Synthesis of New 6-[4-(2-Fluorophenylpiperazine-1-YL)]-3(2H)-Pyridazinone-2-Acethyl-2- (Substitutedbenzal)Hydrazone Derivatives and Evulation of Their Cytotoxic Effects in Liver and Colon Cancer Cell Lines

Article abstract of DOI:10.1007/s11094-019-01927-y

In this study, seven new 3(2H)-pyridazinone derivatives expected to show cytotoxic activity in liver and colon cancer cell lines were synthesized. Their structures were confirmed by the IR, ¹H-NMR, ¹³C-NMR spectra and elementary anal

Full text of DOI:10.1007/s11094-019-01927-y

DOI 10.1007/s11094-019-01927-y
Pharmaceutical Chemistry Journal, Vol. 52, No. 11, February, 2019 (Russian Original Vol. 52, No. 11, November, 2018)
SYNTHESIS OF NEW 6-[4-(2-FLUOROPHENYLPIPERAZINE-
1
-YL)]-3(2H)-PYRIDAZINONE-2-ACETHYL-2-
SUBSTITUTEDBENZAL)HYDRAZONE DERIVATIVES
AND EVULATION OF THEIR CYTOTOXIC EFFECTS
IN LIVER AND COLON CANCER CELL LINES
(
1
,*
2
Zeynep Özdemir, Neºe Baºak-Türkmen, Ýdris Ayhan, Osman Çiftçi, and Mehtap Uysal
3
4
5
Original article submitted July 25, 2018.
In this study, seven new 3(2H)-pyridazinone derivatives expected to show cytotoxic activity in liver and colon
1
13
cancer cell lines were synthesized. Their structures were confirmed by the IR, H-NMR, C-NMR spectra and
elementary analyses. Compunds V -V were tested on HEP3B (liver cancer) and HTC116 (colon cancer) cell
1
7
lines for cytotoxicity by using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium] proliferation assay. Human fibroblast cells were used as safety control in these tests.
-[4-(2-Fluorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(2-chlorobenzal)hydrazone (compo-
6
und V ) was the most active agent with respect to HEP3B and HTC116 cell lines.
3
Keywords: pyridazinone; cytotoxicity; liver cancer; colon cancer.
Abbreviations: HEP3B, liver cancer cell line; HTC116,
colon cancer cell line; MTS, [3-(4,5-dimethylthi-azol-2-yl)-
-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazoli-
problem with the social and economic burden caused by the
incidence of cancer [4 – 6].
5
Hepatocellular carcinoma, which is responsible for the
death of more than 500,000 people per year and is associated
with many etiological factors such as aflatoxin, hepatitis C
and B viruses, is the most common primary carcinoma in the
world. Colon tumors constitute 1% of all cancers and it is the
third most common cause of cancer-related death among
people aged 15-34 [7, 8].
um]; IC50, half maximum inhibitory concentration; HF, hu-
man fibroblast cell line; SHSY5Y, neuroblastoma cell line;
TLC, thin layer chromatography; ATR, attenuated total re-
flection; MM, molecular mass; MP, melting point.
1
. INTRODUCTION
3(2H)-Pyridazinone ring is a six-membered lactam ring
system with C H N O closed formula [9]. It is known that
Cancer, one of the most important problems of today’s
4
4
2
the tautomeric balance is due to the presence of free hydro-
gen in the nitrogen atom in the 3(2H)-pyridazinone deriva-
tives having no substituent at second position [10].
Substituent-bearing derivatives of the pyridazinone ring,
which is known to be aromatic in the ring nitrogen atom, are
weakly acidic and they form salts with strong bases or with
ammonia and amines [11].
medicine, is a disease with invasive features characterized by
uncontrolled proliferation of cells. This disease that changes
according to the tissue from which it was born is the second
most common cause of death in the world today after cardio-
vascular disease [1 – 3]. It is also seen as an important social
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Inonu
University, 44280 Malatya, Turkey.
In recent years, many pharmacological activity studies
have been carried out with compounds bearing the 3(2H)-py-
ridazinone structure and showed that these compounds have
analgesic, anti-inflammatory, antipyretic, antihypertensive,
antiulcer, antioxidant, antiallergic, bronchospasmolytic, anti-
bacterial, antifungal, and anthelmintic properties [12 – 20].
Some compounds were reported in literature to have
anticancer activity due to 3(2H)-pyridazinone structure [18,
2
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Inonu
University, 44280 Malatya, Turkey.
3
Department of Medical Pharmacology, Faculty of Medicine, Yýldýrým
Beyazýt University, 06100 Ankara, Turkey.
4
Department of Medical Pharmacology, Faculty of Medicine, Pamukkale
University, 20160 Denizli, Turkey.
5
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi Uni-
versity, 06100 Ankara, Turkey.
*
e-mail: zeynep.bulut@inonu.edu.tr; zpozdmr@gmail.com
923
0
091-150X/19/5211-0923 © 2019 Springer Science+Business Media, LLC

9
24
Zeynep Özdemir et al.
Scheme 1. Synthesis pathway of 6-(4-(2-fluorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-acetyl-2-(substituted/nonsubstituted benzal)-
hydrazone derivatives.
1
9, 21]. It was also reported that pyridazinones carrying sub-
yl)-3(2H)-pyridazinone-2-ylacetate was obtained by the re-
action of 6-(4-(2-fluorophenyl)piperazine-1-yl)-3(2H)-pyri-
stituents at fourth, fifth and sixth positions and containing
phenyl groups attached to the ring nitrogen exhibit cytostatic
activity [14, 16, 17]. These results suggest that pyridazinone
compounds may be useful in cancer chemotherapy, depending
on the type of cancer, and that derivatives bearing different
substituents may exhibit varying degrees of cytotoxic effect.
This study was aimed to investigate the antiproliferative
effects of seven newly synthesized 3(2H)-pyridazinone de-
rivatives with respect to HEP3B, HTC116, and human
fibroblast (HF) cell lines. The selective killing of cancer cells
by test compounds without damaging healthy cells is very im-
portant in terms of chemotherapy. Therefore, cytotoxic effects
of newly synthesized pyridazinone compounds on cancer cell
lines were investigated in comparison to healthy HF cells.
dazinone with ethyl bromoacetate in the presence of K CO3
2
in acetone [22]. The formation of these compounds was also
confirmed by the IR spectra containing an ester C=O signal
–
1
at about 1750 cm . 6-(4-(2-Fluorophenyl)piperazine-1-yl)-
3(2H)-pyridazinone-2-ylacetohydrazide was synthesized by
the condensation reaction of ethyl 6-(4-(2-fluorophenyl)pi-
perazine-1-yl)-3(2H)-pyridazinone-2-ylacetate with hydra-
zine hydrate (99%) [22]. Eventually, the title compounds
which have benzalhydrazone structure were obtained by the
condensation reaction of 6-(4-(2-fluorophenyl)piperazine-
1-yl)-3(2H)-pyridazinone-2-ylacetohydrazide with substi-
tuted/nonsubstituted benzaldehydes. All these compounds
are reported in this article for the first time. Molecular struc-
1
tures of new compounds were confirmed by IR, H-NMR,
1
3
C-NMR, and LC/MS/MS spectral data and elementary
2
. RESULTS
analyses. Molecular structures, yields, melting points, mo-
lecular weights, and molecular formulas of compounds
V -V are given in Table I.
2
.1.Chemistry
1
7
Compounds V -V were synthesized according to the lit-
1
7
erature methods as outlined in Scheme 1.
2.2.Pharmacological Activity
Target compounds have been synthesized starting from
nucleophilic displacement reaction of commercial 3,6-di-
chloropyridazine with (2-fluorophenyl)piperazine in ethanol
afforded 3-chloro-6-[4-(2-fluorophenyl)piperazine-1-yl]py-
ridazine. The physical and spectral properties of 3-chloro-6-
Cytotoxicty and cell viability
In this study, the cytotoxic effects of seven newly synthe-
sized pyridazinone derivatives on HTC116 and HEP3B can-
cer cell lines were studied in comparison to the HF cell line.
Six of these compounds did not show any toxic effect against
[
4-(2-fluorophenyl)piperazine-1-yl]pyridazine were in accor-
dance with the literature [22]. Then, 6-(4-(2-fluorophenyl)pi-
perazine-1-yl)-3(2H)-pyridazinone was obtained as a result
of hydrolysis of 3-chloro-6-substituted pyridazine by heating
in glacial acedic acide [22]. The formation of these com-
pounds was confirmed by the IR spectra with C=O signal at
HF cells as compared to cancer cells. Compound V is the
3
most active one in HEP3B and HTC116 cell line, but it has
not been considered as candidate anticancer drug since it also
causes high mortality in HF cells. Data on the cell viability
and IC50 of eight pyridazinone compound are presented in
Fig. 1 and Table 2.
–
1
about 1660 cm . Ethyl 6-(4-(2-fluorophenyl)piperazin-1-

Synthesis of New Derivatives
925
TABLE 1. Molecular structures, yields, melting points, molecular weights, and molecular formulas of compounds V -V
1
7
Compound
R
-H
Yield (%)
79.90
78.15
73.19
91.21
85.69
77.45
68.77
MP
MM
Molecular formula
V
V
V
V
V
V
V
1
2
3
4
5
6
7
226 – 228
198 – 200
203 – 205
192 – 194
209 – 211
239 – 241
237 – 239
434.48
448.49
468.91
468.91
513.36
464.49
477.53
C
C
23
24
H
H
23FN
25FN
6
6
O
O
2
-4CH
-2Cl
-4Cl
-4Br
3
2
C
23
H
H
H
22ClFN
23ClFN
6
6
O
2
2
C
23
23
O
C
6 2
22BrFN O
-2CH
3
O
C
24
25
H
H
25FN
28FN
6
7
O
O
3
2
-4N(CH
3
)
2
C
3
. DISCUSSION
Nowadays, investigation of potentially safer new
higher damage to healthy cells. The compounds which have
a halogen atom at the phenyl ring caused a further decrease
of cell viability on L929 cell line as compared to other com-
anticancer agents is the focus of researchs in the area.
Pyridazinone derivatives have been previously synthesized
by our research group and the relationship between structure
and anticancer activity has been investigated. Apoptosis and
gene expression studies were carried out in order to elucidate
the mechanism of action of the five most active compounds
in these studies, in which a series of compounds with
nonsubstitued phenyl and 4-chlorophenyl bound to the
piperazine ring at sixths position of the pyridazinone ring
were synthesized. According to the results of our previous
study, the IC50 values of all compounds were lower than IC50
values for the HF cell line, and the substitution on the meta
position of the benzalhydrazone phenyl ring at second posi-
tion of 3(2H)-pyridazinone with strong electron-donating
pounds. The pyridazinone derivative V , in which the methyl
2
substituent was used, such as compound 4, which was the
most active compound in the previous study, did not cause a
decrease in the number of cancerous cells but causes a de-
crease in the number of healthy cells. As a result of this
study, it was found that the cell viability was decreased with
V , V and V treatment in both HEP3B and HTC116 cells
3
4
5
lines, when different concentrations were applied.
It is suggested that the synthesized pyridazinone deriva-
tives may be used in the development of new chemothera-
peutic agents, provided that the compounds are modified to
increase their selectivity to cancer cells in order to reduce the
toxicity.
group such as -OCH improved the cytotoxic effect in
3
4. EXPERIMENTAL
HEP3B cell line. In terms of IC , it was found that com-
50
pound 4 was effective at lower concentrations for both
SHSY5Y (24.3 mM) and HEP3B cells (23.2 mM) in our pre-
vious study [23].
4
.1.Chemicals and Methods
All chemicals used in this study were purchased from
In this study, which we have designed to develop more
active and less toxic compounds, the structure-activity rela-
tionship has focused on the effect of substituent at the phenyl
groups at second position of 3(2H)-pyridazinone ring. There
were more halogen groups than previously, and these com-
pounds caused higher cell death in cancer cell lines as well as
Aldrich, Fluka AG, and E. Merck. 3-Chloro-6-substituted
pyridazines, 3-chloro-6-(4-(2-fluorophenyl)piperazine-1-yl)-
pyridazine, 6-(4-(2-fluorophenyl)piperazine-1-yl)-3(2H)-py-
ridazinone, ethyl 6-(4-(2-fluorophenyl)piperazine-1-yl)-
3(2H)-pyridazinone-2-yl acetate and 6-(4-(2-fluorophenyl)-
piperazine-1-yl)-3(2H)-pyridazinone-2-yl
acetohydrazide
TABLE 2. The IC50 Values of Pyridazinone Dertvatives V -V
1
7
IC50 (mM)
Cell line
HEP3B
V
1
V
2
V
3
V
4
V
5
V
6
V
7
111,8
110,8
130,2
150,5
110,8
35,3
27,7
32,7
62,7
65,8
26,7
66,5
62,2
26,9
94,7
110,6
120,6
103,4
105,9
102,6
HTC116
HF
27,05

9
26
Zeynep Özdemir et al.
Fig. 1. The effect of compounds V -V on cell viability: (1) cells were seeded in 96-well flat-bottomed tissue culture plates at a concentration
3 7
3
of 5 ´ 10 cells/well; (2) cells were treated with DMEM medium containing various concentrations (25, 50, 100, 200, and 300 mM) of the test
compounds for different incubation periods (24, 48 and 72 h); (3) fresh complete medium containing 10 µL of MTS solution was added at the
end of the each time point, and incubated 2 h in incubator; (4) cell proliferation was assessed by measuring the absorbance with an ELISA
microplate reader.

Synthesis of New Derivatives
927
were synthesized according to literature methods [14, 17, 24,
4.3.General procedure for the synthesis of title compounds
-V
2
5]. The purity of all compounds was checked by TLC with
V
1
7
Merck Kieselgel F254 plates. Melting points were determined
on Electrothermal 9200 melting points apparatus and the ob-
tained values are uncorrected. IR spectra were recorded on a
0.01 mol
6-(4-(2-fluorophenyl)piperazine-1-yl)-3(2H)-
pyridazinone-2-ylacetohydrazide and 0.01 mol substi-
tuted/nonsubstituted benzaldehyde were stirred in ethanol
(15 mL) and refluxed for 6 h. At the and of the reaction, the
mixture was poured into ice water. The precipitate was fil-
tered, dried and crystallized from methanol-water mixture.
All spectral data of the seven title compounds were in ac-
cordance with the assigned structures as shown below.
1
Perkin Elmer spectrometer by ATR technique. H-NMR and
1
3
C-NMR spectra were recorded on a Bruker Avance
Ultrashield FT-NMR spectrometer in DMSO at 600 MHz
and 75 MHz, respectively. Mass spectra (LC/MS-MS) were
obtained using Agilent 6400 Series Triple Quadrupole
B.08.00 (B8023.0). Elemental analyses were performed with
Leco CHNS-932 instrument in the Inonu University
6-[4-(2-Fluorophenyl)piperazine-1-yl]-3(2H)-pyridazi
none-2-acetyl-2-benzalhydrazone (V ). White crystals
1
(
methanol/water); yield 79.90%; m.p. 226 – 228°C; IR (n,
(
Malatya).
.2.Synthesis of Intermediate Compounds
-1
cm , ATR): 3061 (C-H aromatic), 2853 (C-H aliphatic),
1693, 1651 (C=O), 1571 (C=N), 1239 (C-N), 1069 (C-O),
4
1
46 (C-F). H-NMR (DMSO-d , 600 MHz): d 3.12 (4H; t;
8
6
3
-Chloro-6-[4-(2-fluorophenyl)piperazine-1-yl]pyrida
zine (I). 0.01 mol of 3,6-dichloropyridazine and 0.01 mol of
2-fluorophenyl)piperazine are stirred in 15 mL of ethanol
CH N; b+b¢), 3.38 (4H; t; CH N; a+a¢), 5.06 (2H; s;
2
2
5
CH CO), 6.92 (1H; d; J = 4.15 Hz, pyridazinone H ), 6.94
2
(
4
1H; d; J = 4.16 Hz, pyridazinone H ), 7.07 – 8.02 (9H; m;
(
under reflux with heating for 6 h according to the literature
procedure. The reaction medium is poured into ice-cold wa-
ter; the precipitate obtained by filtration is purified by crys-
tallization from ethanol. The melting points of non-original
compounds were in accordance with the literature data [22].
phenyl protons), 8.72 (1H; s; -N=CH-) and 11.65 (1H; s;
1
3
NH-N). C-NMR (DMSO-d , 600 MHz), d 46.61 (2C,
-
6
CH -N; b+b¢), 50.13 (2C, CH -N; a+a¢), 53.00 (1C;
2
2
-N-CH -C=O), 116.53 (1C; =CH), 119.89 (1C; pyridazinone
2
5
6
C ), 123.14 (1C; phenyl C ), 125.35 (1C; phenyl C ), 126.96
2
3
(1C; phenyl C ), 127.36 (1C; phenyl C ), 128.84 (1C; phenyl
5
6
-(4-(2-Fluorophenyl)piperazine-1-yl)-3(2H)-pyridazi
4
C ), 129.30 (1C; pyridazinone C ), 130.44 (1C;
4
none (II). A solution of 0.05 mol of 3-chloro-6-[4-(2-fluoro-
phenyl)piperazine-1-yl]pyridazine in 30 mL glacial acetic
acid was refluxed for 6 h. Acetic acid was removed under re-
duced pressure; the residue was dissolved in water and ex-
tracted with chloroform. The organic phase was dried over
sodium sulphate and evaporated under reduced pressure. The
residue was purified by recrystallization from ethanol. The
melting points of non-original compounds were in accor-
dance with the literature [22].
5
-fluorophenyl C ), 131.01 (1C; 2-fluorophenyl C ), 134.43
4
2
6
1C; 2-fluorophenyl C ), 140.04 (1C; 2-fluorophenyl C ),
5
(
1
1
44.34 (1C; 2-fluorophenyl C ), 147.44 (1C; phenyl C ),
1
1
6
49.03 (1C; pyridazinone C ), 154.63 (1C; 2-fluorophenyl
2
C ), 158.26 (1C; CH -N-C=O) and 168.45 (1C; pyridazinone
2
3
+
C ); LC/MSMS (ESI-) m/z 433.0 [M] ; Anal. Calcd. for
C H FN O .1/2 H O: C, 62.29; H, 5.46; N, 18.95. Found:
2
3
23
6
2
2
C, 62.46; H, 5.579; N, 18.69 %.
6-[4-(2-Fluorophenyl)piperazine-1-yl]-3(2H)-pyridaz-
Ethyl 6-(4-(2-fluorophenyl)piperazine-1-yl)-3(2H)-py-
ridazinone-2-ylacetate (III). A mixture of 0.01 mol
inone-2-acetyl-2-(4-methylbenzal)hydrazone (V ). White
2
crystals (methanol/water); yield 78.15%; m.p. 198 – 200°C;
-
1
6
-(4-(2-fluorophenyl)piperazine-1-yl)-3(2H)-pyridazinone,
.02 mol ethyl bromoacetate, and 0.02 mol potassium car-
IR (n, cm , ATR): 3034 (C-H aromatic), 2851 (C-H
0
aliphatic), 1651 (C=O), 1583 (C=N), 1240 (C-N), 1081
1
(C-O), 846 (C-F). H-NMR (DMSO-d , 600 MHz): d 2.34
bonate in acetone (40 mL) was refluxed overnight. After the
mixture was cooled, the organic salts were filtered off, the
solvent evaporated, and the residue was purified by
recrystallization from n-hexane to give the esters. The melt-
ing points of non-original compounds were in accordance
with the literature [22].
6
(3H; s; -CH ), 3.11 (4H; t; CH N; b+b¢), 3.38 (4H; t; CH N;
3
2
2
a+a¢), 5.05 (2H; s; CH CO), 6.99 (1H; d; J = 4.19 Hz,
5
pyridazinone H ), 7.01 (1H; d; J = 4.21 Hz, pyridazinone
4
H ), 7.06 – 7.98 (8H; m; phenyl protons), 8.67 (1H; s;
1
N=CH-) and 11.57 (1H; s; -NH-N). C-NMR (DMSO-d6,
2
3
-
6
00 MHz), d 21.48 (1C; -CH ), 46.61 (2C; CH -N; b+b¢),
6
-(4-(2-Fluorophenyl)piperazine-1-yl)-3(2H)-pyridaz-
inone-2-ylacetohydrazide (IV). Hydrazine hydrate (99%,
ml) was added to the solution of 0.01 mol ethyl
-(4-(2-fluorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-
3
2
50.11 (2C, CH -N; a+a¢), 53.00 (1C; -N-CH -C=O), 116.53
2
2
5
1C; =CH), 119.89 (1C; pyridazinone C ), 123.19 (1C;
(
3
6
-methylphenyl C ), 125.36 (1C; 4-methylphenyl C ),
2
4
6
3
26.95 (1C; 4-methylphenyl C ), 127.34 (1C; 4-methylphe-
1
ylacetate in 25 mL methanol and stirred for 3 h at room tem-
perature. The precipitate obtained was filtered off, washed
with water, dried, and recrystallized from ethanol. The melt-
ing points of non-original compounds were in accordance
with the literature [22].
5
nyl C ), 127.55 (1C; 4-methylphenyl C ), 128.79 (1C; pyri-
4
4
3
dazinone C ), 130.00 (1C; 2-fluorophenyl C ), 131.01 (1C;
4
-fluorophenyl C ), 131.74 (1C; 2-fluorophenyl C ), 140.25
5
2
1
(1C; 4-methylphenyl C ), 144.40 (1C; 2-fluorophenyl C ),
6
1
149.01 (1C; 2-fluorophenyl C ), 154.63 (1C; 2-fluorophenyl

9
28
Zeynep Özdemir et al.
2
C ), 156.25 (1C; pyridazinone C ) 158.25 (1C; CH -N-C=O)
3
and 168.33 (1C; pyridazinone C ); LC/MSMS (ESI-) m/z
6
6-[4-(2-Fluorophenyl)piperazine-1-yl]-3(2H)-pyridaz-
2
inone-2-acetyl-2-(4-bromobenzal)hydrazon (V ). White
5
+
47.0 [M] ; Anal. Calcd. for C H FN O .1/3 H O: C,
4
6
1
crystals (methanol/water); yield 85.69%; m.p. 209 – 211°C;
24
25
6
2
2
-1
3.42; H, 5.69; N, 18.49. Found: C, 63.66; H, 5.708; N,
8.26%.
-[4-(2-Fluorophenyl)piperazine-1-yl]-3(2H)-pyridaz-
IR (n, cm , ATR): 3023 (C-H aromatic), 2849 (C-H
aliphatic), 1690, 1657 (C=O), 1584 (C=N), 1241 (C-N),
1
1068 (C-O), 834, 812 (C-Br, C-F). H-NMR (DMSO-d ,
6
6
inone-2-acetyl-2-(2-chlorobenzal)hydrazone (V ). White
3
600 MHz): d 2.51 (4H; t; CH N; b+b¢), 3.10 (4H; t; CH N;
2
2
crystals (methanol/water); yield 73.19%; m.p. 203 – 205°C;
a+a¢), 5.06 (2H; s; CH CO), 6.91 (1H; d; J = 4.14 Hz,
5
pyridazinone H ), 6.94 (1H; d; J = 4.16 Hz, pyridazinone
2
-1
IR (n, cm , ATR): 3175 (C-H aromatic), 2851 (C-H
aliphatic), 1690, 1656 (C=O), 1585 (C=N), 1242 (C-N), 881,
1
35 (C-Cl, C-F). H-NMR (DMSO-d , 600 MHz): d 2.51
4
H ), 6.99 – 7.99 (8H; m; phenyl protons), 8.70 (1H; s;
1
N=CH-) and 11.71 (1H; s; -NH-N). C-NMR (DMSO-d6,
3
8
-
6
(
4H; t; CH N; b+b¢), 3.11 (4H; t; CH N; a+a¢), 5.06 (2H; s;
2 2
6
00 MHz), d 46.60 (2C, CH -N; b+b¢), 50.11 (2C, CH -N;
2
2
5
CH CO), 6.91 (1H; d; J = 4.15 Hz, H ), 6.93 (1H; d;
4
J = 4.16 Hz, H ), 6.99 – 7.91 (8H; m; phenyl protons), 8.72
2
a+a¢), 52.99 (1C; -N-CH -C=O), 116.39 (1C; =CH), 119.87
2
5
1C; pyridazinone C ), 123.14 (1C; 2-fluorophenyl C ),
4
(
13
(
1H; s; -N=CH-) and 11.71 (1H; s; -NH-N). C-NMR
5
23.63 (1C; 4-bromophenyl C ), 125.35 (1C; 2-fluorophenyl
1
5
C ), 126.96 (1C; 4-bromophenyl C ), 129.25 (1C; 4-bromo-
2
(
DMSO-d , 600 MHz), d 46.47 (2C, N-(CH ) ), 46.60 (2C,
6
3 2
6
phenyl C ), 132.26 (1C; pyridazinone C ), 133.73 (1C;
4
CH N; b+b¢), 50.11 (2C, CH N; a+a¢), 52.98 (1C;
2
2
6
2-fluorophenyl C ), 140.03 (1C; 4-bromophenyl C ), 143.15
3
-
N-CH -C=O), 116.39 (1C; =CH), 119.88 (1C; pyridazinone
2
5
C ), 123.14 (1C; 2-chlorophenyl C ), 125.33 (1C; 2-chloro-
6
3
(1C; 2-fluorophenyl C ), 146.23 (1C; 4-bromophenyl C ),
1
5
4
phenyl C ), 126.97 (1C; 2-chlorophenylphenyl C ), 129.03
1
149.03 (1C; 2-fluorophenyl C ), 154.62 (1C; 4-bromophenyl
5
1C; 2-fluorophenyl C ), 129.20 (1C; 2-fluorophenyl C ),
4
4
C ), 156.24 (1C; pyridazinone C ), 158.25 (1C; 2-flu-
6
(
4
29.57 (1C; pyridazinone C ), 130.50 (1C; 2-fluorophenyl
2
orophenyl C ), 163.88 (1C; CH -N-C=O), 168.53 (1C;
1
2
6
C ), 131.05 (1C; 2-fluorophenyl C ), 133.40 (1C; 2-chloro-
3
3
+
pyridazinone C ); LC/MSMS (ESI-) m/z 512.9 [M] , 514.9
3
phenyl C ), 140.09 (1C; 2-fluorophenyl C ), 143.05 (1C;
1
+
[M+2] ; Anal. Calcd. for C H BrFN O .H O: C, 51.99; H,
2
3
22
6
2
2
1
-chlorophenyl C ), 146.14 (1C; 2-chlorophenyl C ), 149.03
2
2
4.55; N, 15.82. Found: C, 52.23; H, 4.477; N, 15.47%.
6-[4-(2-Fluorophenyl)piperazine-1-yl]-3(2H)-pyridaz-
inone-2-acetyl-2-(2-methoxybenzal)hydrazone (V6).
White crystals (methanol/water); yield 77.45%; m.p.
6
1C; pyridazinone C ), 156.24 (1C; 2-fluorophenyl C ),
2
(
3
61.08 (1C; CH -N-C=O) and 168.53 (1C; pyridazinone C );
1
2
+
+
LC/MSMS (ESI-) m/z 467.0 [M] ; 469.0 [M+2] ; Anal.
-
1
Calcd. for C H ClFN O .2/3 H O: C, 57.44; H, 4.89; N,
2
39 – 241°C; IR (n, cm , ATR): 3022 (C-H aromatic), 2840
2
3
22
6
2
2
1
7.47. Found: C, 57.48; H, 5.047; N, 17.06%.
-[4-(2-Fluorophenyl)piperazine-1-yl]-3(2H)-pyridaz-
(
C-H aliphatic), 1683, 1652 (C=O), 1567 (C=N), 1240
1
C-N), 1098 (C-O), 846 (C-F). H-NMR (DMSO-d6,
6
(
inone-2-acetyl-2-(4-chlorobenzal)hydrazone (V ). White
4
600 MHz): d 2.51 (4H; t; CH N; b+b¢), 3.11 (4H; t; CH N;
2 2
crystals (methanol/water); yield 91.21%; m.p. 192 – 194°C;
a+a¢), 5.05 (2H; s; CH CO), 6.91 (1H; d; J = 4.15 Hz,
5
pyridazinone H ), 6.93 (1H; d; J = 4.16 Hz, pyridazinone
-1
2
IR (n, cm , ATR): 3044 (C-H aromatic), 2852 (C-H
aliphatic), 1689, 1656 (C=O), 1584(C=N), 1242(C-N), 834,
1
13 (C-Cl, C-F). H-NMR (DMSO-d , 600 MHz): d 2.51
4
H ), 6.98 – 7.86 (8H; m; phenyl protons), 8.57 (1H; s;
1
3
-N=CH-) and 11.60 (1H; s; -NH-N). C-NMR (DMSO-d6,
8
6
(
4H; t; CH N; b+b¢), 3.10 (4H; t; CH N; a+a¢), 5.06 (2H; s;
2 2
600 MHz), 46.60 (2C, CH N; b+b¢), 50.12 (2C, CH N; a+a¢),
2
2
5
CH CO), 6.99 (1H; d; J = 4.19 Hz, pyridazinone H ), 7.00
2
53.03 (1C; -N-CH -C=O), 56.17 (1C; -OCH ), 112.27 (1C;
2 3
5
CH), 116.52 (1C; pyridazinone C ), 121.20 (1C;
4
1H; d; J = 4.20 Hz, pyridazinone H ), 7.06 – 7.91 (8H; m;
(
=
6
-methoxyphenyl C ), 122.43 (1C; 2-methoxyphenyl C ),
4
123.18 (1C; 2-methoxyphenylphenyl C ), 125.96 (1C; 2-flu-
5
phenyl protons), 8.72 (1H; s; -N=CH-) and 11.71 (1H; s;
2
1
NH-N). C-NMR (DMSO-d , 600 MHz), d 46.60 (2C,
3
-
6
5
orophenyl C ), 127.01 (1C; 2-fluorophenyl C ), 131.07 (1C;
4
CH -N; b+b¢), 50.11 (2C, CH -N; a+a¢), 52.99 (1C;
2
2
4
6
pyridazinone C ), 132.10 (1C; 2-fluorophenyl C ), 139.92
-
N-CH -C=O), 116.39 (1C; =CH), 119.87 (1C; pyridazinone
2
5
C ), 123.19 (1C; 2-fluorophenyl C ), 125.33 (1C;
4
3
(1C; 2-fluorophenyl C ), 142.95 (1C; 2-methoxyphenyl C ),
3
5
-chlorophenyl C ), 127.96 (1C; 2-fluorophenyl C ), 129.35
5
1
149.00 (1C; 2-fluorophenyl C ), 154.62 (1C; 2-methoxyphe-
4
2
6
1C; 4-chlorophenyl C ), 131.00 (1C; 4-chlorophenyl C ),
1
nyl C ), 156.24 (1C; 2-methoxyphenyl C ), 158.08 (1C;
2
(
4
33.39 (1C; pyridazinone C ), 134.85 (1C; 2-fluorophenyl
6
2
pyridazinone C ), 159.22 (1C; 2-fluorophenyl C ), 163.58
1
6
C ), 136.51 (1C; 4-chlorophenyl C ), 140.03 (1C;
3
3
(1C; CH -N-C=O) and 168.32 (1C; pyridazinone C );
+
LC/MSMS (ESI-) m/z 463.0 [M] ; Anal. Calcd. for
2
3
1
-fluorophenyl C ), 143.05 (1C; 4-chlorophenyl C ), 149.03
2
1
1C; 2-fluorophenyl C ), 154.62 (1C; 4-chlorophenyl C ),
4
(
C H FN O .H O: C, 60.88; H, 5.53; N, 17.75. Found: C,
2
4
25
6
3
2
6
56.24 (1C; pyridazinone C ), 158.25 (1C; 2-fluorophenyl
1
60.74; H, 5.337; N, 17.57%.
2
C ), 163.87 (1C; CH -N-C=O), 168.53 (1C; pyridazinone
6-[4-(2-Fluorophenyl)piperazine-1-yl]-3(2H)-pyridazi
none-2-acetyl-2-(4-dimethylaminobenzal)hydrazone (V7).
Yellow crystals (methanol/water); yield 68.77%; m.p.
2
3
C ); LC/MSMS (ESI-) m/z 466.9 [M] , 468.2 [M+2] ; Anal.
+
+
Calcd. for C H ClFN O .1/3 H O: C, 58.17; H, 4.81; N,
2
3
22
6
2
2
-
1
1
7.70. Found: C, 58.36; H, 4.949; N, 17.82%.
237 – 239°C; IR (n, cm , ATR): 3122 (C-H aromatic), 2857

Synthesis of New Derivatives
929
(
(
(
C-H aliphatic), 1648 (C=O), 1585 (C=N), 1241 (C-N), 1182
1
C-O), 846 (C-F). H-NMR (DMSO-d , 600 MHz): d 2.50
CONFLICT OF INTERESTS
6
No conflict of interests was reported by all authors.
6H; s; N(CH ) ), 2.97 (4H; t; CH N; b+b¢), 3.38 (4H; t;
3
2
2
CH N; a+a¢), 5.01 (2H; s; CH CO), 6.73 (1H; d;
2
2
5
J = 4.03 Hz, pyridazinone H ), 6.75 (1H; d; J = 4.05 Hz,
REFERENCES
4
pyridazinone H ), 6.98 – 7.88 (8H; m; phenyl protons), 8.06
1
3
(
1H; s; -N=CH-) and 11.34 (1H; s; -NH-N). C-NMR
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(
DMSO-d , 600 MHz), d 46.58 (2C, N(CH ) ), 46.62 (2C,
6
3 2
2
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CH -N; b+b¢), 50.12 (2C, CH -N; a+a¢), 52.98 (1C;
2
2
-
N-CH -C=O), 112.25 (1C; =CH), 116.39 (1C; pyridazinone
2
5
C ), 119.88 (1C; 2-fluorophenyl C ), 121.80 (1C; 4-dime-
4
5
thylaminophenyl C ), 123.13 (1C; 2-fluorophenyl C ),
5
3. R. Khoogar, B. C. Kim, J. Morris, and M. J. Wargovich,
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2
25.35 (1C; 4-dimethylaminophenyl C ), 126.89 (1C; 4-di-
1
6
methylaminophenyl C ), 128.63 (1C; pyridazinone C ),
4
6
31.07 (1C; 2-fluorophenyl C ), 140.10 (1C; 4-dimethylami-
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3
nophenyl C ), 145.11 (1C; 2-fluorophenyl C ), 148.97 (1C;
3
1
-dimethylaminophenyl C ), 151.88 (1C; 2-fluorophenyl
4
5
. F. A. Sloan and H. Gelband, Cancer Control Opportunities in
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Washington, DC (2007).
1
4
C ), 154.63 (1C; 4-dimethylaminophenyl C ), 156.24 (1C;
6
pyridazinone C ), 158.26 (1C; 2-fluorophenyl C ), 163.11
2
3
1C; CH -N-C=O), 167.83 (1C; pyridazinone C );
+
LC/MSMS (ESI-) m/z 476.1 [M] ; Anal. Calcd. for
(
6. X. Ma and H. Yu, Yale J. Biol. Med., 79(3 – 4), 85–94 (2006).
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7
. A. G. Vaiopoulos, I. D. Kostakis, M. Koutsilieris, and
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C H FN O .1/2H O: C, 61.71; H, 6.01; N, 20.15. Found:
2
5
28
7
2
2
8
9
C, 61.92; H, 6.097; N, 20.08%.
.4. Activity Studies
.4.1. Cell cultures and incubation. HTC116, HEP3B
4
4
10. E. Matrai, J. Mol. Struct., 408/409, 467 – 472 (1997).
11. A. R. Katritzky and A. J. Boulton, Advances in Heterocyclic
Chemistry, Vol. 9, Academic Press, New York – London
and HF (BJ, CRL-2522) cells were obtained from the Ameri-
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DMEM containing 10% fetal bovine serum and 1% antibiot-
ics (100 µg/mL streptomycin and 10000 U /mL penicillin).
Media were changed twice a week until cells reached
(
1968).
1
1
2. A. A. Siddiqui, R. Mishra, and M. Shaharyar, Eur. J. Med.
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7
0 – 80% confluency.
.4.2. Cell viability assay. The MTS [3-(4,5-Dime-
4
14. S. Utku, M. Gökçe, G. Aslan, et al., Arzneim Forsch, 61, 1 – 7
(92011).
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04 – 309 (2012).
thylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium] assay was performed to determine
viable cell ratio according to the manufacturer¢s instructions
1
1
1
1
(
Promega). Briefly, cells were seeded in 96-well flat-bot-
9
tomed tissue culture plates at a concentration of 5 ´
–
3
1
0
cells/well. After 24 h, the cells were treated with
DMEM medium containing different concentrations (25, 50,
00, 200, and 300 mM) of the test compounds for different
3
1
1
9. N. F. Abd El-Ghaffar, M. K. Mohamed, M. S. Kadah, J. Chem.
incubation periods (24, 48, and 72 h). At the end of the each
time point, fresh complete medium containing 10 mL of MTS
solution was added and further incubated 2 h in incubator.
Cell proliferation was assessed by measuring the absorbance
with an ELISA microplate reader (Weida). Each experiment
was performed quadruplet and results are expressed as the
percentage growth inhibition with respect to the untreated
cells.
Pharm. Res., 3, 248 – 259 (2011).
2
0. M. Asif, Int. J. Adv. Chem., 2(2), 148 – 161 (2014).
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225 – 233 (2012).
2
22. Z. Özdemir, M. Gökçe, H. Uslu., et al., Intern. Multidisciplinary
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60(7), 452 – 458 (2010).
2
2
2
3
Statistical deviations for the viability were calculated au-
tomatically by the Excel 2007 software (SE ? 5%), and for
the IC50, values, by the ‘’Origin Pro 7.5” and “Origin 6.1”
5
(
for 7Crf) PC programs.