Synthesis and biological evaluation of new 3(2H)-pyridazinone derivatives as non-toxic anti-proliferative compounds against human colon carcinoma HCT116 cells

Article abstract of DOI:10.1080/14756366.2020.1755670

Novel 3(2H)-pyridazinone derivatives were designed, synthesised in satisfactory yields and evaluated in different experimental assays to assess their preliminary toxicity in vivo and anti-proliferative effects against HCT116 cell lines in vitro. Artemia salina lethality test provided LC₅₀ values >100 μg/mL for all compounds. Successive assays revealed that some compounds were endowed with a promising anti-proliferative effect against HCT116 cells, alone or stimulated by serotonin as a pro-inflammatory factor in order to mimick an inflamed model in vivo of cancer cell microenvironment. Moreover, the kinurenic acid level after treatment with these newly synthesised compounds was monitored as a marker of anti-proliferation in colon carcinoma models. The IC₅₀ values obtained for the best-in-class compounds were comparable to that of daunorubicin as a reference drug. Conversely, these compounds were not able to counteract the spontaneous migration of human cancer HCT116 cell line in the wound healing paradigm.

Full text of DOI:10.1080/14756366.2020.1755670

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis and biological evaluation of new 3(2H)-
pyridazinone derivatives as non-toxic anti-
proliferative compounds against human colon
carcinoma HCT116 cells
Zeynep Özdemir, Semra Utku, Bijo Mathew, Simone Carradori, Giustino
Orlando, Simonetta Di Simone, Mehmet Abdullah Alagöz, Azime Berna
Özçelik, Mehtap Uysal & Claudio Ferrante
To cite this article: Zeynep Özdemir, Semra Utku, Bijo Mathew, Simone Carradori, Giustino
Orlando, Simonetta Di Simone, Mehmet Abdullah Alagöz, Azime Berna Özçelik, Mehtap Uysal
& Claudio Ferrante (2020) Synthesis and biological evaluation of new 3(2H)-pyridazinone
derivatives as non-toxic anti-proliferative compounds against human colon carcinoma
HCT116 cells, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 1100-1109, DOI:
10.1080/14756366.2020.1755670
To link to this article: https://doi.org/10.1080/14756366.2020.1755670
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1100–1109
https://doi.org/10.1080/14756366.2020.1755670
BRIEF REPORT
Synthesis and biological evaluation of new 3(2H)-pyridazinone derivatives as
non-toxic anti-proliferative compounds against human colon carcinoma
HCT116 cells
a
b
c
d
, Giustino Orlandod, Simonetta Di Simoned,
€
Zeynep Ozdemir , Semra Utku , Bijo Mathew , Simone Carradori
Mehmet Abdullah Alagoz , Azime Berna Ozc¸elik , Mehtap Uysal and Claudio Ferranted
a
e
e,f
€
€
a
b
_
€ €
Department of Pharmaceutical Chemistry, Inonu University, Malatya, Turkey; Department of Pharmaceutical Chemistry, Mersin University,
c
Mersin, Turkey; Department of Pharmaceutical Chemistry, Division of Drug Design and Medicinal Chemistry Research Lab, Ahalia School of
Pharmacy, Palakkad, India; Department of Pharmacy, “G. d’Annunzio” University of Chieti-Pescara, Chieti, Italy; Department of Pharmaceutical
Chemistry, Gazi University, Ankara, Turkey; Department of Pharmaceutical Chemistry, Erzincan Binali Yıldırım University, Erzincan, Turkey
d
e
f
ABSTRACT
ARTICLE HISTORY
Received 12 March 2020
Accepted 8 April 2020
Novel 3(2H)-pyridazinone derivatives were designed, synthesised in satisfactory yields and evaluated in dif-
ferent experimental assays to assess their preliminary toxicity in vivo and anti-proliferative effects against
HCT116 cell lines in vitro. Artemia salina lethality test provided LC50 values >100mg/mL for all compounds.
Successive assays revealed that some compounds were endowed with a promising anti-proliferative effect
against HCT116 cells, alone or stimulated by serotonin as a pro-inflammatory factor in order to mimick an
inflamed model in vivo of cancer cell microenvironment. Moreover, the kinurenic acid level after treatment
with these newly synthesised compounds was monitored as a marker of anti-proliferation in colon carcin-
oma models. The IC50 values obtained for the best-in-class compounds were comparable to that of dauno-
rubicin as a reference drug. Conversely, these compounds were not able to counteract the spontaneous
migration of human cancer HCT116 cell line in the wound healing paradigm.
KEYWORDS
Serotonin; anti-proliferative
agents; pyridazinone;
kynurenic acid; HCT116;
wound healing; Artemia
salina lethality test
1. Introduction
that are biologically active. Literature survey revealed that substituted
pyridazinones have reported to possess pharmacological activities,
Cancer consists of an uncontrolled proliferation of cells in different
tissues and organs; it is a disease whose clinical appearance, treat-
ment and approach are different from each other. Cancer is a
major global health problem and it is currently the second leading
cause of death in the world being expected to surpass cardiovas-
cular diseases in the next few years1,2. Many factors, from bacteria
to viruses, from radiation to heredity, from environmental factors
to nutritional habits and chemicals, are accused of cancer forma-
tion. In the data announced by the World Health Organisation
(WHO), approximately 18 million people were diagnosed with can-
cer in 2018, and around 10 million people died from cancer.
According to data of Global Cancer Obervatory (GLOBOCAN), the
most common types are lung (2.1 million), breast (2.09 million),
colorectal (1.8 million), prostate (1.3 million), stomach (1 million)
cancer. According to cancer-related deaths, lung (1.8 million), colo-
rectal (881 thousand), stomach (783 thousand), liver (782 thou-
sand) and breast (627 thousand) are listed. Colorectal carcinomas
(CRC) are one of the most common types of cancer in the world
that cause death. CRC metastases account for 40–50% of recently
6–12
which can be rationalised in the SAR study reported in Figure 1
.
There are also compounds which were shown to have anti-cancer or
cytostatic activity in the literature against HEP3B (liver cancer cells),
HCT116 (colon cancer cells), SH-SY5Y (neuroblastoma cells) and
promising selectivity index with respect to human fibroblasts13–16
.
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.
Pursuing our efforts to discover novel anti-cancer com-
pounds17–19 and with the aim of enlarging the SAR knowledge
within this chemical scaffold, we designed fifteen new 3(2H)-pyri-
dazinones investigating the anti-proliferative effects against the
human HCT116 cell line, their toxicity in the Artemia salina lethal-
ity assay in vivo, the HCT116 viability after serotonin challenging
and compound treatment, the release of kynurenic acid after com-
pound treatment and, lastly, the capability to limit the spontan-
eous migration of HCT116 cells in the wound healing paradigm.
diagnosed cases and are correlated with high morbidity3,4
.
In medicinal chemistry pyridazinones have been the subject of
intensive synthetic investigations, because they possess a wide spec-
trum of pharmacological activities and gained importance in recent
years5. A number of compounds such as zardaverine/imidazole,
2. Experimental protocols
The fine chemicals and all solvents used in this study were pur-
chased locally from Merck (Darmstadt, Germany) and Aldrich
bemoradan, indolindan, pimobendan are examples of pyridazinones Chemical Co. (Steinheim, Germany).
CONTACT Simone Carradori
simone.carradori@unich.it
Department of Pharmacy, “G. d’Annunzio” University of Chieti-Pescara, Chieti 66100, Italy; Bijo Mathew
Department of Pharmaceutical Chemistry, Division of Drug Design and Medicinal Chemistry Research
bijovilaventgu@gmail.com, bijo.mathew@ahalia.ac.in
Lab, Ahalia School of Pharmacy, Palakkad 678557, Kerala, India.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1101
Figure 1. Structure-activity relationships (SARs) within the 3(2H)-pyridazinone derivatives reported in the literature.
2.1. Chemical studies
crystallised from ethanol. M.P.: 182 ꢀC. (Lit: M.P. 180–182 ꢀC). Yield:
58%, C11H12FN2O2 Calcd.: 223.2270. Found: 223.2854. NMR spectra
are in accordance with literature data.
Melting points of the compounds were determined on
Electrothermal 9200 melting points apparatus (Southent, Great
Britain) and the values given are uncorrected. The IR spectra of
the compounds were recorded on
a Bruker Vector 22 IR
Spectrophotometer (Bruker Analytische Messtechnik, Karlsruhe,
Germany). The 1H-NMR and 13C-NMR spectra of the compounds
2.1.3. Synthesis of 6-(3-fluoro-4-methoxyphenyl)-3(2H)-pyridazinone (III)
A solution of 0.043 mol of bromine in 25 mL of glacial acetic acid
was added dropwise to a solution of 0.039 mol of 6-(3-fluoro-4-
methoxyphenyl)-4,5-dihydro-3(2H)-pyridazinone (II) in 100 mL of
glacial acetic acid at 60–70 ꢀC. Then, the reaction mixture was
refluxed for 3 h. After cooling to 5 ꢀC, it was poured into ice water
and converted to free base with ammonium hydroxide. The pre-
cipitate was collected by filtration, washed with cold water until
neutral, dried, and crystallised from ethanol-water mixture. M.P.:
221 ꢀC. (Lit: M.P. 220–222 ꢀC). Yield: 76%, C11H10FN2O2 Calcd.:
221.0726, Found: 221.0721. NMR spectra are in accordance with
literature data.
were recorded on
a
Bruker 400 MHz-NMR Spectrometer
(Rheinstetten, Karlsruhe, Germany) using tetramethylsilane as an
internal standard. All the chemical shifts were recorded as d
(ppm). The mass spectra (HRMS) of the compounds were recorded
on Waters Acquity Ultra Performance Liquid Chromatograpy
Micromass which combined LCT PremierTM XE UPLC/MS TOFF
spectrophotometer (Waters Corp, Milford, USA) by ESI þ and
ESI– techniques.
2.1.1. Synthesis of 4-(3-fluoro-4-methoxyphenyl)-4-oxobutanoic
acid (I)
A mixture of 0.275 mol aluminium chloride, 20 mL carbon disul-
phide and 0.25 mol succinic anhydride was added portionwise in
standard conditions to a mixture of 0.25 mol 2-fluoroanisole and
50 mL of carbon disulphide. Then, the mixture was refluxed for 4 h
at 40–50 ꢀC. After cooling, the residue was poured onto ice water
and the precipitate was collected, dried and recrystallized from
water. M.P.: 164 ꢀC. (Lit: M.P. 168 ꢀC). Yield: 78%, C11H12FO4 Calcd.:
226.0720, Found: 227.0724. NMR spectra are in accordance with
literature data.
2.1.4. Synthesis of ethyl 6-(3-fluoro-4-methoxyphenyl)-3(2H)-pyri-
dazinone-2-yl-acetate (IV)
A
mixture of required 6-substituted-3(2H)-pyridazinones (III)
(0.01 mol), ethyl 3-bromo-acetate (0.02 mol) and potassium carbon-
ate (0.02 mol) 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
ethanol to give the ester. M.P.: 126 ꢀC. Yield: 69%, C15H16FN2O4
Calcd.: 307.1094, Found: 307.1074. 1H-NMR (400 MHz) (DMSO-d6):
d 8.09 (d, 1H, pyridazinone H5), 7.05–7.82 (m, 3H, phenyl H2, H5,
H6), 7.31 (t, 1H, pyridazinone H4), 4.88 (s, 2H, CH2COOCH2CH3),
4.10 (q, 2H, CH2COOCH2CH3), 3.91 (s, 3H, OCH3), 1.22 (t, 3H,
2.1.2. Synthesis of 6-(3-fluoro-4-methoxyphenyl)-4,5-dihydro-3(2H)-
pyridazinone (II)
0.01 Mol of 4-(3-fluoro-4-methoxyphenyl)-4-oxobutanoic acid and
0.015 mol of hydrazine hydrate (0.85 mL; 55%) in 30 mL of ethanol
were refluxed for 4 h. The reaction mixture was cooled and the
precipitate thus formed was collected by filtration, dried, CH2COOCH2CH3).

1102
Z. ÖZDEMIR ET AL.
2.1.5. Synthesis of 6-(3-fluoro-4-methoxyphenyl)-3(2H)-pyridazi- –N–CH2–C¼O), 7.10 (1H; d; pyridazinone H5), 7.26 (1H; d; pyridazi-
none H4), 7.12–7.97 (7H; m; phenyl protons), 8.14 (1H; s; –N¼CH–),
none-2-yl-acetohydrazide (V)
To the ethanolic solution of ethyl 6-substituted-3(2H)-pyridazi-
11.96 (1H; s; –NH–N); 13C-NMR (DMSO-d6, 300 MHz): d 53.9 (1C;
CH3O), 56.7 (1C; –N–CH2–C¼O), 113.4 (1C; CF3), 113.6 (1C; ¼CH),
none-2-yl-acetate (IV) (25 mL, 0.01 mol) hydrazine hydrate (99%)
114.3 (1C; pyridazinone C5), 114.5 (2C; 4-fluorophenyl C3,5), 126.0
(3 mL) was added and stirred for 3 h at room temperature. The
(2C; 4-fluorophenyl C2,6), 127.5 (1C; pyridazinone C4), 128.1 (2C; 3-
obtained precipitate was filtered off, washed with water, dried
fluoro-4-methoxyphenyl C2,6), 131.4 (1C; 4-fluorophenyl C1), 138.3
(2C; 3-fluoro-4-methoxyphenyl C3,5), 142.8 (1C; 3-fluoro-4-methoxy-
phenyl C1), 151.2 (1C; pyridazinone C6), 152.9 (1C; 4-fluorophenyl
C4), 159.4 (1C; 3-fluoro-4-methoxyphenyl C4), 163.8 (1C;
CH2–N–C¼O), 168.5 (1C; pyridazinone C3); C21H17F4N4O3 MS (ESIþ)
Calcd.: 449.1237, Found: m/z 449.1237 (Mþ; 100.0%).
and recrystallized from ethanol. M.P.: 206 ꢀC. Yield: 76%,
C13H13FN4O3 Calcd.: 293.1050, Found: 293.1060.
2.1.6. Synthesis of substituted/nonsubstituted benzalhydrazone
derivatives of 6-(3-fluoro-4-methoxyphenyl)-3(2H)-pyridazinone-2-
yl-acetohydrazide (via-o)
A mixture of 6-(3-fluoro-4-methoxyphenyl)-3(2H)-pyridazinone-2-yl-
2.1.6.4. N’-(2-fluorobenzylidene)-2–(3-(3-fluoro-4-methoxyphenyl)-
6-oxopyridazin-1(6H)-yl)acetohydrazide (VId). White crystals; yield:
68%; M.P.: 222 ꢀC; IR (ꢀ cmꢁ1, ATR): 1696 (C¼O; hydrazone), 1676
(C¼O; pyridazinone ring), 1596 (C¼N); 1H-NMR (DMSO-d6,
300 MHz): d 3.87 (3H; s; CH3O), 5.34 (2H; s; –N–CH2–C¼O), 7.09
(1H; d; pyridazinone H5), 7.12 (1H; d; pyridazinone H4), 7.10–8.26
(7H; m; phenyl protons), 8.40 (1H; s; –N¼CH–), 12.00 (1H; s;
–NH–N); 13C-NMR (DMSO-d6, 300 MHz): d 53.9 (1C; CH3O), 56.6 (1C;
–N–CH2–C¼O), 113.5 (1C; CF3), 113.7 (1C; ¼CH), 114.4 (1C; pyrida-
zinone C5), 127.4 (1C; 2-trifluoromethylphenyl C5), 130.0 (1C; 2-tri-
fluoromethylphenyl C4), 130.5 (1C; 2-trifluoromethylphenyl C6),
132.2 (1C; 2-trifluoromethylphenyl C3), 133.3 (1C; pyridazinone C4),
139.8 (1C; 3-fluoro-4-methoxyphenyl C6), 142.7 (1C; 3-fluoro-4-
methoxyphenyl C5), 142.9 (1C; 2-trifluoromethylphenyl C1), 148.6
(1C; 3-fluoro-4-methoxyphenyl C2), 148.6 (1C; 3-fluoro-4-methoxy-
phenyl C1), 151.2 (1C; 2-trifluoromethylphenyl C2), 152.9 (1C; pyri-
dazinone C6), 159.3 (1C; 3-fluoro-4-methoxyphenyl C3), 159.4 (1C;
3-fluoro-4-methoxyphenyl C4), 163.8 (1C; CH2–N–C¼O), 168.5 (1C;
pyridazinone C3); C21H17F4N4O3 MS (ESIþ) Calcd.: 449.1237, Found:
m/z 449.1258 (Mþ; 100.0%).
acetohydrazide
V
(0.01 mol) and appropriate benzaldehyde
(0.01 mol) was refluxed in ethanol (15 mL) for 6 h. Then, the mix-
ture was poured into ice water. The formed precipitate was recrys-
tallized from the appropriate solvent. The compounds were
identified by IR, 1H-NMR, 13C-NMR and mass spectra. All spectral
data of the compounds were in accordance with the assigned
structures as shown below.
2.1.6.1. N’-benzylidene-2-(3-(3-fluoro-4-methoxyphenyl)-6-oxopyri-
dazin-1(6H)-yl)acetohydrazide (VIa). White crystals; yield: 80%;
M.P.: 132 ꢀC; IR (ꢀ cmꢁ1, ATR): 1698 (C¼O; hydrazone), 1648 (C¼O;
pyridazinone ring), 1587 (C¼N); 1H-NMR (DMSO-d6, 300 MHz): d
3.90 (3H; s; CH3O), 5.30 (2H; s; –N–CH2–C¼O), 7.09 (1H; d; pyridazi-
none H5), 7.27 (1H; d; pyridazinone H4), 7.11–8.14 (8H; m; phenyl
protons), 8.24 (1H; s; –N¼CH–), 11.76 (1H; s; –NH–N). 13C-NMR
(DMSO-d6, 300 MHz): d 53.9 (1C; CH3O), 56.7 (1C; –N–CH2–C¼O),
113.4 (1C; ¼CH), 113.8 (1C; pyridazinone C5), 114.5 (1C; phenyl C4),
127.3 (2C; phenyl C3,5), 129.1 (2C; phenyl C2,6), 134.3 (1C; pyridazi-
none C4), 142.8 (2C; 3-fluoro-4-methoxyphenyl C2,6), 144.5 (1C;
phenyl C1), 147.6 (2C; 3-fluoro-4-methoxyphenyl C3,5), 148.6 (1C; 3-
fluoro-4-methoxyphenyl C1), 151.2 (1C; pyridazinone C6), 159.3 (1C;
3-fluoro-4-methoxyphenyl C4), 163.5 (1C; CH2–N–C¼O), 168.2 (1C;
pyridazinone C3); C20H17FN4O3 MS (ESIþ) Calcd.: 381.1348, Found:
m/z 381.1348 (Mþ; 100.0%).
2.1.6.5. N’-(2-methylbenzylidene)-2–(3-(3-fluoro-4-methoxyphenyl)-
6-oxopyridazin-1(6H)-yl)acetohydrazide (VIe). White crystals; yield:
96%; M.P.: 210 ꢀC; IR (ꢀ cmꢁ1, ATR): 1700 (C¼O; hydrazone), 1640
(C¼O; pyridazinone ring), 1588 (C¼N); 1H-NMR (DMSO-d6,
300 MHz): d 2.45 (3H; s; CH3), 3.90 (3H; s; CH3O), 5.29 (2H; s;
–N–CH2–C¼O), 7.08 (1H; d; pyridazinone H5), 7.11 (1H; d; pyridazi-
none H4), 7.09–8.13 (7H; m; phenyl protons), 8.31 (1H; s; –N¼CH–),
11.69 (1H; s; –NH–N); 13C-NMR (DMSO-d6, 300 MHz): d 20.1 (1C;
CH3), 53.9 (1C; CH3O), 56.5 (1C; –N–CH2–C¼O), 113.6 (1C; ¼CH),
114.3 (1C; pyridazinone C5), 126.5 (1C; 2-methylphenyl C5), 126.6
(1C; 2-methylphenyl C4), 127.1 (1C; 2-methylphenyl C6), 130.0 (1C;
2-methylphenyl C3), 131.3 (1C; pyridazinone C4), 131.4 (1C; 3-flu-
oro-4-methoxyphenyl C6), 132.3 (1C; 3-fluoro-4-methoxyphenyl C5),
137.1 (1C; 2-methylphenyl C1), 142.8 (1C; 2-methylphenyl C2),
143.9 (1C; 3-fluoro-4-methoxyphenyl C2), 151.2 (1C; 3-fluoro-4-
methoxyphenyl C1), 152.9 (1C; pyridazinone C6), 159.3 (1C; 3-flu-
oro-4-methoxyphenyl C3), 159.4 (1C; 3-fluoro-4-methoxyphenyl C4),
163.4 (1C; CH2–N–C¼O), 168.1 (1C; pyridazinone C3); C21H17F4N4O3
MS (ESIþ) Calcd.: 449.1237, Found: m/z 449.1258 (Mþ; 100.0%).
2.1.6.2. N’-(4-fluorobenzylidene)-2–(3-(3-fluoro-4-methoxyphenyl)-
6-oxopyridazin-1(6H)-yl)acetohydrazide (VIb). White crystals; yield:
83%; M.P.: 238 ꢀC; IR (ꢀ cmꢁ1, ATR): 1693 (C¼O; hydrazone), 1652
(C¼O; pyridazinone ring), 1514 (C¼N); 1H-NMR (DMSO-d6,
300 MHz): d 3.90 (3H; s; CH3O), 5.30 (2H; s; –N–CH2–C¼O), 7.08
(1H; d; pyridazinone H5), 7.11 (1H; d; pyridazinone H4), 7.27–7.82
(7H; m; phenyl protons), 8.10 (1H; s; –N¼CH–), 11.76 (1H; s;
–NH–N); 13C-NMR (DMSO-d6, 300 MHz): d 53.7 (1C; CH3O), 56.7 (1C;
–N–CH2–C¼O), 113.4 (1C; ¼CH), 113.6 (1C; pyridazinone C5), 114.3
(2C; 4-fluorophenyl C3,5), 116.3 (2C; 4-fluorophenyl C2,6), 127.5 (1C;
pyridazinone C4), 129.5 (2C; 3-fluoro-4-methoxyphenyl C2,6), 129.6
(1C; 4-fluorophenyl C1), 131.4 (2C; 3-fluoro-4-methoxyphenyl C3,5),
142.8 (1C; 3-fluoro-4-methoxyphenyl C1), 148.6 (1C; pyridazinone
C6), 152.9 (1C; 4-fluorophenyl C4), 159.3 (1C; 3-fluoro-4-methoxy-
phenyl C4), 164.3 (1C; CH2–N–C¼O), 168.3 (1C; pyridazinone C3);
C20H17F2N4O3 MS (ESIþ) Calcd.: 399.1269, Found: m/z 399.1285
(Mþ; 100.0%).
2.1.6.6.
N’-(2-methoxybenzylidene)-2–(3-(3-fluoro-4-methoxy-
phenyl)-6-oxopyridazin-1(6H)-yl)acetohydrazide (VIf). White crys-
tals; yield: 80%; M.P.: 214 ꢀC; IR (ꢀ cmꢁ1, ATR): 1693 (C¼O;
hydrazone), 1649 (C¼O; pyridazinone ring), 1589 (C¼N); 1H-NMR
2.1.6.3. N’-(4-trifluoromethylbenzylidene)-2–(3-(3-fluoro-4-methoxy-
phenyl)-6-oxopyridazin-1(6H)-yl)acetohydrazide (VIc). White crys- (DMSO-d6, 300 MHz): d 3.86 (3H; s; CH3O), 3.90 (3H; s; CH3O), 5.29
tals; yield: 84%; M.P.: 252 ꢀC; IR (ꢀ cmꢁ1, ATR): 1704 (C¼O; (2H; s; –N–CH2–C¼O), 7.00 (1H; d; pyridazinone H5), 7.09 (1H; d;
hydrazone), 1699 (C¼O; pyridazinone ring), 1587 (C¼N); 1H-NMR pyridazinone H4), 7.08–8.13 (7H; m; phenyl protons), 8.38 (1H; s;
(DMSO-d6, 300 MHz):
d
3.90 (3H; s; CH3O), 5.33 (2H; s; –N¼CH–), 11.71 (1H; s; –NH–N); 13C-NMR (DMSO-d6, 300 MHz): d

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1103
53.9 (2C; CH3O), 56.7 (1C; –N–CH2–C¼O), 113.6 (1C; ¼CH), 114.5 (1C; 4-nitrophenyl C3), 114.3 (1C; 4-nitrophenyl C5), 124.4 (1C; 4-
(1C; pyridazinone C5), 121.1 (1C; 2-methoxyphenyl C5), 122.3 (1C; nitrophenyl C2), 124.4 (1C; 4-nitrophenyl C6), 127.5 (1C; pyridazi-
2-methoxyphenyl C4), 126.0 (1C; 2-methoxyphenyl C6), 127.5 (1C; none C4), 128.3 (1C; 3-fluoro-4-methoxyphenyl C2), 130.0 (1C; 3-flu-
2-methoxyphenyl C3), 131.4 (1C; pyridazinone C4), 132.1 (1C; 3-flu- oro-4-methoxyphenyl C6), 140.6 (1C; 4-nitrophenyl C1), 142.9 (1C;
oro-4-methoxyphenyl C6), 140.1 (1C; 3-fluoro-4-methoxyphenyl C5), 3-fluoro-4-methoxyphenyl C5), 148.2 (1C; 3-fluoro-4-methoxy-
140.2 (1C; 2-methoxyphenyl C1), 142.8 (1C; 3-fluoro-4-methoxy- phenyl C1), 151.2 (1C; 3-fluoro-4-methoxyphenyl C3), 152.8 (1C;
phenyl C2), 148.6 (1C; 3-fluoro-4-methoxyphenyl C1), 151.2 (1C; pyridazinone C6), 159.3 (1C; 4-nitrophenyl C4), 159.4 (1C; 3-fluoro-
2-methoxyphenyl C2), 152.9 (1C; pyridazinone C6), 158.1 (1C; 3-flu- 4-methoxyphenyl C4), 164.0 (1C; CH2–N–C¼O), 168.7 (1C; pyridazi-
oro-4-methoxyphenyl C3), 159.3 (1C; 3-fluoro-4-methoxyphenyl C4), none C3); C20H17FN5O5 MS (ESIþ) Calcd.: 426.1214, Found: m/z
159.4 (1C; CH2–N–C¼O), 168.1 (1C; pyridazinone C3); C21H20FN4O4 426.1205 (Mþ; 100.0%).
MS (ESIþ) Calcd.: 411.1469, Found: m/z 411.1468 (Mþ; 100.0%).
2.1.6.10.
N’-(4-isopropylbenzylidene)-2–(3-(3-fluoro-4-methoxy-
2.1.6.7. N’-(4-methylbenzylidene)-2–(3-(3-fluoro-4-methoxyphenyl)-
phenyl)-6-oxopyridazin-1(6H)-yl)acetohydrazide (VIj). White crys-
6-oxopyridazin-1(6H)-yl)acetohydrazide (VIg). White crystals; yield: tals; yield: 71%; M.P.: 164 ꢀC; IR (ꢀ cmꢁ1, ATR): 1700 (C¼O;
74%; M.P.: 220 ꢀC; IR (ꢀ cmꢁ1, ATR): 1696 (C¼O; hydrazone), 1656 hydrazone), 1656 (C¼O; pyridazinone ring), 1584 (C¼N); 1H-NMR
(C¼O; pyridazinone ring), 1586 (C¼N); 1H-NMR (DMSO-d6, (DMSO-d6, 300 MHz): d 1.21 (6H; d; CH3), 2.90–2.95 (1H; q; –CH–),
300 MHz): d 2.34 (3H; s; CH3), 3.90 (3H; s; CH3O), 5.28 (2H; s; 3.90 (3H; s; CH3O), 5.28 (2H; s; –N–CH2–C¼O), 7.09 (1H; d; pyridazi-
–N–CH2–C¼O), 7.07 (1H; d; pyridazinone H5), 7.10 (1H; d; pyridazi- none H5), 7.26 (1H; d; pyridazinone H4), 7.11–8.13 (7H; m; phenyl
none H4), 7.08–8.10 (7H; m; phenyl protons), 8.13 (1H; s; –N¼CH–), protons), 8.20 (1H; s; –N¼CH–), 11.71 (1H; s; –NH–N); 13C-NMR
11.69 (1H; s; –NH–N); 13C-NMR (DMSO-d6, 300 MHz): d 20.1 (1C; (DMSO-d6, 300 MHz): d 24.1 (2C; CH3), 39.6 (1C; CH), 53.9 (1C;
CH3), 53.9 (1C; CH3O), 56.5 (1C; –N–CH2–C¼O), 113.6 (1C; ¼CH), CH3O), 56.5 (1C; –N–CH2–C¼O), 114.4 (1C; ¼CH), 127.1 (1C; pyrida-
114.3 (1C; pyridazinone C5), 126.5 (1C; 4-methylphenyl C5), 126.6 zinone C5), 127.2 (2C; 4-isopropylphenyl C3,5), 127.3 (1C; 4-isopro-
(1C; 4-methylphenyl C4), 127.1 (1C; 4-methylphenyl C6), 130.0 (1C; pylphenyl C4), 127.4 (2C; 4-isopropylphenyl C2,6), 127.5 (1C;
4-methylphenyl C3), 131.3 (1C; pyridazinone C4), 131.4 (1C; 3-flu- pyridazinone C4), 132.1 (2C; 3-fluoro-4-methoxyphenyl C5,6), 142.8
oro-4-methoxyphenyl C6), 132.3 (1C; 3-fluoro-4-methoxyphenyl C5), (1C; 4-isopropylphenyl C1), 144.6 (1C; 3-fluoro-4-methoxyphenyl
137.1 (1C; 4-methylphenyl C1), 142.8 (1C; 4-methylphenyl C2), C2), 148.6 (1C; 3-fluoro-4-methoxyphenyl C1), 151.1 (1C; pyridazi-
143.9 (1C; 3-fluoro-4-methoxyphenyl C2), 151.2 (1C; 3-fluoro-4- none C6), 152.9 (1C; 3-fluoro-4-methoxyphenyl C3), 159.3 (1C; 3-flu-
methoxyphenyl C1), 152.9 (1C; pyridazinone C6), 159.3 (1C; 3-flu- oro-4-methoxyphenyl C4), 159.4 (1C; CH2–N–C¼O), 168.1 (1C;
oro-4-methoxyphenyl C3), 159.4 (1C; 3-fluoro-4-methoxyphenyl C4), pyridazinone C3); C23H23FN4O3 MS (ESIþ) Calcd.: 423.1832, Found:
163.4 (1C; CH2–N–C¼O), 168.1 (1C; pyridazinone C3); C21H20FN4O3 m/z 423.1817 (Mþ; 100.0%).
MS (ESIþ) Calcd.: 395.1519, Found: m/z 395.1513 (Mþ; 100.0%).
2.1.6.11.
N’-(2-bromobenzylidene)-2–(3-(3-fluoro-4-methoxy-
2.1.6.8.
N’-(4-methoxybenzylidene)-2–(3-(3-fluoro-4-methoxy-
phenyl)-6-oxopyridazin-1(6H)-yl)acetohydrazide (VIk). White crys-
phenyl)-6-oxopyridazin-1(6H)-yl)acetohydrazide (VIh). White crys- tals; yield: 89%; M.P.: 189 ꢀC; IR (ꢀ cmꢁ1, ATR): 1705 (C¼O;
tals; yield: 85%; M.P.: 207 ꢀC; IR (ꢀ cmꢁ1, ATR): 1687 (C¼O; hydrazone), 1667 (C¼O; pyridazinone ring), 1588 (C¼N); 1H-NMR
hydrazone), 1662 (C¼O; pyridazinone ring), 1599 (C¼N); 1H-NMR (DMSO-d6, 300 MHz):
d 3.89 (3H; s; CH3O), 5.32 (2H; s;
(DMSO-d6, 300 MHz): d 3.81 (3H; s; CH3O), 3.90 (3H; s; CH3O), 5.28 –N–CH2–C¼O), 7.09 (1H; d; pyridazinone H5), 7.27 (1H; d; pyridazi-
(2H; s; –N–CH2–C¼O), 7.00 (1H; d; pyridazinone H5), 7.02 (1H; d; none H4), 7.37–8.38 (7H; m; phenyl protons), 8.58 (1H; s; –N¼CH–),
pyridazinone H4), 7.03–7.98 (7H; m; phenyl protons), 8.11 (1H; s; 11.95 (1H; s; –NH–N); 13C-NMR (DMSO-d6, 300 MHz): d 53.9 (1C;
–N¼CH–), 11.63 (1H; s; –NH–N); 13C-NMR (DMSO-d6, 300 MHz): d CH3O), 56.6 (1C; –N–CH2–C¼O), 113.6 (1C; ¼CH), 114.3 (1C;
53.9 (1C; CH3O), 54.1 (1C; CH3O), 56.7 (1C; –N–CH2–C¼O), 113.4 pyridazinone C5), 123.8 (1C; 2-bromophenyl C5), 127.5 (1C; 2-bro-
(1C; ¼CH), 113.6 (1C; pyridazinone C5), 113.6 (1C; 4-methoxy- mophenyl C3), 128.4 (1C; 2-bromophenyl C4), 130.0 (1C; 2-bromo-
phenyl C3), 113.8 (1C; 4-methoxyphenyl C5), 114.3 (1C; 4-methoxy- phenyl C6), 131.4 (1C; pyridazinone C4), 132.2 (2C; 3-fluoro-4-
phenyl C2), 114.5 (1C; 4-methoxyphenyl C6), 126.9 (1C; methoxyphenyl C2,6), 133.1 (1C; 3-fluoro-4-methoxyphenyl C5),
pyridazinone C4), 127.6 (1C; 3-fluoro-4-methoxyphenyl C2), 127.6 133.5 (1C; 2-bromophenyl C1), 142.8 (1C; 3-fluoro-4-methoxy-
(1C; 3-fluoro-4-methoxyphenyl C6), 129.1 (1C; 4-methoxyphenyl phenyl C1), 148.6 (1C; 2-bromophenyl C2), 151.2 (2C; 3-fluoro-4-
C1), 142.8 (1C; 3-fluoro-4-methoxyphenyl C5), 144.3 (1C; 3-fluoro-4- methoxyphenyl C3, pyridazinone C6), 152.8 (1C; 3-fluoro-4-methox-
methoxyphenyl C1), 152.9 (1C; 3-fluoro-4-methoxyphenyl C3), yphenyl C4), 159.4 (1C; CH2–N–C¼O), 168.4 (1C; pyridazinone C3);
159.3 (1C; pyridazinone C6), 159.4 (1C; 4-methoxyphenyl C4), 161.2 C20H16BrFN4O3 MS (ESIþ) Calcd.: 459.0468, Found: m/z 459.0467
(1C; 3-fluoro-4-methoxyphenyl C4), 163.2 (1C; CH2–N–C¼O), 168.0 (Mþ; 100.0%).
(1C; pyridazinone C3); C21H20FN4O4 MS (ESIþ) Calcd.: 411.1469,
Found: m/z 411.1483 (Mþ; 100.0%).
2.1.6.12. N’-(2-fluorobenzylidene)-2–(3-(3-fluoro-4-methoxyphenyl)-
6-oxopyridazin-1(6H)-yl)acetohydrazide (VIl). White crystals; yield:
72%; M.P.: 224 ꢀC; IR (ꢀ cmꢁ1, ATR): 1689 (C¼O; hydrazone), 1648
2.1.6.9. N’-(4-nitrobenzylidene)-2–(3-(3-fluoro-4-methoxyphenyl)-6-
1
oxopyridazin-1(6H)-yl)acetohydrazide (VIi). White crystals; yield: (C¼O; pyridazinone ring), 1585 (C¼N); H-NMR (DMSO-d6, 300 MHz):
38%; M.P.: 265 ꢀC; IR (ꢀ cmꢁ1, ATR): 1705 (C¼O; hydrazone), 1647 d 3.90 (3H; s; CH3O), 5.31 (2H; s; –N–CH2–C¼O), 7.09 (1H; d; pyridazi-
(C¼O; pyridazinone ring), 1581 (C¼N); 1H-NMR (DMSO-d6, none H5), 7.11 (1H; d; pyridazinone H4), 7.26–8.13 (7H; m; phenyl pro-
300 MHz): d 3.90 (3H; s; CH3O), 5.35 (2H; s; –N–CH2–C¼O), 7.10 tons), 8.46 (1H; s; –N¼CH–), 11.87 (1H; s; –NH–N); 13C-NMR (DMSO-d6,
(1H; d; pyridazinone H5), 7.26 (1H; d; pyridazinone H4), 7.12–8.29 300 MHz): d 53.7 (1C; CH3O), 56.4 (1C; –N–CH2–C¼O), 113.4 (1C;
(7H; m; phenyl protons), 8.31 (1H; s; –N¼CH–), 12.06 (1H; s; ¼CH), 113.6 (1C; pyridazinone C5), 114.3 (1C; 2-fluorophenyl C5),
–NH–N); 13C-NMR (DMSO-d6, 300 MHz): d 53.8 (1C; CH3O), 56.4 (1C; 116.3 (1C; 2-fluorophenyl C3), 121.9 (1C; 2-bromophenyl C4), 126.9
–N–CH2–C¼O), 113.4 (1C; ¼CH), 113.6 (1C; pyridazinone C5), 113.7 (1C; 2-fluorophenyl C6), 127.6 (1C; pyridazinone C4), 131.4 (1C; 3-

1104
Z. ÖZDEMIR ET AL.
Scheme 1. Synthesis of compounds VIa-o. Reagents and conditions: (i) AlCl3, CS2; (ii) H2NNH2, EtOH, reflux (6h); (iii) Br2, CH3COOH, reflux (overnight); (iv)
.
BrCH2COOCH2CH3, K2CO3, acetone, reflux (overnight); (v) H2NNH2 H2O, MeOH, rt; (vi) EtOH, reflux (6 h), nonsubstituted/substitutedbenzaldehyde.
fluoro-4-methoxyphenyl C2), 137.4 (1C; 3-fluoro-4-methoxyphenyl C6), 2.1.6.14.
N’-(4-hydroxybenzylidene)-2–(3-(3-fluoro-4-methoxy-
142.8 (1C; 3-fluoro-4-methoxyphenyl C5), 148.6 (1C; 2-fluorophenyl
phenyl)-6-oxopyridazin-1(6H)-yl)acetohydrazide (VIn). White crys-
tals; yield: 42%; M.P.: 251 ꢀC; IR (ꢀ cmꢁ1, ATR): 1683 (C¼O;
hydrazone), 1653 (C¼O; pyridazinone ring), 1581 (C¼N); 1H-NMR
C1), 151.2 (1C; 3-fluoro-4-methoxyphenyl C1), 152.9 (1C; 2-fluoro-
phenyl C2), 159.4 (1C; 3-fluoro-4-methoxyphenyl C3), 160.3 (1C; pyri-
dazinone C6), 161.9 (1C; 3-fluoro-4-methoxyphenyl C4), 163.6 (1C;
CH2–N–C¼O), 168.4 (1C; pyridazinone C3); C20H16F2N4O3 MS (ESIþ)
Calcd.: 399.1269, Found: m/z 399.1257 (Mþ; 100.0%).
(DMSO-d6, 300 MHz):
d 3.90 (3H; s; CH3O), 5.25 (2H; s;
–N–CH2–C¼O), 6.81 (1H; d; pyridazinone H5), 6.83 (1H; d; pyridazi-
none H4), 7.08–8.12 (7H; m; phenyl protons), 9.92 (1H; s; –N¼CH–),
11.55 (1H; s; –NH–N); 13C-NMR (DMSO-d6, 300 MHz): d 53.8 (1C;
CH3O), 56.5 (1C; –N–CH2–C¼O), 113.5 (1C; ¼CH), 113.7 (1C; pyrida-
zinone C5), 114.4 (1C; 4-hydroxyphenyl C3), 116.1 (1C; 4-hydroxy-
phenyl C5), 116.1 (1C; 4-hydroxyphenyl C2), 122.9 (1C;
4-hydroxyphenyl C6), 125.4 (1C; pyridazinone C4), 127.6 (1C; 3-flu-
oro-4-methoxyphenyl C2), 129.1 (1C; 3-fluoro-4-methoxyphenyl C6),
131.3 (1C; 4-hydroxyphenyl C1), 142.8 (1C; 3-fluoro-4-methoxy-
phenyl C5), 144.8 (1C; 3-fluoro-4-methoxyphenyl C1), 148.5 (1C; 3-
fluoro-4-methoxyphenyl C3), 151.2 (1C; pyridazinone C6), 152.9 (1C;
4-hydroxyphenyl C4), 159.8 (1C; 3-fluoro-4-methoxyphenyl C4),
159.9 (1C; CH2–N–C¼O), 167.9 (1C; pyridazinone C3); C20H17FN4O4
MS (ESIþ) Calcd.: 397.1312, Found: m/z 397.1309 (Mþ; 100.0%).
2.1.6.13.
N’-(4-bromobenzylidene)-2–(3-(3-fluoro-4-methoxy-
phenyl)-6-oxopyridazin-1(6H)-yl)acetohydrazide (VIm). White crys-
tals; yield: 69%; M.P.: 256 ꢀC; IR (ꢀ cmꢁ1, ATR): 1701 (C¼O;
hydrazone), 1651 (C¼O; pyridazinone ring), 1584 (C¼N); 1H-NMR
(DMSO-d6, 300 MHz):
d 3.90 (3H; s; CH3O), 5.30 (2H; s;
–N–CH2–C¼O), 7.09 (1H; d; pyridazinone H5), 7.26 (1H; d; pyridazi-
none H4), 7.11–8.11 (7H; m; phenyl protons), 8.21 (1H; s; –N¼CH–),
11.82 (1H; s; –NH–N); 13C-NMR (DMSO-d6, 300 MHz): d 53.9 (1C;
CH3O), 56.4 (1C; –N–CH2–C¼O), 113.4 (1C; ¼CH), 113.8 (1C; pyrida-
zinone C5), 114.3 (1C; 4-bromophenyl C3), 114.5 (1C; 4-bromo-
phenyl C5), 123.7 (1C; 4-bromophenyl C2), 127.5 (1C;
4-bromophenyl C6), 129.4 (1C; pyridazinone C4), 131.4 (1C; 3-flu-
oro-4-methoxyphenyl C2), 132.3 (1C; 3-fluoro-4-methoxyphenyl C6),
133.6 (1C; 4-bromophenyl C1), 142.8 (1C; 3-fluoro-4-methoxy-
phenyl C5), 143.4 (1C; 3-fluoro-4-methoxyphenyl C1), 148.5 (1C; 3-
2.1.6.15.
N’-(2,4-dichlorobenzylidene)-2–(3-(3-fluoro-4-methoxy-
phenyl)-6-oxopyridazin-1(6H)-yl)acetohydrazide (VIo). White crys-
tals; yield: 91%; M.P.: 229 ꢀC; IR (ꢀ cmꢁ1, ATR): 1705 (C¼O;
fluoro-4-methoxyphenyl C3), 151.2 (1C; pyridazinone C6), 152.9 (1C; hydrazone), 1645 (C¼O; pyridazinone ring), 1582 (C¼N); 1H-NMR
4-bromophenyl C4), 159.4 (1C; 3-fluoro-4-methoxyphenyl C4), 163.6 (DMSO-d6, 300 MHz):
d 3.90 (3H; s; CH3O), 5.32 (2H; s;
(1C; CH2–N–C¼O), 168.3 (1C; pyridazinone C3); C20H16BrFN4O3 MS –N–CH2–C¼O), 7.08 (1H; d; pyridazinone H5), 7.11 (1H; d; pyridazi-
(ESIþ) Calcd.: 459.0468, Found: m/z 459.0446 (Mþ; 100.0%).
none H4), 7.10–8.13 (6H; m; phenyl protons), 8.35 (1H; s; –N¼CH–),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1105
Table 1. Molecular structures, yields and melting points of VIa–o.
conditions and after challenging with serotonin (5-HT) at 1 ng/mL.
The anti-proliferative effects were compared to that induced by
daunorubicin (0.1–20 mg/mL), used as reference drug.
Finally, the effects of the most potent compounds were eval-
uated on the spontaneous migration of HCT116 cells, in the 48 h
following the experimental lesion of cell monolayer (wound heal-
ing paradigm). The detailed protocol related to wound healing
experimental model was described in our previous article20.
Compound
R1
R2
Yield (%)
mp (ꢀC)
VIa
VIb
VIc
VId
VIe
VIf
VIg
VIh
VIi
VIj
VIk
VIl
VIm
VIn
VIo
H
H
H
CF3
CH3
OCH3
H
H
H
H
Br
F
H
H
H
F
CF3
H
H
80
83
84
68
96
80
74
85
38
71
89
72
69
42
91
132
238
252
222
210
214
220
207
265
164
189
224
256
251
229
2.2.3. Statistical analysis
Results of in vitro studies were expressed as means standard
error (SE) of three experiments performed in triplicate. Statistical
analysis was determined through analysis of variance (ANOVA),
followed by post hoc Newman-Keuls comparison multiple test. The
level of significance was set at p < 0.05.
H
CH3
OCH3
NO2
i-C3H7
H
H
Br
OH
Cl
3. Results and discussion
In order to enlarge our SAR on this chemical scaffold, we investi-
gated the presence of different substituents on the hydrazone
moiety to explore the chemical space in terms of electronic and
steric effects. Moreover, we deleted the piperazine linker attached
to the pyridazinone core nucleus aiming at limiting the conform-
ational freedom of our compounds. The title compounds (VIa–o)
were synthesised according to the literature methods as outlined
in Scheme 1.
Cl
11.98 (1H; s; –NH–N); 13C-NMR (DMSO-d6, 300 MHz): d 53.7 (1C;
CH3O), 56.7 (1C; –N–CH2–C¼O), 113.4 (1C; ¼CH), 113.5 (1C; pyrida-
zinone C5), 113.7 (1C; 4-chlorophenyl C3), 114.4 (1C; 4-chloro-
phenyl C5), 129.9 (1C; 4-chlorophenyl C6), 130.7 (1C; pyridazinone
C4), 131.4 (1C; 3-fluoro-4-methoxyphenyl C2), 134.1 (1C; 3-fluoro-4-
methoxyphenyl C6), 135.5 (1C; 4-chlorophenyl C1), 139.5 (1C; 3-flu-
oro-4-methoxyphenyl C5), 142.9 (1C; 3-fluoro-4-methoxyphenyl C1),
148.6 (1C; 3-fluoro-4-methoxyphenyl C3), 151.2 (1C; pyridazinone
C6), 152.8 (1C; 4-chlorophenyl C2), 159.3 (1C; 4-chlorophenyl C4),
159.4 (1C; 3-fluoro-4-methoxyphenyl C4), 163.7 (1C; CH2–N–C¼O),
168.5 (1C; pyridazinone C3); C20H15Cl2FN4O3 MS (ESIþ) Calcd.:
449.0583, Found: m/z 449.0574 (Mþ; 100.0%).
Synthesis of the compounds was initiated by obtaining benzoyl
propanoic acid derivative (I) in the presence of succinic anhydride
and 2-fluoroanisole by anhydrous aluminium chloride catalysis.
Subsequently, the reaction of this compound with hydrazine
hydrate led to the formation of 4,5-dihydro-3(2H)-pyridazinone (II).
6-Substituted-3(2H)-pyridazinone derivative (III) was obtained by
oxidation of II with bromine in glacial acetic acid. Ethyl 6-substi-
tuted-3(2H)-pyridazinone-2-ylacetate derivative (IV) was obtained
by the reaction of III with ethyl bromoacetate in the presence of
K2CO3 in acetone. Then, 6-substituted-3(2H)-pyridazinone-2-ylace-
tohydrazide derivative (V) was synthesised by the condensation
reaction of IV with hydrazine hydrate. Ultimately, the title com-
pounds bearing benzylidenhydrazide structure were obtained by
the reaction of V with substituted/nonsubstituted benzaldehydes.
All of the title compounds were reported for the first time in this
study. The reaction yields ranged approximately from 38% to 96%.
Compound VIe was synthesised with the highest yield (96%),
while compound VIi with the lowest yield (38%). The physical and
spectral properties of the starting compounds were in accordance
with the literature. Molecular structures of title compounds were
confirmed by IR, 1H-NMR, 13C-NMR, and mass spectral data. Their
molecular structures, yields, and melting points are given in
Table 1.
Firstly, the biocompatibility limit of the compounds was deter-
mined through the A. salina brine shrimp lethality test in vivo. The
nauplii were stimulated with compounds VIa–o, in the range
0.01–10 mg/mL. The lethality test showed LC50 values >100 mg/mL
for all the compounds. Based on our previous investigations20,21, a
10-fold lower concentration (10 mg/mL) was selected for the subse-
quent in vitro cell-based tests. In this regard, the human colon
cancer HCT116 cell line was selected and treated with the afore-
mentioned molecules. The HCT116 viability was stimulated
through 5-HT challenging. 5-HT has long been described as a pro-
inflammatory factor, particularly in the gut22, with in vitro studies
2.2. Pharmacology
2.2.1. Artemia salina lethality test in vivo
In Artemia salina lethality bioassay, brine shrimp larvae were incu-
bated for 24 h with compounds VIa-o (0.01–10 mg/mL) dissolved
in the incubation medium (artificial sea water). The detailed proto-
col was described in our previous article20.
2.2.2. Human colon cancer HCT116 cell culture and experi-
ments in vitro
HCT116 cell line (ATCC CCL-247TM) was cultured in DMEM
R
V
(Euroclone) supplemented with 10% (v/v) heat-inactivated foetal
bovine serum and 1.2% (v/v) penicillin G/streptomycin in 75 cm2
tissue culture flask (n¼5 individual culture flasks for each condi-
tion) as previously reported with or without serotonin
treatment20.
In the same condition, the kynurenic acid (KA) extracellular
level was determined through a validated high performance liquid
chromatography (HPLC)-fluorimetric method21. To assess the cyto-
toxicity of synthesised compounds (VIa–o), a viability assay was
performed on 96 microwell plates, using 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) test. Cells were incu-
bated with compounds (10 mg/mL) for 24 h. An aliquot of 10 mL of
MTT (5 mg/mL) was added to each well and incubated for 3 h. The substantiating a mitogen role, mediated by different receptor
viability of HCT116 cell line was evaluated both in basal types towards multiple cell lines23. According to these findings, a

1106
Z. ÖZDEMIR ET AL.
Figure 2. Effects of serotonin (5-HT) in the range 10 pg/mL ꢁ 1 mg/mL on colon cancer HCT116 cell viability (MTT test). Data are means SE and analysed through
ꢂ
analysis of variance (ANOVA), followed by post hoc Newman-Keuls test. ANOVA, p < 0.01; post hoc, p < 0.05 vs. CTR (control) group.
Figure 3. Effects of compounds VIa–o at 10mg/mL on serotonin (5-HT)-induced colon cancer HCT116 cell viability (MTT test). Data are means SE and analysed
ꢂ
ꢂꢂ
ꢂꢂꢂ
p < 0.001 vs. 5-HT (sero-
through analysis of variance (ANOVA), followed by post hoc Newman-Keuls test. ANOVA, p < 0.0001; post hoc, p < 0.05, p < 0.01,
tonin) group.
preliminary study was carried out in order to optimise the experi- as
a reliable proliferative stimulus for the following tests.
mental conditions that could demonstrate a cell viability-stimulat- Specifically, compounds VIc–e and VIh–m were able to inhibit
ing effect of 5-HT, in a wide range of concentrations (10 pg/mL – 5-HT-stimulated viability of HCT116 cells (Figure 3), thus substanti-
1 mg/mL). We observed that HCT116 cell viability increased in a ating the potential anti-proliferative effect of the compounds in
concentration-dependent manner, in the range 0.1–1 mg/mL,
although it remained constant, at upper tested concentrations
(Figure 2) given. Considering our previous ex vivo and in vitro
studies focussed on inflamed colon specimens and hypothalamic
the real in vivo colon cancer cell microenvironment, characterised
by the up-regulated production of multiple pro-inflammatory and
anti-apoptotic/mitogen factors, including 5-HT24,25
.
Furthermore, compounds were assayed for evaluating modula-
cells, respectively, reporting 5-HT concentrations in the order of tory effects on the extracellular level of KA, one of the two main
1 ng/mL20,21, we have chosen the 5-HT concentration of 1 ng/mL kynurenine metabolites. Kynurenic acid was reported to be

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1107
Figure 4. Effects of compounds VIa–o at 10 mg/mL on serotonin (5-HT)-induced reduction of kynurenic acid (KA) release from colon cancer HCT116 cells. Data are
ꢂ
ꢂꢂ
p < 0.01,
means SE and analysed through analysis of variance (ANOVA), followed by post hoc Newman-Keuls test. ANOVA, p < 0.0001; post hoc, p < 0.05,
ꢂꢂꢂ
p < 0.001 vs. 5-HT (serotonin) group.
Figure 5. Effects of compounds VIc, VIe, VIk and daunorubicin at 0.1–20 mg/mL on HCT116 cell viability. Data are means SE and analysed through analysis of vari-
ꢂꢂ ꢂꢂꢂ
ꢂ
ance (ANOVA), followed by post hoc Newman-Keuls test. ANOVA, p < 0.0001; post hoc, p < 0.05, p < 0.01, p < 0.001 vs CTR (control) group.

1108
Z. ÖZDEMIR ET AL.
Finally, considering their potencies in reducing HCT116 cell via-
bility, compounds VIe and VIk were further studied in order to
evaluate their effects on the spontaneous migration of HCT116
cells, through the wound healing paradigm. In this experiment,
HCT116 cells were treated with compounds VIe and VIk at their
respective IC50 values. The spontaneous migration of HCT116 cells
was monitored in the 48 h following the experimental lesion of
cell monolayer. The null effects on sponstaneous migration
(Figure 6) rule out any involvement of the tested compounds in
altering the invasion capacity of human colon cancer cells.
4. Conclusion
Fifteen new 3(2H)-pyridazinone derivatives were synthesised and
studied for their ability to limit the proliferation of HCT116 cell
line (colon carcinoma), alone or after stimulation with serotonin, a
well-recognized pro-inflamamtory factor in the gut. In particular,
compound VIc induced a strong release of kynurenic acid after
treatment, thus representing a strong chemopreventive agent in
this model. Moreover, all compounds resulted non-toxic up to
100 mg/mL in the A. salina lethality assay, whereas three of them
(VIc, VIe and VIk) displayed a promising inhibitory action compar-
Figure 6. Effects of compounds VIe (3.09 mg/mL) and VIk (2.73 mg/mL) on the
spontaneous migration of human colon cancer HCT116 cell line (wound healing
paradigm). The spontaneous migration was monitored in the 48 h following treat-
ment. Data are expressed as percentage scratch area relative to the untreated
CTR group.
able to that of daunorubicin as
basal conditions.
a
standard drug at
produced in multiple tissues, including brain and peripheral
organs26, although pharmacokinetic studies excluded any possibil-
ity of the peripheral pool to cross blood-brain barrier27. In the
brain, the kynurenine-derived kynurenic acid was described as a
reliable marker of neuroprotection28,29, whereas it seems to be
involved in inflammatory response at the peripheral level30.
Kynurenic acid was also described as an anti-proliferative factor
towards colon, renal, and glioma cells31. Specifically, this marker
was considered as a potential chemopreventive agent against
colon cancer32,33. The assessment of KA levels showed that the
sole compound VIc was able to blunt 5-HT-induced KA depletion
(Figure 4) after treatment. Additionally, the KA level after com-
pound VIc stimulation was even higher compared to basal condi-
tion (CTR group). Conversely, the other tested compounds (VIa,
VIb and VId–m) failed to prevent the inhibition of KA level follow-
ing the stimulation with 5-HT. They were able in potentiating 5-
HT-induced KA depletion, as well. The results of this pharmaco-
logical screening suggest a minor role exerted by the compounds
VIa, VIb and VId–m as anti-proliferative agents.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This work was supported by intramural grants by Ministero
ꢀ
Italiano dell’Universita e della Ricerca (MIUR) FAR 2019 (ex 60%),
held by Dr. Simone Carradori.
ORCID
Simone Carradori
http://orcid.org/0000-0002-8698-9440
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