Synthesis, antitumor, cytotoxic and antioxidant evaluation of some new pyrazolotriazines attached to antipyrine moiety

Article abstract of DOI:10.1016/j.ejmech.2012.08.034

Iminopropanehydrazonoyl cyanide 4 was achieved upon reaction of antipyrine diazonium salt 2 with 3-iminobutanenitrile (3) in EtOH/AcONa. 3-Aminopyrazole derivative 5 was obtained upon reaction of 4 with hydrazine hydrate. Diazodization of 5 afforded the d

Full text of DOI:10.1016/j.ejmech.2012.08.034

European Journal of Medicinal Chemistry 56 (2012) 254e262
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antitumor, cytotoxic and antioxidant evaluation of some new
pyrazolotriazines attached to antipyrine moiety
,
a,
M.A. Metwally a , M.A. Gouda b, Ammar N. Harmal a, A.M. Khalil a
*
a Department of Chemistry, Faculty of Science, Mansoura University, Mansoura, Egypt
b Department of Chemistry, Faculty of Science and Arts, Ulla, Taibah University, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
Iminopropanehydrazonoyl cyanide 4 was achieved upon reaction of antipyrine diazonium salt 2 with 3-
iminobutanenitrile (3) in EtOH/AcONa. 3-Aminopyrazole derivative 5 was obtained upon reaction of 4
with hydrazine hydrate. Diazodization of 5 afforded the diazonium salt 6 which coupled with active
methylene compounds 7e10, 19, 20, 25, 29 and 32 in pyridine to give aryl hydrazone derivatives 11e14,
21, 22, 26, 30 and 33, respectively. Refluxing of compounds 11e14, 21, 22, 26 and 33 in acetic acid
afforded the pyrazolotriazines 15e18, 23, 24, 28 and 35, respectively. The newly synthesized compounds
were screened for their cytotoxic and antioxidant activities. The results showed clearly that compounds
4, 5, 13, 22, and 24 displayed promising in vitro anticancer activity against four different cell lines (HepG2,
WI 38, VERO and MCF-7). Compounds 4 and 22 are the more potent antioxidant and anticancer agents.
On the other hand, most of the compounds exhibited good cytotoxic activity toward (EAC).
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 14 October 2011
Received in revised form
11 June 2012
Accepted 22 August 2012
Available online 31 August 2012
Keywords:
Iminobutanentirile
Cyclohexane-1,3-dione
Diazonium salt
Cyclocondensation
Antitumor and antioxidant activities
1. Introduction
compounds that interrupt radical-chain oxidation processes, causing
thus a high scientific interest [11,12]. Antipyrine derivatives are
It is well known that nitriles are widely used as intermediates
for a large number of heterocyclic compounds. Aminopyrazole
compounds can be readily obtained by the reaction of nitrile
derivatives with hydrazine hydrate [1e4].
strong inhibitors of cycloxygenase isoenzymes, platelet tromboxane
and prostanoids synthesis [8,13]. The biological activity of these
compounds has also been attributed to its scavenging activity
against reactive oxygen and nitrogen species, as well as to the
inhibition of neutrophil’s oxidative burst. However, besides its well
recognized benefits, antipyrine derivatives have been associated
with potential adverse effects characterized by leukopenia, most
commonly of neutrophils, causing neutropenia in the circulating
blood (agranulocytosis). It is worth to mention that there are studies
demonstrating that this adverse effect might be exaggerated [14,15].
Moreover, several pyrazole ring systems are associated with
antifungal, antitubercular, antibacterial, antiviral anticancer and
antioxidant activities [16e18] as well as the biological activities of
pyrazolotriazine ring systems are well documented [19e21]. It has
been used as adenine analogs, antagonists, antischistosomal, anti-
tumor and antibacterial agents [22e27]. Therefore, it is a real
challenge to combine the above mentioned boilable rings together
in a molecular framework to see the additive effect of these rings
toward the antioxidant and antitumor activities.
Also, aminopyrazole is often used for construction of pyrazolo-
triazine heterocyclic systems via diazodization and diazocoupling
with active methylene moieties [5e7]. Antipyrine or phenazone
derivatives are well known compounds used mainly as analgesic and
antipyretic drugs [8]. One of the best known antipyrine derivatives is
4-aminoantipyrine which is used for the protection against oxidative
stress as well as prophylactic of some diseases including cancer and
these are important directions in medicine and biochemistry [9,10].
Reactive oxygen species (ROS), including free radicals led to
a decrease in the antioxidant capacity and may generate other
reactive species that damage the living cell. Oxidative stress may
arise in a biological system after an increased exposure to oxidants,
so the antioxidants play a major role in protecting biological systems
against such threats. Different types of antioxidants (vitamins C and
E, glutathione, lipoic acid and butylated phenols, etc.) have been
widely used in different fields of industry and medicine as
2. Result and discussion
The synthetic strategies adopted to obtain the corresponding
pyrazolotriazines are depicted in Schemes 1e4. The starting
* Corresponding author. Tel.: þ20 502212966; fax: þ20 502222993.
E-mail address: mamegs@mans.edu.eg (M.A. Metwally).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.08.034

M.A. Metwally et al. / European Journal of Medicinal Chemistry 56 (2012) 254e262
255
H3C
NH
CH3
N
CN
CH3
NH2
CH3
H3C
Ph
N
NaNO2
HCl
3
N
CN
NH
Ph
N
H
EtOH/AcONa
N2+Cl-
O
H3C
4
2
NH2NH2.H2O
Dioxane
X
H3C
CN
NC
CH3
N
H3C
N
Ph
N
N
HN
Ph
N
CH3
CH3
X
N
N
N2+Cl-
N
NH2
NaNO2
7-10
Ph
N
O
N
N
N
N
HCl/AcOH
Pyridine
N
H
H3C
11-14
N
O
N
N
H
H3C
H3C
H
6
5
X
X= CN, COOEt
H3C
H3C
CN
CH3
CH3
N
N
X= NH=C-CH3
N
N
N
X
N
N
Ph
N
Ph
N
AcOH
N
N
N
N
X=
N
N
O
O
S
NH2
N
N
H3C
H3C
15-18
H
Scheme 1. Synthetic route for 3-substituted-7-methylpyrazolo[5,1-c][1,2,4]triazines 15e18.
material,
4-((3-amino-5-methyl-1H-pyrazol-4-yl)diazenyl)-1,5-
In similar manner, the diazonium salt 6 reacted with acetyla-
cetone (19) or cyclohexane-1,3-dione (20) in pyridine to afford
products that may be formulated as hydrazone derivatives 21 and
22, respectively based on both elemental analyses and spectral
data. Cyclizations of the resulted hydrazones 21 and 22 under acidic
condition gave the desired pyrazolotriazines 23 and 24, respec-
tively (Scheme 2).
The synthetic potency of 6 was investigated to develop a facile
and convenient route to polysubstituted pyrazolotriazine deriva-
tives of expected biological activity [30]. Thus, coupling of
compound 6 with 2-aminoprop-1-ene-1,1,3-tricarbonitrile (25)
[31] in pyridine afforded hydrazone derivative 26 which gave pyr-
azolopyridotriazine derivative 28 upon refluxing in acetic acid
through the intermediate 27. Furthermore, we have investigated
the reactivity of 6 with 3-methyl-1H-pyrazol-5(4H)-one (29) [32] to
synthesize dipyrazolo[5,1-c:30,40-e][1,2,4]triazine ring system 31.
Thus, coupling of diazonium salt 6 with compound 29 in pyridine
furnished the hydrazone derivative 30 which cyclized in POCl3/
DMF to afford the target compound 31 (Scheme 3).
dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (5) was prepared by
coupling of 1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-
4-diazonium chloride (2) with 3-iminobutanenitrile (3) [28] in
ethanol containing sodium acetate followed by heating the formed
N0-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-
iminopropane hydrazonoyl cyanide (4) under reflux with hydrazine
hydrate in dioxane. Diazotization of aminopyrazole 5 with sodium
nitrite in a mixture of acetic acid and hydrochloric acid led to the
corresponding diazonium salt [intermediate] 6 which coupled with
malononitrile (7), ethyl cyanoacetate (8), 3-iminobutanenitrile (9) or
2-(benzo[d]thiazol-2-yl)acetonitrile (10) [29] in pyridine to give the
corresponding hydrazones 11e14, respectively. Cyclizations of
compounds 11e14 afforded the desired pyrazolotriazines 15e18
under the influence of acetic acid (Scheme 1).
R
R
O
O
O
H3C
CH3
N
R
R
O
Treatment of 6 with 1-phenyl-2-thiocyanatoethanone (32) in
pyridine gave hydrazone derivative 33. The preparation of
compound 34 through cyclization of 33 in acetic acid was failed and
we obtain the iminothiadiazole 35 (Scheme 4). Assignment of the
new synthesized compounds was based on elemental analyses and
spectral data (IR, 1H NMR and mass spectra) (C.f. Experimental Part).
N
HN
19, 20
Ph
N
NaNO2
5
[6]
N
N
HCl/AcOH
Pyridine
N
O
N
H
H3C
21, 22
R
R
H3C
O
H3C
CH3
N
CH3
AcOH
N
O
O
2.1. Biological studies
N
N
Ph
N
N
N
Ph
N
N
N
N
N
R
N
O
2.1.1. Antitumor activity
N
O
R
N
H3C
N
H
H3C
2.1.1.1. Effect of drugs on the viability of Ehrlich ascites cells (EAC)
in vitro. Twenty antipyrine derivatives were tested for cytotoxicity
against a well known established model EAC in vitro [33]. Results for
the ED100, ED50, ED25 and IC50 values of the active compounds are
summarized in Table 1. The data showed clearly that most of
compounds have good activities. Thus, it would appear that the
23, 24
19, 21, 23, R= CH3,
20, 22, 24, R,R= -CH2CH2CH2-
Scheme 2. Synthesis of 3,4-disubstituted-7-methylpyrazolo[5,1-c][1,2,4]triazines 23
and 24.

256
M.A. Metwally et al. / European Journal of Medicinal Chemistry 56 (2012) 254e262
NC
H2N
NC
H2N
CN
CN
CN
CN
CN
CN
H3C
H3C
NC
CH3
CH3
N
N
NH2
N
N
N
HN
Ph
N
Ph N
25
[6]
N
N
N
N
Pyridine
N
N
O
O
N
H
N
H3C
H3C
H
26
AcOH
H3C
CH3
CH3
N
NH2
NH2
CN
N
N
N
Ph
N
N
Ph
N
N
N
N
CN
NH2
N
N
28
N
N
27
O
N
CN
NH2
H3C
H3C
CH3
N
N
NH
H3C
H3C
H3C
N
CH3
CH3
O
O
N
N
H
29
POCl3
DMF
N
N
N
HN
CH3
Ph
N
Ph
N
Pyridine
N
N
N
N
N
N
N
O
O
N
H
N
N
H
H3C
H3C
31
30
Scheme 3. Synthesis of pyrazolo[5,1-c]pyrido[2,3-e][1,2,4]triazine 28 and dipyrazolo[5,1-c:30,40-e][1,2,4]triazine 31.
presence of a basic skeleton of antipyrine, pyrazole or triazine
moieties are necessary for the good results of cytotoxic activity
against (EAC). By comparing the results obtained of the investigated
compounds to their structures the following structure activity rela-
tionships (SAR’s) were postulated: (i) Antipyrine derivative 4 is less
potent than pyrazole 5 which may be attributed to the conversion of
iminobutanenitrile moiety into aminopyrazole. (ii) Most of the
hydrazone derivatives are less potent than the corresponding pyr-
azolotriazine which may be attributable to the cyclization of
hydrazone into triazine moiety. (iii) Compounds 26 and 28 showed
very high cytotoxic activity (IC50 ¼ 77.1 and 78, respectively) which
may be due to presence of hydrazono-malononitrile dimer and
triazine moiety, respectively. (iv) Compound 24 is less potent than 23
which may be due to presence of cyclohexane moiety.
standard MTT method [34e36], against a panel of four human
tumor cell lines namely; heptacelluar carcinoma (HepG2), lung
fibroblasts (WI 38), kidney of a normal adult African green monkey
(VERO) and breast cancer (MCF-7). The cell lines were obtained
from ATCC via the Holding company for biological products and
vaccines (VACSERA) (Cairo, Egypt). 5-Fluorouracil (5-FU) was used
as a standard anticancer drug. MTT assay is a standard colorimetric
assay for measuring cell growth. It is used to determine cytotoxicity
of potential medicinal agents and other toxic materials. In brief,
yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) is reduced to purple formazan by mitochondrial dehy-
drogenases of living cells. A suitable solvent is added to dissolve the
insoluble purple formazan product into a colored solution. The
absorbance of this colored solution can be quantified by measuring
at a certain wavelength. When the amount of purple formazan
produced by cells treated with an agent is compared with that
produced by unreacted control cells, the effectiveness of the agent
in causing death of cells can deduced through the production of
2.1.2. Cytotoxicity and in vitro anticancer evaluation
Out of the newly synthesized compounds, twenty analogs were
selected to be evaluated for their in vitro anticancer effect via the
Ph
O
Ph
O
NCS
NCS
H3C
H3C
CH3
CH3
SCN
N
N
N
N
N
HN
N
Ph
O
32
Ph
N
[6]
N
N
N
N
Pyridine
N
N
O
O
N
N
H
H3C
H3C
H
33
AcOH
Ph
O
H3C
S
CH3
CH3
N
N
N
N
Ph
N
N
Ph
NH
SCN
Ph
N
N
N
N
N
O
N
N
H3C
34
N
H
H3C
35
Scheme 4. Synthesis of 4-((3-(5-benzoyl-2-imino-1,3,4-thiadiazol-3(2H)-yl)-5-methyl-1H-pyrazol-4-yl)diazenyl)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (35).

M.A. Metwally et al. / European Journal of Medicinal Chemistry 56 (2012) 254e262
257
Table 1
highest cytotoxic activity for the percentage viability IC50 at
24.8 mg/mL against VERO cell line. The remaining two active
compounds 5 and 24 showed moderate activity against the same
cell line at IC50 80.3 and 61.1 mg/mL, respectively. Furthermore,
interpretation of the results revealed that compounds 4, 5, 13, 22
and 24 vary from very strong to moderate anticancer activity
against MCF-7 cell line with percentage inhibition IC50 at 18.1, 29.9,
84.7, 69 and 68.2 mg/mL, respectively.
In vitro antitumor assay for 5-fluorouracil and the investigated compounds.
Compound no.
IC50
97.3
100
m
g
ED100
m
g
ED50 m
g
ED25
m
g
5-FU
4
5
68
38.6
84.3
58
32.26
0.46
1.71
97.2
79.3
64
68.7
80
70.8
62
65.3
70.1
64
97
11
12
13
14
15
16
17
18
21
22
23
24
26
28
30
31
33
35
94.3
94.9
97.5
95
91.9
94
95.5
93.8
95
98.8
96.7
99
77.1
78
93
92.1
94.3
90.7
48
27.85
22.53
1.30
11.17
34.26
28.02
14.38
26.63
15.59
0.88
49.8
60.1
56.4
42.8
47
2.1.3. Antioxidant activity assay
The antioxidant activities of twenty pyrazolotriazine compounds
were evaluated same as reported by Lissi et al. [37]. The data showed
clearly that, compounds 4 has good activity, while compound 22 and
24 exhibited moderate activities. On the other hand, the other
compounds showed weak activities (Table 3 and Fig. 1). Thus, it
would appear that introducing of aminopyrazole moiety enhances
the antioxidant properties of aminoantipyrine ring system.
55
49
70
80
54.1
63.3
55.5
80.2
21
22
45.5
43
73.9
91.4
38
40.6
63.3
61
11.01
0.67
64.48
62.53
26.63
34.69
26.92
39.36
2.1.4. Bleomycin-dependent DNA damage
65.2
60.5
48.1
38
The bleomycine is a family of glycopeptide antibiotics that is
used routinely as antitumor agents. The bleomycin assay has been
adopted for assessing the pro-oxidant effects of food antioxidants.
The antitumor antibiotic bleomycin binds iron ions and DNA. The
bleomycine iron complex degrades DNA that, upon heating with
thiobarbituric acid (TBA), yields a pink chromogen. Upon the
addition of suitable reducing agents antioxidants compete with
DNA and diminish chromogen formation [38]. To show the mech-
anism of action of our potent compounds 4, 5, 13, 22 and 24, their
protective activity against DNA damage induced by the bleomycine
iron complex were examined. The results in Table 4 showed that
compounds 4, 22, 24 and 33 exhibited high protection against DNA
damage induced by the bleomycine iron complex, thus diminishing
chromogen formation between the damaged DNA and TBA
molecules.
Where, ED100, ED50, and ED25 are the effective doses at 25, 50, and 100
respectively, of the compounds used. The dead % refers to the % of the dead tumor
cells and 5-FU is 5-fluorouracil as a well known cytotoxic agent.
m
g,
a dose response curve. The obtained results (Table 2) revealed that
compounds 4, 5, 13, 22 and 24 exhibited variable degrees of
inhibitory activity toward the four tested human tumor cell lines.
As for activity against HepG2 cell line, the very highest cytotoxic
activity was displayed by compound
4 which showed the
percentage viability IC50 at 8.3 mg/mL, whereas, the highest cyto-
toxic activity was displayed by compound 22 which showed the
percentage viability IC50 at 16.2 mg/mL and weak inhibitory activity
was also demonstrated by compounds 5, 13 and 24. The WI 38 cell
line showed highest sensitivity toward compounds 4 and 22 which
have IC50 at 13.8 and 19.8 mg/mL, respectively, whereas,
compounds 5, 13 and 24 displayed weak cytotoxic activities. On the
other hand, compound 4 showed very highest cytotoxic activity
IC50 at 10.0 mg/mL toward VERO cell line, compound 22 showed the
2.2. Structure activity relationship’s (SAR’s)
By comparing the experimental cytotoxicity of the compounds
reported in this study to their structures, the following structure
activity relationship’s (SAR’s) were postulated. (i) The presence of
a basic skeleton of antipyrine moiety is necessary for the broad
Table 2
Cytotoxicity (IC50) of tested compounds on different cell lines.
Table 3
Compound no.
IC50 (mg/mL)*
Antioxidant activity assay (ABTS) of some new pyrazolotriazine compounds.
HepG2
WI 38
VERO
MCF-7
Entry
Compound no.
% Inhibition
5-FU
4
5
9.1
8.3
79.1
4.0
13.8
73.1
6.4
10.0
80.3
2.5
15.1
84.7
1
Control of ABTS
0.0
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
22
23
Ascorbic acid
4
5
92.30
80.93
28.38
17.16
21.61
28.60
19.27
15.88
17.58
24.36
17.16
25.84
40.04
27.54
52.54
9.86
11
12
13
14
15
16
17
18
21
22
23
24
26
28
30
31
33
35
182.6
153.2
70.8
178.9
254.1
268.4
138.7
225.2
174.0
16.2
197.2
169.2
70.2
169.3
377.0
367.1
175.1
198.7
188.6
19.8
208.7
179.2
734.6
180.8
301.1
284.5
164.2
243.6
199.2
24.8
221.2
150.0
69.0
164.1
451.7
420.2
151.8
185.7
214.3
29.9
11
12
13
14
15
16
17
18
21
22
23
24
26
28
30
31
33
35
39.3
55.3
40.3
53.0
33.1
61.1
30.6
68.2
147.9
203.4
202.0
209.8
95.3
175.9
303.3
299.7
310.8
92.8
212.3
220.2
213.7
276.5
96.8
191.8
418.8
393.2
411.3
100.7
209.6
8.28
16.10
16.10
17.58
15.67
186.3
171.3
210.3
IC50, (
mg/mL): 1e10 (very strong), 11e25 (strong), 26e50 (moderate), 51e100
(weak), 100e200 (very weak), above 200 (non-cytotoxic).

258
M.A. Metwally et al. / European Journal of Medicinal Chemistry 56 (2012) 254e262
Fig. 1. Structure activity relationships (SAR’s) of the more potent antitumor and antioxidant compounds.
spectrum of cytotoxic activity toward different cell lines (HepG2,
WI 38, VERO and MCF-7). (ii) Antipyrine derivative 4 is more potent
than 5-florouracil toward HepG2 which may be attributed to the
replacement of pyrimidine with the antipyrine moiety and the
presence of iminobutanenitrile moiety. (iii) Compound 5 is less
potent than compound 4 which may be attributed to the conver-
sion of the iminobutanenitrile into pyrazole moiety. (iv) Most of the
hydrazone derivatives are more potent than pyrazolotriazine
compounds which may be attributable to the presence of hydra-
zone moiety. (v) Compound 24 is less potent than 23 which may be
due to cyclization of hydrazone moiety into pyrimidine ring system.
(vi) Cyclization of thioamide 9 to 2,3-dihydrothiazole derivative 7a
enhances the antitumor activity against different cell lines.
and antioxidant activities. Most of the tested compounds exhibited
good cytotoxic activity toward (EAC), so, there are relationships
between the cancer and oxidant results [9,10].
4. Experimental
4.1. General
All melting points are in degree centigrade (uncorrected) and
were determined on Gallenkamp electric melting point apparatus.
Elemental analyses (C, H and N) were carried out at the Microan-
alytical Center of Cairo Univ., Giza, Egypt. The IR spectra were
recorded (KBr) on a Mattson 5000 FTIR Spectrophotometer at the
Microanalytical Center (Faculty of Science; Mansoura University).
The 1H NMR spectra were measured on a Varian Spectrophotom-
eter at 300 MHz, using TMS as an internal reference and DMSO-d6
as solvent at the Microanalytical Center (Faculty of Science, Cairo
University). The mass spectra were recorded on Shimadzu Qp-2010
plus at the Microanalytical Center; (Faculty of Science; Cairo
University). Biological activity was carried in Pharmacognosy
Department, Faculty of Pharmacy, Mansoura University, Mansoura,
Egypt.
3. Conclusion
The objective of the present study was to synthesize and eval-
uate the antitumor and antioxidant activity of some novel pyr-
azolotriazines with the hope of discovering new structure leads
serving as antitumor and antioxidant gents. The data showed
clearly that compounds 4, 5, 13, 22 and 24 displayed promising
in vitro antitumor (against four cell lines) and antioxidant activities,
while compounds 4 and 22 exhibited broad spectrum of antitumor
4.1.1. Synthesis of N0-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-
1H-pyrazol-4-yl)-2-iminopropanehydrazonoyl cyanide (4)
Table 4
A well stirred solution of 4-aminoantipyrine (1) (1.02 g, 5 mmol)
in conc. HCl (3 mL) and 2 mL H2O was cooled in ice bath and
diazotized with the solution of NaNO2 (0.35 g, 5.1 mmol in 5 mL
H2O). The cold diazonium solution was added slowly to a well
stirred solution of 3 (0.41 g, 5 mmol) in ethanol (20 mL) containing
sodium acetate (1.64 g, 20 mmol). The reaction mixture was stirred
for another 2 h. The crude product was filtered off, dried well and
recrystallized from ethanol/benzene mixture to give 4. Yield (78%);
Results of bleomycin-dependent DNA damage assay on some selected
compounds.
Compound no.
Absorbance of samples
Ascorbic acid
4
5
13
22
24
33
0.098
0.063
0.124
0.117
0.080
0.089
0.101
orange crystals; m.p. 218e220 ꢀC; IR (KBr):
n
/cmꢁ1 ¼ 3299, 3203
(2NH), 2190 (CN), 1646 (CO), 1612 (C]N), 1490 (N]N); 1H NMR

M.A. Metwally et al. / European Journal of Medicinal Chemistry 56 (2012) 254e262
259
(DMSO-d6)
d
(ppm): 2.23 (s, 3H, CH3, pyrazole), 2.29 (s, 3H, CH3e
149 (12.3), 137 (4.2), 119 (18.0), 109 (7.0), 91 (26.9), 77 (23.1), 56
(100.0), 55 (17.0). Anal. Calcd. for C20H21N9O3 (435.44): C, 55.17; H,
4.86; N, 28.95%. Found: C, 55.24; H, 4.93; N, 29.03%.
C]N), 3.22 (s, 3H, CH3eN), 7.32e7.54 (m, 5H, AreH), 7.64 (br, 1H,
C]NH), 7.97 (br, 1H, NH, hydrazo); MS (EI, 70 eV) m/z (%) ¼ 298
(Mþ þ 2, 2.1), 297 (Mþ þ 1, 8.9), 296 (Mþ,1.1), 202 (9.2), 91 (10.8), 83
(43.0), 77 (10.3), 56 (100.0), 55 (8.1). Anal. Calcd. for C15H16N6O
(296.33): C, 60.80; H, 5.44; N, 28.36%. Found: C, 60.86; H, 5.49; N,
28.46%.
4.1.3.3. N0-(4-((E)-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)diazenyl)-5-methyl-1H-pyrazol-3-yl)-2-
iminopropanehydrazonoyl cyanide (13). Yield (82.0%); black crys-
tals; m.p. <300 ꢀC; IR (KBr):
n
/cmꢁ1 ¼ 3423, 3255, 3224 (3NH),
4.1.2. Synthesis of 4-((3-amino-5-methyl-1H-pyrazol-4-yl)
2200 (CN), 1737 (br, CO, C]N); 1H NMR (DMSO-d6)
d (ppm): 2.3 (s,
diazenyl)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (5)
To a solution of 4 (1.48 g, 5 mmol) in dioxane (20 mL) and
hydrazine hydrate (0.25 g; 98%, 5 mmol) was added. After refluxing
for 4 h, the reaction mixture was cooled then the formed precipitate
was filtered off, dried and recrystallized from DMF/EtOH to give 5.
3H, CH3, antipyrine), 2.49 (s, 3H, CH3eC]N), 2.66 (s, 3H, CH3,
pyrazole), 3.3 (s, 3H, CH3eN), 7.36e7.58 (m, 7H, 2NH, AreH), 12.15
(br, 1H, NH, hydrazone); MS (EI, 70 eV) m/z (%) ¼ 405 (Mþ þ 1, 1.1),
404 (Mþ, 0.8), 387 (29.7), 203 (7.5), 188 (28.7), 173 (3.3), 147 (18.2),
138 (3.3), 120 (25.7), 109 (4.2), 91 (17.4), 77 (17.9), 56 (100.0), 52
(9.3). Anal. Calcd. for C19H20N10O (404.43): C, 56.43; H, 4.98; N,
34.63%. Found: C, 56.47; H, 5.02; N, 34.67%.
Yield (90%); orange powder; m.p. 288e290 ꢀC; IR (KBr):
n/
cmꢁ1 ¼ 3451, 3262, 3201 (NH2, NH), 1641 (CO), 1610 (C]N), 1484
(N]N); 1H NMR (DMSO-d6)
d (ppm): 2.29 (s, 3H, CH3-antipyrine),
2.33 (s, 3H, CH3-pyrazole), 3.19 (s, 3H, CH3-antipyrine), 6.08 (br, 2H,
NH2), 7.35e7.56 (m, 6H, AreH, NH-pyrazole); MS (EI, 70 eV) m/z
(%) ¼ 312 (Mþ þ 1, 7.1), 311 (Mþ, 37.0), 296 (2.1), 119 (5.5), 77 (9.2),
56 (100.0). Anal. Calcd. for C15H17N7O (311.34): C, 57.87; H, 5.50; N,
31.49%. Found: C, 57.96; H, 5.57; N, 31.53%.
4.1.3.4. N0-(4-((E)-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)diazenyl)-5-methyl-1H-pyrazol-3-yl)benzo[d]thiazole-
2-carbohydrazonoyl cyanide (14). Yield (65.0%); yellow crystals;
m.p. 208e210 ꢀC; IR (KBr):
n
/cmꢁ1 ¼ 3426, 3208 (2NH), 2250 (CN),
1648 (CO), 1515 (N]N); 1H NMR (DMSO-d6)
d (ppm): 2.41 (s, 3H,
CH3, antipyrine), 2.69 (s, 3H, CH3, pyrazole), 3.27 (s, 3H, CH3eN),
7.35e8.27 (m, 10H, AreH, NH, pyrazole), 12.88 (br, s, 1H, NH-
hydrazone); MS (EI, 70 eV) m/z (%) ¼ 498 (Mþ þ 2, 7.2), 497
(Mþ þ 1, 4.3), 496 (Mþ, 10.4), 482 (6.3), 297 (16.2), 282 (10.7), 255
(14.8), 229 (10.5), 174 (23.1), 161 (54.1), 146 (15.9), 135 (13.5), 119
(30.2), 109 (27.7), 91 (33.3), 77 (35.9), 56 (100.0), 54 (19.0). Anal.
Calcd. for C24H20N10OS (496.55): C, 58.05; H, 4.06; N, 28.21%.
Found: C, 58.10; H, 4.14; N, 28.26%.
4.1.3. Synthesis of 4-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-
1H-pyrazol-4-yllazo)-5-methyl-pyrazol-3-yl-hydrazones 11e14, 21,
22, 26, 30 and 33
General procedure: A well stirred solution of 5 (1.56 g, 5 mmol) in
a mixture of acetic acid (10 mL) and conc. HCl (3 mL) was cooled in
an ice bath and then a solution of NaNO2 (0.35 g, 5.1 mmol in 5 mL
H2O) was added dropwise to it. The above cooled diazonium
solution was added slowly to a well stirred solution of active
methylene compounds namely; malononitrile (7) (0.33 g, 5 mmol)
4.1.3.5. 3-(2-(4-((1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyr-
azol-4-yl)diazenyl)-5-methyl-1H-pyrazol-3-yl)hydrazono)pentane-
2,4-dione (21). Yield (76.0%); dark brown crystals; m.p. > 300 ꢀC; IR
or ethyl-2-cyanoacetate (8) (0.57 g,
5
mmol) or 3-
iminobutanenitrile (9) (0.41 g, 5 mmol) or 2-(benzo[d]thiazol-2-
yl)acetonitrile (10) (0.87 g, 5 mmol) or acetylacetone (19) (0.5 g,
5 mmol) or cyclohexane-1,3-dione (20) (0.56 g, 5 mmol) or 2-
iminopropane-1,1,3-tricarbonitrile (25) (0.66 g, 5 mmol) or 3-
methyl-1H-pyrazol-5(4H)-one (29) (0.49 g, 5 mmol) or 1-phenyl-
2-thiocyanatoethanone (32) (0.89 g, 5 mmol) in pyridine (15 mL).
The reaction mixture was stirred for 2 h. The crude product was
filtered off, dried well and crystallized from the appropriate solvent
to give compounds 11e14, 21, 22, 26, 30 and 33, respectively.
(KBr):
n
/cmꢁ1 ¼ 3405, 3233 (2NH), 1680, 1637 (3CO), 1494 (N]N);
1H NMR (DMSO-d6)
d (ppm): 2.36 (s, 3H, COeCH3), 2.37 (s, 3H,
CH3eCO), 2.44 (s, 3H, CH3-antipyrine), 2.60 (s, 3H, CH3-pyrazole),
3.28 (s, 3H, NeCH3), 7.39-7.58 (m, 6H, AreH, NH-pyrazole), 11.2 (br,
NH, NH-hydrazone); MS (EI, 70 eV) m/z (%) ¼ 406 (Mþ ꢁ H2O, 6.5),
405 (10.0), 404 (36.1), 205 (12.4), 91 (17.7), 83 (17.0), 77 (14.3), 67
(24.0), 56 (100.0), 51 (6.5). Anal. Calcd. for C20H22N8O3 (422.44): C,
56.86; H, 5.25; N, 26.53%. Found: C, 56.96; H, 5.36; N, 26.59%.
4.1.3.1. (4-((1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-
4-yl)diazenyl)-5-methyl-1H-pyrazol-3-yl)carbonohydrazonoyl dicya-
nide (11). Yield (67.0%); orange crystals; m.p. 226e228 ꢀC; IR (KBr):
4.1.3.6. 2-(2-(4-((1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyr-
azol-4-yl)diazenyl)-5-methyl-1H-pyrazol-3-yl)hydrazono)cyclo-
hexane-1,3-dione (22). Yield (64.0%); dark brown crystals; m.p.
n
/cmꢁ1 ¼ 3442, 3423, 3410 (NH2, 2NH), 2220 (CN), 1646 (CO), 1496
223e225 ꢀC; IR (KBr):
n
/cmꢁ1 ¼ 3386, 3206 (2NH), 1675, 1632
(N]N); 1H NMR (DMSO-d6)
d
(ppm): 2.49 (s, 3H, CH3-antipyrine),
(3C]O), 1496 (N]N); 1H NMR (DMSO-d6)
d (ppm): 1.85e1.93 (m,
2.66 (s, 3H, CH3-pyrazole), 3.37 (s, 3H, CH3eN), 7.36e7.59 (m, 6H,
NH-pyrazole, AreH), 9.40 (br,1H, NH-hydrazone); MS (EI, 70 eV) m/
z (%) ¼ 389 (Mþ þ 1, 3.7), 388 (Mþ, 6.3), 374 (4.8), 311 (9.1), 296
(4.6), 281 (4.3), 214 (24.4), 202 (14.1), 189 (39.5), 174 (29.8), 148
(24.6), 132 (2.6), 121 (40.2), 93 (45.5), 77 (45.0), 56 (100.0), 53
(38.9). Anal. Calcd. for C18H16N10O (388.39): C, 55.66; H, 4.15; N,
36.06%. Found: C, 55.71; H, 4.26; N, 36.18%.
2H, CH2, cyclohexane) 2.4e2.7 (m, 10H, 2CH3, 2CH2, cyclohexa-
none), 2.74 (s, 1H, CH, cyclohexanone), 3.21 (s, 3H, CH3eN), 7.36e
7.60 (m, 5H, AreH), 9.00 (s, 1H, NH); MS (EI, 70 eV) m/z (%) ¼ 434
(Mþ, 65.0), 279 (79.0), 239 (77.1), 227 (85.4), 210 (95.5), 207 (31.9),
189 (65.0), 174 (8.9), 160 (77.7), 144 (74.0), 134 (17.8), 124 (13.4), 107
(19.1), 71 (20.4), 59.95 (100.0). Anal. Calcd. for C21H22N8O3 (434.45):
C, 58.06; H, 5.10; N, 25.79%. Found: C, 58.12; H, 5.14; N, 25.83%.
4.1.3.2. Ethyl
2-cyano-2-(2-(4-((E)-(1,5-dimethyl-3-oxo-2-phenyl-
4.1.3.7. 2-Amino-3,3-dicyano-N0-(4-((E)-(1,5-dimethyl-3-oxo-2-
phenyl-2,3-dihydro-1H-pyrazol-4-yl)diazenyl)-5-methyl-1H-pyrazol-
3-yl)acrylohydrazonoyl cyanide (26). Yield (76.0%); dark red crys-
2,3-dihydro-1H-pyrazol-4-yl)diazenyl)-5-methyl-1H-pyrazol-3-yl)
hydrazono)acetate (12). Yield (77.0%); orange crystals; m.p. 175e
177 ꢀC; IR (KBr):
n
/cmꢁ1 ¼ 3222 (br, 2NH), 2206 (CN), 1718, 1668
tals; m.p. >300 ꢀC; IR (KBr):
n
/cmꢁ1 ¼ 3388, 3299, 3197 (3NH), 2198
(2CO), 1448 (2N]N); 1H NMR (DMSO-d6)
d
(ppm): 1.31 (t, 3H, CH3,
(CN), 1650 (CO), 1623 (C]N), 1498 (N]N); 1H NMR (DMSO-d6)
J ¼ 7.2 Hz), 2.43 (s, 3H, CH3), 2.66 (s, 3H, CH3-pyrazole), 3.30 (s, 3H,
CH3eN), 4.28 (q, 2H, OCH3, J ¼ 7.2 Hz), 7.43e7.95 (m, 6H, AreH, NH-
pyrazole), 12.5 (br, 1H, NH-hydrazone); MS (EI, 70 eV) m/z (%) ¼ 436
(Mþ þ 1, 4.2), 435 (Mþ,11.7), 363 (4.5), 236 (6.3), 214 (5.0), 202 (6.2),
d (ppm): 2.05 (s, 3H, CH3, antipyrine), 2.64 (s, 3H, CH3, pyrazole),
2.67 (s, 2H, NH2), 3.22 (s, 3H, CH3eN), 7.35e7.53 (m, 5H, NH, AreH),
8.42 (br, 1H, NH, pyrazole), 8.52 (br, 1H, NH, hydrazone); MS (EI,
70 eV) m/z (%) ¼ 455 (Mþ þ 1, 21.1), 454 (Mþ, 25.2), 436 (26.1), 428

260
M.A. Metwally et al. / European Journal of Medicinal Chemistry 56 (2012) 254e262
(23.6), 406 (22.7), 390 (17.1), 207 (23.6), 187 (24.2), 178 (21.7), 156
(37.6), 148 (24.2), 137 (40.1), 121 (30.1), 106 (19.6), 95 (39.4), 81
(77.3), 69 (100.0), 52 (14.6). Anal. Calcd. for C21H18N12O (454.45): C,
55.50; H, 3.99; N, 36.99%. Found: C, 55.52; H, 4.07; N, 37.03%.
antipyrine), 2.64 (s, 3H, CH3, pyrazole), 3.35 (s, 3H, CH3eN), 7.35e7.51
(m, AreH, NH2); MS (EI, 70 eV) m/z (%) ¼ 404 (Mþ, 0.8), 403 (Mþ ꢁ 1,
2.4),175 (100.0),146 (16.3),130(4.9),119 (12.8),105(21.9), 96(52.1), 77
(68.9), 67 (61.9), 52 (28.1). Anal. Calcd. for C19H20N10O (404.43): C,
56.43; H, 4.98; N, 34.63%. Found: C, 56.48; H, 5.04; N, 34.67%.
4.1.3.8. 1,5-Dimethyl-4-((E)-(5-methyl-3-((E)-2-(3-methyl-5-oxo-
1H-pyrazol-4(5H)-ylidene)hydrazinyl)-1H-pyrazol-4-yl)diazenyl)-2-
phenyl-1H-pyrazol-3(2H)-one (30). Yield (65.0%); red crystals; m.p.
4.1.4.4. 4-((4-Amino-3-(benzo[d]thiazol-2-yl)-7-methylpyrazolo[5,1-
c][1,2,4]
triazin-8-yl)diazenyl)-1,5-dimethyl-2-phenyl-1H-pyrazol-
278 ꢀC; IR (KBr):
n
/cmꢁ1 ¼ 3380, 3210, 3151 (3NH), 1654, 1600 (2C]
3(2H)-one (18). Yield (63.0%); yellow crystals; m.p. 252e254 ꢀC; IR
O), 1498 (N]N); 1H NMR (DMSO-d6)
d
(ppm): 2.12 (s, 3H, CH3,
(KBr):
n
/cmꢁ1 ¼ 3446, 3424 (br, NH), 1650 (CO), 1629 (C]N), 1492
antipyrine), 2.44 (s, 3H, CH3, pyrazolone), 2.66 (s, 3H, CH3, pyr-
azolone), 3.29 (s, 3H, CH3eN), 7.34e7.56 (m, 5H, AreH, NH, pyr-
azole), 11.60 (br, 1H, NH, hydrazone), 12.9 (s, 1H, NH, pyrazolone);
MS (EI, 70 eV) m/z (%) ¼ 421 (Mþ þ 1, 23.7), 420 (Mþ, 24.5), 77
(27.3), 56 (100.0). Anal. Calcd. for C19H20N10O2 (420.43): C, 54.28; H,
4.79; N, 33.32%. Found: C, 54.33; H, 4.84; N, 33.37%.
(N]N); 1H NMR (DMSO-d6)
d (ppm): 2.58 (s, 3H, CH3), 2.69 (s, 3H,
CH3), 3.35 (s, 3H, CH3eN), 7.38e8.19 (m, 11H, NH2, AreH); MS (EI,
70 eV) m/z (%) ¼ 496 (Mþ, 14.5), 255 (20.9), 84 (49.2), 77 (100.0), 57
(86.3). Anal. Calcd. for C24H20N10OS (496.55): C, 58.05; H, 4.06; N,
28.21%. Found: C, 58.11; H, 4.15; N, 28.28%.
4.1.4.5. 4-((3-Acetyl-4,7-dimethylpyrazolo[5,1-c][1,2,4]triazin-8-yl)
4.1.3.9. Cyanic N0-(4-((E)-(1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazol-4-yl)diazenyl)-5-methyl-1H-pyrazol-3-yl)-2-
oxo-2-phenylacetohydrazonic thio-anhydride (33). Yield (68.0%);
diazenyl)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one
(23).
Yield (67.0%); dark brown crystals; m.p. >300 ꢀC; IR (KBr):
n/
cmꢁ1 ¼ 1670, 1637 (2CO), 1492 (N]N); 1H NMR (DMSO-d6)
dark brown crystals; m.p. 137 ꢀC; IR (KBr):
n
/cmꢁ1 ¼ 3380, 3332
d (ppm): 2.18 (s, 3H, CH3eCO), 2.6 (s, 3H, CH3, triazine), 2.69 (s, 3H,
(2NH), 2150 (CN), 1660 (C]O), 1488 (2N]N); 1H NMR (DMSO-d6)
CH3, antipyrine), 2.85 (s, 3H, CH3, pyrazole), 3.38 (s, 3H, CH3eN),
7.39e7.59 (m, 5H, AreH); MS (EI, 70 eV) m/z (%) ¼ 406 (Mþ þ 2,
4.5), 405 (Mþ þ 1, 11.1), 404 (Mþ, 40.8), 77 (12.0), 56 (100.0), 55
(9.2). Anal. Calcd. for C20H20N8O2 (404.43): C, 59.40; H, 4.98; N,
27.71%. Found: C, 59.47; H, 5.03; N, 27.76%.
d
(ppm): 2.29 (s, 3H, CH3, antipyrine), 2.46 (s, 3H, CH3, pyrazole),
3.28 (s, 3H, CH3eN), 7.38e7.56 (m, 7H, AreH, 2NH); MS (EI, 70 eV)
m/z (%) ¼ 480 (Mþ ꢁ H2O, 36.2), 221 (34.2), 167 (3.4), 135 (34.5), 77
(100.0), 51 (32.2). Anal. Calcd. for C24H21N9O2S (499.55): C, 57.70; H,
4.24; N, 25.23%. Found: C, 57.64; H, 4.22; N, 25.18%.
4.1.4.6. 3-((1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-
yl)diazenyl)-2-methyl-8,9-dihydrobenzo[e]pyrazolo[5,1-c][1,2,4]tri-
azin-6(7H)-one (24). Yield (66.0%); black crystals; m.p. >300 ꢀC; IR
4.1.4. General procedure for the synthesis of pyrazolotriazines 15e
18, 23, 24, 28 and thiadiazole 35
A suspension of arylazo derivatives 11e14, 21, 22, 26 and 33
(5 mmol) in acetic acid (10 mL) was refluxed for 7 h. The reaction
mixture was poured into ice cold water then the formed precipitate
was filtered off, dried and recrystallized from DMF/ethanol to give
compounds 15e18, 23, 24, 28 and 35, respectively.
(KBr):
d6)
n
/cmꢁ1 ¼1646, 1637 (2C]O), 1500 (N]N); 1H NMR (DMSO-
d
(ppm): 1.65e1.68 (m, 2H, CH2, cyclohexanone), 2.45e2.72 (m,
10H, 2CH3, 2CH2, cyclohexanone), 3.42 (s, 3H, CH3eN), 7.36e7.58
(m, 5H, AreH); MS (EI, 70 eV) m/z (%) ¼ 400 (Mþ ꢁ CH4, 42.2),
296 (48.8), 212 (43.8), 203 (47.1), 72 (100.0), 59 (17.8), 51 (16.5).
Anal. Calcd. for C21H20N8O2 (416.44): C, 60.57; H, 4.84; N, 26.91%.
Found: C, 60.62; H, 4.88; N, 26.96%.
4.1.4.1. 4-Amino-8-((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)diazenyl)-7-methylpyrazolo[5,1-c][1,2,4]triazine-3-
carbonitrile (15). Yield (65.0%); orange crystals; m.p. 301 ꢀC; IR
4.1.4.7. 6,8-Diamino-3-((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-
(KBr):
n
/cmꢁ1 ¼ 3407, 3386, 3380 (NH, NH2), 2210 (CN), 1658 (CO),
1H-pyrazol-4-yl)
diazenyl)-2-methylpyrazolo[5,1-c]pyrido[2,3-e]
1603 (C]N), 1492 (N]N); 1H NMR (DMSO-d6)
d
(ppm): 2.49 (s, 3H,
[1,2,4]triazine-7-carbonitrile (28). Yield (68.0%); dark brown crys-
CH3, antipyrine), 2.62 (s, 3H, CH3, pyrazole), 3.24 (s, 3H, CH3eN),
7.34e7.58 (m, 7H, AreH, NH2); MS (EI, 70 eV) m/z (%) ¼ 385
(Mþ ꢁ 3H, 6.7), 297 (10.9), 161 (16.4), 132 (9.7), 93 (100.0), 77 (61.2),
56 (75.3), 55 (55.5). Anal. Calcd. for C18H16N10O (388.39): C, 55.66;
H, 4.15; N, 36.06%. Found: C, 55.72; H, 4.23; N, 36.13%.
tals; m.p. 300 ꢀC; IR (KBr):
n
/cmꢁ1 ¼ 3294, 3151 (NH2), 2199 (CN),
1650 (CO), 1637 (CN); 1H NMR (DMSO-d6)
d (ppm): 2.05 (s, 3H, CH3,
antipyrine), 2.61 (s, 3H, CH3, pyrazole), 3.23 (s, 3H, CH3eN), 3.34 (br,
4H, 2NH2), 7.35e7.53 (m, 5H, AreH); MS (EI, 70 eV) m/z (%) ¼ 452
(Mþ ꢁ 2H, 28.7), 436 (34.4), 418 (32.3), 79.9 (100.0), 63.90 (99.0), 54
(49.5). Anal. Calcd. for C21H18N12O (454.45): C, 55.50; H, 3.99; N,
36.99%. Found: C, 55.53; H, 4.05; N, 37.06%.
4.1.4.2. Ethyl 4-amino-8-((1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazol-4-yl)
diazenyl)-7-methylpyrazolo[5,1-c][1,2,4]
triazine-3-carboxylate (16). Yield (74.3%); dark brown crystals; m.p.
4.1.4.8. 4-((3-(5-Benzoyl-2-imino-1,3,4-thiadiazol-3(2H)-yl)-5-
methyl-1H-pyrazol-4-yl)diazenyl)-1,5-dimethyl-2-phenyl-1H-pyr-
azol-3(2H)-one (35). Yield (65.0%); orange crystals; m.p. 214e
227e230 ꢀC; IR (KBr):
n
/cmꢁ1 ¼ 3386, 3280, 3259 (NH2, NH), 1716,
1689 (2C]O),1616 (N]C),1494 (N]N); 1H NMR (DMSO-d6)
d (ppm):
1.36 (t, 3H, CH3, J ¼ 7.5 Hz), 2.50 (s, 3H, CH3, antipyrine), 2.67 (s, 3H,
CH3, pyrazole), 3.37 (s, 3H, CH3eN), 4.27 (q, 2H, OeCH2, J ¼ 7.5 Hz),
7.39e7.57 (m, 7H, AreH, NH2), 8.3(br,1H, NH, pyrazole);MS(EI, 70eV)
m/z (%) ¼ 389 (Mþ eEtOH,16.6), 388 (25.2), 373 (24.3), 350 (23.3), 339
(19.2), 331 (19.2), 311 (25.2), 302 (18.2), 292 (23.6), 279 (22.4), 71
(53.4), 57 (100.0), 55 (59.1). Anal. Calcd. for C20H21N9O3 (435.44): C,
55.17; H, 4.86; N, 28.95%. Found: C, 55.24; H, 4.92; N, 28.97%.
216 ꢀC; IR (KBr):
n
/cmꢁ1 ¼ 3420 (br, 2NH), 1716 (CO), 1635 (br, CO,
C]N); 1H NMR (DMSO-d6)
d (ppm): 2.54 (s, 3H, CH3, antipyrine),
2.67 (s, 3H, CH3, pyrazole), 3.35 (s, 3H, CH3eN), 7.38e7.56 (m, 10H,
AreH), 8.61 (br, 1H, NH, pyrazole), 9.34 (s, 1H, ¼NH); MS (EI, 70 eV)
m/z (%) ¼ 500 (Mþ þ 1, 6.3), 464 (15.8), 77 (22.9), 56 (100.0), 53
(13.3). Anal. Calcd. for C24H21N9O2S (499.55): C, 57.70; H, 4.24; N,
25.23%. Found: C, 57.78; H, 4.27; N, 25.28%.
4.1.4.3. 4-((4-Amino-3-(1-iminoethyl)-7-methylpyrazolo[5,1-c]
[1,2,4]triazin-8-yl)diazenyl)-1,5-dimethyl-2-phenyl-1H-pyrazol-
3(2H)-one (17). Yield (75.0%); dark brown crystals; m.p. >300 ꢀC; IR
4.1.5. Synthesis of 4-((3,7-dimethyl-1H-dipyrazolo[5,1-c:30,40-e]
[1,2,4]triazin-6-yl)diazenyl)-1,5-dimethyl-2-phenyl-1H-pyrazol-
3(2H)-one (31)
To a suspension of 30 (0.42 g, 1 mmol) in DMF (6 mL), phos-
phorous oxychloride (1 g, 6 mmol) was added, the reaction mixture
(KBr):
n
/cmꢁ1 ¼3440, 3426(NH2, 2NH),1633 (br, CO, C]N),1490 (N]
N); 1H NMR(DMSO-d6)
d (ppm): 2.49(s, 3H, CH3eCO), 2.51 (s, 3H, CH3,

M.A. Metwally et al. / European Journal of Medicinal Chemistry 56 (2012) 254e262
261
was refluxed on a water bath for 12 h. The reaction mixture was
poured into sodium carbonate solution then the formed precipitate
was filtered off, dried and recrystallized from DMF to give 31. Yield
of doseeresponse data (e.g., drug concentrations x1, x2, xn and
growth inhibition y1, y2, yn). The values of y are in the range of 0e1.
(63.0%); black crystals; m.p. >300 ꢀC; IR (KBr):
n
/cmꢁ1 ¼ 3423 (NH),
4.3.4. Linear regression
1625 (br, C]O, C]N), 1454 (N]N); MS (EI, 70 eV) m/z (%) ¼ 403
(Mþ þ 1, 0.2), 81 (100.0), 63 (79.9), 55 (10.4), 50 (3.2). Anal. Calcd.
for C19H18N10O (402.41): C, 56.71; H, 4.51; N, 34.81%. Found: C,
56.74; H, 4.56; N, 34.87%.
The simplest estimate of IC50 is to plot x ꢁ y and fit the data with
a straight line (linear regression). IC50 value is then estimated using
the fitted line, i.e.,
Y ¼ a*X þ b; IC50 ¼ ð0:5 ꢁ bÞ=a
4.2. Antitumor activity
4.4. Antioxidant assay
Different concentrations of the tested compounds were
prepared (ED100, ED50 and ED25 mg/DMSO). The amount of DMSO
4.4.1. Antioxidant activity screening assay; ABTS method
was adjusted to give a final concentration of 0.1%. Ascites fluid was
obtained from the peritoneal cavity of the donor animal from
(National Cancer Institute, Cairo, Egypt) contain Ehrlich cell was as
aseptically aspirated. The cells were grown partially floating and
attach in a suspension culture (RPMI 1660 medium, Sigma Chem-
ical Co. St. Louis, USA), supplemented with 10% fetal bovine serum
(GIBCO, UK). They were maintained at 37 ꢀC in humidified atmo-
sphere with 5% CO2 for 2 h. The viability of the cell used in control
experiments (DMSO only without drug) exceeded 95% as deter-
mined by microscopically examination using a hemocytometer and
trypan blue stain (stain only the dead cells).
Antioxidant activity determinations were evaluated from the
bleaching of ABTS derived radical cations. The radical cation was
derived from ABTS [2,20-azino-bis (3-ethyl benzothiazoline-6-
sulfonic acid)] was prepared by reaction of ABTS (60
MnO2 (3 mL, 25 mg/mL) in phosphate buffer solution (10
m
l) with
m
M, pH 7,
5 mL) After shaking the solution for a few minutes, it was centri-
fuged and filtered. The Absorbance (A control) of the resulting
greeneblue solution (ABTS radical solution) was recorded at lmax
734 nm. The absorbance (A test) was measured upon the addition
of (20 ml of 1 mg/mL) solution of the tested sample in spectroscopic
grade MeOH/buffer (1:1 v/v) to the ABTS solution. The decrease in
the absorbance is expressed as % inhibition which calculated from
this equation:
4.3. Cytotoxicity and antitumor evaluation
% Inhibition ¼ ½AðcontrolÞ ꢁ AðtestÞ=AðcontrolÞꢃ ꢂ 100
4.3.1. Materials and methods
The reagents RPMI-1640 medium (Sigma Co., St. Louis, USA)
Fetal Bovine serum (GIBCO, UK) and the cell lines HepG2, WI 38,
VERO, MCF-7 were obtained from ATCC.
Ascorbic acid (20
ml, 2 mM) solution was used as standard
antioxidant (positive control). Blank sample was run using solvent
without ABTS (Table 2).
4.3.2. Procedure
4.4.2. Bleomycin-dependent DNA damage assay [38e40]
The stock samples were diluted with RPMI-1640 medium to
desired concentrations ranging from 10 to 1000 mg/mL. The final
concentration of dimethylsulfoxide (DMSO) in each sample did not
exceed 1% v/v. The cytotoxic activity of the compounds was tested
against HepG2, WI 38, MCF-7 and VERO. The % viability of cell was
examined visually. 5-Fluorouracil was used as a standard anti-
cancer drug for comparison. Briefly, cell were batch cultured for
10 d, then seeded in 96-well plates of 10 ꢂ 103 cells/well in fresh
complete growth medium in 96-well microtiter plastic plates at
37 ꢀC for 24 h under 5% CO2 using a water jacketed carbon dioxide
incubator (Shedon.TC2323. Cornelius, OR, USA). The medium
(without serum) was added and cells were incubated either alone
(negative control) or with different concentrations of sample to
give a final concentrations of (1000, 500, 200, 100, 50, 20, 10 mg/
mL). Cells were suspended in RPMI-1640 medium, 1% antibiotice
To the reaction mixtures in a final volume of 1.0 mL, the
following reagents at the final concentrations stated were added:
DNA (0.2 mg/mL), bleomycin (0.05 mg/mL), FeCl3 (0.025 mM),
magnesium chloride (5 mM), KH2PO4/KOH buffer pH 7.0 (30 mM)
and ascorbic acid (0.24 mM) or the test fractions diluted in MeOH to
give a concentration of (0.1 mg/mL). The reaction mixtures were
incubated in a water bath at 37 ꢀC for 1 h. At the end of the incu-
bation period, 0.1 mL of ethylenediaminetetraacetic acid (EDTA)
(0.1 M) was added to stop the reaction (the iron EDTA complex is
unreactive in the bleomycin assay). DNA damage was assessed by
adding 1 mL 1% (w/v) thiobarbituric acid (TBA) and 1 mL of 25% (v/
v) hydrochloric acid (HCl) followed by heating in a water-bath
maintained at 80 ꢀC for 15 min. The chromogen formed was
extracted into 1-butanol, and the absorbance was measured at
532 nm.
antimycotic mixture (104
mg/mL potassium penicillin, 104 mg/mL
streptomycin sulfate and 25 mg/mL Amphotericin B) and 1%
L
-
glutamine in 96-well flat bottom microplates at 37 ꢀC under 5% CO2.
After 96 h of incubation, the medium was again aspirated, trays
were inverted onto a pad of paper towels, the remaining cells
rinsed carefully with medium, and fixed with 3.7% (v/v) formal-
dehyde in saline for at least 20 min. The fixed cells were rinsed with
water, and examined. The cytotoxic activity was identified as
confluent, relatively unaltered mono-layers of stained cells treated
with compounds.
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