Cytotoxicity of new pyridazin-3(2H)-one derivatives orchestrating oxidative stress in human triple-negative breast cancer (MDA-MB-468)

Article abstract of DOI:10.1002/ardp.201800128

Triple-negative breast cancer (TNBC) is a complex and aggressive subtype of breast cancer characterized by high morbidity and mortality. In the absence of targeted therapy, only chemotherapy is available in this case of cancer. The current study investigated the antitumor effect of new pyridazin-3(2H)-one derivatives on the human TNBC cell line, MD-MB-468. The in vitro cytotoxic activities were investigated using the tetrazolium-based MTT assay. Lipid peroxidation, H₂O₂ content, and the specific activities of antioxidant enzymes were also determined. Two molecules, 6f and 7h, were found to be selectively highly active against tumor cells with IC₅₀ values of 3.12 and 4.9 μM, respectively. Furthermore, cells exposed to 6f showed a significant increase in H₂O₂ and lipid peroxidation levels, accompanied by a decrease in the enzyme activities of glutathione reductase (GR) and thioredoxin reductase (TrxR). The cytotoxicity of the compound 6f may improve the therapeutic efficacy of the current treatment for TNBC via the inhibition of GR and TrxR activities.

Full text of DOI:10.1002/ardp.201800128

Received: 23 April 2018
Revised: 18 September 2018
Accepted: 26 September 2018
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DOI: 10.1002/ardp.201800128
FULL PAPER
Cytotoxicity of new pyridazin-3(2H)-one derivatives
orchestrating oxidative stress in human triple-negative breast
cancer (MDA-MB-468)
Najat Bouchmaa1,2
Reda Ben Mrid3
Youness Boukharsa2
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Mohamed Nhiri3
Abdelmajid Zyad1
Hassan Ait Mouse1
Jamal Taoufik2
M'hammed Ansar2
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1 Team of Experimental Oncology and Natural
Substances, Cellular and Molecular Immuno-
pharmacology, Faculty of Sciences and
Techniques, Sultan Moulay Slimane
Abstract
Triple-negative breast cancer (TNBC) is a complex and aggressive subtype of breast
cancer characterized by high morbidity and mortality. In the absence of targeted
therapy, only chemotherapy is available in this case of cancer. The current study
investigated the antitumor effect of new pyridazin-3(2H)-one derivatives on the
human TNBC cell line, MD-MB-468. The in vitro cytotoxic activities were investigated
using the tetrazolium-based MTT assay. Lipid peroxidation, H2O2 content, and the
specific activities of antioxidant enzymes were also determined. Two molecules, 6f and
7h, were found to be selectively highly active against tumor cells with IC50 values of
3.12 and 4.9 µM, respectively. Furthermore, cells exposed to 6f showed a significant
increase in H2O2 and lipid peroxidation levels, accompanied by a decrease in the
enzyme activities of glutathione reductase (GR) and thioredoxin reductase (TrxR). The
cytotoxicity of the compound 6f may improve the therapeutic efficacy of the current
treatment for TNBC via the inhibition of GR and TrxR activities.
University, Beni-Mellal, Morocco
2 Laboratory of Medicinal Chemistry, Faculty
of Medicine and Pharmacy,
Mohammed V University, Rabat, Morocco
3 Laboratory of Biochemistry and Molecular
Genetics, Faculty of Sciences and Techniques,
Tangier, Morocco
Correspondence
Prof. Abdelmajid Zyad, Team of Experimental
Oncology and Natural Substances, Cellular
and Molecular Immuno-pharmacology, Faculty
of Sciences and Techniques, Sultan Moulay
Slimane University, B.P: 523 Beni Mellal,
Morocco.
Email: a.zyad@usms.ma, ab.zyad2@gmail.com
Funding information
Lalla Salma Foundation: Prevention and
Treatment of Cancer, Grant number: 09/AP
2013
K E Y W O R D S
anticancer agent, cytotoxicity, oxidative stress, pyridazin-(2H)-3-ones, triple-negative breast
cancer
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1
INTRODUCTION
therapies since they do not express the relevant receptors (ER, PR,
HER).[2,4–6] Currently, conventional-targeted breast treatment offers
Triple-negative breast cancers (TNBC), which account for 10–17% of
all breast cancers, tend to grow more aggressively than other subtypes,
show relatively early recurrence and intrinsically have poor
prognosis.[1]
only limited benefits and might cause additional side-effects; such
treatment leaves them associated with a high rate of local and systemic
relapse.[7] In addition, TNBC is more aggressive than other disease
subtypes with no effective therapeutic targeted has been achieved
neither no specific molecular targets agents are currently available for
their treatment.[8] Indeed, the absence of chemotherapeutic vulner-
abilities have been confirmed by the fact that women with TNBC have
the lower survival rate and the highest incidence of metastatic disease
This subtype of breast cancer is characterized by a lack of
expression of both oestrogen and progesterone receptors with
simultaneous absence of human epidermal growth factor 2 (HER2)
receptors.[2,3] Indeed, TNBC tumors are not sensitive to standard
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Arch Pharm Chem Life Sci. 2018;1–11.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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BOUCHMAA ET AL.
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of all breast cancer subtypes.[4,9,10] Consequently, there is a need to
develop new alternative therapies for TNBC to overcome the systemic
cytotoxicity of existing chemotherapy and the tumor cell resistance to
the anticancer effect of these molecules.
4a–f were prepared according to the methods described in the
literature.[26] Then, treatment of substituted aldehydes 4a–f with
levulinic acid in the presence of HCl (chlorhydric acid) in acetic acid
gave adducts 5a–f, which were heated by hydrazine hydrate to afford
the pyridazin-3(2H)-ones 6a–f. Finally, the mixture of substituted 6-
methylpyridazin-3(2H)-ones 6 and diphosphore pentasulfure solution
in pyridin gave 7g and 7h.[27]
Reactive oxygen species (ROS) are a group of molecules (hydroxyl
radicals, peroxides, H2O2, nitric oxide) that are maintained, at an
optimal level, through enzymatic and non-enzymatic systems.[10] The
increase of ROS production or the decrease of the antioxidant defense
system efficiency is responsible for the cell's oxidative stress.[11] In
cancer cells, the intracellular antioxidant capacity is mainly conferred
by the glutathione- and thioredoxin-dependent systems. In fact, the
important enzymes implicated in the regulation of redox homeostasis
such as peroxidases and thiol reductases, are limited to the pool of GSH
and thioredoxin as source of reducing equivalents. Glutathione
reductase (GR) which catalyzes the reduction of GSSG to GSH and
thioredoxin reductase (TrxR) which is responsible for the reduction of
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2.2 Biological assays
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2.2.1 Cytotoxicity
The cytotoxic activity of pyridazin-3(2H)-one derivatives 6a–f and 7g,
7h was evaluated toward the tumor cell line (MDA-MB-468) using the
MTT assay. Figure 1 showed the effect of increasing concentrations of
each compound on the viability of tumor cells. It is shown in this figure
that this effect is dose dependent and is different from a compound to
another. Table 1 showed the IC50 values of each component. It is
depicted in this table that 6f, 7h, and 6e exhibited a potent cytotoxicity
with IC50 values equal to 3.125 7.02 μM, 4.9 5.30 μM, and
7.85 3.99 μM, respectively. However, the three compounds 6a, 6d,
and 7g showed a lesser cytotoxic activity with IC50 values ranging from
9.01 0.76 μM (6a) to 11.5 2.16 μM (7g). For 6b and 6c compounds,
only a moderate cytotoxicity was observed (IC50: 15 0.73 and
17.44 2.66 μM, respectively) compared to the positive control
methotrexate (MTX). Given together, the eight compounds can be
classified on the basis of their cytotoxic level against the MDA-MB-468
cell line, respectively, 6f > 7h > 6e > 6a > 7g > 6d > 6b > 6c. Interest-
ingly, the compound 6f bearing a Br and Cl at the radical positions was
the most cytotoxic compound as showed by the lowest IC50 values.
thioredoxin is reported to be up-regulated in
a number of
cancers.[12,13] Therefore, reducing GSH and thioredoxin pool in cancer
cells, through the inhibition of GR and TrxR enzymes, may be
considered as an effective strategy to combat these tumor cells by
altering their ability to eliminate ROS and to cope with oxidative
damage.
Pyridazin-3(2H)-one derivatives constitute an interesting class of
heterocyclic lead compounds for drug discovery and research into
anticancer molecules.[14–16] Recently, it was reported that some
pyridazin-3(2H)-one derivatives specifically interact with and inhibit
PIM (potential tumor targets) kinases with low nanomolar
potency.[17–20] In addition, pyridazin-3(2H)-ones are endowed with
many biological activities.[21–23] In this context, the current study was
aimed at developing novel pyridazin-3(2H)-one derivatives with
promising cytotoxic activity. The obtained molecules were tested
for their in vitro cytotoxic effect against the aggressive TNBC cell line
MDA-MB-468. Thereafter, two highly cytotoxic molecules were
chosen and compared for their ability to induce oxidative stress in
the same cell line in an attempt to reveal the likely mechanisms
involved in this cytotoxic effect.
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2.2.2 Cytotoxicity against normal cells (PBMCs)
The majority of clinically approved anticancer drugs are characterized
by a narrow therapeutic window that results mainly from a high
systemic toxicity of drugs. A drug can be interesting only if it is toxic
against tumor cells and its effects on normal cells are very weak or null
which limits the systemic cytotoxicity. Thus, we tested the most
important compounds (which are more cytotoxic against tumor cells)
against the human peripheral blood mononuclear cells (PBMCs) from
healthy donors in order to determine their effects against normal cells.
Interestingly our results presented in Figure 2 depicted a very low
cytotoxic effect of these molecules on normal PBMCs indicating their
selective cytotoxicity.
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2
RESULTS
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2.1 Chemistry
As described in our paper Bouchmaa et al.,[24] the pyridazinone
derivatives incorporating benzofuran moiety studied in this work
(compounds 6a–f and 7g, 7h) were synthesized according to the
general procedures outlined in Scheme 1. In the first step,
salicylaldehydes 2a–f were synthesized via Reimer Thieman[25]
formylation of the appropriate substituted phenol 1a–f with CHCl3
and NaOH. Aldehydes 2a–f have been previously synthesized with
very low yield. Briefly, reaction of intermediates 2a–f with bromace-
taldehyde diethyl acetal in the presence of potassium carbonate in
DMF (dimethylformamide) yielded compounds 3a–f. Next, these
compounds 3a–f were cyclized to benzo[b]furan-2-ylcarboxaldehydes
4a–f by heating in concentrated acetic acid. Benzo[b]furanaldehydes
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2.2.3 Hydrogen peroxide content (H2O2)
As indicated in Figure 3A, both 7h molecule and the positive control
were associated with a significant decrease of H2O2 content as
compared to the negative control. However, cell exposure to the 6f
compound showed a significant increase of H2O2. In fact, 6f had the
ability to increase the H2O2 contents by more than 30-fold compared
either to the negative or the positive control (Figure 3A).

BOUCHMAA ET AL.
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SCHEME 1 Synthesis of target compounds 6a–f, 7g and 7h. Reagents: (a) CHCl3/NaOH 10 N, reflux 2 h; (b) BrCH2CH(OC2H5)2/K2CO3/
DMF, reflux 4 h; (c) CH3COOH, reflux 24 h; (d) H3CCOCH2CH2COOH/CH3COOH, reflux 24 h; (e) H2N-NH2/EtOH, reflux 2 h; (f) P2S5/pyridin,
reflux 4 h
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2.2.4 Lipid peroxidation
increased the MDA content to very high levels compared to the
negative control.
The lipid peroxidation, which is an indicator of oxidative stress, was
quantified by measuring the MDA content (Figure 3B). The two
pyridazin-3(2H)-one derivatives tested here were able to induce an
increase in the MDA content compared to the negative control.
However, the effect of the 6f compound was more pronounced and
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2.2.5 Antioxidant enzyme activity
The effect of the two compounds, 6f and 7h, on the phase I antioxidant
enzymes (SOD, CAT, and GPx) is represented in Figure 4. The figure

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FIGURE 1 Cytotoxicity toward MDA-MB-468 cell line after treatment with 6a–6f, 7g and 7h pyridazin-3(2H)-one derivatives for 48 h.
Each value represents the mean of three independents measurement. Bars represent the standard error. Different letters indicate significant
differences among treatments. p-Value <0.05 indicates significant difference
showed that 6f and 7h selectively increased the enzyme activities of
SOD (Figure 4A), CAT (Figure 4B), and GPx (Figure 4C). In fact, 7h
caused a slight increase of the three enzymes activities which was
almost not significant compared to the negative control. However,
treatment with 6f leads to a significant increase in the enzymes
activities. This increase was time dependent and was almost doubled
after exposure to 6f for 48 h compared to 24 h.
In the same order of ideas, MDA-MB-468 tumor cells exposure to
7h and 6f differentially affected the GR and TrxR activities. On the one
hand, the two pyridazin-3(2H)-one derivatives decreased the GR

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TABLE 1 Inhibitory concentration 50 (IC50) in μM of the pyridazin-3(2H)-one derivatives tested against MDA-MB-468 tumor cell line
Molecules series
6a
6b
6c
6d
6e
6f
7g
7h
MTX
IC50 [μM]
9.01 0.76a
15 0.73b
17.44 2.66b
10 2.80a
7.85 3.99a
3.1 7.02c
11.5 2.16a
4.9 5.30c
4.6 5.62c
Each value represents the mean of three independents measurement. Different letters indicate significant differences among treatments. p-Value <0.05
indicates significant difference.
activity compared to the untreated control cells (Figure 5A). The
change caused by the 7h molecule was comparable both at 24 and 48 h
exposure. However, after the profound decrease observed after 24 h
exposure to 6f, GR activity increased to reach a value similar to that
observed for the 7h molecule. On the other hand, the 7h molecule
caused a slight decrease in TrxR activity after 24 h while a significant
increase was observed after 48 h treatment. However, the effect of
the 6f compound was totally different after 48 h treatment. In fact, 6f
FIGURE 2 Effect of pyridazin-3(2H)-one derivatives on the viability of PBMCs treated with 6a, 6d–6f, 7g and 7h for 48 h. Cell viability was
calculated as described in Materials and Methods section. Each value represents the mean of three independents measurement. Bars
represent the standard error. Different letters indicate significant differences among treatments. p-Value <0.05 indicates significant difference

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BOUCHMAA ET AL.
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FIGURE 3 Concentration of hydrogen peroxide (H2O2) and
malondialdehyde (MDA) in the MDA-MB-468 cell line after
treatment with 6f or 7h pyridazin-3(2H)-one derivatives, NT
(negative control) and MTX (positive control). Each value represents
the mean of six replicates issued from two independent
experiments. Bars represent the standard error. Different letters
indicate significant differences among treatments. p-Value <0.05
indicates significant difference
FIGURE 4 Activities of antioxidative enzymes; superoxide
dismutase (SOD, A), catalase (CAT, B) and glutathione peroxidase
(GPx, C) in the MDA-MB-468 cell line after treatment with 6f or 7h
pyridazin-3(2H)-one derivatives, NT (negative control) and MTX
(positive control). Each value represents the mean of six replicates
issued from two independent experiments. Bars represent the
standard error. Different letters indicate significant differences
among treatments. p-Value <0.05 indicates significant difference
decreased the TrxR activity by 73% after 48 h exposure compared to
the untreated cells (Figure 5B).
cells of the control group retain their typical morphology (data not
shown).Interestingly, oneofthemostimportantresultsofthisstudywas
that pyridazin-3(2H)-one molecules especially 6f and 7h did not show a
significant cytotoxicity toward human normal PBMCs (Figure 2). Recall
that PBMCs are the first normal cell populations that come into contact
with antitumor drugs used in conventional chemotherapy and that
collapse from the first week of intravenous treatment of patients
resulting in significant immune deficiency and increased side effects.
These findings suggest a selective killing ability of these molecules
against tumor cells (MDA-MB-468) without impacting normal cells.
In this approach, understanding biochemical differences between
cancer and normal cells can be highly beneficial. Cancer cells as
compared to normal cells are under greater intrinsic oxidative stress
due to alterations in metabolism. Furthermore, it was reported that
cancer cells have an elevated level of ROS compared to normal cells.
This elevation of ROS may be the result of an abnormal mitochondrial
oxidative metabolism and can be responsible for the initiation and
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3
DISCUSSION
During the last decade, many interesting functional structures of
pharmacophore moieties in pyridazinones made them an attractive
source of new active derivatives.[28,29] Combination of benzofuran and
pyridazinones has led to good results for the first time. From the results
of cytotoxicity, we have shown that newly synthesized pyridazin-3(2H)-
one derivatives exhibit much higher inhibition of viability toward the
triple negative human breast adenocarcinoma MDA-MB-468 tumor
cells (Figure 1) with IC50 values around 4 μM for both 6f and 7h
derivatives (Table 1). These results corroborate recent findings showing
a cytotoxic activity of other pyridazin-3(2H)-one derivatives against
different cell lines.[30–32] Furthermore, exposure of tumor cells to 6f or
7h derivatives led to a significant reduction in the number of living cells.
Tumor cells from treated groups appeared small with less aggregates
and deformed with the presence of apoptotic bodies while the tumor

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considered as a normal strategy from cells to face the oxidative stress
caused by the two pyridazin-3(2H)-one derivatives tested here.
However, the impact of the oxidative stress seems to be more
pronounced after exposure to the 6f molecule. In fact, the
determination of lipid peroxidation levels in the cells, measured as
MDA content, showed a profound increase in the lipid peroxidation
after cell exposure to 6f for 48 h compared to 24 h. This result indicates
that despite the increase of antioxidant enzyme activities (SOD, CAT,
and GPx), the oxidative damage was more pronounced that cells failed
to stop.
In cancer cells, the intracellular antioxidant capacity is mainly
conferred by the glutathione- and thioredoxin-dependent systems. In
fact, the important enzymes implicated in the regulation of redox
homeostasis such as peroxidases and thiol reductases, are limited
to the pool of GSH and thioredoxin as source of reducing
equivalents.[44–46] Thus, to determine the likely cause of the increase
in ROS and the reason why cells failed to cope with the oxidative
damage, especially after treatment with the 6f molecule, the effect of
the two molecules, 6f and 7h, on TrxR and GR was evaluated. The data
obtained showed that while 6f and 7h compounds decreased the GR
activity, the effect on TrxR activity was different. In fact, the 7h
molecule caused a slight decrease in TrxR activity after 24 h, however,
after 48 h, TrxR activity significantly increased. Nevertheless, 6f seems
to be an important inhibitor of TrxR. In fact, 6f decreased the TrxR
activity by 73% after 48 h. Previous studies have shown that the
inhibition of reduced GSH and thioredoxin synthesis might be used to
sensitize cancer cells to chemotherapy agents.[47–49] Recently,
Rodman et al.[50] showed that the depletion of GSH and the inhibition
of TrxR activity increased radiation responses in human breast and
pancreatic cancer cells. Consequently, the results presented in this
study suggest that 6f and 7h have an important in vitro anticancer
capacity, partially through the increasing of intracellular ROS. These
results indicated also that the anticancer activity of the 6f compound
could be due to the inhibition of GR and TrxR activities.
FIGURE 5 Activities of antioxidative enzymes; glutathione
reductase (GR, A) and thioredoxin reductase (TrxR, B) in the MDA-
MB-468 cell line after treatment with 6f or 7h pyridazin-3(2H)-one
derivatives, NT (negative control) and MTX (positive control). Each
value represents the mean of six replicates issued from two
independent experiments. Bars represent the standard error.
Different letters indicate significant differences among treatments.
p-Value <0.05 indicates significant difference
progression of different types of cancer such as breast, prostate, lung,
and colon cancers.[33–35] However, the elevation of ROS to a certain
level may be lethal for tumor cells themselves. In fact, several studies
outlined the implication of elevated levels of H2O2 in the induction of
apoptosis[36,37] and their low concentration enhanced proliferation
and migration of mammalian cells.[38] Therefore, killing cancer cells
through ROS or oxidative stress causing-agents represent one of the
theories proposed in cancer therapy.[39–41] In the current study, we
showed a significant increase in the content of H2O2 in cells exposed to
6f compared to negative control. 6f had the ability to increase the
H2O2 contents by more than 30-fold compared either to the negative
or MTX. Superoxide dismutase is recognized as the primary defense
barrier against ROS which catalyzes the dismutation of superoxide
anion radicals (O2•−) to hydrogen peroxide (H2O2).[42] Hydrogen
peroxide, generated by the activity of SOD, is eliminated by its
conversion to H2O in subsequent reactions by CAT and GPx.[43] The
current study demonstrated that the pyridazin-3(2H)-one derivatives
selectively increased the enzyme activities of SOD, CAT, and GPx. In
fact, the two molecules tested here (6f and 7h) affect differentially
these enzymes. 7h induced a small increase in the activities of SOD and
GPx, however, 6f has the ability to increase, by at least twofold, the
activities of SOD, CAT, and GPx after 24 h and fourfold after 48 h of
exposure. This increment was even higher compared to the positive
control. The increased levels of antioxidant enzyme activities are
Considering the structure–activity relationship, the substitution at
C5 in the benzofuran ring was of interest. Taken together, results of
our experiments confirmed that molecules possessing Br or Cl in the
radical 1 or 2 in the C5 of benzofuran are stronger cytotoxic agents of
pyridazin-3(2H)-one derivatives against the TNBC cell line (MDA-MB-
468) which is associated with induction of oxidative stress. The
interesting cytotoxic effect of 6f can probably be attributed to the Br in
position 1 (R1) and/or Cl in position 2 (R2). These results are in
agreement with data demonstrating that compounds substituted with
this radical are relatively potent as cytotoxic agents.[51] Finally, these
compounds may be used as anticancer agents.
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4
CONCLUSION
Among the newly developed approaches to the search for new anti-
tumor molecules, we are interested in the one based on the
induction of cytotoxicity via orchestrating oxidative stress. To our
knowledge, this is the first study investigating specific activities of

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antioxidant enzymes (SOD, CAT, GPx, GR, and TR activities) on the
TNBC cell line (MDA-MB-468) by synthesized pyridazin-3(2H)-one
molecules family. Taking all the data into account, two molecules, 6f
and 7h were found to be selectively highly active against tumor cells
with IC50 value of 3.12 and 4.9 µM, respectively. Accordingly,
manipulating ROS generation by pyridazin-3-one molecules may be a
potential way to kill TNBC (MDA-MB-468) cells effectively and
selectively without any toxicity against normal cells. It is concluded
that the cytotoxicity of the 6f compound may improve the
therapeutic efficacy of the current treatment for TNBC via the
inhibition of GR and TrxR.
5-[(7-Chlorobenzo[b]furan-2-yl)methyl]-6-methylpyridazin-3
(2H)-thione 7h
This compound was obtained as yellow solid, yield 62%, mp 188–
190°C (ethanol); IR (KBr νmax cm−1), 1606 (CN), 1082 (CS); 1H NMR
(DMSO-d6, 300 MHz, J Hz), δ = 2.33 (s, 3H, ─NC─CH3), 4.19 (s, 2H,
─CH2─), 6.86 (s, 1H, H3′), 7.20–7.57 (m, 4H, H4, H4′, H5′, and H6′),
14.51 (ls, 1H, NH). MS: m/z; 290.7 [M−H]+, 313.10 [M−Na]+.
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5.2 Biological assays
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5.2.1 Chemicals and cell lines
All chemicals and biochemicals reagents were purchased from
Sigma–Aldrich except RPMI 1640 medium and L-glutamin were from
Lonza, fetal bovine serum was from Gibco BRL (Cergy Pontoise,
France), Bradford reagent was obtained from Bio-Rad (Hercules, CA,
USA), and β-nicotinamide adenine dinucleotide 2′-phosphate
reduced tetrasodium salt hydrate (NADPH) was purchased from
Roche Diagnostics (Mannheim, Germany). The human breast
adenocarcinoma cell line (MDA-MB-468) was from the stock
cultures of the Laboratory of Experimental Oncology and Natural
Substances, Faculty of Sciences and Technology, Sultan Moulay
Slimane University, Morocco.
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5
EXPERIMENTAL
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5.1 Chemistry
The materials and methods for the synthesis of the investigated
compounds and their structural characterization are provided as
Supporting Information, as are the InChI codes and some biological
activity data.
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5.1.1 Synthesis of the drug molecules
Melting points were determined on a Büchi SMP 20 apparatus and are
not corrected. Infrared (IR) spectra were recorded with an IR VERTEX
70 FT-IR (Bruker Optics) spectrometer. 1H Nuclear magnetic reso-
nance (1H NMR) spectra were recorded on a Bruker Avance (300 MHz)
spectrometer, using tetramethylsilane (TMS) as internal standard and,
CDCl3 and DMSO-d6 as solvent. Mass spectra was recorded on a API
3200 LC/MS/MS mass spectrometer using electrospray ionization
(ESI) in positive polarity.
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5.2.2 Cell culture
MDA-MB-468 (triple negative human breast carcinoma) cell line was
cultured in RPMI 1640 medium supplemented with 5% heat-
inactivated fetal bovine serum, 1% penicillin G-streptomycin, and
0.2% of L-glutamine. Incubation was performed at 37°C in humidified
atmosphere containing 5% CO2.
General procedures for the formylation of phenols 2a–f, the
synthesis of 2-formylphenoxyacetadehyde diethyl acetals 3a–f, the
synthesis of substituted benzo[b]furan-2-carbaldehydes 4a–f,
the synthesis of substituted 3-benzo[b]furan-2-ylmethylene-levulinic
acids 5a–f, the synthesis of substituted 5-(benzo[b]furan-2-ylmethyl)-
6-methylpyridazin-3(2H)-ones 6a–f have been described in our paper
Bouchmaa et al.[24]
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5.2.3 Cytotoxic activity evaluation
MDA-MB-468 cell line was harvested from starting cultures at the
exponential growth phase. After PBS wash, adherent cells were
harvested from sub-confluent cultures using cell scraper and
suspended in RMPI. The harvested cells were poured in flat-bottomed
96-well microliter plates containing 100 μL of complete medium per
well (4.3 × 104 cells/mL). After overnight incubation, several concen-
trations of pyridazin-3(2H)-one derivatives in DMSO completed to
100 μL with complete RPMI were added. Control cells were treated
with DMSO alone. In all cases, the final concentration of DMSO never
exceeded 0.1%. After 48 h incubation in humidified atmosphere at
37°C and 5% CO2, 100 μL of medium was carefully removed from each
well and replaced with 20 μL MTT solution (5 mg/mL PBS). After 4 h
incubation under the same conditions, the cleavage of MTT to
formazan by metabolically active cells was quantified by scanning the
plates at 540 nm using a Multiskan EX (Finland) apparatus. Three
independent sets of experiments performed in duplicate were
evaluated. The % of cell viability was calculated by the following
formula:
General procedures for the synthesis of substituted 5-(benzo[b]-
furan-2-ylmethyl)-6-methylpyridazin-3(2H)-thiones 7g and 7h: the
mixture of substituted 6-methylpyridazin-3(2H)-ones 6 and diphos-
phorpentasulfur solution in pyridin was refluxed for 4 h; the precipitate
formed is filtered and recrystallized from an appropriate solvent.
5-[(5-Chlorobenzo[b]-furan-2-yl)methyl]-6-methylpyridazin-3-
(2H)-thione 7g
This compound was obtained as yellow solid, yield 65%, mp 165–
167°C (ethanol); IR (KBr νmax cm−1), 1605 (C N), 1080 (C S);


1

H NMR (DMSO-d6, 300 MHz, J Hz), δ = 2.30 (s, 3H, ─N C─CH3),
4.15 (s, 2H, ─CH2─), 6.76 (s, 1H, H3′), 7.28 (dd, 1H, J6′-7′ = 8.70 and J6′-
4′ = 2.40, H6′), 7.32 (s, 1H, H4), 7.58 (d, 1H, J7′-6′ = 8.70, H7′), 7.65 (d,
1H, J4′-6′ = 2.40, H4′), 14.51 (ls, 1H, NH).

BOUCHMAA ET AL.
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5.2.7 Preparation of cell extracts for antioxidant
% Cell viability ¼ 100 ꢀ A=A0
enzyme assays
MDA-MB-468 cells were treated with 6f and 7h for 24 and 48 h. Then,
after washing once with PBS (10 mM, pH 7.4), the cells were harvested
and centrifuged 1200×g for 10 min. The pellet was suspended in
500 µL of lysis buffer composed of 50 mM Tris-HCl, 1 mM phenyl-
methanesulfonyl (PMSF), 0.1% (v/v) Triton X-100, in 1.5 mL Eppendorf
tubes and maintained in constant agitation at 4°C for 30 min. The
homogenate was then centrifuged (1600×g, 20 min) at 4°C. The
supernatant (enzyme extract solution) was kept at −80°C or used for
the determination of SOD, CAT, GPx, GR, and TrxR activities.
where A0 and A are the absorbance of negative control and test culture,
respectively.
The cytotoxic effects of pyridazin-3(2H)-one derivatives against
the cell line were compared using their IC50 values (concentration of
tested molecules leading to 50% inhibition of cell viability).
|
5.2.4 Cytotoxic effect against PBMCs
This test was realized in order to evaluate the effect of synthesized
pyridazin-3(2H)-one derivatives against normal cells using the MTT
colorimetric assay described above. To isolate the human PBMCs,
blood samples were collected from human healthy donors in
heparinized tubes and the PBMCs were isolated using standard
Ficoll-Hypaque density centrifugation. The interface lymphocytes
were washed twice with phosphate buffer solution (PBS). Cells were
incubated in 96-well microtiter plates in the presence of the same
concentrations of synthesized pyridazin-3(2H)-one derivatives in the
same conditions as tumor cells. The % of viability was calculated using
the following formula:
|
5.2.8 Antioxidant enzyme assays
SOD activity was assayed according to the method of Sun et al.[54] with
some modifications. Briefly, the reaction mixture was composed of
0.05 M phosphate buffer, pH 7.5, 10 mM methionine, 0.1 µM EDTA,
2 µM riboflavin, 75 µM nitro blue tetrazolium (NBT), and the enzyme
extract. The SOD activity was measured at 560 nm. One unit of SOD
activity was defined as the quantity of SOD required to obtain a 50%
inhibition of the reduction of NBT. The activity was expressed as units
per mg of protein content.
CAT activity was measured by the method of Aebi[55] with some
modifications. The final reaction volume of 0.5 mL included 0.1 M
phosphate buffer, pH 7.0, 15 mM H2O2, and enzyme extract. The
decomposition of H2O2 was monitored at 240 nm for 2 min at 25°C.
The extinction coefficient (43.6 M/cm) was used to calculate the
catalase activity, expressed as µmol of H2O2 decomposed/min/mg
protein.
% Cell viability ¼ 100 ꢀ A=A0
where A0 and A are the absorbance of negative control and test culture,
respectively.
|
5.2.5 Hydrogen peroxide (H2O2) content
GPx activity was measured by the method of Lawrence and
Burk[56] with some modifications. The reaction mixture contained
0.1 M potassium phosphate, pH 7.0, 1 mM EDTA, 1 mM sodium azide,
1 mM GSH, GR (10 μg/mL), 0.25 mM NADPH, and enzyme extract.
The mixture was incubated at 25°C for 3 min and completed by adding
0.25 mM of H2O2. The rate of NADPH oxidation was monitored at
340 nm for 5 min. GPx activity was calculated and expressed as nmol of
NADPH oxidized/min/mg protein by using the extinction coefficient
of 6.2 mM/cm.
[52]
H2O2 content was determined following the protocol of Ref.
.
Briefly, cells (0.7 × 106 per dish) were seeded in 6-well plates and
incubated in complete medium with IC50 of both 6f and 7h
molecules. After 48 h of incubation, cells were crushed in 0.1%
trichloroacetic acid (TCA). The homogenate was centrifuged at
12000×g for 15 min at 4°C, the layer was kept in dark for 1 h after
mixing with phosphate buffer (10 mM, pH 7.0) and potassium iodide
(1 M). The absorbance of the resulting solution was measured at
390 nm. H2O2 concentrations were calculated using a standard
curve.
GR activity was estimated by a modified method of Carlberg and
Mannervik.[57] Briefly, the reaction mixture contained 0.1 M phos-
phate buffer, pH 7.6, 1 mM GSSG, 0.2 mM NADPH. The contents were
incubated at 25°C for 3 min and the reaction was initiated by adding
enzyme extract. The rate of NADPH oxidation was monitored at
340 nm. GR activity was expressed as nmol of NADPH oxidized/min/
mg protein by using the extinction coefficient of 6.2 mM/cm.
TrxR was measured as the reduction of DTNB (5,5′-dithiobis-
(2-nitrobenzoic acid)) in the presence of NADPH.[58] The reaction
mixture contained 0.1 M phosphate buffer, pH 7.6, 1 mM EDTA,
0.25 mM NADPH, 1 mM DTNB, and enzyme extract. The increase in
the absorbance at 412 nm was monitored at 25°C. TrxR activity was
expressed as nmol of DTNB reduced/min/mg protein by using the
extinction coefficient of 13.6 mM/cm.
|
5.2.6 Malondialdehyde (MDA) content
Lipid peroxidation measured as MDA content in MDA-MB-468 cells
was determined using thiobarbituric acid (TBA) according to the
method described previously[53] with slight modifications. In brief,
cell homogenate, in different conditions described above, was mixed
with trichloroacetic acid (20%) and TBA (0.67%). The mixture was
heated at 95°C for 1 h. After cooling, 1 mL n-butanol was added to
the mixture followed by centrifugation at 12000×g for 10 min.
Organic supernatant was collected to measure the absorbance at
532 nm.

10
BOUCHMAA ET AL.
|
|
5.2.9 Protein content determination
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2009, 53, 1048.
Total protein content of the samples was determined following the
method of Bradford[59] using BSA as a protein standard.
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5.2.10 Statistical analysis
The used data are mean values SD. Results were subjected to a one-
way analysis of variance (ANOVA) followed by the Tukey test using
PASW statistics (version 18). Different letters indicate significant
differences at the level of 5%.
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ACKNOWLEDGMENTS
The authors would like to thank Professor Jamal Koubali, Department
of English Language, Sultan Moulay Slimane University, Béni Mellal
(Morocco), for reading and correcting the English language of the
paper. This work was supported by the Lalla Salma Foundation:
Prevention and Treatment of Cancer. Research project No. 09/AP
2013 (Rabat-Morocco).
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https://doi.org/10.1002/ardp.201800128