Synthesis and antioxidant activity of a novel series of pyrazolotriazine, coumarin, oxoazinone, and pyrazinopyrimidine derivatives

Article abstract of DOI:10.1002/ardp.201300128

A series of pyrazolotriazone derivatives 8-10 were obtained via coupling of 3-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxopropanenitrile (1) with 3-pyrazole diazonium chlorides 2-4, followed by heating of the formed hydrazones 5-7 in acetic acid, respectively. Moreover, coupling reaction of 1 with the aryl diazonium salts 15a-c afforded hydrazones 16a-c. Furthermore, treatment of 1 with 2-hydroxy-1-aldehydes 18-20 afforded the corresponding coumarins 21-23, respectively. Finally, compound 1 reacted with 1-nitrosonaphthalen-2-ol (26) and 6-amino-5-nitroso-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (28) to give 3-oxo-3H-naphtho[2,1-b][1,4]oxazine-2-carbonitrile (25) and pyridazinopyrimidine (29), respectively. The newly synthesized compounds were screened for their ABTS antioxidant activity. Compounds 7 (90.39%), 10 (85.88%), and 16a (91.95%) exhibited promising activities. The most potent compound, 16a, has the ability to protect DNA from the damage induced by bleomycin.

Full text of DOI:10.1002/ardp.201300128

Arch. Pharm. Chem. Life Sci. 2013, 346, 1–9
1
Full Paper
Synthesis and Antioxidant Activity of a Novel Series
of Pyrazolotriazine, Coumarin, Oxoazinone, and
Pyrazinopyrimidine Derivatives
Moustafa A. Gouda1,2
1
Faculty of Science, Department of Chemistry, Mansoura University, Mansoura, Egypt
Faculty of Science and Arts, Department of Chemistry, Ulla, Taibah University, Medina, KSA
2
A series of pyrazolotriazone derivatives 8–10 were obtained via coupling of 3-(3,5-dimethyl-1H-
pyrazol-1-yl)-3-oxopropanenitrile (1) with 3-pyrazole diazonium chlorides 2–4, followed by heating
of the formed hydrazones 5–7 in acetic acid, respectively. Moreover, coupling reaction of 1 with
the aryl diazonium salts 15a–c afforded hydrazones 16a–c. Furthermore, treatment of 1 with
2-hydroxy-1-aldehydes 18–20 afforded the corresponding coumarins 21–23, respectively. Finally,
compound 1 reacted with 1-nitrosonaphthalen-2-ol (26) and 6-amino-5-nitroso-2-thioxo-2,3-
dihydropyrimidin-4(1H)-one (28) to give 3-oxo-3H-naphtho[2,1-b][1,4]oxazine-2-carbonitrile (25)
and pyridazinopyrimidine (29), respectively. The newly synthesized compounds were screened for
their ABTS antioxidant activity. Compounds 7 (90.39%), 10 (85.88%), and 16a (91.95%) exhibited
promising activities. The most potent compound, 16a, has the ability to protect DNA from the
damage induced by bleomycin.
Keywords: ABTS antioxidant assay / Bleomycin-dependent DNA damage / Coumarin / Oxopropanenitrile /
Pyrazolotriazone / Pyrimidine
Received: April 11, 2013; Revised: May 10, 2013; Accepted: May 24, 2013
DOI 10.1002/ardp.201300128
Introduction
RGD mimetics [16, 17], and the neurokinin-2 receptor
ligand [18].
Various substituted 1,2,4-triazin-5-one derivatives have
great importance in the medicinal and agricultural
fields [1–5]. Recently, significant biological activities of
this class of compounds have been observed, in particular
of 4-amino-1,2,4-triazin-5(2H)-one derivatives working as
herbicidal [6, 7], antimicrobial [8–10], anti-HIV [11], and
anticancer agents [12].
The piperazinone ring has largely been used as a rigid
template in the construction of novel receptor ligands [13].
For example, these heterocyclic compounds are of great
biological interest since they represent the structural core of
several biologically active compounds, such as Leu-enkepha-
lin analogs [14], cholecystokinin receptor antagonists [15],
Coumarin (2H-chromen-2-one) and its derivatives are widely
distributed in nature and have been reported to exhibit
diverse pharmacological properties such as anticancer [19],
anti-coagulant, estrogenic, dermal, photosensitizing, anti-
microbial, vasodilatory, molluscicidal, antihelminthic, seda-
tive, hypnotic, analgesic [20, 21], hypothermic [20, 21], and
free radical-scavenging activities, especially of the superoxide
anions generated by activated neutrophils [22].
Furthermore, 1,4-benzoxazinone-based compounds have
a wide range of biological activities and pharmaceutical
actions, e.g., as inhibitors of bacterial histidine protein
kinase [23, 24], in treating heart diseases [25], and as inhibitors
of nitric oxide synthase (NOS), thus representing potential
drugs for the treatment of neurodegenerative, inflammatory,
autoimmune, and cardiovascular disorders [26, 27].
On the other hand, 3-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxo-
propanenitrile (1) is a very handy and cheap cyanoacetylation
reagent, which was first synthesized and introduced in the
late 1950s by Ried and Schleimer [28]. It was successfully
Correspondence: Dr. Moustafa A. Gouda, Faculty of Science, Depart-
ment of Chemistry, Mansoura University, El-Gomhoria Street, Mansoura
35516, Egypt.
E-mail: dr_mostafa_chem@yahoo.com
Fax: þ20 50 2246781
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2
M. A. Gouda
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–9
applied for the synthesis of various N-alkyl and N-aryl
cyanoacetamides [29, 30]. Recently, Gorobets et al. [31]
prepared a series of cyanoacetamides via refluxing of 1 with
the appropriate amine in toluene. We recently reported the syn-
thesis of 2-(2-cyanoacetylamino)-4,5,6,7-tetrahydrobenzo[b]-
thiophene-3-carboxylic acid ethyl ester from compound 1 [32].
Herein, compound 1 was used as a key intermediate for the
synthesis of coumarin, oxazine, pyrazolotriazinone, and
pyrazinopyrimidine derivatives in high yield and purity, in
order to investigate their antioxidant activities.
pyridine-3-diazonium chloride 3 was coupled with ethyl
cyanoacetate to give the corresponding hydrazono derivative
11. Compound 11 gave the corresponding aminopyrazolo-
triazine derivative 12 upon refluxing in acetic acid and did
not afford the pyrazolotriazinone 9 (Scheme 2).
Furthermore, Metwally et al. [33] reported that coupling of
the diazonium salt of 4-((3-amino-5-methyl-1H-pyrazol-4-yl)-
diazenyl)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (2) with
ethyl cyanoacetate gave the corresponding hydrazono
derivative 13. Compound 13 gave the corresponding amino-
prazolotriazine derivative 14 upon refluxing in acetic acid
and did not afford the pyrazolotriazinone 8 (Scheme 3).
The coupling of 1 with the corresponding aryl diazonium
salts 15a–c afforded the hydrazine derivatives 16a–c. Attempts
to prepare 4-oxo-3,4-dihydrocinnoline-3-carbonitrile deriva-
tives 17a–c (bioisosters of 8–10) via cyclization of 16a–c
were unsuccessful (Scheme 4).
Results and discussion
Chemistry
Compound 1 was used to synthesize the pyrazolotriazone
derivatives 8–10 via coupling with the corresponding 3-
pyrazole diazonium chlorides 2–4 [33–35], followed by
heating the formed hydrazones 5–7 in acetic acid (Scheme 1).
An analogous reaction has been reported by El-Deen
et al. [36], where as the 4,6-dimethyl-1H-pyrazolo[3,4-b]-
The reactivity of 1 towards 2-hydroxy carboxaldehydes was
studied in order to prepare the corresponding coumarin
derivatives in a one-pot reaction with high yields. Thus,
CH3
H3C
CH3
N
H3C
H3C
Ph
N
N2+Cl-
NH
N
CH3
N
N
N
N
N
H3C
N
Ph
N
N
O
O
CN
CN
N
N
CH3
N
N
H3C
2
AcOH
O
N
Ph
N
HN
O
N
N
N
EtOH/ AcONa
H3C
H
reflux, 12 h
O
NH
°
stirrig at 0-5 C, 2 h
N
8
H3C
yield 73%
5
yield 75%
CH3
H3C
N2+Cl-
CH3
N
N
N
CH3
N
CH3
O
N
N
NH
CN
CN
N
H3C
N
3
AcOH
CH3
N
HN
N
N
H3C
O
EtOH/ AcONa
°
N
reflux, 12 h
CN
H3C
N
H
NH
stirrig at 0-5 C, 2 h
O
N
9
H3C
1
yield 70%
6
yield 63%
CH3
N
N2+Cl-
NH
H3C
N
N
N
O
HN
NH
CN
N
CN
N
N
O
N
H
N
H
4
AcOH
EtOH/ AcONa
N
H
N
reflux, 12 h
°
stirrig at 0-5 C, 2 h
N
H
10
yield 80%
7
yield 63%
Scheme 1. Synthesis of the pyrazolotriazone derivatives 8–10.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 1–9
Bioactive Pyrazolotriazines and Pyrazinopyrimidines
3
NC
NC
COOEt
COOEt
CH3
N
CH3
N
N
CH3
N
N
N
N
N
HN
COOEt
NH2
AcOH
N
N
N
N
N
N
H
H3C
H3C
OEt
H3C
reflux, 7 h
H
12
11
yield 78%
yield 89%
EtOH/CH3COONa
O
CN
°
stirrig at 0-5 C, 2 h
3
Scheme 2. Synthesis of the aminopyrazolo-
triazine derivative 12.
OEt
O
OEt
N
H3C
H3C
H3C
N
O
CN
CH3
CN
CH3
N
CH3
N
N
N
Ph N
N
Ph N
AcOH
N
Ph N
HN
COOEt
N
N
N
N
N
N
N
O
reflux, 7 h
O
NH
O
NH
NH2
N
H
N
H3C
N
H3C
H3C
yield 67%
14
13
yield 65%
OEt
EtOH/CH3COONa
O
CN
°
stirrig at 0-5 C, 2 h
2
Scheme 3. Synthesis of the aminopyrazolotriazine derivative 14.
CH3
N2+Cl-
N
N
CH3
N
H3C
CN
AcOH
N
O
R
15
N
N
NH
N
H3C
CN EtOH / AcONa
R
CN
reflux, 12 h
O
O
°
stirrig at 0-5 C, 2 h
1
17
R
16
_
15 17; a, R = OCH3; b, R = CH3; c, R = Cl
16a, yield 85%; 16b, yield 73%; yield 73%
16c,
Scheme 4. Synthesis of the hydrazone deriv-
atives 16a–c.
treatment of 1 with salicylaldehyde (18), 2-hydroxy-1-naphth-
aldehyde (19) or 7-hydroxy-5-methoxy-2-methyl-4-oxo-4H-
chromene-6-carboxaldehyde (20) afforded the corresponding
coumarin derivatives 21–23 (Scheme 5). Yamashita et al. [37]
prepared the coumarin derivative 21 in a multistep synthesis
as outlined in Scheme 6 via Knoevenagel reaction of
salicyladehyde with malononitrile using isopropyl alcohol
as solvent in Ti(O-i-Pr)4, followed by aqueous hydrolysis for
the formed iminocoumarin 25.
Furthermore, the synthesis of compound 27, a bioisoster of
22, was also investigated. Condensation of 1 with 1-nitro-
sonaphthalen-2-ol 26 in ethanol catalyzed by piperidine was
carried out to obtain 3-oxo-3H-naphtho[2,1-b][1,4]oxazine-2-
carbonitrile 27. Moreover, the synthesis of compound 29, a
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M. A. Gouda
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–9
OH
OH
O
N
O
O
CHO
O
NC
NC
O
NC
18
O
N
EtOH/ piperidine
reflux, 1 h
26
EtOH/ piperidine
reflux, 12 h
21
yield 82%
OH
CH3
N
CHO
27
yield 62%
CH3
N
O
N
H3C
CN
19
O
N
O
O
N
H3C
CN
EtOH/ piperidine
reflux, 2 h
1
NH
O
O
H2N
N
H
S
NC
O
N
1
22
NH
28
OH
yield 83%
DMF / TEA
reflux, 12 h
OHC
N
H
N
H
S
O
NC
29
O
H3CO
O
yield 53%
O
CH3
20
H3CO
O
O
EtOH/ piperidine
reflux, 1.5 h
Scheme 7. Synthesis of naphtho[2,1-b][1,4]oxazine 27 and the
pyridazinopyrimidine derivative 29.
CH3
23
yield 68%
stable free radical (X•) will be determined by the following
reaction:
Scheme 5. Synthesis of the coumarin derivatives 21–23.
XH þ Y• ! X• þ YH
bioisoster of 17, was also investigated. Thus, compound 1
reacted with 6-amino-5-nitroso-2-thioxo-2,3-dihydropyrimi-
din-4(1H)-one (28) in dimethylformamide (DMF) catalyzed by
triethylamine in order to obtain the corresponding pyrida-
zinopyrimidine derivative 29 (Scheme 7). The formation of 29
can be explained according to the postulated mechanism
shown in Fig. 1.
where XH is the tested compound, Y• is the radical released
from ABTS, and YH is scavenging the free radical X•.
The rate and/or the extent of the process is measured in
terms of the decrease in X• concentration, which is related to
the ability of the added compounds to trap free radicals. The
decrease in color intensity of the free radical solution due to
scavenging of the free radical by the antioxidant material
is measured calorimetrically at a specific wavelength (e.g.,
734 nm). The assay employs the radical cation derived from
2,20-azino-bis(3-ethyl benzthiazoline-6-sulfonic acid; ABTS) as a
stable free radical to assess the antioxidant potential of the
investigated compounds.
Biological evaluation
ABTS antioxidant assay
The newly synthesized compounds were tested for their
antioxidant activity using the ABTS method as reported by
Lissi et al. [38]. The antioxidant activity assay employed herein
is one of several assays that depend on measuring the
consumption of stable free radicals; that is, it evaluates the
free radical-scavenging activity of the investigated compo-
nent. The methodology assumes that the consumption of the
Some of the compounds displayed antioxidant activity
compared with ascorbic acid, as shown in Table 1. Com-
pounds 7, 10, and 16a displayed high antioxidant potency,
while compounds 16b, 21, and 27 showed moderate
CN
NH
O
OH
NC
NC
CN
O
O
CHO
aq. HCl
24
0.1 equiv.
Ti(O-i-Pr)4
18
21
25
i-PrOH
°
25 C, 8 h
yield 70%
Scheme 6. Multistep synthesis of the coumarin
derivative 21.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 1–9
Bioactive Pyrazolotriazines and Pyrazinopyrimidines
5
O
N
O
NH
CH3
N
O
N
O
NC
O
_
H2N
N
H
S
H2O
NH
28
29
N
H3C
CN
N
H
N
H
S
DMF / TEA
O
1
Figure 1. The postulated mechanism of for-
mation of compound 29.
antioxidant activity; the remaining compounds showed
weak antioxidant activity. Compound 16a exhibited the
highest antioxidant activity comparable to that of ascorbic
acid.
antioxidants. The antitumor antibiotic bleomycin binds iron
ions and DNA. The bleomycin–iron complex degrades DNA
which, upon heating with thiobarbituric acid (TBA), yields a
pink chromogen. Upon the addition of suitable reducing
agents, antioxidants compete with the DNA and diminish
chromogen formation [39]. The protective activity against
DNA damage induced by the bleomycin–iron complex was
examined in order to show the action of the more potent
compounds 7, 10, 16a, and 21. The results in Table 2 show that
compound 16a exhibits high protection against DNA damage
induced by the bleomycin–iron complex, thus diminishing
chromogen formation between the damaged DNA and TBA
molecules.
Bleomycin-dependent DNA damage assay
Bleomycins are a family of glycopeptide antibiotics that were
used routinely as antitumor agents. The bleomycin assay
was adopted for assessing the pro-oxidant effects of food
Table 1. ABTS antioxidant activity assay of the new compounds.
Compound no.
Absorbance of
% Inhibition
samples (l)
Structure–activity relationships (SAR) of the tested
compounds for antioxidant activity
Control of ABTSa)
Ascorbic acid
5
6
7
8
9
10
16a
16b
16c
21
22
23
27
29
0.510
0.042
0.439
0.466
0.049
0.465
0.483
0.072
0.043
0.211
0.405
0.168
0.488
0.434
0214
0%
91.76%
13.92%
8.62%
90.39%
8.82%
5.29%
85.88%
91.95%
58.62%
20.58%
67.05
Comparing the results obtained for the antioxidant properties
of the compounds reported in this study with their structures,
the following SAR are postulated (Fig. 2): (i) It seems that
the hydrazone and pyrazole moieties within 5–7 play an
important role in the antioxidant activity, as their elimina-
tion through cyclization of the hydrazone derivatives 5–7 into
the triazine derivatives 8–10 leads to a decrease in activity.
(ii) Compounds 7 and 10 are more potent than compounds 5,
6, 8, and 9, which may be due to the presence of the indole
moiety. (iii) The antioxidant activities of compounds 16a–c
follow the order 16a > 16b > 16c, which may be explained by
the electron-donating character of the substituents in para-
position to the hydrazone moiety in the following order:
OCH3 > –CH3 > –Cl. (iv) Compound 21 exhibited better
antioxidant activity than compounds 22 and 23, which
may be explained by the extra ring/substituent in 22 and 23
decreasing the activity. (v) Compound 27 has higher
antioxidant activity than compound 22, which may be
attributed to the replacement of the naphthoxazine by the
benzocoumarin ring system.
4.31
14.90
52.74
20.01
0.362
a)
ABTS: The method used for antioxidant activity
ð%Þ Inhibition ¼ ½ðAcontrol ꢀ AtestÞ=Acontrolꢁ ꢂ 100.
Table 2. Bleomycin-dependent DNA damage of the investigated
compounds.
Compound no.
Absorbance of samples
Ascorbic acid
7
10
16a
21
0.087
0.099
0.110
0.085
0.091
Conclusion
The objective of the present study was to synthesize and evaluate
the antioxidant activities of some novel coumarin, oxazine,
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M. A. Gouda
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–9
CH3
H3C
N
N
N
N
CN
O
HN
NH
N
CN
O
Absence of pyrazole ring system
N
N
H
and hydrazone moiety
decrease the activity
N
H
N
10
N
H
7
CH3
N
N
O
H3C
O
O
CN
NC
NC
O
O
N
NH
N
Replacement of carbon atom
by nitrogen atom enhances
the antioxidant activity
R
_
16a c
27
22
Presence of pyrazole ring system
and p-methoxy-phenyl hydrazone enhance
the activity of 1
b > 1
s
6a
1
6
a > 1
6
6
c, due to the electronic effect
Figure 2. Structure–activity relationships of
the more potent antioxidant compounds.
pyrazolotriazinone, and pyrazinopyrimidine derivatives, with
the hope of discovering new structures serving as antioxidant
agents. The data clearly showed that compounds 7, 10, 16a, and
21 displayed promising in vitro antioxidant activities, by using
the ABTS method. Also, compound 16a exhibited high
protection against the DNA damage induced by the bleomycin–
iron complex, comparable to ascorbic acid.
General procedure for the synthesis of 3-(3,5-dimethyl-1H-
pyrazol-1-yl)-2-arylhydrazono)-3-oxopropanenitrile
derivatives 5–7
A well-stirred solution of 4-((5-amino-3-methyl-1H-pyrazol-4-yl)
diazenyl)-2,3-dimethyl-1-phenyl-1,2-dihydropyrazol-5-one (1.60 g,
5 mmol), 3-amino-4,6-dimethyl-2H-pyrazolo[3,4-b]pyridine (0.81 g,
5 mmol) or 5-amino-3-(1H-indol-3-yl)-1H-pyrazole (0.99 g, 5 mmol)
in a mixture of acetic acid (10 mL) and concentrated HCl
(3 mL) was cooled in an ice bath, and then a solution of
sodium nitrite (0.4 g, 5.8 mmol in 5 mL H2O) was added
dropwise under stirring. The above cooled diazonium solution
was added slowly to a well-stirred solution of 1 (0.82 g, 5 mmol)
in ethanol (25 mL) containing sodium acetate (2.4 g, 30 mmol).
Experimental
Chemistry
Instruments and methods
The reaction mixture was stirred for
a further 2 h. The
All melting points are given in degree Celsius (uncorrected) and
were determined on a Gallenkamp electric melting point
apparatus. Thin-layer chromatography (TLC) was carried out on
silica gel 60 F254 precoated aluminum sheets. The IR spectra
were recorded (KBr) on a Mattson 5000 FTIR spectrophotometer
(l, cmꢀ1) at the Microanalytical Unit, Faculty of Science,
Mansoura University. The 1H NMR spectra were carried out on
a Varian spectrophotometer at 300 MHz using tetramethyl silane
(TMS) as internal reference and DMSO-d6 and chloroform as
solvents; they were recorded at the Microanalytical Center, Cairo
University. The mass spectra (EI) were recorded on a Varian Star
3400 Cx ion trap GC/MS Shimadzu GCMS-QP 5050 A EI (70 eV) at
the Regional Center for Mycology and Biotechnology, Al-Azhar
University. Elemental analyses (C, H, and N) were carried out at
the Microanalytical Center, Cairo University, Giza, Egypt. The
results of the elemental analysis were found to agree favorably
with the calculated values. Biological activity determinations
were carried out at the Pharmacognosy Department, Faculty of
Pharmacy, Mansoura University, Mansoura, Egypt.
crude product was filtered-off, dried well, and crystallized from
a mixture of EtOH/DMF to give compounds 5, 6, and 7,
respectively.
3-(3,5-Dimethyl-1H-pyrazol-1-yl)-3-oxo-2-[2-{4-((1,5-
dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-
diazenyl)-5-methyl-1H-pyrazol-3-yl}hydrazono]-
propanenitrile (5)
Yield (1.82 g, 75%); orange powder; m.p. 212°C; IR (KBr):
n/cm ¼ 3442 (OH), 2242 (CN), 1743, 1643 (2CO); 1H NMR
(300 MHz, DMSO-d6) d (ppm): 2.58 (s, 3H, CH3, antipyrine), 2.66
(s, 3H, CH3 pyrazole), 2.93 (s, 3H, N–CH3 antipyrine), 3.31 (br,
6H, 2CH3), 3.94 (s, 1H, CH), 7.35–7.61 (m, 5H, Ar–H), 13.41 (s, 1H,
OH); MS (EI, 70 eV) m/z (%) ¼ 485 (3.3), 298 (3.6), 260 (4.0), 238
(5.7), 209 (4.2), 193 (6.2), 178 (8.9), 149 (7.3), 122 (33.2), 111 (24.2),
98 (17.1), 78 (25.5), 66 (46.7), 54 (100). Anal. calcd. for
C23H23N11O2 (485.2): C, 56.90; H, 4.77; N, 31.73%. Found: C,
56.88; H, 4.72; N, 31.68%.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 1–9
Bioactive Pyrazolotriazines and Pyrazinopyrimidines
7
3-(3,5-Dimethyl-1H-pyrazol-1-yl)-2-(2-(4,6-dimethyl-2H-
pyrazolo[3,4-b]pyridin-3-yl)hydrazono)-3-
oxopropanenitrile (6)
Yield (1.05 g, 63%); yellow powder; m.p. >320°C; IR (KBr): n/
4,6-Dihydro-7-(1H-indol-3-yl)-4-oxopyrazolo[5,1-c][1,2,4]-
triazine-3-carbonitrile (10)
Yield (1.10 g, 80%); pale green powder; m.p. 240°C; IR (KBr): n/
cm ¼ 3245 (br, 2NH), 2241 (CN), 1640 (CO); 1H NMR (300 MHz,
DMSO-d6) d (ppm): 4.48 (s, 1H, CH), 7.09–8.35 (m, 5H, Ar–H), 12.26
(s, 1H, NH), 14.62 (s, 1H, NH); MS (EI, 70 eV) m/z (%) ¼ 278 (2.0), 231
(0.2), 184 (21.0), 144 (100), 116 (40.7), 88 (31.9), 62 (26.0), 50 (16.8).
Anal. calcd. for C14H8N6O (276.08): C, 60.87; H, 2.92; N, 30.42%.
Found: C, 60.84; H, 2.88; N, 30.40%.
–
cm ¼ 3310, 3172 (2NH), 2221 (CN), 1683 (CO), 1633 (C N), 1529
–
þ
–
–
(N N); MS (EI, 70 eV) m/z (%) ¼ 335 (M ꢀH, 0.2), 240 (26.1), 212
(16.0), 160 (21.2), 147 (21.3), 131 (36.0), 118 (21.0), 104 (19.6), 86
(71.7), 77 (22.0), 67 (14.0), 56 (100). Anal. calcd. for C16H16N8O
(336.14): C, 57.13; H, 4.79; N, 33.31%. Found: C, 57.10; H, 4.75; N,
33.25%.
General procedure for the synthesis of 3-(3,5-dimethyl-1H-
pyrazol-1-yl)-2-arylhydrazono)-3-oxopropanenitrile
derivatives 16a–c
2-(2-(3-(1H-Indol-3-yl)-1H-pyrazol-5-yl)hydrazono)-3-(3,5-
dimethyl-1H-pyrazol-1-yl)-3-oxopropanenitrile (7)
Yield (1.17 g, 63%); pale green powder; m.p. 232°C; IR (KBr):
A well-stirred solution of 4-anisidine (0.62 g, 5 mmol), p-
toluidine (0.54 g, 5 mmol) or 4-chloroaniline (0.64 g, 5 mmol)
in concentrated HCl (3 mL) was cooled in an ice bath, and
then a solution of sodium nitrite (0.4 g, 5.8 mmol in 5 mL H2O)
was added dropwise. The above cooled diazonium solution
was added slowly to a well-stirred solution of 1 (0.82 g,
5 mmol) in ethanol (25 mL) containing sodium acetate (2.4 g
30 mmol). The reaction mixture was stirred for a further 2 h.
The obtained crude product was filtered-off, dried well, and
crystallized from ethanol to give compounds 16a–c,
respectively.
–
n/cm ¼ 3244, 3192 (2NH), 2240 (CN), 1640 (CO), 1522 (N N);
–
1H NMR (300 MHz, DMSO-d6) d (ppm): 3.37 (br, 6H, 2CH3);
4.48 (s, 1H, CH); 7.22–8.37 (m, 5H, Ar–H, NH-indole), 12.21
(br, s, 1H, NH-pyrazole), 12.28 (br, s, 1H, NH-hydrazone);
MS (EI, 70 eV) m/z (%) ¼ 261 [MþꢀHþ(3,5-dimethyl-1H-pyrazole-
1-carbonyl), 0.2], 244 (0.3), 213 (2.4) 184, (10.6), 144 (100), 116
(32.9), 88 (42.5), 62 (31.3). Anal. calcd. for C19H16N8O (372.14):
C, 61.28; H, 4.33; N, 30.09%. Found: C, 57.10; H, 4.75;
N, 33.25%.
General procedure for the synthesis of pyrazolotriazines 8,
9, and 10
2-(2-(4-Methoxyphenyl)hydrazono)-3-(3,5-dimethyl-1H-
A suspension of arylazo derivatives 5 (2.42 g, 5 mmol), 6 (1.68 g,
5 mmol), and 7 (1.86 g, 5 mmol) in acetic acid (10 mL) was
refluxed for 12 h. The reaction mixture was poured into ice-cold
water and then neutralized with sodium bicarbonate. The
formed precipitate was filtered-off, dried, and crystallized from
a mixture of DMF/ethanol to give compounds 8, 9, and 10,
respectively.
pyrazol-1-yl)-3-oxopropanenitrile (16a)
Yield (1.26 g, 85%); orange powder; m.p. 142°C; IR (KBr):
–
n/cm ¼ 3160 (NH), 2224 (CN), 1684 (CO), 1610 (C N), 1512
–
1
–
(N N); H NMR (300 MHz, DMSO-d ) d (ppm): 3.35 (br, 6H, 2CH ),
–
6
3
3.80 (s, 3H, OCH3), 6.95–7.48 (m, 5H, Ar–H, CHpyrazole), 12.19
(s, 1H, NH); MS (EI, 70 eV) m/z (%) ¼ 298 (Mþþ1, 0.7), 251 (5.5), 233
(28.4), 178 (34.3), 152 (12.4), 136 (26.0), 121 (84.9), 106 (29.4),
91 (28.4), 77 (50.7), 63 (65.7), 51 (100). Anal. calcd. for C15H15N5O2
(297.31): C, 60.60; H, 5.09; N, 23.56%. Found: C, 60.66; H, 5.05;
N, 23.60%.
8-((2,3-Dimethyl-5-oxo-1-phenyl-2,5-dihydro-1H-pyrazol-
4-yl)diazenyl)-7-methyl-4-oxo-4,6-dihydropyrazolo[5,1-c]-
[1,2,4]triazine-3-carbonitrile (8)
2-(2-(4-Tolyl)hydrazono)-3-(3,5-dimethyl-1H-pyrazol-1-yl)-
Yield (1.41 g, 73%); orange powder; m.p. 236°C; IR (KBr):
n/cm ¼ 3442 (br, NH), 2242 (CN), 1743, 1643 (2CO), 1488
3-oxopropanenitrile (16b)
(N N); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.57 (s, 3H, CH3-
–
–
Yield (1.17 g, 83%); red powder; m.p. 129°C; IR (KBr): n/cm ¼ 3167
(NH), 2222 (CN), 1683 (CO), 1593 (C C), 1534 (N N); 1H NMR
antipyrine), 2.67 (s, 3H, CH3-pyrazole), 3.13 (s, 3H, N–CH3), 7.40–
7.60 (m, 5H, Ar–H), 12.42 (s, 1H, NH); MS (EI, 70 eV) m/z (%) ¼ 389
(2.0), 343 (1.4), 280, (1.0), 252 (6.3), 233 (5.4), 208 (1.4), 178 (5.8), 149
(14.8), 122 (11.8), 111 (18.2), 95 (24.6), 76 (41.0), 70 (52.1), 56 (100).
Anal. calcd. for C11H8N6O (240.08): C, 55.52; H, 3.88; N, 32.38%.
Found: C, 55.48; H, 3.81; N, 32.40%.
–
–
–
–
(300 MHz, DMSO-d6) d (ppm): 2.28 (s, 3H, CH3), 3.32 (br, 6H, 2CH3),
7.20–7.42 (m, 5H, Ar–H, CHpyrazole), 12.19 (s, 1H, NH);
MS (EI, 70 eV) m/z (%) ¼ 281 (Mþ, 0.6), 251 (91.9), 217 (57.5),
208 (33.7), 179 (10.0), 157 (28.9), 125 (6.8), 105 (87.1), 91 (45.1),
76 (100), 65 (38.1), 50 (82.3). Anal. calcd. for C15H15N5O
(281.31): C, 64.04; H, 5.37; N, 24.90%. Found: C, 64.06; H, 5.35;
N, 24.85%.
3,4,8,9,9a-Pentaza-5,7-dimethyl-1-oxo-9,9a-dihydro-1H-
fluorene-2-carbonitrile or pyrido[2,3:3,4]-4-oxo-4,6-
2-(2-(4-Chlorophenyl)hydrazono)-3-(3,5-dimethyl-1H-
pyrazol-1-yl)-3-oxopropanenitrile (16c)
dihydropyrazolo[5,1-c][1,2,4]triazine-3-carbonitrile (9)
Yield (0.84 g, 70%); orange powder; m.p. >320°C; IR (KBr):
n/cm ¼ 3444 (NH), 2253 (CN), 1727 (CO), 1639 (C N); 1H NMR
–
Yield (1.13 g, 75%); reddish brown powder; m.p. 176°C;
IR (KBr): n/cm ¼ 3180 (NH), 2224 (CN), 1686 (CO), 1591
–
(300 MHz, DMSO-d6) d (ppm): 2.70 (s, 3H, CH3), 2.94 (s, 3H, CH3),
3.37 (s, 1H, CH), 7.17 (s, 1H, Ar–H), 12.50 (s, 1H, NH); MS (EI, 70 eV)
m/z (%) ¼ 240 (10.4), 212 (8.0), 172 (11.5), 146 (24.9), 131 (31.1), 119
(38.2), 104 (67.9), 85 (18.6), 76 (68.9), 65 (68.4), 56 (100). Anal. calcd.
for C11H8N6O (240.08): C, 55.00; H, 3.36; N, 34.98%. Found: C,
55.05; H, 3.31; N, 34.95%.
–
–
–
(C C), 1526 (N N), 780 (C–Cl; MS (EI, 70 eV) m/z (%) ¼ 301
–
(Mþ, 0.2), 237 (19.7), 177 (34.6), 125 (73.0), 111 (46.8), 99 (70.4),
89 (52.5), 74 (100), 62 (38.1), 50 (42.6). Anal. calcd. for
C
14H12ClN5O (301.73): C, 55.73; H, 4.01; N, 23.21%. Found: C,
55.66; H, 4.05; N, 23.15%.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
M. A. Gouda
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–9
General procedure for the synthesis of coumarin
derivatives 21–23 and oxazinone 27
refluxed for 12 h. The reaction mixture was poured into ice-
cold water; then, the formed precipitate was filtered-off, dried,
and crystallized from the DMF/EtOH solvent to give compound 29.
Yield (0.52 g, 53%); washing from hot DMF; brown powder
>315°C; IR (KBr): n/cm ¼ 3300–3190 (br, 3NH), 2193 (þCN), 1715,
A suspension of 1 (0.82 g, 5 mmol) and 2-hydroxybenzaldehyde
18 (0.61 g, 5 mmol), 2-hydroxy-1-naphthaldehyde 19 (0.86 g,
5 mmol), or 7-hydroxy-5-methoxy-2-methyl4-oxo-4H-chromene-6-
carbaldehyde 20 (1.17 g, 5 mmol) or 1-nitroso naphthalen-2-ol 26
(0.86 g, 5 mmol) in ethanol (25 mL) containing piperidine (0.42 g,
6 mmol) was refluxed for the appropriate time. The reaction
mixture was poured into ice-cold water; then, the formed
precipitate was filtered-off, dried, and recrystallized from the
proper solvent to give compounds 21–23 and 27, respectively.
–
1651 (2CO), 1610 (C N); MS (EI, 70 eV) m/z (%) ¼ 221 (M , 0.5), 216
–
(6.1), 178 (23.6), 151 (15.4), 136 (26.9), 119 (39.1), 105 (15.2), 90
(40.9), 77 (61.1), 63 (69.6), 54 (100). Anal. calcd. for C7H3N5O2S
(221.2): C, 38.01; H, 1.37; N, 31.66%. Found: C, 37.99; H, 1.32;
N, 31.61%.
Pharmacology
ABTS screening assay [38]
3-Oxo-2H-chromene-3-carbonitrile (21)
Antioxidant activities were evaluated from the bleaching of ABTS-
derived radical cations. The radical cations derived from ABTS
(2,20-azino-bis(3-ethyl benzthiazoline-6-sulfonic acid) were pre-
pared by reaction of ABTS (60 mL) with MnO2 (3 mL, 25 mg/mL) in
sodium phosphate buffer (5 mL, pH 7). After shaking the solution
for a few minutes, it was centrifuged and filtered. The absorbance
Acontrol of the resulting green-blue solution (ABTS radical solution)
was recorded at lmax ¼ 734 nm. The absorbance Atest was
measured upon the addition of 20 mL of a 1 mg/mL solution
of the tested sample in spectroscopic-grade MeOH/buffer (1:1 v/v)
to the ABTS solution. The inhibition ratio (%) was calculated using
the following formula:
Yield (0.70 g, 82%); reaction time 1 h, crystallization from
ethanol; yellow powder; m.p. 187°C [lit. 184°C]; IR (KBr):
n/cm ¼ 2207 (CN), 1740 (CO), 1610 (C C); 1H NMR (300 MHz,
–
–
DMSO-d6) d (ppm): 7.02–8.25 (m, 4H, Ar–H), 9.44 (s, 1H, C4-H); MS
(EI, 70 eV) m/z (%) ¼ 172 (Mþ, 0.2), 154 (4.1), 143 (0.9), 127 (1.6), 115
(9.4), 101 (8.4), 88 (100.0), 62 (35.6), 50 (37.3). Anal. calcd. for
C10H5NO2 (171.03): C, 70.18; H, 2.94; N, 8.18%. Found: C, 70.13;
H, 2.95; N, 8.13%.
3-Oxo-3H-benzo[f]-chromene-3-carbonitrile (22)
Yield (0.92 g, 83%); reaction time 2 h, crystallization from
ethanol/benzene; yellow powder; m.p. 303°C; IR (KBr):
ꢀ
ꢁ
n/cm ¼ 2226 (CN), 1731 (CO), 1601 (C C); 1H NMR (300 MHz,
–
–
Acontrol ꢀ Atest
ð%ÞInhibition ¼
ꢂ 100
DMSO-d6) d (ppm): 7.63–8.65 (m, 6H, Ar–H), 9.78 (s, 1H, C1-H); MS
(EI, 70 eV) m/z (%) ¼ 221 (Mþ, 100), 193 (66.8), 164 (58.1), 138
(28.9), 113 (9.7), 96 (7.9), 87 (147), 74 (9.9), 62 (17.2). Anal. calcd. for
Acontrol
Ascorbic acid (20 mL, 2 mM) solution was used as a standard
antioxidant (positive control). Blank samples were run using
solvent without ABTS (Table 1).
C14H7NO2 (221.21): C, 76.01; H, 3.19; N, 6.33%. Found: C, 76.05; H,
3.15; N, 6.30%.
Bleomycin-dependent DNA damage assay [40, 41]
To the reaction mixtures in a final volume of 1.0 mL, the following
reagents were added at the final concentrations stated: DNA
(0.2 mg/mL), bleomycin (0.05 mg/mL), FeCl3 (0.025 mM), magne-
sium chloride (5 mM), KH2PO4/KOH buffer pH 7.0 (30 mM), and
ascorbic acid (0.24 mM), and 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
incubation period, 0.1 mL 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 TBA and 1 mL 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.
5-Methoxy-8-methyl-2,6-dioxo-6,9-dihydro-2H-pyrano-
[2,3-g]chromene-3-carbonitrile (23)
Yield (0.96 g, 68%); reaction time 1.5 h, crystallization from ethanol/
benzene; pale yellow powder; m.p. 273°C; IR (KBr): n/cm ¼ 2219
1
–
(CN), 1776, 1664 (2CO), 1615 (C C); H NMR (300 MHz, DMSO-d )
–
6
d (ppm): 2.35 (s, 3H, CH3), 3.96 (s, 3H, OCH3), 6.19 (s, 1H, CH), 7.39
(s, 1H, CH), 8.67 (s, 1H, CH); MS (EI, 70 eV) m/z (%) ¼ 284 (Mþþ1,
25.9), 257 (31.4), 242 (14.0), 228 (100), 200 (49.4), 172 (41.5), 157
(21.7), 143 (29.0), 114 (30.7), 101 (38.4), 86 (38.4), 68 (1.6), 58 (5.5), 62
(17.2). Anal. calcd. for C15H9NO5 (283.05): C, 63.61; H, 3.20; N, 4.94%.
Found: C, 63.60; H, 3.15; N, 4.93%.
3-Oxo-3H-naphtho[2,1-b[1,4]]oxazine-3-carbonitrile (27)
Yield (0.69 g 62%); reaction time 12 h, crystallization from ethanol/
benzene; black powder; m.p. >315°C; IR (KBr): n/cm ¼ 2201 (CN),
1
–
1726 (CO), 1621 (C N); H NMR (300 MHz, DMSO-d ) d (ppm): 7.45–
–
6
This work was supported by Taibah University, Medina, KSA, project
No. 433/1921, year: 2012.
7.94 (m, 6H, Ar–H,); MS (EI, 70 eV) m/z (%) ¼ 222 (Mþ, 1.5), 208
(20.4), 164 (100), 151 (39.5), 143 (13.1), 129 (24.2), 115 (16.1), 97
(12.3), 77 (19.4), 69 (29.5), 55 (81.9). Anal. calcd. for C13H6N2O2
(222.04): C, 70.27; H, 2.72; N, 12.61%. Found: C, 70.23; H, 2.69;
N, 12.66%.
The author has declared no conflict of interest.
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www.archpharm.com