Synthesis and in-vitro antioxidant and antitumor evaluation of novel pyrazole-based heterocycles

Article abstract of DOI:10.1007/s13738-018-1566-x

Abstract: Hydrazide-hydrazone, namely, N′-((5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)-2-cyanoacetohydrazide 3, was utilized as a key starting material for the construction of a variety of novel heterocyclic moieties. The newly synthesized compounds were characterized by elemental analyses and spectral data (IR, ¹H-NMR, and mass spectra). Some of the newly synthesized compounds were screened for their antitumor and antioxidant activities. The results showed clearly that compounds 6, 11, 13, 22, and 29 displayed promising in-vitro antitumor activity against two cell lines (HepG2 and MCF-7). Among these compounds, compounds 13 and 29 exhibited the highest activity as antitumor agents. On the other hand, compounds 13, 22, and 29 exhibited the highest inhibitory antioxidant activity using ABTS method. While, in case of erythrocytes hemolysis, compounds 4, 13, and 29 proved to exhibit potent antioxidative activity as vitamin C. Meanwhile, compounds 6, 11, 16, 21, and 22 showed a remarkable promising activity. In light of the highest potency of our novel compounds in targeting both antitumor and antioxidant activities, compounds 13 and 29 warrant persistent preclinical development as antitumor and antioxidant agents.

Full text of DOI:10.1007/s13738-018-1566-x

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-018-1566-x
ORIGINAL PAPER
Synthesis and in-vitro antioxidant and antitumor evaluation of novel
pyrazole-based heterocycles
Mahmoud F. Ismail1 · Amira A. El‑sayed1
Received: 18 August 2018 / Accepted: 30 November 2018
©
Iranian Chemical Society 2018
Abstract
Hydrazide-hydrazone, namely, N′-((5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)-2-cyanoacetohydrazide 3, was
utilized as a key starting material for the construction of a variety of novel heterocyclic moieties. The newly synthesized
1
compounds were characterized by elemental analyses and spectral data (IR, H-NMR, and mass spectra). Some of the newly
synthesized compounds were screened for their antitumor and antioxidant activities. The results showed clearly that compounds
6
, 11, 13, 22, and 29 displayed promising in-vitro antitumor activity against two cell lines (HepG2 and MCF-7). Among these
compounds, compounds 13 and 29 exhibited the highest activity as antitumor agents. On the other hand, compounds 13, 22, and
2
9 exhibited the highest inhibitory antioxidant activity using ABTS method. While, in case of erythrocytes hemolysis, com-
pounds 4, 13, and 29 proved to exhibit potent antioxidative activity as vitamin C. Meanwhile, compounds 6, 11, 16, 21, and 22
showed a remarkable promising activity. In light of the highest potency of our novel compounds in targeting both antitumor and
antioxidant activities, compounds 13 and 29 warrant persistent preclinical development as antitumor and antioxidant agents.
Graphical abstract
Keywords Pyrazole · Diarylidene hydrazine · Iminocoumarin · 1,3-Thiazole · Antioxidant · Antitumor
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s13738-018-1566-x) contains
supplementary material, which is available to authorized users.
Extended author information available on the last page of the article
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1
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3

Journal of the Iranian Chemical Society
Introduction
on Shimadzu GC-MS QP1000EX apparatus in central
analytical lab, Ain Shams University. All reactions were
monitored by TLC (thin layer chromatography, Merck) and
spots were detected using a UV lamp (254 nm). Biologi-
cal activity was tested at the drugs department, Faculty of
pharmacy, Mansoura University, Egypt.
The pyrazole nucleus is a ubiquitous feature of a pharma-
cophoric scaffold, which represents a class of heterocyclic
compounds with a wide range of biological applications.
Many of them are widely used as potent analgesic, antiar-
rhythmic and anticonvulsant [1], antidiabetic [2], anticancer
[
3], anti-inflammatory [4, 5], anti-conceptive and antipy-
N′-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)-2-cyanoacetohydrazide 3
retic [6], muscle relaxing [7], antifungal [8, 9], antibacterial
activities [8], insecticidal [10], herbicidal [11], anti HIV,
antagonist of OPL1, BACE inhibitors for the treatment of
Alzheimer’s disease, hepatitis C virus, and hypoxia induc-
ible factor [12].
A mixture of 5-chloro-3-methyl-1-phenyl-1H-pyrazole-
4
-carbaldehyde 1 (2.2 g, 0.01 mol) and cyanoacetohy-
drazide 2 (0.99 g, 0.01 mol) in absolute ethanol and/or
dioxane (30 ml) was heated under reflux for 4 h. The pale
yellow solid which separated after slow evaporation was
collected by filtration and then recrystallized from ethanol
to give 3 as pale yellow crystals; m.p.: 190 °C, yield: 87%.
After profound investigation, various methods are avail-
able for the synthesis of the pyrazole moiety such as utility
of the catalytic performance of Fe O @HNTs-PEI [13], util-
3
4
ity of a cellulose-based nanobiocomposite decorated with
Fe O nanoparticles in water as a green solvent at ambient
3
4
IR (KBr): 3192, 3126 (NH), 2961, 2922, 2876 (CH , CH ),
temperature [14], and via multicomponent reactions (MCRs)
are powerful synthetic tools [15].
2
3
H
−
1. 1
2
263 (C≡N), 1681 (C=O), 1646, 1618 (C=N) cm
NMR (400 MHz, DMSO-d ) δ (ppm): 7.57–7.47 (m, 5H,
From synthetic aspect, since cyanoacetohydrazide deriva-
tives possess both electrophilic and nucleophilic properties
6
Ar–H), for anti-isomer [11.69 (s, 1H, NH, exchangeable
with D O), 7.96 (s, 1H, HC=N), 4.12 (s, 2H, CH CN),
[
16]. They have been used to design diverse heterocyclic
2
2
2
.41 (s, 3H, CH )], for syn-isomer [11.63 (s, 1H, NH,
moieties such as pyrazole, 1,3-dithiolane, thiadiazole,
3
exchangeable with D O), 8.13 (s, 1H, HC=N), 3.76 (s,
1
,3-thiazolidinone, iminocoumarin, and pyridazine deriva-
2
·
+
2
1
H, CH CN), 2.04 (s, 3H, CH )]. MS m/z (%): 301 (M ;
2 3
tives [17–19].
0.4), 265 (11.7), 141 (5.0), 128 (33.8), 118 (19.0), 77
Due to all of the aforementioned facts and in continu-
ation our previous work, N′-((5-chloro-3-methyl-1-phenyl-
(
100), 51 (24.2). Anal. Calcd. for C H ClN O (301.73):
14 12 5
C 55.73; H 4.01; Cl 11.75; N 23.21. Found: C 55.71; H
1
H-pyrazol-4-yl)methylene)-2-cyanoacetohydrazide 3 as a
3
.99; Cl 11.74; N 23.18.
readily available precursor for the synthesis of such new
heterocyclic systems which might show pharmacological
effects.
1
,2-Bis((5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)meth-
ylene)hydrazine 4
Experimental
Chemistry
Method A A mixture of 5-chloro-3-methyl-1-phenyl-
1
H-pyrazole-4-carbaldehyde 1 (2.2 g, 0.01 mol) and
cyanoacetohydrazide 2 (0.99 g, 0.01 mol) in ethanol (30 ml)
containing drops of piperidine was heated under reflux for
General information
2
h. The light brown solid which separated after cooling was
collected by filtration and then recrystallized from ethanol
Melting points are uncorrected and were determined
in open capillary tubes using an electric melting point
to give 4, yield: 79%.
−
1
apparatus (G-K). The IR spectra (400–4000 cm ) were
recorded on the Nicolet iS10 FT-IR spectrometer using
Method B Compound 3 (0.5 g, 1.7 mmol) was heated under
reflux in pyridine (10 ml) for 4 h. The light brown solid
which separated after cooling was collected by filtration and
then recrystallized from ethanol to give 4, yield: 44%.
1
KBr Wafer technique. H-NMR spectrum was recorded
with a Varian Gemini spectrometer at 400 MHz with TMS
as the internal standard and chemical shifts were reported
on a δ scale (ppm) using CDCl or DMSO-d as solvents,
3
6
Method C A mixture of compound 3 (0.5 g, 1.7 mmol) and
anhydrous potassium carbonate (1.38 g, 0.01 mol) in dry
acetone (30 ml) was heated under reflux in water bath for
while the coupling constants (J values) are given in Hz.
Elemental analyses (C, H, N, and S) were carried out at
the Microanalytical Data Centre at the Faculty of Science,
Cairo University, Egypt. Mass spectra were performed
2
4 h. After cooling, poured onto cooled water to afford light
1
3

Journal of the Iranian Chemical Society
brown solid that was recrystallized from ethanol to give 4,
to precipitate, the product obtained was filtered and then
recrystallized from benzene to give 6, yield 33%, while
the insoluble fractional in benzene was recrystallized
from ethanol to afford 5, yield: 46%.
yield: 41%.
Method D A mixture of 3 (1.0 g, 3.3 mmol) and hydrazine
monohydrate (0.16 ml 3.3 mmol) in dioxane (20 ml) was
heated under reflux for 3 h. After cooling, the solid depos-
ited was collected by filtration and recrystallized from etha-
nol to give 4, yield: 67%.
5: As brown crystals; m.p.: > 360 °C. IR (KBr): 3374,
3217 (NH), 2927, 2857 (CH ), 2213 (C≡N), 1672 (C=O)
3
−
1. 1
cm
H NMR (400 MHz, DMSO-d ) δ (ppm): 11.82 (br.s,
6
1H, NH, exchangeable with D O), 11.19 (br.s, 1H, NH,
2
exchangeable with D O), 7.81–7.24 (m, 5H, Ar–H), 2.20
2
+
Method E A mixture of 9 (1.0 g, 2.3 mmol) and hydrazine
monohydrate (0.12 ml, 2.3 mmol) in ethanol (20 ml) was
heated under reflux for 3 h. After slow evaporation, pour
onto acidified cold water, the solid deposited was collected
by filtration and recrystallized from ethanol to give 4, yield:
(s, 3H, CH ). MS m/z (%): 299 (M. ; 16.1), 217 (13.2),
3
188 (100), 182 (9.0), 174 (35.9), 105 (25.7), 91 (33.8), 77
(64.9). Anal. Calcd. for C H ClN O (299.72): C 56.10;
1
4
10
5
H 3.36; Cl 11.83; N 23.37. Found: C 56.08; H 3.34; Cl
11.86; N 23.39.
6
4%.
6 (As lactam–lactim tautomer): as orange crystals;
m.p.: 210–211 °C. IR (KBr): 3418, 3294, 3120 (NH ,
2
H
−
1. 1
Method F A mixture of 23 (1.0 g, 2.2 mmol) and hydrazine
monohydrate (0.11 ml, 2.2 mmol) in ethanol (20 ml) was
heated under reflux for 3 h. After slow evaporation, the solid
deposited was recrystallized from ethanol to give 4, yield:
NH), 2926, 2850 (CH, CH ), 1687, 1641 (C=O) cm
3
NMR (400 MHz, DMSO-d ) δ (ppm): 13.31 (s, 1H, OH,
6
exchangeable with D O for lactim tautomer), 12.20 (s,
2
2H, NH , exchangeable with D O for lactam tautomer),
2
2
6
1%.
10.65 (s, 1H, NH, exchangeable with D O for lactam tau-
2
4
: As buff crystals; m.p.: 250 °C. IR (KBr): 2998, 2932
tomer), 10.40 (s, 2H, NH , exchangeable with D O for
2
2
−
1. 1
(
CH ), 1632 (C=N) cm
H NMR (400 MHz, DMSO-d )
lactim tautomer), 7.69 (d, 2H, Ar–H, J = 8.8 Hz), 7.50 (t,
3
6
δ (ppm): 8.57 (s, 2H, HC=N), 7.62–7.51 (m, 10H, Ar–H),
2H, Ar–H, J = 7.6, J = 8.4 Hz), 7.34–7.28 (m, 1H, Ar–H),
·
+
2
1
.51 (s, 6H, 2CH ). MS m/z (%): 217 (M –C H ClN ;
3.46 (s, 1H, CHCO), 2.15, 2.05 (2 s, 3H, CH ). MS m/z
3
11
9
3
3
·
+
00), 182 (62.4), 156 (52.1), 141 (33.7), 105 (11.9) 77
(%): 317 (M ; 15.0), 316 (71.3), 299 (25.0), 281 (100),
274 (8.1), 265 (27.3), 77 (91.1), 51 (30.2). Anal. Calcd.
for C H ClN O (317.73): C 52.92; H 3.81; Cl 11.16; N
(
55.9), 55 (23.8). Anal. Calcd. for C H Cl N (437.33): C
2
2
18
2
6
6
0.42; H 4.15; Cl 16.21; N 19.22. Found: C 60.40; H 4.16;
1
4
12
5
2
Cl 16.23; N 19.19.
22.04. Found: C 52.92; H 3.83; Cl 11.13; N 22.01.
5
4
′-Chloro-3′-methyl-5-oxo-1′-phenyl-2,5-dihydro-1H,1′H-[3,
′-bipyrazole]-4-carbonitrile 5 and 5′-chloro-3′-methyl-5-o
N′-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)-2-cyano-3-(4-nitrophenyl)acrylohydrazide 9 (as
lactam-lacim tautomer)
xo-1′-phenyl-4,5-dihydro-1H,1′H-[3,4′-bipyrazole]-4-carbox
amide 6 (as lactam-lacim tautomer)
A mixture of 3 (1.0 g, 3.3 mmol), p-nitrobenzaldehyde
(0.50 g, 3.3 mmol) and sodium ethoxide (15 ml) in absolute
ethanol (20 ml) was stirred at ambient temperature for 4 h.
The resulting mixture was acidified with cold dilute acetic
acid. The precipitated solid was filtered off, washed several
times with cold water, and recrystallized from ethanol to
give 9 as yellow crystals; m.p.: 216–218 °C, yield: 71%.
Method A Compound 1 (0.5 g, 2.3 mmol) and cyanoace-
tohydrazide 2 (0.23 g, 2.3 mmol) are mixed together in
absolute ethanol (15 ml) containing few drops of glacial
acetic acid (0.5 ml). The reaction mixture was refluxed for
1
0 h and left to cool, the solid formed was filtered and then
recrystallized from ethanol to give 5, yield: 73%.
IR (KBr): 3273, 3194 (NH), 2962, 2923, 2854 (CH ), 2210
3
Method B Compound 3 (0.5 g, 1.7 mmol) was heated
under reflux in pyridine (10 ml) for 4 h. The light brown
solid which separated after cooling was collected by filtra-
tion and then recrystallized from ethanol to give 4. After
acidification of the mother liquor, the solid deposited was
filtrate and then recrystallized from ethanol to give 5,
yield: 31%.
(C≡N), 1680, 1668 (C=O), 1626 (C=N), 1604 (C=C), 1529,
−
1. 1
1346 (NO ) cm
HNMR (400 MHz, DMSO-d6) δ (ppm):
2
11.60 (s, 1H, OH, exchangeable with D O for lactim tau-
2
tomer), 8.57 (s, 1H, NH, exchangeable with D O for lactam
2
tautomer), 8.47 (s, 1H, HC=N), 8.24–8.22 (d, 2H, Ar–H,
J=8.4 Hz), 8.01 (s, 1H, HC
), 7.73–7.71 (d, 2H, Ar–H,
(olefinic)
J = 8.4 Hz), 7.59–7.49 (m, 5H, Ar–H), 2.51 (s, 3H, CH ).
3
+
MS m/z (%): 434 (M. ; 4.4), 342 (100), 324 (16.0), 316
Method C Compound 3 (0.5 g, 1.7 mmol) was heated
under reflux in glacial acetic acid (10 ml) for 10 h. The
reaction mixture was then partially evaporated and left
(27.5), 299 (7.7), 281 (5.4), 265 (10.5), 77 (57.9). Anal.
Calcd. for C H ClN O (434.84): C 58.01; H 3.48; Cl 8.15;
21
15
6
3
N 19.33. Found: C 57.97; H 3.46; Cl 8.13; N 19.36.
1
3

Journal of the Iranian Chemical Society
N′-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)-2-cyano-2-(2-oxoindolin-3-ylidene)acetohy-
drazide 11
m.p.: 192–194 °C, yield: 61%. IR (KBr): 3341, 3216 (NH ),
2
2935, 2837 (CH ), 2212 (C≡N), 1676 (C=O), 1628 (C=N),
3
−
1. 1
1606 (C=C) cm H NMR (400 MHz, DMSO-d ) δ (ppm):
6
8
.25 (br.s, 2H, NH , exchangeable with D O), 7.79 (s, 1H,
2 2
A mixture of 3 (1.0 g, 3.3 mmol), isatin (0.49 g, 3.3 mmol),
and piperidine (0.5 ml) in absolute ethanol (20 ml) was
heated under reflux for 4 h. After cooling, the solid depos-
ited was collected by filtration, and recrystallized from etha-
nol to give 11 as dark brown crystals; m.p.: 284–285 °C,
yield: 53%. IR (KBr): 3273, 3194 (NH), 2961, 2923, 2876
HC=N), 7.65–7.50 (m, 5H, Ar–H), 7.34 (s, 1H, Ar–H),
7.08–7.06 (d, 2H, Ar–H), 3.85 (s, 3H, OCH ), 3.82 (s, 3H,
3
OCH ), 3.79–3.77 (d, 1H, COCHCN, J=6.8 Hz), 3.69–3.67
3
(d, 1H, CHAr, J=8 Hz), 2.27 (s, 3H, CH ). MS m/z (%): 343
3
·
+
(M –[C H (OMe) +Cl]; 17.1), 341 (100), 327 (15.3), 301
6
3
2
(3.4), 77 (28.8). Anal. Calcd. for C H ClN O (515.96):
2
6
22
7
3
(
(
(
CH ), 2202 (C≡N), 1728, 1680 (C=O), 1621 (C=N), 1604
C 60.53; H 4.30; Cl 6.87; N 19.00. Found: C 60.55; H 4.31;
3
−1. 1
C=C) cm H NMR (400 MHz, DMSO-d ) δ (ppm): 12.35
Cl 6.85; N 18.98.
6
br.s, 1H, NH, exchangeable with D O), 11.26 (br.s, 1H,
2
NH, exchangeable with D O), 8.55 (d, 1H, Ar–H
,
indolinone
N′-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)-2-imino-2H-chromene-3-carbohydrazide 15a
2
J=10 Hz), 8.52 (s, 1H, HC=N), 7.67–7.49 (m, 5H, Ar–H),
7
.43 (d,d, 1H, Ar–H , J = 8.4 Hz), 7.31 (t, 1H,
indolinone
Ar–H
, J=7.6, J=7.2 Hz), 6.88 (d, 1H, Ar–H
,
A mixture of 3 (1.0 g, 3.3 mmol) and salicylaldehyde 14a
(0.35 ml, 3.3 mmol) in absolute ethanol (20 ml) in the
presence of piperidine (0.5 ml) was heated under reflux
for 3 h. After cooling, the solid deposited was collected
by filtration and recrystallized from ethanol to give 15a
as light yellow crystals; m.p.: 210–211 °C, yield: 85%. IR
indolinone
indolinone
·
+
J = 8 Hz), 2.60 (s, 3H, CH ). MS m/z (%): 431 (M + 1;
3
1
3.6), 395 (25.6), 217 (100), 183 (19.7), 77 (57.9). Anal.
Calcd. for C H ClN O (430.85): C 61.33; H 3.51; Cl 8.23;
22
15
6
2
N 19.51. Found: C 61.35; H 3.49; Cl 8.21; N 19.53.
1-(((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)methyl-
(KBr): 3312 (NH), 2958, 2921, 2854 (CH ), 1682 (C=O),
3
−
1. 1
ene)amino)-4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-car-
bonitrile 12
1632 (C=N), 1617 (C=C) cm
H NMR (400 MHz,
DMSO-d ) δ (ppm): 13.46 (s, 1H, NH, exchangeable with
6
D O), 9.17 (s, 1H, NH, exchangeable with D O), 8.49
2
2
A mixture of 3 (0.3 g, 1 mmol) and acetylacetone (0.10 ml,
(s, 1H, HC=C), 8.29 (s, 1H, HC=N), 7.81(d, 1H, Ar–H,
1
mmol) in ethanol (15 ml) containing a few drops of piperi-
J = 7.6 Hz), 7.59–7.49 (m, 7H, Ar–H),7.29–7.23 (d,d, 1H,
dine (four drops) was refluxed for 6 h. After cooling, the
reaction mixture was acidified with cold dilute acetic acid.
The solid that separated out was filtered off, washed sev-
eral times with cold water and recrystallized from ethanol
to give 12 as buff crystals; m.p.: 243–245 °C, yield: 55%.
Ar–H, J = 7.6, J = 8.4 Hz), 2.65 (s, 3H, CH ). MS m/z
3
+
(%): 339 (M. –C H O; 23.6), 338 (100), 321 (32.6), 303
4
2
(44.9), 261 (2.8), 218 (14.7), 217 (22.0), 182 (15.0), 155
(9.6), 128 (14.9), 77 (46.9).Anal. Calcd. for C H ClN O
2
1
16
5
2
(405.84): C 62.15; H 3.97; Cl 8.73; N 17.26. Found: C
IR (KBr): 2999, 2928, 2849 (CH ), 2215 (C≡N), 1651
62.19; H 3.98; Cl 8.71; N 17.24.
3
−
1. 1
(
C=O), 1591 (C=N) cm
H NMR (400 MHz, DMSO-d )
6
δ (ppm): 8.76 (s, 1H, HC=N), 7.62–7.51 (m, 5H, Ar–H),
6
3
3
5
.39 (s, 1H, CH
H, CH
), 2.47 (s, 3H, CH
), 2.346 (s,
3pyrazole
N′-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)-2-imino-6,8-dinitro-2H-chromene-3-carbohy-
drazide 15b
pyridinone
), 2.343 (s, 3H, CH ). MS m/z (%):
3pyridinone
3
pyridinone
·
+
65 (M ; 5.8), 218 (51.2), 183 (27.3), 156 (16.8), 77 (100),
1 (28.6). Anal. Calcd. for C H ClN O (365.82): C 62.38;
1
9
16
5
H 4.41; Cl 9.69; N 19.14. Found: C 62.35; H 4.43; Cl 9.66;
A mixture of 3 (1.0 g, 3.3 mmol) and 3,5-dinitrosalicylal-
dehyde 14b (0.7 g, 3.3 mmol) in absolute ethanol (20 ml)
in the presence of piperidine (0.5 ml) was heated under
reflux for 3 h. After cooling, the solid deposited was col-
lected by filtration and recrystallized from dioxane to
give 15b as yellow crystals; m.p.: 248 °C, yield: 88%. IR
N 19.10.
6
-Amino-1-(((5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)amino)-4-(3,4-dimethoxyphenyl)-2-oxo-1,2,3,4
tetrahydropyridine-3,5-dicarbonitrile 13
-
(
KBr): 3206 (NH), 2962, 2926, 2863 (CH ), 1683 (C=O),
3
−
1. 1
A mixture of 3 (0.3 g, 1 mmol), 2-(3,4-dimethoxyben-
1619 (C=N), 1550, 1339 (NO ) cm
H NMR (400 MHz,
2
zylidene)malononitrile (0.21 g, 1 mmol) in absolute ethanol
DMSO-d ) δ (ppm): 11.81 (s, 1H, NH, exchangeable
6
(
15 ml) containing few drops of piperidine (four drops) was
with D O), 8.98 (s, 1H, NH, exchangeable with D O),
2
2
heated under reflux for 6 h. After evaporation of the solvent
in vacuo, the solid obtained was collected and recrystal-
lized from benzene to give 13 as reddish brown crystals;
8.50 (s, 1H, Ar–H), 8.30 (s, 1H, HC=C), 8.19 (s, 1H,
HC=N), 8.13(s, 1H, Ar–H), 7.59–7.47 (m, 5H, Ar–H),
+
2.45(s, 3H, CH ). MS m/z (%): 219 (M. –C H N O ;
3
10
4
4
6
1
3

Journal of the Iranian Chemical Society
1
00), 156 (13.3), 77 (25.6), 51 (12.6). Anal. Calcd. for
GC-MS: 2 peaks; 1st peak; percentage=16.88%; MS m/z
+
C H ClN O (495.84): C 50.87; H 2.85; Cl 7.15; N
(%): 219 (M. –C H N O ; 100), 217 (8.2), 182 (5.4), 155
2
1
14
7
6
11
5
4
7
1
9.77. Found: C 50.89; H 2.82; Cl 7.17; N 19.79.
(20.7), 143 (6.2), 77 (37.0), 51 (17.6).
2
nd peak; percentage = 83.12%; MS m/z (%): 219
+
(
M. –C H N O ; 92.8), 217 (100), 182 (100), 155 (70.5),
13 9 4 7
N-(3-(2-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)hydrazine-1-carbonyl)-2H-chromen-2-ylidene)
formamide 16
141 (100), 114 (29.5), 77 (100), 51 (68.3).
4-Amino-N′-((5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)-3-phenyl-2-thioxo-2,3-dihydrothiazole-5-car-
bohydrazide 18
A solution of compound 15a (0.25 g, 0.62 mmol) in a
mixture of triethylorthoformate (5 ml) and acetic anhy-
dride (5 ml) was heated under reflux for 3 h. After cool-
ing, the yellow precipitated was collected by filtration and
recrystallized from toluene to give 16 as yellow crystals;
m.p.: 241–243 °C, yield: 65%. IR (KBr): 3209 (NH), 2958,
To a solution of compound 3 (3.01 g, 0.01 mol) in ethanol
(30 ml) containing triethylamine (0.5 ml), elemental sul-
fur (0.32 g, 0.01 mol) and phenyl isothiocyanate (1.2 ml,
0.01 mol) were added. The reaction mixture was heated at
60 °C for 1 h with continuous stirring. The grey solid depos-
ited, while hot was collected by filtration, washed several
times with ethanol and recrystallized from toluene to give
18 as buff crystals; m.p.: 261–262 °C, yield: 80%. IR (KBr):
3382, 3247, 3178 (NH , NH), 2958, 2925, 2857 (CH ), 1632
2
1
923, 2854 (CH ), 1710 (C=O
), 1677 (C=O
),
amide
6
3
aldehyde
−
1. 1
616 (C=N) cm
H NMR (400 MHz, DMSO-d ) δ
(
(
ppm): 11.69 (s, 1H, NH, exchangeable with D O), 8.83
2
s, 1H, CHO), 8.43 (s, 1H, HC=C), 8.27 (s, 1H, HC=N),
7
.98(d, 1H, Ar–H, J = 7.6 Hz), 7.80–7.74 (d,d, 1H, Ar–H,
2
3
−1. 1
J = 8.4, J = 8.4 Hz) 7.60–7.50 (m, 5H, Ar–H), 7.47–7.45
(C=O), 1602 (C=N), 1242 (C=S) cm H NMR (400 MHz,
(
d, 1H, Ar–H, J = 7.6 Hz), 7.43–7.37 (d,d, 1H, Ar–H,
CDCl ) δ (ppm): 8.52 (br.s, 1H, NH, exchangeable with
3
J = 8.4, J = 7.6 Hz), 2.43 (s, 3H, CH ). MS m/z (%): 432
D O), 7.74 (s, 1H, HC=N), 7.66–7.35 (m, 10H, Ar–H),
3
2
+
(
M. -1; 11.7), 362 (27.1), 217 (100), 77 (54.3). Anal.
6.31 (br.s, 2H, NH , exchangeable with D O), 2.69 (s, 3H,
2
2
+
Calcd. for C H ClN O (433.85): C 60.91; H 3.72; Cl
CH ). MS m/z (%): 468 (M. ; 12.6), 433 (17.6), 392 (37.4),
2
2
16
5
3
3
8
.17; N 16.14. Found: C 60.93; H 3.69; Cl 8.14; N 16.17.
250 (100), 218 (46.7), 218 (47.4), 77 (33.7). Anal. Calcd.
for C H ClN OS (468.98): C 53.78; H 3.65; Cl 7.56; N
2
1
17
6
2
N-(3-(2-((5-Chloro-3-methyl-1-phenyl-1H-pyra-
zol-4-yl)methylene)hydrazine-1-carbonyl)-6,8-di-
nitro-2H-chromen-2-ylidene)formamide 17a
and 3-(((5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)amino)-2-ethoxy-7,9-dinitro-2,3-dihy-
dro-4H-chromeno[2,3-d]pyrimidin-4-one 17b
17.92; S 13.67. Found: C 53.80; H 3.66; Cl 7.53; N 17.91;
S 13.65.
6-(((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)methyl-
ene)amino)-3-phenyl-2-thioxo-2,3-dihydrothiazolo[4,5-d]
pyrimidin-7(6H)-one 19
A solution of compound 15b (0.25 g, 0.51 mmol) in a mix-
A solution of compound 16 (0.16 g, 0.34 mmol) in a mix-
ture of triethylorthoformate (3 ml) and acetic anhydride
(3 ml) was heated under reflux for 9 h. After cooling, the
solid deposited was collected by filtration and recrystal-
lized from toluene to give 19 as off white crystals; m.p.:
258–259 °C, yield: 83%. IR (KBr): 2959, 2922, 2853
ture of triethylorthoformate (5 ml) and acetic anhydride
(
5 ml) was heated under reflux for 3 h. After cooling, the
solid deposited was collected by filtration and recrystal-
lized from toluene to give 17a,b as yellow crystals; m.p.:
2
03–205 °C, yield: 67%.
IR (KBr): 3443 (NH), 2973, 2927, 2861 (C–H .), 1719
(CH ), 1666 (C=O
), 1592 (C=N), 1243 (C=S)
aliph
3
Pyrimidinone
−
1. 1
(
C=O
), 1678 (C=O
), 1631 (C=N), 1613 (C=C),
cm
H NMR (400 MHz, DMSO-d ) δ (ppm): 9.31 (s, 1H,
aldehyde
528, 1346 (NO ) cm
amide
6
−
1. 1
1
H NMR (400 MHz, DMSO-d ) δ
Ar–H
), 8.22 (s, 1H, HC=N), 7.65–7.35 (m, 10H,
2
6
Pyrimidinone
+
(
ppm):
Ar–H), 2.57 (s, 3H, CH ). MS m/z (%): 478 (M. ; 8.4),450
3
1
7a: 9.03 (s, 1H, CHO), 8.79 (s, 1H, Ar–H), 8.72 (s, 1H,
(12.2), 443 (23.9), 402 (19.8), 343 (35.4), 261 (24.1), 217
NH, exchangeable with D O), 8.59 (s, 1H, HC=C), 8.48 (s,
(66.3), 77 (100). Anal. Calcd. for C H ClN OS (478.97):
2
22 15
6
2
1
2
H, HC=N), 8.32 (s, 1H, Ar–H), 7.67–7.52 (m, 5H, Ar–H),
C 55.17; H 3.16; Cl 7.40; N 17.55; S 13.39. Found: C 55.19;
.51(s, 3H, CH ).
H 3.14; Cl 7.41; N 17.52; S 13.36.
3
1
7b: 8.69 (s, 1H, Ar–H), 8.58 (s, 1H, HC=C), 8.41 (s,
1
5
H, HC=N), 8.30 (s, 1H, Ar–H), 7.67–7.52 (m, 5H, Ar–H),
.83 (s,1H, CH ), 3.66–3.56 (q, 2H, CH CH
pyrimidinone
2
3,
J=7.2 Hz), 2.50 (s, 3H, CH ), 1.03–1.00 (t, 3H, CH CH ,
3
2
3
J=6.8 Hz).
1
3

Journal of the Iranian Chemical Society
N′-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)-2-cyano-3-mercapto-3-(phenylamino)acrylo-
hydrazide 21
N′-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)-2-cyano-3-(ethylthio)-3-(phenylamino)acrylo-
hydrazide 23
To a stirred solution of potassium hydroxide (0.56 g,
To a stirred solution of potassium hydroxide (0.56 g,
0.01 mol) in DMF (20 ml) compound 3 (3.01 g, 0.01 mol)
was added. After stirring for 30 min, phenyl isothiocyanate
(1.2 ml, 0.01 mol) was added to the resulting mixture. Stir-
ring was persistent at ambient temperature for 12 h, and
then, ethyl iodide (0.62 ml, 0.01 mol) was added and stirring
was persistent for additional 6 h. The reaction mixture was
poured onto ice cold water, and the resulting precipitate was
filtered off, washed several times with cold water and recrys-
tallized from benzene to give 23 as reddish brown crystals;
m.p.: 178–180 °C, yield: 71%. IR (KBr): 3275 (NH), 2963,
2925, 2858 (CH , CH ), 2193 (C≡N), 1637 (C=O), 1621
0
.01 mol) in DMF (20 ml) compound 3 (3.01 g, 0.01 mol)
was added. After stirring for 30 min, phenyl isothiocyanate
1.2 ml, 0.01 mol) was added to the resulting mixture. Stir-
(
ring was persistent at ambient temperature for 12 h. The
reaction mixture was acidified with cold dilute HCl. The
solid product that separated was filtered, washed with water,
and recrystallized from benzene to give 21 as buff crystals;
m.p.: 136–138 °C, yield: 72%. IR (KBr): 3273, 3194 (NH),
2
958, 2925, 2853 (CH ), 2225 (C≡N), 1676 (C=O), 1630
3
−1. 1
(
C=N) cm H NMR (400 MHz, DMSO-d ) δ (ppm): 12.70
6
(
s, 1H, NH, exchangeable with D O), 10.53 (br.s, 1H, NH,
2
2
3
−
1. 1
exchangeable with D O), 8.06 (s, 1H, HC=N), 7.59–7.34 (m,
(C=N), 1590 (C=C) cm
H NMR (400 MHz, CDCl ) δ
2
3
1
0H, Ar–H), 2.43 (s, 3H, CH ), 1.21 (s, 1H, SH, exchange-
(ppm): 12.54 (s, 1H, NH, exchangeable with D O), 9.04
3
2
+
able with D O). MS m/z (%): 436 (M. ; 0.9), 401 (1.1),
(s, 1H, NH, exchangeable with D O), 8.03 (s, 1H, HC=N),
2
2
3
59 (20.3), 357 (100), 343 (23.5), 316 (5.7), 281 (8.4), 128
7.54–7.28 (m, 10H, Ar–H), 2.65–2.60 (q, 2H, CH CH
2
3,
(
(
9.6), 77 (24.2), 51 (7.5). Anal. Calcd. for C H ClN OS
J = 7.2), 2.54 (s, 3H, CH ), 1.22–1.18 (t, 3H, CH CH ,
2
1
17
6
3 2 3
+
436.92): C 57.73; H 3.92; Cl 8.11; N 19.24; S 7.34. Found:
J=7.6). MS m/z (%): 404 (M. –C H S; 1.3), 389 (3.5), 369
2 4
C 57.72; H 3.89; Cl 8.09; N 19.27; S 7.35.
(100), 354 (12.6), 339 (10.3), 77 (9.0), 51 (2.4). Anal. Calcd.
for C H ClN OS (464.97): C 59.41; H 4.55; Cl 7.62; N
2
3
21
6
N′-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)-2-cyano-2-(5-oxo-3-phenylthiazoli-
din-2-ylidene)acetohydrazide 22
18.07; S 6.90. Found: C 59.39; H 4.53; Cl 7.64; N 18.11;
S 6.89.
N′-((5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)-2-cyano-2-(4-hydroxy-1,3-dithiol-2-ylidene)
acetohydrazide 29
To a stirred solution of potassium hydroxide (0.56 g,
0
.01 mol) in DMF (20 ml) compound 3 (3.01 g, 0.01 mol)
was added. After stirring for 30 min, phenyl isothiocyanate
1.2 ml, 0.01 mol) was added to the resulting mixture. Stir-
(
To a stirred solution of potassium hydroxide (0.56 g,
0.01 mol) in DMF (20 ml), compound 3 (3.01 g, 0.01 mol)
was added. After stirring for 30 min, the resulting mixture
ring was persistent at ambient temperature for 12 h, and
then, chloroacetyl chloride (0.8 ml, 0.01 mol) was added
and stirring was persistent for additional 6 h. The reac-
tion mixture was acidified with cold dilute acetic acid. The
separated solid was filtered off, washed several times with
cold water, and recrystallized from toluene to give 22 as
green crystals; m.p.: 140–142 °C, yield: 79%. IR (KBr):
was cooled at 10 °C in an ice bath, and then, CS (0.6 ml,
2
0.01 mol) was added slowly. Stirring was persistent for
12 h at ambient temperature, and then, chloroacetyl chlo-
ride (0.8 ml, 0.01 mol) and/or ethyl chloroacetate (1.07 ml,
0.01 mol) were added and stirring was persistent for addi-
tional 6 h. The reaction mixture was acidified with cold
dilute acetic acid. The separated solid was filtered off,
washed several times with cold water, and recrystallized
from toluene to give 29 as brown crystals; m.p.: 254 °C,
3
1
334 (NH), 2968, 2924, 2854 (CH , CH ), 2195 (C≡N),
2
3
745 (C=O
), 1664 (C=O
), 1618 (C=N), 1595
thiazolidinone
amide
−
1. 1
(
(
C=C) cm
H NMR (400 MHz, CDCl ) δ (ppm): 9.03
3
s, 1H, NH, exchangeable with D O), 7.99 (s, 1H, HC=N),
2
7
3
.66–7.30 (m, 10H, Ar–H), 3.89 (s, 2H, CH S), 2.58 (s,
yield: 58%. IR (KBr): 3424 (OH), 3254 (NH), 2923, 2851
2
+
−1.
H, CH ). MS m/z (%): 259 (M. –C H ClN ; 100), 244
(CH ), 2205 (C≡N), 1620 (br.s) (C=O), 1603 (C=N) cm
3
11
9
3
3
1
(
29.4), 218 (17.9), 190 (4.7), 77 (7.9), 51 (4.9). Anal. Calcd.
H NMR (400 MHz, DMSO-d ) δ (ppm): 11.83 (s, 1H, OH,
6
for C H ClN O S (476.94): C 57.92; H 3.59; Cl 7.43; N
exchangeable with D O), 9.89 (s, 1H, NH, exchangeable
2
3
17
6
2
2
1
7.62; S 6.72. Found: C 57.94; H 3.57; Cl 7.39; N 17.60;
with D O), 8.63 (s, 1H, HC=N), 7.64–7.46 (m, 5H, Ar–H),
2
S 6.74.
6.69 (s, 1H, HC=C), 2.43 (s, 3H, CH ). MS m/z (%): 416
3
+
(
M. -1; 5.3), 399 (10.2), 375 (15.4), 218 (30.1), 200 (100),
7
4
7 (29.5). Anal. Calcd. for C H ClN O S (417.89): C
17 12 5 2 2
8.86; H 2.89; Cl 8.48; N 16.76; S 15.34. Found: C 48.84;
H 2.90; Cl 8.45; N 16.77; S 15.36.
1
3

Journal of the Iranian Chemical Society
Cytotoxicity and antitumor evaluation
Materials and methods
ꢀ
− A(test)/A(control)
ꢁ × 100,
control)
%
Inhibition = A
(
where the standard antioxidant (positive control) was ascor-
bic acid solution (20 µl, 2 mM) and blank sample was car-
ried out by solvent without ABTS.
Cell lines Hepatocellular carcinoma (HepG-2) and mam-
mary gland (MCF-7) were obtained from ATCC via Hold-
ing company for biological products and vaccines (VAC-
SERA), Cairo, Egypt.
Antioxidant activity screening assay for erythrocyte
hemolysis Rats’ blood was obtained by cardiac puncture
then collected in heparinized tubes. Separation of eryth-
rocytes from plasma was occurred and the buffy coat was
washed three times with ten volumes of 0.15 M NaCl. The
erythrocytes were centrifuged in the last wash at 2500 rpm
for 10 min to obtain a constantly packed cell preparation.
In this assay [23], erythrocyte hemolysis was mediated
by peroxyl radicals. The erythrocytes were suspended
in phosphate buffered saline (PBS) pH 7.4. The suspen-
sion (10%) was added to the same volume of 200 mM
Chemical reagents The reagents RPMI-1640 medium,
MTT, and DMSO (sigma co., St. Louis, USA), Fetal Bovine
serum (GIBCO, UK).
Doxorubicin was used during comparison as a standard
anticancer drug.
MTT assay The different cell lines mentioned above were
used to determine the inhibitory effects of compounds on
cell growth using the MTT assay [20, 21]. This colorimetric
test is based on the change of the yellow tetrazolium bromide
2
,2′-azobis(2-amidinopropane)dihydrochloride (AAPH)
solution (in PBS) containing samples to be tested at vari-
ous concentrations. The reaction mixture was shaken gen-
tly while being incubated at 37 °C for 2 h. Then diluted
with eight volumes of PBS and centrifuged at 2500 rpm
for 10 min. The absorbance A of the supernatant was
recorded at 540 nm. Similarly, the reaction mixture was
treated with eight volumes of distilled water to achieve
complete hemolysis, and the absorbance B of the superna-
tant obtained after centrifugation was recorded at 540 nm:
(
MTT) to a purple formazan derivative by mitochondrial
succinate dehydrogenase in viable cells. The cells were cul-
tured in RPMI-1640 medium with 10% fetal bovine serum.
Antibiotics added were 100 units/ml penicillin and 100 µg/
ml streptomycin at 37 °C in a 5% CO incubator. The cells
2
4
were seeded in a 96-well plate at a density of (1.0×10 cells/
well) at 37 °C for 48 h under 5% CO . After incubation, the
2
cells were treated with various concentrations of compounds
and put in the incubator for 24 h; then, 20 µl of MTT solution
at 5 mg/ml was added and incubated for 4 h. DMSO (100 µl)
was added into each well to dissolve the purple formazan
formed. At 570 nm absorbance, the colorimetric assay was
measured and recorded using a plate reader (EXL 800, USA):
Calculation of the percentage hemolysis = (1 − A/B) × 100%,
where the data were indicated mean standard deviation and
the positive control was l-ascorbic acid.
Results and discussion
Calculation of the relative cell viability (%)
=
(A of treated samples/A of untreated sample) × 100.
Chemistry
Antioxidant assay
Herein, we report the study of the reactions of the requisite
5
-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde 1
Antioxidant activity screening assay; ABTS method Deter-
minations of antioxidant activities were estimated from
the bleaching of ABTS-derived radical cations. The radi-
cal cation was derived from ABTS [2,2′-azino-bis(3-ethyl
benzothiazoline-6-sulfonic acid)] was prepared by reaction
[
24] with cyanoacetohydrazide 2 in different conditions to
obtain variant products.
Condensation of pyrazole aldehyde derivative 1 with
cyanoacetohydrazide 2 in boiling absolute ethanol and/or
dioxane tolerated the hydrazide-hydrazone derivative 3. Dur-
ing refluxing compound 3 in absolute ethanol containing a
few drops of piperidine afforded the diarylidene hydrazine
derivative 4 [25] as light brown crystal. The same product
of ABTS (60 µl) with MnO (3 ml, 25 mg/ml) in phosphate
2
buffer solution (10 µM, pH 7, 5 ml). After the solution was
shaking for 3 min, centrifuged and filtered, the Absorbance
A
of the resulting ABTS radical solution (green–blue)
control)
(
4
was isolated and identified when the reaction executed in
was recorded at λ
734 nm. Upon the addition of (20 µl
max
refluxing compound 1 with cyanoacetohydrazide 2 in etha-
nol containing a few drops of piperidine directly (Scheme 1).
Heterointra cyclization of compound 3 via refluxing it
with glacial acetic acid for 10 h to give pyrazolinone deriv-
ative 5, beside that the amide derivative 6 was obtained.
of 1 mg/ml) solution of the tested sample in spectroscopic
grade MeOH/buffer (1:1 v/v) to the ABTS solution, the
absorbance A
was measured. The decrease in the absorb-
test)
(
ance is expressed as % inhibition which calculated from this
equation [22]:
1
3

Journal of the Iranian Chemical Society
Scheme 1 Formation and reactions of compound 3 under different conditions
The spectral and microanalytical data for compounds
–6 were fit with their chemical structures. The structure
of N′-((5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)-2-cyanoacetohydrazide 3 was elucidated by
elemental analysis and spectroscopic data. The IR spec-
trum showed the appearance of stretching absorption bands
3
−
1,
−1
for υ at 3192, 3126 cm υ ≡ at 2263 cm and υ
C=O
Fig. 1 Diasteriomeric isomers of compound 3
NH
C
N
−
1
1
at 1681 cm . Furthermore, H NMR spectrum of 3 is in
accordance with its proposed structure, as it showed signals
for NH, methyl, methylene, methine, and aromatic protons.
Compound 3 exists as a diasteriomeric mixture (syn/anti)
in the ratio 11.5:88.5. The higher ratio of the anti-isomer
is due to its higher stability, since it underwent less steric
hindrance because of both hetroaryl and cyanoacetamido
groups in the opposite sides. The down field value for the
signal of NH proton as well as the upfield value of the signal
of imino methine (CH=N) proton of the anti-isomer may
be attributed to extension of the conjugation as a result of
Thus, alternative synthetic route for pyrazolinone derivative
5
could be solely obtained in one step by the reaction of
compound 1 with cyanoacetohydrazide 2 in boiling absolute
ethanol containing a few drops of glacial acetic acid. On the
other hand, refluxing of compound 3 in dry pyridine afforded
4
and 5. Unfortunately, refluxing of compound 3 with anhy-
drous potassium carbonate in dry acetone, the diarylidene
hydrazine derivative 4, was obtained as the sole product
instead of the desired pyrazolo[3,4-c]pyrazole 7 (Scheme 1).
Noteworthy, compound 4 was obtained when we used
hard base such as piperidine and K CO and borderline base
coplanarity of hetroaryl group and –NH–CO–CH CN moi-
2
ety, as shown in Fig. 1.
2
3
as pyridine were classified according to HSAB theory (Pear-
son concept). Meanwhile, in the case of using hard acid as
acetic acid, the cyclized pyrazolinone derivatives 5 and 6
were obtained.
The mechanistic scenarios for the formation of com-
pounds 4–6 are illustrated, as shown in Scheme 2.
1
The H NMR spectrum of compound 6 in DMSO solu-
tion revealed the presence of four peaks exchangeable with
1
3

Journal of the Iranian Chemical Society
Scheme 2 Mechanistic path-
ways to compounds 4–6
due to intramolecular chelated H-bond in six-membered ring
Fig. 2).
(
Focusing on reactivity of β-ketonitriles towards some
nucleophilic and electrophilic agents led us to explore the
utilization of the hydrazide-hydrazone derivative 3 as a pre-
cursor to construct more heterocycles.
Therefore, compound 3 was allowed to react with hydra-
zine hydrate to constructing pyrazole derivative 8, as
described in the literature [26, 27]. However, its reaction
with hydrazine hydrate produced the diarylidene hydrazine
derivative 4 instead of 8 (Scheme 3).
Fig. 2 Diastereomeric mixture of compound 6
D O at δ=13.31, 12.20, 10.65, and 10.40 ppm correspond-
Plausible mechanistic pathways to demonstrate the forma-
tion of compound 4 is illustrated in Scheme 4. The pathway
b was extrapolated from the anterior mechanism.
2
1
ing to OH, NH , NH, and NH , respectively. H NMR of 6
2
2
revealed its existence as a diasteromeric mixture in the ratio
of 51:49. The down field shifts of OH and NH protons are
The proclivity of 3 towards some electrophilic reagents
such as aromatic aldehydes, heterocyclic ketone, aliphatic
2
1
3

Journal of the Iranian Chemical Society
Scheme 3 Hydrazinolysis of
compound 3
Scheme 4 Mechanistic illustra-
tion of 4 formation
ketone, arylidene malononitrile, and phenyl isothiocyanate
was also investigated as follow in Scheme 5.
study, the diarylidene hydrazine derivative 4 was obtained
instead. As an alternative way, when a harder base such
as sodium ethoxide used at room temperature instead of
piperidine, the reaction was smoothly achieved to afford
9 (Scheme 5). The IR spectrum of compound 9 exhibited
In our previous work [17, 18], when β-ketonitrile was
refluxed with p-nitrobenzaldehyde in boiling ethanol in
the presence of piperidine, the corresponding condensa-
tion product 9 was afforded (Scheme 5). Meanwhile, in this
−
1
stretching absorption bands at 2210 cm (C≡N) and 1680,
1
3

Journal of the Iranian Chemical Society
Scheme 5 Synthetic pathway to compounds 9–13
Fig. 3 Lactam–lactim tautomers
of compound 9
1
1
668 cm− (C=O). Moreover, IR spectrum indicated the
yield as dark brown crystals (Scheme 5). The structure 11
was substantiated from the microanalytical and spectro-
scopic data.
presence of the nitro group by displaying two bands due
to asymmetrical and symmetrical stretching bands at 1529
−
1
1
and 1346 cm . In DMSO solution, the H NMR spectrum
of compound 9 exhibited two singlet signals for NH and OH
protons properly represented to its existence as a lactam-
lactim dynamic equilibrium in the ratio of 39:61. The higher
proportion of the lactim tautomer is due to its more conju-
gated pattern, as shown in Fig. 3.
The synthesis of 4,6-dimethyl pyridinone derivative 12
was commenced from the reaction of cyanoacetamide 3
with acetylacetone in ethanolic solution containing drops
1
of piperidine. As a proof for the proposed structure of 12, H
NMR spectrum of 12 showed characteristics singlet peaks
for HC=N, CH
, CH
, and two CH
at
3pyridinone
pyridinone
3pyrazole
Unfortunately, hydrazinolysis of the condensation prod-
uct 9 in boiling ethanol yielded the diarylidene hydrazine
derivative 4 rather than the desired pyrazole derivative 10
δ = 8.76, 6.39, 2.47, 2.346, and 2.343 ppm, respectively,
besides multiplet peak for Ph at δ=7.62–7.51 ppm.
Heating a mixture of compound 3 with 2-(3,4-dimeth-
oxybenzylidene)malononitrile in absolute ethanol containing
a catalytic amount of piperidine afforded 6-amino-2-oxo-
1,2,3,4-tetrahydropyridine derivative 13 (Scheme 5). The
[
28] (Scheme 5).
Refluxing of an ethanolic solution of isatin with the
active methylene group of compound 3 afforded 11 in good
1
3

Journal of the Iranian Chemical Society
Scheme 6 Synthetic pathway to compounds 15a,b–19
proposed structure of 13 was confirmed by spectral and
elemental analyses.
The IR spectrum of compound 16 refers to the existence
−
1
of an aldehydic carbonyl group at 1710 cm . Moreover, the
1
Following the same literately reported a synthesis of
iminocoumarin derivatives [17, 18], herein, novel imino-
coumarin derivatives were built upon refluxing compound
H NMR spectrum indicated the presence of a singlet peak
at δ 8.83 ppm (aldehydic proton).
For compounds 17a,b, the IR spectrum showed the pres-
ence of a stretching absorption band for the carbonyl groups
3
with an ethanolic solution of salicylaldehyde and/or
−
1
1
3
,5-dinitrosalicylaldehyde 14a,b (1:1 molar ratio) in the
at 1719 and 1678 cm . As well, the H NMR spectrum
revealed the existence of a mixture of the two compounds
17a,b due to the presence of methine proton as singlet at
δ 5.83 ppm and ethoxy protons as quartet and triplet at
3.66–3.56 and 1.01 ppm, respectively. Besides, the presence
of aldehydic proton as singlet peak at δ 9.03 ppm.
presence of a catalytic amount of piperidine.(Scheme 6).
The structures of iminocoumarin derivatives 15a,b
were unambiguously ascertained on the basis of analyti-
cal and spectroscopic data. For example, the IR spectrum
−
1
−1
of 15a displayed υ at 3312 cm , υ
at 1682 cm
C=O
NH
−
1
and υ
at 1632 cm . At the same time, the IR spectrum
Refluxing a three component, an ethanolic mixture of 3
with phenyl isothiocyanate and elemental sulfur afforded
2-thioxo-2,3-dihydrothiazole derivative 18 under basic con-
C=N
−
1
−1
of 15b displayed υ at 3206 cm , υ
at 1683 cm ,
NH
C=O
−
1
−1
υ
at 1619 cm and υ
at 1550 and 1339 cm , it
C=N
NO2
is also worthy to mentioned that IR of both compounds
showed the absence of a stretching absorption band for
nitrile group.
dition (Et N) (Scheme 6).
3
The structure of 18 was unequivocally proven via inves-
tigating the IR spectrum which exhibited the absence of
1
Afterwards, compounds 15a,b were individually refluxed
with triethyl orthoformate in the presence of distilled acetic
anhydride to afforded 16 and 17a,b (Scheme 6).
Characterizations of the newly synthesized compounds
cyano group. Furthermore, H NMR displayed broad singlet
at δ 6.31 ppm exchangeable with D O corresponding to NH
2
2
protons.
2-Thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidin-7(6H)-one
derivative 19 was furnished when 18 was subjected to react
1
6, 17a,b were based on spectral and elemental analyses.
1
3

Journal of the Iranian Chemical Society
Scheme 7 Synthetic pathway to
compounds 21–23
with triethyl orthoformate in the presence of distilled acetic
anhydride (Scheme 6). The proposed structure of 19 was
confirmed by spectral and elemental analyses. In particular,
spectrum of 22 which revealed the presence of a singlet
peak at δ=3.89 ppm corresponding to CH protons of the
2
thiazolidinone ring.
1
H NMR spectrum of 19 showed a characteristic peak for
Moreover, curing of the nonisolable potassium sulfide
salt 20 with ethyl iodide afforded N′-((5-chloro-3-methyl-1-
phenyl-1H-pyrazol-4-yl)methylene)-2-cyano-3-(ethylthio)-
3-(phenylamino)acrylohydrazide 23 (Scheme 7).
Unfortunately, when the novel ketene N,S-acetal 23 was
refluxed with hydrazine hydrate in ethanol, the sulfur-free
compound which was characterized as the diarylidene
hydrazine derivative 4 was obtained as the sole product
instead of the desired pyrazole derivative 24 [19]. No clue
for the formation of 3-aminopyrazole derivative 24 was
detected neither from spectroscopic nor elemental analyses
(Scheme 7).
CH
at δ 9.31 ppm and the absence of peak of NH2
pyrimidinone
protons.
The reactivity of the active methylene moiety of com-
pound 3 was investigated. Thus, when compound 3 was
allowed to react with phenyl isothiocyanate in dry DMF con-
taining a catalytic amount of potassium hydroxide at room
temperature [18, 19], it yielded the nonisolable intermedi-
ate potassium sulfide salt 20, followed by acidification with
dilute HCl to isolate compound 21 in good yield (Scheme 7).
The microanalytical and spectroscopic data were in agree-
ment with the proposed structure 21.
Otherwise, treatment of the nonisolable potassium
sulfide salt 20 in situ with chloroacetyl chloride afforded
Finally, compound 3 with carbon disulfide in the pres-
ence of potassium hydroxide in DMF afforded the intermedi-
ate dipotassium dithiolate salts 25 which in situ underwent
S-acylation with chloroacetyl chloride and/or S-alkylation
with ethyl chloroacetate as different intermediates 26 and 27,
respectively. In both cases, the 4-oxo-1,3-dithiolane deriva-
tive 28 was anticipated. However, unexpectedly, its enol
tautomer 29 was obtained instead. The chemical structure
of 29 was supported on the basis of elemental analysis and
spectral data (Scheme 8).
1
,3-thiazolidin-5-one derivative 22 (Scheme 7). Suppos-
edly, the reaction mechanism is assumed to proceed via
S-acylation followed by closure of the thiazole ring upon
N-alkylation of the remaining CH Cl group to award thia-
2
zolidinone derivative 22. Elemental analyses and spectral
data were in favor of this proposed 1,3-thiazolidin-5-one
structure. The IR spectrum of 22 showed absorption band at
−
1
1
745 cm due to carbonyl group of the thiazolidinone ring.
1
The structure of 22 was also ascertained from H NMR
1
3

Journal of the Iranian Chemical Society
Scheme 8 Synthetic pathways
to 4-hydroxy-1,3-dithiole
derivative 28
Pharmacology
respectively. Whereas, the strongest cytotoxic activity was
exhibited by compounds 6, 11, and 22 which showed the
percentage viability IC at 15.90 ± 1.4, 19.02 ± 1.6, and
Cytotoxicity and antitumor evaluation
5
0
1
1.79± 1.2 µM, respectively. Meanwhile, compounds 15a,
Out of the newly synthesized compounds, 14 analogs were
selected to be evaluated for their in-vitro anticancer effects
via the standard MTT method [20, 21], against a panel of
two human tumor cell lines; (HepG2) hepatocellular carci-
noma and (MCF-7) mammary gland breast cancer. Doxoru-
bicin was used during comparison as a standard anticancer
drug. The results of cytotoxic activity of compounds are
summarized in Table 1 and Fig. 4.
15b, 4, and 18 showed moderate activity, while other com-
pounds showed weak activities. On the other hand, the
activity against MCF-7 cell line revealed that compounds
13 and 29 have the very highest percentage viability IC
5
0
at 5.48 ± 0.4 and 8.26 ± 0.7 µM, respectively. Both com-
pounds 6 and 22 showed the strongest activities. Whereas,
compounds 4, 11, 15a, 15b, and 18 showed moderate activi-
ties. Meanwhile, the other tested compounds exhibited weak
activities.
The obtained results revealed that the tested compounds,
namely, 4, 6, 9, 11, 13, 15a, 15b, 16, 18, 19, 21, 22, 23,
and 29 exhibited variable degrees of inhibitory activ-
ity towards the two tested human tumor cell lines. As for
activity against HepG2, the very strongest cytotoxic activity
was exhibited by compounds 13 and 29 which showed the
percentage viability IC at 6.48 ± 0.6 and 9.16 ± 0.9 µM,
Structure activity relationships (SARs) By comparing the
cytotoxicity assessment of the reported compounds, in this
study, we conclude the following essential structural fea-
tures for activity enhancement.
5
0
1
3

Journal of the Iranian Chemical Society
Table 1 Cytotoxic activity of some new compounds against human
ABTS method
tumor cells
1
00.00%
0.00%
80.00%
9
Compounds
In-vitro cytotoxicity IC50 (µM)
7
6
5
4
3
2
1
0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
HepG2
MCF-7
DOX
4.50± 0.3
43.41± 2.8
15.90± 1.4
52.96± 3.4
19.02± 1.6
6.48± 0.6
31.74± 2.3
36.22± 2.5
69.53± 3.9
24.86± 1.9
64.78± 3.7
83.45± 4.8
11.79± 1.2
74.63± 4.2
9.16± 0.9
4.17± 0.2
48.03± 3.0
12.71± 1.1
61.34± 4.1
23.34± 1.8
5.48± 0.4
35.82± 2.5
40.85± 2.8
72.82± 4.5
29.28± 2.2
58.96± 3.6
91.75± 5.2
18.36± 1.5
51.40± 3.4
8.26± 0.7
4
6
9
1
1
1
1
1
1
1
2
2
2
2
1
3
5a
5b
6
Fig. 5 Antioxidant activity of some new compounds by ABTS
method
8
9
Erythrocyte haemolysis
1
6.00%
5.00%
4
3
2
2
.00%
.00%
.00%
3
9
IC50 (µM): 1–10 (very strong), 11–20 (strong), 21–50 (moderate),
1–100 (weak) and above 100 (non-cytotoxic)
DOX Doxorubicin
1.00%
0.00%
5
1
00
Fig. 6 Antioxidant activity of some new compounds by erythrocyte
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
HePG2
MCF-7
hemolysis method
Table 2 Antioxidant activity of some new compounds by ABTS
method
Method
ABTS
Abs
−Abs(test)/Abs(control) ×100
(
control)
Compounds
Absorbance of samples % Inhibition
Control of ABTS
Ascorbic acid
0.525
0.061
0.408
0.332
0.409
0.363
0.262
0.404
0.406
0.411
0.384
0.410
0.419
0.281
0.412
0.281
–
88.4
22.3
36.8
22.1
30.8
50.1
23.0
22.7
21.7
26.8
21.9
20.2
46.5
21.5
46.5
Fig. 4 Cytotoxic activity of some new compounds against human
4
6
9
tumor cells
1
1
1
1
1
1
1
2
2
2
2
1
3
5a
5b
6
8
9
1
2
(
(
1) The presence of pyrazole skeleton is necessary for the
broadening of cytotoxic activity towards both cell lines
(
HepG2 and MCF-7).
2) Since compound 13 has a remarkable potency closer to
doxorubicin against both cell lines (HepG2 and MCF-
7
) which may be attributed to the presence of 3,4-dihy-
dropyridinone moiety, beside incorporating of amino
and two methoxy groups (electron donating groups).
3) 4-Hydroxy-1,3-dithiole ring in 29 enhances the antitu-
mor activity against both cell lines.
(
3
9
1
3

Journal of the Iranian Chemical Society
Table 3 Antioxidant activity of some new compounds by erythrocyte
In case of erythrocytes hemolysis, compounds 4, 13, and
9 proved to exhibit potent antioxidative activity as vitamin
hemolysis method
2
C, while compounds 6, 11, 16, 21, and 22 showed very high
activities.
Method
Erythrocyte hemolysis
A/B×100
From Tables 2 and 3 and Figs. 5 and 6 and the above-
mentioned results, we may include the following structure
activity relationships (SARs).
Compounds
Absorbance of
samples
% Hemolysis
Absorbance of H O (B)
Vit-C
0.850
0.031
0.031
0.033
0.039
0.034
0.031
0.043
0.042
0.034
0.046
0.038
0.032
0.034
0.046
0.031
–
2
3.64
3.64
3.88
4.58
4.00
3.64
5.05
4.94
4.00
5.41
4.47
3.76
4.00
5.41
3.64
(
(
1) The presence of 3,4-dihydropyridinone, 1,3-thiazo-
lidin-5-one and 4-hydroxy-1,3-dithiole moieties in
compounds 13, 22, and 29, respectively, besides the
incorporation of pyrazole ring bearing methyl, phe-
nyl groups, and chlorine atom proved to be vital for
remarkable potential inhibitory antioxidant activity
using ABTS method.
4
6
9
1
1
1
1
1
1
1
2
2
2
2
1
3
5a
5b
6
8
9
1
2
2) Introduction of heterocyclic moieties as in compounds
4
, 13, and 29 enhanced the antioxidant activity in
erythrocytes hemolysis to the same extent of vitamin
C. Those compounds are currently under more evalua-
tion as a potential candidate drugs.
3
9
Literately, some previous reports showed that structurally
similar but they are different in pyrazole moiety which mani-
fested different results in antitumor and antioxidant activities
than our results [17, 29].
(
(
(
(
4) Cyclization of compound 3 into 4,5-dihydropyrazolone
derivative 6 incorporating amide group in position 4
enhances the antitumor activity against both cell lines.
5) Introduction of indolin-2-one moiety into compound
Conclusion
1
1 increases the antitumor activity against HepG2 cell
The goal of the present study is too utilizing some pyrazole
derivatives as scaffold for constructing different heterocy-
clic moieties on it. As an ultimate goal, those synthesized
compounds were screened and evaluated as potent agents
against the antitumor and antioxidant activity. The data col-
lection showed clearly that compounds 6, 11, 13, 22, and 29
displayed promising in-vitro antitumor activity against two
cell lines (HepG2 and MCF-7). Among these compounds,
compounds 13 and 29 exhibited broad spectrum of antitumor
agent. On the other hand, compounds 13, 22, and 29 exhib-
ited the highest inhibitory antioxidant activity using ABTS
method. Meanwhile, in case of erythrocytes hemolysis, com-
pounds 4, 13, and 29 proved to exhibit potent antioxidative
activity as vitamin C. Noteworthy, Compounds 13 and 29
manifested the highest potency in targeting both antitumor
and antioxidant activities.
line.
6) Compound 22 is more potent than 21 and 23 which may
be due to conversion of compound 3 into 1,3-thiazoli-
din-5-one moiety.
7) Compound 16 is less potent than compound 15a which
may be attributed to the conversion of the iminocou-
marin into N-formyl moiety. In addition, compound 19
is less potent than compound 18 which may be attrib-
uted to the conversion of 4-amino thiazol-2-thione into
2
-thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidin-7(6H)-
one moiety. As a conclusion, it is obvious that in these
two cases, disappearance of NH and NH diminishes
2
the potency which could be refused to declining in
hydrophobicity character.
Antioxidant activity screening
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Affiliations
Mahmoud F. Ismail1 · Amira A. El‑sayed1
1
*
Mahmoud F. Ismail
Department of Chemistry, Faculty of Science, Ain Shams
fawzy2010@sci.asu.edu.eg
University, Cairo, Abbassia 11566, Egypt
1
3