H5; b, 4-CH3C6H4; c, 4-ClC6H4; d, 4-NO2C6H4
Scheme 1. Synthesis of pyrazoles 2–6.
pyrazole ring. In a similar manner pyrazole derivatives 6a–d were
prepared following the same sequence of reactions as for products
4 (cf. Scheme 1). Thus, reaction of each of 4a–d with dime-
thylformamide–dimethylacetal in dry toluene under reflux for 10 h
afforded the enaminones 5a–d. The structures of the latter products
were established based on microanalyses and spectral data (see
Experimental). For example, 1H NMR spectra of products 5a–
d showed in each case two characteristic singlet signals near
depicted in Scheme 2, it is suggested that the reaction of 3 with 1
starts with a regioselective 1,3-dipolar cycloaddition of the N-
phenyl-C-acetylnitrilimine intermediate 7, generated in situ by
base-catalyzed dehydrohalogenation of the hydrazonoyl chloride
1a, to the carbon–carbon double bond of the enaminones 3a–d to
afford the non-isolable cycloadducts 8a–d. Elimination of dime-
thylamine from the latter intermediates 8 yielded the corresponding
pyrazole derivatives 4a–d as end products and the other isomers
10a–d were discarded. This is because the 1H NMR spectra of such
products revealed, in each case, two singlet signals for the pyrazole
d
5.87–5.81 and 7.78–7.68 ppm assignable to the olefinic protons
(CH]CH) of the enaminone residue, in addition to the signals of the
two protons at position-5 of pyrazole rings. Reaction of compounds
5a–d with N-phenyl 2-oxo-propane-hydrazonoyl chloride 1a in
dioxane under reflux for 8 h in the presence of triethylamine
afforded pyrazole derivatives 6a–d in high yields. The structures of
the latter products were evidenced by their microanalyses and
spectroscopic (mass, IR, 1H NMR) data (see Experimental).
ring proton in the region d 9.18–9.05 and 8.94–8.86 ppm assignable
to pyrazole-5H. Literature reports [39,40] indicated that the 1H
NMR spectra of 5-unsubstituted pyrazoles exhibit the characteristic
singlet signals of 5-CH at
d 8.12–9.0 ppm, while, the 4-unsub-
stituted pyrazoles showed the 4-CH signal at
d 5.81–5.89 [41,42].
It is interesting to suggest a mechanism for the reaction of
enaminones with hydrazonoyl chlorides to know the regiose-
lectivity of such reaction. To achieve this, we have taken reaction of
enaminones 3a–d with N-phenyl 2-oxo-propane-hydrazonoyl
chloride 1a as a typical example for the prepared compounds. As
3. Pharmacology
Some pyrazole derivatives have anticancer activities [43]. So,
we devoted our study to investigate the anticancer, anti-HCV and

1044
S.M. Riyadh et al. / European Journal of Medicinal Chemistry 45 (2010) 1042–1050
Cl
Dioxane / Et3N
N
NHPh
Ph
H3COC
N
H3COC
N
7
1a
O
N(CH3)2
PhOC
N
O
N
3a-d
Ar
O
H
H
COCH3
PhOC
PhOC
Ph
N
N
N
N
N
H
H
N
N
N
Me2N
Me2N
COCH3
9a-d
Ph
Ar
8a-d
Ar
- NHMe2
- NHMe2
O
O
PhOC
COCH3
PhOC
Ph
N
N
N
N
N
N
N
N
COCH3
Ar
Ph
Ar
10a-d
4a-d
Ar : a, C6H5; b, 4-CH3C6H4; c, 4-ClC6H4; d, 4-NO2C6H4
Scheme 2. The mechanism of reaction of hydrazonoyl halide with enaminone.
antioxidant activities of some representative examples of the newly
synthesized compounds 2–6.
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.
Antioxidant activity of the tested compounds using DPPH method
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
Table 1
In order to monitor the potential of fifteen 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 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 positive
control. Table 1 showed the MIC values for the tested compounds
and ribaverin against HCV and SSPE. The results showed that all the
tested compounds are highly effective at very low concentration
compared to ribaverin in the following ascending order 2d, 4c, 6b,
3b, 6c, 4d, 2b, 2c, 2a, 6a, 5b, 5a, 3a, 4b and 5c. It is worth to mention
that all tested compounds are more potent than Ribaverin.
MIC of Ribaverin and the tested compounds against HCV and SSPE.
Tested compound
MIC
HCV
mg/ml
Subacute Sclerosing
Panencephalitis (SSPE)
Ribaverin
2a
2b
2c
2d
3a
3b
4b
4c
4d
5a
5b
5c
6a
6b
6c
16.15
77.89
3.615
3.424
3.516
3.089
4.00
3.178
4.120
3.111
3.216
3.992
3.821
4.150
3.714
3.123
3.198
0.162
0.159
0.161
0.144
0.32
0.150
0.322
0.147
0.155
0.291
0.181
0.323
0.171
0.149
0.151
3.1.2. DPPH radical scavenging activity
Radical scavenging activities of new synthesized compounds
were measured using model colorimetric test: DPPH radical scav-
enging test. The results are summarized in Table 2. The stable

S.M. Riyadh et al. / European Journal of Medicinal Chemistry 45 (2010) 1042–1050
1045
Table 2
Decrease of DPPH absorbance (%) by the tested compounds.
Table 4
Antitumor activities of the tested compounds.
Compound number
Decrease of DPPH absorbance (%)
Compound No
Average GI50
Relative potency
[mean ꢁ SD (n ¼ 3)]
to etoposide
Ascorbic acid (standard)
79.042 ꢁ 2.928
95.16 ꢁ 0.031
95.80 ꢁ 0.045
95.55 ꢁ 0.023
98.19 ꢁ 0.024
94.55 ꢁ 0.044
96.60 ꢁ 0.066
94.00 ꢁ 0.089
97.70 ꢁ 0.043
95.98 ꢁ 0.066
94.65 ꢁ 0.033
94.90 ꢁ 0.056
93.00 ꢁ 0.054
95.00 ꢁ 0.033
96.90 ꢁ 0.056
96.11 ꢁ 0.055
2a
2b
2c
2d
3a
3b
4b
4c
4d
5a
5b
5c
6a
6b
6c
0.0178
0.0159
0.0168
0.0115
0.022
0.01152
0.078
0.0115
0.0154
0.021
0.02
0.079
0.0199
0.01151
0.0153
168.5393258
188.6792453
178.5714286
260.8695652
136.3636364
260.4166667
38.46153846
260.8695652
194.8051948
142.8571429
150
2a
2b
2c
2d
3a
3b
4b
4c
4d
5a
5b
5c
6a
6b
6c
37.97468354
150.7537688
260.6429192
196.0784314
3.1.4. In vivo efficacy study
exhibited good scavenging activity against the DPPH radical in the
following descending order 2d, 4c, 6b, 3b, 6c, 4d, 2b, 2c, 2a, 6a, 5b,
5a, 3a, 4b and 5c. It is worth to mention that all tested compounds
are more potent than ascorbic acid.
The in vivo activity of the tested compounds and Etoposide
against murine colon cancer Colon 38 was tested (Table 4). Treat-
ment of Colon 38 implanted into mice with ER-37328 induced
a clear dose-dependent inhibition of tumor 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 with the order of activity in descending order
2d, 4c, 6b, 3b, 6c, 4d, 2b, 2c, 2a, 6a, 5b, 5a, 3a, 4b and 5c. The
cytotoxicity results of the tested compounds compared to the
activity of etoposide where the tested compound active at low
concentration and also at the same level of toxicity of etoposides
where the author had mentioned no death or no weight variation
so the toxicity nearly more or less equal to that of etoposide.
In a progress to determine the mechanism of action of the
antitumor activities, the correlation coefficients with top-
oisomerase I inhibitor, and cisplatin, calculated according to the NCI
COMPARE analysis procedure using the tested compounds as the
benchmark, were measured (Table 5). These calculations culmi-
nated on the newly synthesized compounds act as topoisomerase I
inhibitors. The correlation coefficients are indicative measure to the
possibility of the mechanism of the antitumor activity, where as the
value is high to certain drug, this means that the compound is
similar in mechanism of action to this drug and vice-versa. Upon
this rational the values of correlation coefficients of the tested
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 nitrate lipids
[44], proteins [45], DNA [46,47] and carbohydrates [48] 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 [49,50], there has
been considerable interest in the ability of natural and synthetic
antioxidants to diminish (ONOOꢀ) damage. In the present work, the
ability of the synthetic compounds to prevent (ONOOꢀ)-mediated
damage was examined using the model system: (ONOOꢀ)-medi-
ated tyrosine nitration. When the amino acid tyrosine is exposed to
(ONOOꢀ) at pH 7.4, 3-NT is formed [51]. The relative inhibitory
activity of these compounds is summarized in Table 3 as IC50 values.
Under our experimental conditions the relative potencies of the
tested compounds are in ascending order 2d, 4c, 6b, 3b, 6c, 4d, 2b,
2c, 2a, 6a, 5b, 5a, 3a, 4b and 5c. It is worth to mention that all tested
compounds are more potent than Trolox (Table 3).
Table 5
The correlation coefficients of the newly synthesized compounds with topo-
isomerase I inhibitors.
Table 3
IC50 values of the compounds tested for the inhibition of (ONOOꢀ)-mediated
3-nitrotyrosine formation.
Compound number
The correlation coefficients with (topoisomerase I
inhibitor), and cisplatin, calculated according to the
NCI COMPARE analysis procedure using the tested
compounds as the benchmark
Compound number
IC50
(n ¼ 3)]
(
m
M) [mean ꢁ SD
Etoposide
Doxorubicin
SN-38
Cisplatin
Trolox
2a
2b
2c
2d
3a
3b
4b
4c
4d
5a
5b
5c
6a
6b
6c
58.430 ꢁ 5.9
39.502 ꢁ 0.0376
37.777 ꢁ 0.0399
38.123 ꢁ 0.0388
31.542 ꢁ 0.0212
43.987 ꢁ 0.0334
34.201 ꢁ 0.0245
44.123 ꢁ 0.0356
32.189 ꢁ 0.0212
36.550 ꢁ 0.0289
43.981 ꢁ 0.0333
42.167 ꢁ 0.0343
44.550 ꢁ 0.0466
42.111 ꢁ 0.0354
33.983 ꢁ 0.0233
35.150 ꢁ 0.0267
2a
2b
2c
2d
3a
3b
4b
4c
4d
5a
5b
5c
6a
6b
6c
17.14
20.11
18.16
31.12
5.11
27.18
4.78
30.11
21.15
13.67
15
16.77
19.12
18.11
30.14
4.88
15.98
18.76
17.67
30.000
4.67
14.66
17.24
16.18
29.12
3.66
27.01
4.11
26.15
3.90
25.24
3.16
29.11
21.00
12.13
14.88
4.00
15.88
28.16
22.00
28.16
20.15
11.88
13.99
3.89
14.78
27.14
21.17
27.28
20.00
10.12
12.13
3.14
13.88
26.15
20.16
4.12
16.18
28.56
22.13

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S.M. Riyadh et al. / European Journal of Medicinal Chemistry 45 (2010) 1042–1050
Table 6
University, Giza, Egypt. Hydrazonoyl chloride 1a–d was prepared as
following the literature method [52].
The relative potencies of the newly synthesized compounds to Etoposide.
Compound number
Relative potency to etoposide
5.2. Synthesis of 1-(1-aryl-4-benzoyl-1H-pyrazol-3-yl)-
ethanone 2a–d
2a
2b
2c
2d
3a
3b
4b
4c
4d
5a
5b
5c
6a
6b
6c
168.50
188.60
178.50
260.80
136.30
260.40
38.40
260.80
194.80
142.80
150
To a mixture of equimolar amounts of enaminone and the
appropriate hydrazonoyl chlorides 1a–d (50 mmol of each) in
dioxane (100 mL) was added triethylamine (7 mL, 50 mmol). The
reaction mixture was refluxed for 8 h. The solvent was evaporated
and the residue was washed by petroleum ether and treated with
methanol. The solid that formed was filtered and crystallized from
ethanol to give compounds 2a–d.
37.90
150.70
260.60
196.00
5.2.1. 1-(4-Benzoyl-1-phenyl-1H-pyrazol-3-yl)-ethanone (2a)
Yellow crystals, (11.6 g, 80%), mp 133–135 ꢂC (Lit. mp 132 ꢂC)
[21]; IR (KBr)
y
¼ 1687, 1650 (2 CO), cmꢀ1; 1H NMR (DMSO-d6)
d
¼
2.57 (s, 3H, COCH3), 7.23–7.91 (m, 10H, Ar–H), 9.01 (s, 1H, pyrazole-
H-5) ppm; MS, m/z (%) 290 (Mþ, 25), 213 (20), 105 (50), 77 (100).
Anal. Calcd. for C18H14N2O2 (290.32): C, 74.47; H, 4.86; N, 9.65.
Found: C, 74.29; H, 4.68; N, 9.58%.
agents is high near the side of etoposide and low in other side of
antitumor agents so we can got to the final conclusion is the
confirmation of the mechanism of action of the tested agent is
similar to that of etoposide as topoisomerase II inhibitor.
5.2.2. 1-[4-benzoyl-1-(4-methylphenyl)-1H-pyrazol-3-yl]-
3.1.5. Inhibition of topoisomerase II activity
ethanone (2b)
The effects of the tested compounds and etoposide on top-
oisomerase II activity were analyzed by means of relaxation assay.
The tested compounds and etoposide inhibited topoisomerase II
activity and the relative potency of each compound were deter-
mined and confirmed in the following descending order 2d, 4c, 6b,
3b, 6c, 4d, 2b, 2c, 2a, 6a, 5b, 5a, 3a, 4b and 5c (Table 6).
All the previous mentioned measurements revealed that all the
tested compounds are more active than Etoposide and act similarly
to its mechanism of action as topoisomerase I inhibitor.
Yellow crystals, (11.9 g, 80%), mp 150–152 ꢂC; IR (KBr)
y
¼ 1685,
1649 (2 CO), cmꢀ1 1H NMR (DMSO-d6)
; d
¼ 2.24 (s, 3H, Ar–CH3),
2.53 (s, 3H, COCH3), 7.29–8.05 (m, 9H, Ar–H), 9.02 (s, 1H, pyrazole-
H-5) ppm; MS, m/z (%) 304 (Mþ, 20), 227 (25), 105 (40), 77 (100).
Anal. Calcd. for C19H16N2O2 (304.35): C, 74.98; H, 5.30; N, 9.20.
Found: C, 74.82; H, 5.22; N, 9.34%.
5.2.3. 1-[4-benzoyl-1-(4-chlorophenyl)-1H-pyrazol-3-yl]-
ethanone (2c)
Yellow crystals, (13.3 g, 82%), mp 178–180 ꢂC; IR (KBr)
y
¼ 1688,
1644 (2 CO), cmꢀ1 1H NMR (DMSO-d6)
; d
¼ 2.57 (s, 3H, COCH3),
4. Conclusion
7.49–8.02 (m, 5H, Ar–H), 8.04 (d, 2H, J ¼ 8 Hz, Ar-H), 8.05 (d, 2H, J ¼
8 Hz, Ar–H), 9.03 (s, 1H, pyrazole-H-5) ppm; MS, m/z (%) 326 (Mþ þ
2, 6), 325 (Mþ þ 1, 12), 324 (Mþ, 20), 281 (40), 111 (20), 105 (50), 77
(100). Anal. Calcd. for C18H13ClN2O2 (324.77): C, 66.57; H, 4.03; N,
8.63. Found: C, 66.39; H, 4.12; N, 8.54%.
A series of pyrazole derivatives incorporating one and two
pyrazole rings as substituent were prepared using hydrazonoyl
chlorides and enaminones as starting materials. It is worth
noting that the presence of different substituents on the
aromatic rings in position-1 of parent pyrazole is responsible for
the remarkable difference in the inhibition activity against HCV
and Topoisomerase II.
The unsubstituted phenyl group has the highest activity than
substituted ones, and the 4-nitrogroup greatly reduces the activi-
ties than the 4-methyl one.
Moreover, the newly synthesized compounds were found to
have radical scavenging activity than ascorbic acid. Based on the
results of biological activity, we can conclude that the new
synthetic compounds are more potent than the reference standards
and are promising candidates to further advance drug discovery
efforts.
5.2.4. 1-[4-benzoyl-1-(4-nitrophenyl)-1H-pyrazol-3-yl]-
ethanone (2d)
Brown crystals, (12.6 g, 75%), mp 217–219 ꢂC; IR (KBr)
y
¼ 1693,
1639 (2 CO), cmꢀ1 1H NMR (DMSO-d6)
; d
¼ 2.59 (s, 3H, COCH3),
7.49–8.02 (m, 5H, Ar–H), 8.14 (d, 2H, J ¼ 8 Hz, Ar–H), 8.21 (d, 2H, J ¼
8 Hz, Ar–H), 9.08 (s, 1H, pyrazole-H-5) ppm; MS, m/z (%) 337 (Mþ þ
2, 15), 336 (Mþ þ 1, 30), 335 (Mþ, 40), 258 (50), 105 (60), 77 (100).
Anal. Calcd. for C18H13N3O4 (335.32): C, 64.48; H, 3.91; N, 12.53.
Found: C, 64.39; H, 4.11; N, 12.44%.
5.3. Synthesis of 1-(1-aryl-4-benzoyl -1H-pyrazol-3-yl)-3-
dimethylamino-propenones (3a–d)
5. Experimental protocol
A mixture of 1-(4-benzoyl-1-aryl-1H-pyrazol-3-yl)-ethanones
2a–d (30 mmol of each) and dimethylformamide–dimethylacetal
(DMF–DMA) (7 mL) in dry toluene (50 mL) was refluxed for 10 h.
The solvent was evaporated under reduced pressure and methanol
was added to the reaction mixture. The solid product was collected
by filtration and crystallized from ethanol to give products 3a–d.
5.1. Chemistry
Melting points are uncorrected. IR spectra were recorded (KBr)
on Pye Unicam SP-1000 Spectrophotometer. 1H NMR spectra were
obtained on Varian Gemini 300 MHz spectrometer using TMS as
internal reference and DMSO-d6 as solvent. Chemical shifts are
5.3.1. 1-(4-Benzoyl-1-phenyl-1H-pyrazol-3-yl)-3-dimethylamino-
expressed as
d
(ppm). Mass spectra were recorded on a GCMS-QP
propenone (3a)
1000 EX mass spectrometer operating at 70 eV. The elemental
analyses were performed at the Microanalytical Center at Cairo
Dark orange crystals, (8.8 g, 85%), mp 215–217 ꢂC; IR (KBr)
y
¼
1683, 1640 (2 CO), cmꢀ1; 1H NMR (DMSO-d6)
d
¼ 2.84 (s, 3H, CH3),

S.M. Riyadh et al. / European Journal of Medicinal Chemistry 45 (2010) 1042–1050
1047
2.96 (s, 3H, CH3), 5.84 (d, 1H, J ¼ 13 Hz, CH]), 7.23–7.99 (m, 10H,
Ar–H), 7.65 (d, 1H, J ¼ 13 Hz, CH]), 8.89 (s, 1H, pyrazole-H-5) ppm;
MS, m/z (%) 345 (Mþ, 15), 240 (16), 105 (40), 98 (60), 77 (80), 70
(100). Anal. Calcd. for C21H19N3O2 (345.40): C, 73.03; H, 5.54; N,
12.17. Found: C, 73.11; H, 5.68; N, 12.28%.
pyrazole-H-5), 9.05 (s, 1H, pyrazole-H-5) ppm; MS, m/z (%) 474 (Mþ,
17), 431 (20), 369 (15), 105 (100), 77 (80). Anal. Calcd. for
C29H22N4O3 (474.52): C, 73.40; H, 4.67; N, 11.81. Found: C, 73.52; H,
4.58; N, 11.78%.
5.4.3. 1-{4-[4-benzoyl-1-(4-chlorophenyl)-1H-pyrazole-3-
5.3.2. 1-[4-benzoyl-1-(4-methylphenyl)-1H-pyrazol-3-yl]-3-
dimethylamino-propenone (3b)
carbonyl]-1-phenyl-1H-pyrazol-3-yl}-ethanone (4c)
Brown crystals, (7.9 g, 80%), mp 93–95 ꢂC; IR (KBr)
y
¼ 1698,
Orange crystals, (8.6 g, 80%), mp 187–189 ꢂC; IR (KBr)
y
¼ 1677,
1679, 1643 (3 CO), cmꢀ1 1H NMR (DMSO-d6)
; d
¼ 2.69 (s, 3H,
1641 (2 CO), cmꢀ1
;
1H NMR (DMSO-d6)
d
¼ 2.36 (s, 3H, Ar–CH3),
COCH3), 7.46–7.79 (m, 10H, Ar–H), 7.95 (d, 2H, J ¼ 8 Hz, Ar–H), 8.01
(d, 2H, J ¼ 8 Hz, Ar–H), 8.90 (s, 1H, pyrazole-H-5), 9.15 (s, 1H, pyr-
azole-H-5) ppm; MS, m/z (%) 496 (Mþ þ 2, 12), 495 (Mþ þ 1, 20),
494 (Mþ, 30), 452 (16), 390 (40), 105 (50), 77 (100). Anal. Calcd. for
C28H19ClN4O3 (494.94): C, 67.95; H, 3.87; N, 11.32. Found: C, 67.82;
H, 3.78; N, 11.18%.
2.87 (s, 3H, CH3), 3.08 (s, 3H, CH3), 5.75 (d, 1H, J ¼ 13 Hz, CH]),
7.34–7.50 (m, 5H, Ar–H), 7.65 (d,1H, J ¼ 13 Hz, CH]), 7.78 (d, 2H, J ¼ 8
Hz, Ar–H), 7.85 (d, 2H, J ¼ 8 Hz, Ar-H), 8.81 (s, 1H, pyrazole-H-
5) ppm; MS, m/z (%) 359 (Mþ, 40), 254 (40), 105 (30), 98 (60),
77 (50), 70 (100). Anal. Calcd. for C22H21N3O2 (359.43): C, 73.52;
H, 5.89; N, 11.69. Found: C, 73.38; H, 5.78; N, 11.48%.
5.4.4. 1-{4-[4-benzoyl-1-(4-nitrophenyl)-1H-pyrazole-3-carbonyl]-
5.3.3. 1-[4-benzoyl-1-(4-chlorophenyl)-1H-pyrazol-3-yl]-3-
dimethylamino-propenone (3c)
1-phenyl-1H-pyrazol-3-yl}-ethanone (4d)
Yellow crystals, (7.8 g, 78%), mp 94–96 ꢂC; IR (KBr)
y
¼ 1699,
Orange crystals, (9.12 g, 80%), mp 230–232 ꢂC; IR (KBr)
y
¼ 1680,
1681, 1653 (3 CO), cmꢀ1 1H NMR (DMSO-d6)
; d
¼ 2.69 (s, 3H,
1639 (2 CO), cmꢀ1; 1H NMR (DMSO-d6)
d
¼ 2.88 (s, 3H, CH3), 3.09 (s,
COCH3), 7.48–7.81 (m, 10H, Ar–H), 7.98 (d, 2H, J ¼ 8 Hz, Ar–H), 8.05
(d, 2H, J ¼ 8 Hz, Ar–H), 8.94 (s, 1H, pyrazole-H-5), 9.18 (s, 1H, pyr-
azole-H-5) ppm; MS, m/z (%) 505 (Mþ, 5), 462 (16), 300 (40), 105
(80), 77 (100). Anal. Calcd. for C28H19N5O5 (505.49): C, 66.53; H,
3.79; N, 13.85. Found: C, 66.32; H, 3.71; N, 13.82%.
3H, CH3), 5.89 (d, 1H, J ¼ 13 Hz, CH]), 7.46–7.59 (m, 5H, Ar–H), 7.65
(d, 1H, J ¼ 13 Hz, CH]), 7.99 (d, 2H, J ¼ 8 Hz, Ar–H), 8.03 (d, 2H, J ¼ 8
Hz, Ar–H), 8.90 (s, 1H, pyrazole-H-5) ppm; MS, m/z (%) 381 (Mþ þ 2,
18), 380 (Mþ þ 1, 25), 379 (Mþ, 40), 274 (40), 105 (30), 98 (60), 77
(50), 70 (100). Anal. Calcd. for C21H18ClN3O2 (379.85): C, 66.40; H,
4.78; N, 11.06. Found: C, 66.31; H, 4.68; N, 11.18%.
5.5. Synthesis of 1-[1-aryl-4-(4-benzoyl-1H-pyrazole-3-carbonyl)-
1-phenyl-1H-pyrazol-3-yl]-3-dimethylamino-propenones 5a–d
5.3.4. 1-[4-benzoyl-1-(4-nitrophenyl)-1H-pyrazol-3-yl]-3-
dimethylamino-propenone (3d)
A mixture of 1-[1-aryl-4-(4-benzoyl-1H-pyrazole-3-carbonyl)-
1-phenyl-1H-pyrazol-3-yl]-ethanones 4a–d. (10 mmol of each) and
dimethylformamide–dimethylacetal (DMF–DMA) (5 mL) in dry
toluene (30 mL) was refluxed for 10 h. The solvent was evaporated
under reduced pressure and the residue was triturated with
methanol. The solid product formed was collected by filtration and
crystallized from ethanol to give products 5a–d.
Yellow crystals, (9.12 g, 78%), mp 195–197 ꢂC; IR (KBr)
y
¼ 1677,
1641 (2 CO), cmꢀ1; 1H NMR (DMSO-d6)
d
¼ 2.89 (s, 3H, CH3), 3.11 (s,
3H, CH3), 5.89 (d, 1H, J ¼ 13 Hz, CH]), 7.42–7.64 (m, 5H, Ar–H), 7.78
(d, 1H, J ¼ 13 Hz, CH]), 8.23 (d, 2H, J ¼ 8 Hz, Ar–H), 8.43 (d, 2H, J ¼ 8
Hz, Ar–H), 9.09 (s, 1H, pyrazole-H-5) ppm; MS, m/z (%) 392 (Mþ þ 2,
18), 391 (Mþ þ 1, 25), 390 (Mþ, 40), 285 (40), 105 (30), 98 (60), 77
(50), 70 (100). Anal. Calcd. for C21H18N4O4 (390.40): C, 64.61; H,
4.65; N, 14.35. Found: C, 64.51; H, 4.48; N, 14.19%.
5.5.1. 1-[4-(4-benzoyl-1-phenyl-1H-pyrazole-3-carbonyl)-1-
phenyl-1H-pyrazol-3-yl]-3-dimethylamino-propenone (5a)
5.4. Synthesis of 1-[1-aryl-4-(4-benzoyl-1H-pyrazole-3-carbonyl)-
1-phenyl-1H-pyrazol-3-yl]-ethanone 4a–d
Brown crystals, (4.12 g, 80%), mp 210–212 ꢂC; IR (KBr)
y
¼ 1683,
1674, 1640 (3 CO), cmꢀ1; 1H NMR (DMSO-d6)
d
¼ 2.86 (s, 3H, CH3),
2.98 (s, 3H, CH3), 5.81 (d, 1H, J ¼ 13 Hz, CH]), 7.36–7.92 (m, 15H,
Ar–H), 7.68 (d, 1H, J ¼ 13 Hz, CH]), 8.85 (s, 1H, pyrazole-H-5), 9.09
(s, 1H, pyrazole-H-5) ppm; MS, m/z (%) 515 (Mþ, 25), 417 (16), 309
(10), 105 (40), 77 (100), 70 (35). Anal. Calcd. for C31H25N5O3
(515.58): C, 72.22; H, 4.89; N, 13.58. Found: C, 72.10; H, 4.78; N,
13.38%.
To a mixture of 1-(1-aryl-4-benzoyl-1H-pyrazol-3-yl)-3-dime-
thylamino-propenones (3a–d) (20 mmol of each) and N-phenyl-2-
oxo-propanhydrazonoyl chloride (1a) (3.93 g, 20 mmol) in dioxane
(50 mL), triethylamine (2.8 mL, 20 mmol) was added. The reaction
mixture was refluxed for 8 h. The solvent was evaporated and the
residue was washed by petroleum ether and treated with meth-
anol. The solid that formed was filtered and crystallized from
ethanol to give compounds 4a–d.
5.5.2. 1-{4-[4-benzoyl-1-(4-methylphenyl)-1H-pyrazole-3-carbonyl]-
1-phenyl-1H-pyrazol-3-yl}-3-dimethylamino-propenone (5b)
Brown crystals, (4.2 g, 80%), mp 80–82 ꢂC; IR (KBr)
y
¼ 1684,
5.4.1. 1-[4-(4-benzoyl-1-phenyl-1H-pyrazole-3-carbonyl)-1-
phenyl-1H-pyrazol-3-yl]-ethanone (4a)
1678, 1647 (3 CO), cmꢀ1 1H NMR (DMSO-d6)
; d
¼ 2.36 (s, 3H, Ar–
CH3), 2.84 (s, 3H, CH3), 3.05 (s, 3H, CH3), 5.81 (d, 1H, J ¼ 13 Hz,
CH]), 7.31–8.06 (m, 14H, Ar–H), 7.78 (d, 1H, J ¼ 13 Hz, CH]), 8.96
(s, 1H, pyrazole-H-5), 9.01 (s, 1H, pyrazole-H-5) ppm; MS, m/z (%)
529 (Mþ, 35), 431 (16), 240 (40), 105 (100), 98 (80), 77 (70). Anal.
Calcd. for C32H27N5O3 (529.60): C, 72.57; H, 5.14; N, 13.22. Found: C,
72.48; H, 5.22; N, 13.38%.
Pale yellow crystals, (7.5 g, 82%), mp 127–129 ꢂC; IR (KBr)
y
¼
1685, 1678, 1642 (3 CO), cmꢀ1; 1H NMR (DMSO-d6)
d
¼ 2.65 (s, 3H,
COCH3), 7.38–7.98 (m,15H, Ar–H), 8.86 (s,1H, pyrazole-H-5), 9.14 (s,
1H, pyrazole-H-5) ppm; MS, m/z (%) 460 (Mþ, 25), 417 (16), 355
(40), 105 (40), 77 (100). Anal. Calcd. for C28H20N4O3 (460.50): C,
73.03; H, 4.38; N, 12.17. Found: C, 73.10; H, 4.28; N, 12.28%.
5.5.3. 1-{4-[4-benzoyl-1-(4-chlorophenyl)-1H-pyrazole-3-carbonyl]-
5.4.2. 1-{4-[4-benzoyl-1-(4-methylphenyl)-1H-pyrazole-3-
1-phenyl-1H-pyrazol-3-yl}-3-dimethylamino-propenone (5c)
carbonyl]-1-phenyl-1H-pyrazol-3-yl}-ethanone (4b)
Yellow crystals, (4.4 g, 80%), mp 118–120 ꢂC; IR (KBr)
y
¼ 1689,
Pale yellow crystals, (7.5 g, 80%), mp 145–147 ꢂC; IR (KBr)
y
¼
1677, 1643 (3 CO), cmꢀ1; 1H NMR (DMSO-d6)
d
¼ 2.98 (s, 3H, CH3),
1692, 1673, 1648 (3 CO), cmꢀ1; 1H NMR (DMSO-d6)
Ar–CH3), 2.63 (s, 3H, COCH3), 7.46–7.99 (m, 14H, Ar–H), 8.89 (s, 1H,
d
¼ 2.35 (s, 3H,
3.17 (s, 3H, CH3), 5.85 (d, 1H, J ¼ 13 Hz, CH]), 7.51–7.99 (m, 10H, Ar–
H), 7.68 (d, 1H, J ¼ 13 Hz, CH]), 7.98 (d, 2H, J ¼ 8 Hz, Ar–H), 8.05 (d,

1048
S.M. Riyadh et al. / European Journal of Medicinal Chemistry 45 (2010) 1042–1050
2H, J ¼ 8 Hz, Ar–H), 8.86 (s, 1H, pyrazole-H-5), 9.09 (s, 1H, pyrazole-
H-5) ppm; MS, m/z (%) 552 (Mþ þ 2, 16), 551 (Mþ þ 1, 20), 550 (Mþ,
30), 445 (16), 309 (10), 105 (80), 98 (100), 77 (40). Anal. Calcd. for
C31H24ClN5O3 (550.02): C, 67.70; H, 4.40; N, 12.73. Found: C, 67.58;
H, 4.28; N, 12.68%.
5.6.4. 1-{4-[4-[4-benzoyl-1-(4-nitrophenyl)-1H-pyrazole-3-
carbonyl]-1-phenyl-1H-pyrazole-3-carbonyl]-1-phenyl-1H-
pyrazol-3-yl}-ethanone (6d)
Brown crystals, (2.5 g, 75%), mp 90–92 ꢂC; IR (KBr)
y
¼ 1692,
1687, 1682, 1648 (4 CO), cmꢀ1; 1H NMR (DMSO-d6)
d
¼ 2.69 (s, 3H,
COCH3), 7.06–7.95 (m, 19H, Ar–H), 8.89 (s, 1H, pyrazole-H-5), 9.06
(s, 1H, pyrazole-H-5), 9.19 (s, 1H, pyrazole-H-5) ppm; MS, m/z (%)
675 (Mþ, 12), 570 (20), 240 (30), 105 (80), 77 (100). Anal. Calcd. for
C38H25N7O6 (675.67): C, 67.55; H, 3.73; N, 14.51. Found: C, 67.41; H,
3.68; N, 14.39%.
5.5.4. 1-{4-[4-benzoyl-1-(4-nitrophenyl)-1H-pyrazole-3-carbonyl]-
1-phenyl-1H-pyrazol-3-yl}-3-dimethylamino-propenone (5d)
Dark orange crystals, (4.2 g, 75%), mp 102–104 ꢂC; IR (KBr)
y
¼
1691, 1672, 1644 (3 CO), cmꢀ1; 1H NMR (DMSO-d6)
d
¼ 2.98 (s, 3H,
CH3), 3.18 (s, 3H, CH3), 5.87 (d, 1H, J ¼ 13 Hz, CH]), 7.54–7.99 (m,
10H, Ar–H), 7.69 (d, 1H, J ¼ 13 Hz, CH]), 8.08 (d, 2H, J ¼ 8 Hz, Ar–H),
8.25 (d, 2H, J ¼ 8 Hz, Ar–H), 8.89 (s, 1H, pyrazole-H-5), 9.17 (s, 1H,
pyrazole-H-5) ppm; MS, m/z (%) 560 (Mþ, 30), 455 (26), 311 (10),
240 (20), 105 (100), 98 (40), 77 (50). Anal. Calcd. for C31H24N6O5
(560.57): C, 66.42; H, 4.32; N, 14.99. Found: C, 66.48; H, 4.18; N,
14.89%.
5.7. Determination of minimum inhibitory concentration (MIC), of
ribavirin and different tested compounds in HCV replicon cells was
performed as follow
Briefly, 1 ꢃ 104 replicon cells per well were plated in 96-well
plates. On the following day, replicon cells were incubated at 37 ꢂC
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 cyto-
toxicity of tested compounds was measured under the same
5.6. Synthesis of 1-{4-[1-aryl-4-(4-benzoyl-1H-pyrazole-3-
carbonyl)-1-phenyl-1H-pyrazole-3-carbonyl]-1-phenyl-1H-
pyrazol-3-yl}-ethanones 6a–d
To amixture of1-[4-(1-aryl-4-benzoyl-1H-pyrazole-3-carbonyl)-
1-phenyl-1H-pyrazol-3-yl]-3-dimethylamino-propenones 5a–d (5
mmol of each) and N-phenyl-2-oxo-propane-hydrazonoyl chloride
(1a) (0.98 g, 5 mmol) in dioxane (15 mL) was added triethylamine
(0.7 mL, 5 mmol). The reaction mixture was refluxed for 8 h. The
solvent was evaporated and the residue was washed by petroleum
ether and treated with methanol. The solid that formed was filtered
and crystallized from ethanol to give products 6a–d.
experimental settings using
a tetrazolium (MTS)-based cell
viability assay (Promega, Madison, WI). For the cytotoxicity assay
with human hepatocyte cell lines, 1 ꢃ 104 parental Huh-7 cells per
well or 4 ꢃ 104 HepG2 cells per well were used.
5.8. MIC of the tested compounds in hamster brains for antiviral
chemotherapy for Subacute Sclerosing Panencephalitis (SSPE)
5.6.1. 1-{4-[4-(4-benzoyl-1-phenyl-1H-pyrazole-3-carbonyl)-1-
phenyl-1H-pyrazole-3-carbonyl]-1-phenyl-1H-pyrazol-3-yl}-
ethanone (6a)
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 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 3 g
for 10 min. The supernatant was collected, ethanol was added to
remove proteins, and the mixture was heated at 90 ꢂC to evaporate
the ethanol. The protein-free samples were used to evaluate the
MIC in brain tissue by HPLC and bioassay.
Yellowish brown crystals, (2.5 g, 80%), mp 94–96 ꢂC; IR (KBr)
y
¼ 1692, 1682, 1678, 1643 (4 CO), cmꢀ1
;
1H NMR (DMSO-d6)
d
¼
2.61 (s, 3H, COCH3), 7.26–7.97 (m, 20H, Ar–H), 8.86 (s, 1H, pyrazole-
H-5), 9.02 (s, 1H, pyrazole-H-5), 9.14 (s, 1H, pyrazole-H-5) ppm; MS,
m/z (%) 630 (Mþ, 15), 525 (20), 275 (40), 240 (20), 105 (100), 77 (70).
Anal. Calcd. for C38H26N6O4 (630.67): C, 72.37; H, 4.16; N, 13.33.
Found: C, 72.18; H, 4.23; N, 13.25%.
5.6.2. 1-{4-[4-[4-benzoyl-1-(4-methylphenyl)-1H-pyrazole-3-
carbonyl]-1-phenyl-1H-pyrazole-3-carbonyl]-1-phenyl-1H-
pyrazol-3-yl}-ethanone (6b)
5.9. Radical scavenging assays
Brown crystals, (2.5 g, 80%), mp 86–88 ꢂC; IR (KBr)
y
¼ 1690,
1685, 1679, 1648 (4 CO), cmꢀ1; 1H NMR (DMSO-d6)
d
¼ 2.34 (s, 3H,
5.9.1. DPPH radical scavenging assay
Ar–CH3), 2.65 (s, 3H, COCH3), 7.16–7.95 (m, 19H, Ar–H), 8.89 (s, 1H,
pyrazole-H-5), 9.01 (s, 1H, pyrazole-H-5), 9.12 (s, 1H, pyrazole-H-5)
ppm; MS, m/z (%) 644 (Mþ, 15), 539 (20), 474 (40), 240 (20), 105
(100), 77 (70). Anal. Calcd. for C39H28N6O4 (644.70): C, 72.66; H,
4.38; N, 13.04. Found: C, 72.51; H, 4.42; N, 13.09%.
The antioxidant activity of the tested compounds and the
standard were assessed on the basis of the radical scavenging effect
of the stable DPPHꢄ free radical [30]. Weighed quantities of the
tested compounds were dissolved in distilled DMSO and used.
Solution of ascorbic acid used as standard for this study was
prepared in distilled H2O. All these solutions were serially diluted
5.6.3. 1-{4-[4-[4-benzoyl-1-(4-chlorophenyl)-1H-pyrazole-3-
carbonyl]-1-phenyl-1H-pyrazole-3-carbonyl]-1-phenyl-1H-
pyrazol-3-yl}-ethanone (6c)
with respective solvents to get lower dilutions. 10
compound or standard (from 0.0 M/ml to 100 M/ml) was added
to 90 M) in a 96-well
l of DPPHꢄ in methanol solution (100
ml of each
m
m
m
m
Brown crystals, (2.6 g, 80%), mp 86–88 ꢂC; IR (KBr)
y
¼ 1694,
microtitre plate. After incubation in the dark at 37 ꢂC 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 scavenging activity
1687, 1679, 1648 (4 CO), cmꢀ1; 1H NMR (DMSO-d6)
d
¼ 2.68 (s, 3H,
COCH3), 7.06–7.95 (m, 19H, Ar–H), 8.89 (s, 1H, pyrazole-H-5), 9.03
(s, 1H, pyrazole-H-5), 9.15 (s, 1H, pyrazole-H-5) ppm; MS, m/z (%)
667 (Mþ þ 2, 6), 666 (Mþ þ 1, 12), 665 (Mþ, 15), 560 (20), 466 (20),
240 (40), 105 (80), 77 (100). Anal. Calcd. for C38H25ClN6O4
(665.11): C, 68.62; H, 3.79; N, 12.64. Found: C, 68.51; H, 3.62; N,
12.49%.

S.M. Riyadh et al. / European Journal of Medicinal Chemistry 45 (2010) 1042–1050
1049
was calculated by the following formula: % Reduction of absorbance
5.13.2. Growth-inhibitory effect
¼ [(AB ꢀ AA)/AB] ꢃ 100, where: AB: absorbance of blank sample; AA:
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 ꢂC for 72 h, the cell
number was estimated by sulforhodamine B (SRB) assay, as
described by Skehan et al. Separately, the cell number on day 1 was
also measured. 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. Expo-
nentially growing P388 cells were seeded in 96-well plates on day
0. On day 1,0.1 ml aliquots of medium containing graded concen-
trations of test drugs were added to the wells. After incubation at
37 ꢂC for 72 h, the cell number was determined by MTT assay.
IC50 values were calculated as the drug concentrations that reduced
the number of cells to 50% of the control number.
absorbance of tested extract solution (t ¼ 30 min).
5.10. Reaction with reactive nitrogen species
5.10.1. Synthesis of peroxynitrite
Peroxynitrite (ONOOꢀ) was synthesized as previously described
[42]. 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 ꢂC) and the
top layer of the solution collected for the experiment. Concentra-
tions of stock (ONOOꢀ) were redetermined 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.
5.13.3. In vivo efficacy study
Female C57BL/6 mice (6 weeks of age; Charles River, Atsugi,
Japan) 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
randomized and separated into control and treatment (n ¼ 5 each)
groups when the tumor volume reached approximately 300 mm3
on day 1. The tested compound was dissolved in 5% glucose. Eto-
poside was diluted in saline and doxorubicin was dissolved in
saline. The tested compound was administrated on day 1 (single
dose) by i.v. injection into the tail vein, and etoposide and doxo-
rubicin were given at the maximum tolerated dose on the reported
administration schedule. Control animals received 5% glucose.
Tumor volume and body weight were measured on the days indi-
cated in the figures. Tumor volume was calculated according to the
following equation: tumor volume (mm3) ¼ (lengthꢀwidth2)/2.
5.11. Reaction of tested compounds with peroxynitrite
The ability of tested compounds to inhibit peroxynitrite-
induced tyrosine nitration was investigated via reaction of equi-
molar concentrations (100
presence of increasing concentrations of each tested compound
(0–300
M) in 100 mM phosphate buffer, pH 7.4 at 37 ꢂC for 15
mM) of tyrosine and peroxynitrite in the
m
min. Snap-freezing reaction mixtures prior to HPLC analysis
successfully terminated reactions. The formation of 3-nitro-
tyrosine (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).
5.13.4. Relaxation assay
5.12. HPLC analysis
Topoisomerase II was purified from P388 cells. One unit of the
enzyme was defined as the activity to relax completely 0.125 g of
Reaction mixtures were analyzed using reverse-phase HPLC.
Analysis was performed on an Agilent 1100 system with a Zorbax
supercoiled pBR-322 DNA at 30 ꢂC for 1 h. For the assay, 0.125
m
g of
supercoiled pBR-322 DNA (Takara Shuzo Co., Ltd., Tokyo, Japan)
was relaxed with 1 unit of topoisomerase II in 20 l of the assay
buffer [50 mM Tris–HCl (pH 8.0), 100 mM KCl, 10 mM MgCl2, 0.5
mM DTT, 0.5 M EDTA, 1 mM ATP, and 30 g/ml BSA] in the
ODS C18 column (150 ꢃ 4.6 mm i.d., 4
mm) and guard column (15 ꢃ
m
4.6 mm i.d., 4 m). Mobile phase A consisted of methanol/water/5 N
m
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 system was used
(min/% acetonitrile): 0/0, 5/0, 40/50, 60/100, 65/100, and 65.1/0 with
m
m
presence or absence of an inhibitor at 30 ꢂC for 1 h. Samples were
subjected to electrophoresis in 0.7% agarose gels 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 photographed under UV
light.
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.
5.13. Anticancer activities
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Cells. Murine colon cancer Colon 38, murine leukemia P388 and
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