In Vitro Antitumor Activity of Newly Synthesized Pyridazin-3(2H)-One Derivatives via Apoptosis Induction

Article abstract of DOI:10.1007/s11094-018-1712-x

Systemic toxicity associated with drug resistance continues to be the major obstacle to curative therapy of cancer. Tumor cell resistance to chemotherapeutic drugs often results in coordinate resistance to other structurally and functionally unrelated drugs and the subsequent development of cross resistance phenotype. Therefore, it seems necessary to identify new molecules as anticancer agents. In this process, we synthesized a series of new pyridazin-3(2H)-one derivatives and evaluated their antitumor potential. These cyclic molecules were synthesized and designed as a combination of benzofuran with pyridazinones. All final compounds have been characterized by spectral and elemental analyses to confirm successful synthesis reactions. To evaluate their anticancer activity, all derivatives were assessed against the human breast adenocarcinoma cell line (MCF-7) and the murine mastocytoma cell line (P815) using the methyl tetrazolium Test (MTT assay). The cytotoxic activity was found to be dose-dependent and the IC₅₀ values of the synthesized compounds ranged from 14.5 to 40 μM against MCF-7 and from 35 to 82.5 μM against P815. At the same time, no cytotoxic activity was observed against normal cells. In order to investigate the molecular mechanism of the most cytotoxic product (6f), apoptosis induction was measured against MCF-7 cells. Using the annexin-V FITC staining technique, we showed that the cytotoxic effect of this product is associated with apoptosis induction.

Full text of DOI:10.1007/s11094-018-1712-x

DOI 10.1007/s11094-018-1712-x
Pharmaceutical Chemistry Journal, Vol. 51, No. 10, January, 2018 (Russian Original Vol. 51, No. 10, October, 2017)
IN VITRO ANTITUMOR ACTIVITY OF NEWLY
SYNTHESIZED PYRIDAZIN-3(2H)-ONE
DERIVATIVES VIA APOPTOSIS INDUCTION
Najat Bouchmaa,1, 2 Mounir Tilaoui,2 Youness Boukharsa,1
Abdessalam Jaâfari,2 Hassan Aît Mouse,2 My. Ali Oukerrou,2
Jamal Taoufik,1 M’hammed Ansar,1 and Abdelmajid Zyad2, *
Original article submitted February 25, 2017.
Systemic toxicity associated with drug resistance continues to be the major obstacle to curative therapy of can-
cer. Tumor cell resistance to chemotherapeutic drugs often results in coordinate resistance to other structurally
and functionally unrelated drugs and the subsequent development of cross resistance phenotype. Therefore, it
seems necessary to identify new molecules as anticancer agents. In this process, we synthesized a series of
new pyridazin-3(2H)-one derivatives and evaluated their antitumor potential. These cyclic molecules were
synthesized and designed as a combination of benzofuran with pyridazinones. All final compounds have been
characterized by spectral and elemental analyses to confirm successful synthesis reactions. To evaluate their
anticancer activity, all derivatives were assessed against the human breast adenocarcinoma cell line (MCF-7)
and the murine mastocytoma cell line (P815) using the methyl tetrazolium Test (MTT assay). The cytotoxic
activity was found to be dose-dependent and the IC50 values of the synthesized compounds ranged from 14.5
to 40 mM against MCF-7 and from 35 to 82.5 mM against P815. At the same time, no cytotoxic activity was
observed against normal cells. In order to investigate the molecular mechanism of the most cytotoxic product
(6f), apoptosis induction was measured against MCF-7 cells. Using the annexin-V FITC staining technique,
we showed that the cytotoxic effect of this product is associated with apoptosis induction.
Keywords: Pyridazin-3(2H)-one derivatives; cytotoxicity; tumor cells; MTT assay; apoptosis.
1. INTRODUCTION
serious need for newly synthesized or natural products with
enhanced bioactivity. Pyridazin-3(2H)-one derivatives con-
stitute an interesting class of heterocyclic lead compounds
for drug discovery and research into anticancer molecules [6,
7, 8]. 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 [9, 10, 11, 12, 13]. In addition, pyridazin-3(2H)-ones
are endowed by many biological activities, e.g., antidepres-
sant, antihypertensive, antithrombotic, anticonvulsant, car-
diotonic, antibacterial, diuretic, anti-inflammatory, anti-HIV,
hepatoprotective, and prostaglandin inhibitor [14, 15, 16]. In
this context, the present study was aimed at synthesizing new
pyridazin-3(2H)-one derivatives with cytotoxic activity. The
obtained molecules were tested for their antitumor activity
against two tumor cell lines: MCF-7 (human breast
adenocarcinoma) and P815 (murine mastocytoma). The
structure-activity relationship has been considered.
Cancer, also known as a malignant tumor or malignant
neoplasm, is a frightful disease. As estimated by the World
Health Organization (WHO), more than 21 million new can-
cer cases and 13 million deaths are expected by 2030 [1]. In
particular, breast cancer is now considered the leading cause
of cancer-related death among women worldwide [2]. De-
spite the tremendous success of anticancer drugs such as
cisplatin derivatives, these compounds suffer from two main
disadvantages: they are encumbered by the development of
secondary resistance by cancer cells and they have severe
side effects such as nephrotoxicity [3, 4, 5]. Thus, there is a
1
Laboratory of Medicinal Chemistry, Faculty of Medicine and Pharmacy,
Mohammed V University, Rabat, Morocco.
2
Laboratory of Biological Engineering, Natural Substances, Team of Cellu-
lar and Molecular Immuno-Pharmacology, Immunobiology of Cancer
Cells, Faculty of Sciences and Technologies, Sultan Moulay Slimane Uni-
versity, Beni-Mellal, Morocco.
*
e-mail: ab.zyad2@gmail.com
893
0091-150X/17/5110-0893 © 2017 Springer Science+Business Media, LLC

894
Najat Bouchmaa et al.
Fig. 1. Synthesis of target compounds 6a - 6f. Reagents : (a) CHCl3/NaOH 10N, reflux 2h ; (b) BrCH2CH(OC2H5)2/K2CO3/DMF, reflux 4h ;
(c) CH3COOH, reflux 24h ; (d) H3CCOCH2CH2COOH/CH3COOH, reflux 24h ; (e) H2NNH2/EtOH, reflux 2h.
2. MATERIALS AND METHODS
France. Tumor cell lines MCF-7 and P815 were from the
stock cultures of the Laboratory of Biological Engineering,
Faculty of Science & Technology, Sultan Moulay Slimane
University, Morocco.
2.1. Chemicals and Cell Lines
Dulbecco’s modified Eagle’s medium (DMEM) and fetal
bovine serum were from Gibco BRL, Cergy Pontoise,
France. Penicillin G-streptomycin, methotrexate (MTX), di-
methyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT), trypsin/ethylene-
diaminetetraacetic acid (EDTA), phosphate buffer solution
(PBS), ficoll-hypaque, annexin-V biotin-streptavidin fluore-
scein isothiocyanate (FITC) were from Sigma Aldrich,
2.2. Synthesis of Drug Molecules
The scheme of synthesis of 5-(benzo[b]furan-2-ylme-
thyl)-6-methylpyridazin-3(2H)-one derivatives is outlined in
Fig. 1 and Table 1. Salicylaldehydes 2a - 2f, which are not
commercially available, were synthesized via Reimer -
Thieman formylation of the appropriate substituted phenols
1 with CHCl and NaOH. Aldehydes 2a - 2f had been previ-
3
ously synthesized with very low yield. Briefly, reactions of
intermediates 2a - 2f with bromacetaldehyde diethylacetal in
the presence of potassium carbonate in DMF (dimethylfor-
mamide) yielded compounds 3a - 3f. Compounds 3 were
cyclized to benzo[b]furan-2-ylcarboxaldehydes 4a - 4f by
heating in concentrated acetic acid. Benzo[b]furanaldehydes
4 were prepared according to the method described in litera-
ture [17]. Treatment of substituted aldehydes 4 with levulinic
acid in the presence of acetic acid gave products 5a - 5f,
which were treated by hydrazine hydrate in ethanol at reflux
temperature to afford the target pyridazin-3(2H)-ones 6a - 6f
[18].
TABLE 1. Substituents R in Synthesized Compounds
Compound
R
H
a
b
c
d
e
f
CH3
CH2CH3
CH(CH3)2
OCH3
OCH2CH2CH3

In Vitro Antitumor Activity of Newly Synthesized Pyridazin-3(2H)-one Derivatives
895
Fig. 2. In vitro cytotoxicity of compounds 6a - 6f against P815 tumor cell line. Cells were treated with increasing concentrations of compounds
6a - 6f. After 48-h incubation, cytotoxicity was determined as described in Section 2.3.2. Each value represents the mean ± SD of three inde-
pendent experiments.
2.3. Pharmacological Activity
land) apparatus. Three independent sets of experiments per-
formed in duplicate were evaluated. The relative inhibition
of cell proliferation was calculated by the following formula:
2.3.1. Cell cultures. MCF-7 (human breast adenocarci-
noma) and P815 (murine mastocytoma) cell lines were cul-
tured in Dulbecco’s modified Eagle’s medium (DMEM) sup-
plemented with 5% heat-inactivated fetal bovine serum
(Gibco BRL, CergyPontoise, France), penicillin G-strepto-
mycin (1%), and 0.2% sodium bicarbonate (Sigma). Incuba-
tion was performed at 37°C in humidified atmosphere con-
% Inhibition = 100 ´ (1 – A/A )
0
where A and A are the absorbances of negative control and
0
test culture, respectively.
The cytotoxic effects of pyridazin-3(2H)-one derivatives
against the two cell lines were compared using their IC val-
50
taining 5% CO .
2
ues (concentration of tested molecules leading to 50% inhibi-
tion of cell viability).
2.3.2. Cytotoxic activity evaluation. MCF-7 and P815
cell lines were harvested from starting cultures at the expo-
nential growth phase. After PBS wash, adherent cells were
harvested from sub-confluent cultures using trypsin/EDTA
(0.05%/0.02%) and suspended in DMEM. The harvested
cells were poured in flat-bottomed 96-well microtiter plates
containing 100 mL of complete medium per well (5 ´ 104
cells/mL). Three hours later, several concentrations of
pyridazin-3(2H)-one derivatives in DMSO completed to
100 µL with complete DMEM were added. Control cells
were treated with DMSO alone. In all cases, the final con-
centration of DMSO never exceeded 2%. After 48 h incuba-
2.3.3. Cytotoxic effect against peripheral blood
mononuclear cells (PBMCs). This test was realized in order
to evaluate the effect of synthesized pyridazin-3(2H)-one de-
rivatives against normal cells using the MTT colorimetric as-
say described above. To isolate the human PBMCs, blood
samples were collected from 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 pres-
ence of various concentrations of synthesized pyrida-
zin-3(2H)-one derivatives.
tion in humidified atmosphere at 37°C and 5% CO , 100 mL
2
of medium was carefully removed from each well and re-
placed with 20 mL 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 (Fin-
2.4. Apoptosis Assay
Apoptosis induction by the most cytotoxic compound
(6f) was evaluated using the annexin-V biotin-streptavidin
FITC assay to examine the occurrence of phosphatidylserine

896
Najat Bouchmaa et al.
Fig. 3. In vitro cytotoxicity of compounds 6a - 6f against MCF-7 tumor cell line. Cells were treated with increasing concentrations of com-
pounds 6a - 6f. After 48-h incubation, cytotoxicity was determined as described in Section 2.3.2. Each value represents the mean ± SD of three
independent experiments.
externalization and loss of membrane integrity. Briefly,
MCF-7 tumor cells (2 ´ 105 cells/well), treated with IC of
spectrometer using tetramethylsilane (TMS) as internal stan-
dard and CDCl and DMSO-d as solvent. The mass spectra
3
6
50
were recorded on an API 3200 LC/MS/MS mass spectrome-
ter using electrospray ionization (ESI) in positive polarity.
3.1.2. General procedure for the synthesis of phenols
2a - 2f (Method A).
compound 6f, were incubated at 37°C under 5% CO for 6 h.
2
After incubation in the same culture conditions as above, cell
pellets were washed twice with PBS, resuspended in
Annexin-V biotin, and treated with streptavidin conjugated
to FITC for 30 min. The fluorescence was visualized using
an Olympus BX51 microscope equipped with an appropriate
fluorescence filter in order to detect apoptosis induction
[19, 20].
A solution of substituted phenol 1 (0.5 mol) in 300 mL of
10 N NaOH (3 mol) was heated to 65°C. Then, 80 mL of
CHCl was added in three portions over 15 min. The mixture
3
was heated under reflux in chloroform for 2 h. After cooling,
the mixture was acidified to pH 1 with 12 N HCl, the organic
layer was collected and the aqueous layer was extracted with
chloroform. The combined chloroform solution was dried
and evaporated to give a crude product which was distilled or
recrystallized from an appropriate solvent.
2.5. Statistical Analysis
Experiments were performed in duplicate and data were
expressed as the mean ± SD of three experimental runs. The
statistical Student’s or ANOVA tests were performed on
Graph Pad Prism6 Software and considered significant at
p < 0.05.
2-Hydroxybenzaldehyde (2a). This compound was ob-
tained as yellowish oil; yield, 20%; bp, 75 – 77°C
1
(0.4 mmHg); IR (KBr; n , cm-1): 1655 (C=O); H NMR
max
(CDCl , 400 MHz; d, ppm): 7.00 – 8.10 (m, 4H, H , H , H ,
3
3
4
5
3. RESULTS AND DISCUSSION
H ), 9.85 (s,1H, -CHO), 10.85 (s, 1H, exch D O, -OH).
6
2
2-Hydroxy-5-methylbenzaldehyde (2b). This com-
3.1. Chemistry
pound was obtained as white solid; yield, 95%; mp,
48 – 50°C (petroleum ether); IR (KBr; n , cm-1): 1650
3.1.1. Physicochemical analysis. Melting points were
determined on a Büchi SMP 20 apparatus and remained un-
corrected. Infrared (IR) spectra were recorded with a
max
1
(C=O), 2900 – 3000 (C-H); H NMR (CDCl , 400 MHz; d,
3
ppm): 2.37 (s, 3H, -CH ), 6.90 (d, 1H, J = 8.60 Hz, H ),
3
3
1
VERTEX 70 FT-IR (Bruker Optics) spectrometer. The H
7.25 – 7.50 (m, 2H, H H ), 9.80 (s, 1H, -CHO), 10.75 (s,
4,
6
NMR spectra were recorded on a Bruker Avance (400 MHz)
1H, exch D O, -OH).
2

In Vitro Antitumor Activity of Newly Synthesized Pyridazin-3(2H)-one Derivatives
897
2-Hydroxy-5-ethylbenzaldehyde (2c). This compound
J = 3.20 Hz, H ), 7.64 (d, 1H, J = 3.20 Hz, H ), 10.50 (s,
2 5 3
was obtained as yellow oil; yield, 36%; bp, 74 – 76°C
(0.3 mmHg); IR (KBr; n , cm-1): 1651 (C=O), 2900 – 3000
1H, -CHO).
(2-Formyl-4-isopropylphenoxy) acetaldehyde diethyl-
acetal (3d).This compound was obtained as yellow oil;
max
1
(C-H); H NMR (CDCl , 400 MHz; d, ppm): 1.25 (t, 3H,
3
yield, 80%; bp, 160 – 162°C (0.3 mmHg); IR (KBr; n
1
,
J = 7.41 Hz, -CH -CH ), 2.70 (q, 2H, J = 7.41 Hz,
max
2
3
cm-1): 1700 (C=O), 2900 – 3000 (C-H); H NMR (CDCl ,
-CH -CH ), 6.83 (d, 1H, J = 9.50 Hz, H ), 7.26 – 7.52 (m,
3
2
3
3
400 MHz; d, ppm): 1.00 – 1.30 (m, 12H, (-OCH -CH ) ,
2H, H H ), 9.87 (s, 1H, -CHO), 10.88 (s, 1H, exch D O,
2
3 2
4,
6
2
H C-CH-CH ), 2.92 (h, 1H, J = 6.41Hz, H C-CH-CH ),
-OH).
3
3
3
3
2-Hydroxy-5-isoprpylbenzaldehyde (2d).This com-
3.50 – 3.90 (m, 4H, (-OCH -CH ) ), 4.10 (d, 2H,
2 3 2
pound was obtained as yellow oil; yield, 32%; bp, 78 – 80°C
(0.3 mmHg); IR (KBr; n , cm-1): 1650 (C=O), 2900 – 3000
J = 5.30 Hz, -CH -CH), 4.88 (t, 1H, J = 5.30 Hz, -CH -CH),
2 2
6.90 (d, 1H, J = 8.51 Hz, H ), 7.40 (dd, 1H, J = 2.70,
6
1
max
1
(C-H); H NMR (CDCl , 400 MHz; d, ppm): 1.25 (d, 6H,
J = 8.50 Hz, H ), 7.70 (d, 1H, J = 2.70 Hz, H ), 10.50 (s,
2
5
3
3
1H, -CHO).
J = 6.70 Hz, H C-CH-CH ), 2.95 (h, 1H, J = 6.70 Hz,
3
3
(2-Formyl-4-methoxyphenoxy) acetaldehyde diethyl-
H C-CH-CH ), 6.88 – 7.38 (m, 3H, H H , H ), 9.87 (s, 1H,
3
3
3,
4
6
acetal (3e).This compound was obtained as yellow oil; yield,
68%; bp, 154 – 156°C (0.3 mmHg); IR (KBr; n , cm-1):
-CHO), 10.88 (s, 1H, exch D O, -OH).
2
2-Hydroxy-5-methoxybenzaldehyde (2e) and 2-hyd-
roxy-5 propoxybenzaldehyde (2f). These compounds are
commercially available products.
max
1700 (C=O), 2900 – 3000 (C-H); 1H NMR (CDCl ,
3
400 MHz; d, ppm): 1.25 (t, 6H, J = 6.40 Hz, (-OCH -CH ) ),
2
3 2
3.1.3. General procedure for the synthesis of 2-formyl-
phenoxyacetadehyde diethyl acetals 3a - 3f (Method B). To
a stirred suspension containing substituted 2-hydroxybenz-
aldehyde 2 (0.15 mol) and potassium carbonate (0.16 mol) in
100 mL of DMF, bromoacetaldehyde diethyl acetal
(0.16 mol) was added dropwise and the mixture was refluxed
for 4 h. After cooling, the precipitate was filtered off, the sol-
vent was evaporated under reduced pressure, and the oily
residue was distilled.
2.38 (s, 3H, -OCH ), 3.45 – 4.30 (m, 4H, (-OCH -CH ) ),
3 2 3 2
4.42 (d, 2H, J = 5.10 Hz, -CH -CH), 4.97 (t, 1H, J = 5.10 Hz,
2
-CH -CH), 6.95 (d, 1H, J = 8.21 Hz, H ), 7.45 (dd, 1H,
2
6
J = 2.11, J = 8.21 Hz, H ), 7.65 (d, 1H, J = 2.11 Hz, H ),
1
2
5
3
10.56 (s, 1H, -CHO).
(2-Formyl-4-propoxyphenoxy) acetaldehyde diethyl-
acetal (3f).This compound was obtained as yellow oil; yield,
67%; bp, 180 – 183°C (0.5 mmHg); IR (KBr; n , cm-1):
max
1700 (C=O), 2900 – 3000 (C-H); 1H NMR (CDCl ,
3
2-Formylphenoxy acetaldehyde diethylacetal (3a).
400 MHz; d, ppm): 1.01 (t, 3H, J = 7.21 Hz,
This compound was obtained as yellowish oil; yield, 85%;
bp, 181 – 183°C (5 mmHg); IR (KBr; n , cm-1): 1700
-OCH -CH -CH ), 1.24 (t, 6H, J = 6.91 Hz, (-OCH -CH ) ),
2
2
3
2
3 2
1.78 (m, 2H, -OCH -CH -CH ), 3.69 (m, 4H,
max
2
2
3
1
(C=O), 2900 – 3000 (C-H); H NMR (CDCl , 400 MHz; d,
(-OCH -CH ) ), 3.90 (t, 2H, J = 6.21 Hz, -OCH -CH -CH ),
3
2
3 2
2
2
3
ppm): 1.23 (t, 6H, J = 6.42 Hz, (-OCH -CH ) ), 3.51 – 4.12
4.07 (d, 2H, J = 5.11 Hz, -CH -CH), 4.83 (t, 1H, J = 5.11 Hz,
2
3 2
2
(m, 4H, (-OCH -CH ) ), 4.14 (d, 2H, J = 5.13 Hz,
-CH -CH), 6.76 – 7.31 (m, 3H, H , H , H ), 10.46 (s, 1H,
2
3 2
2
3
5
6
-CH -CH), 4.89 (t, 1H, J = 5.13 Hz, -CH -CH), 6.95 – 7.82
-CHO).
3.1.4. General procedure for the synthesis of substi-
tuted benzo[b]furan-2-yl carboxaldehydes 4a 4f
2
2
(m, 4H, H , H , H , H ), 10.48 (s, 1H, -CHO).
3
4
5
6
(2-Formyl-4-methylphenoxy) acetaldehyde diethyl-
acetal (3b). This compound was obtained as yellow oil;
-
(Method C). A stirred solution of compound 3 (0.1 mol) in
35 mL of concentrated acetic acid was refluxed for 24 h. Af-
ter cooling, the solution was evaporated to dryness and the
crude product was distilled or recrystallized from an appro-
priate solvent.
yield, 80%; bp, 192 – 194°C (5 mmHg); IR (KBr; n
1
,
max
cm-1): 1700 (C=O), 2900 – 3000 (C-H); H NMR (CDCl ,
3
400 MHz; d, ppm): 1.25 (t, 6H, J = 6.40 Hz, (-OCH -CH ) ),
2
3 2
2.30 (s, 3H, -CH ), 3.50 – 4.00 (m, 4H, (-OCH -CH ) ), 4.12
3
2
3 2
2-Formylbenzo[b]furan (4a).This compound was ob-
tained as yellow oil; yield, 75%; bp, 121 – 123°C (3 mmHg);
IR (KBr; n , cm-1): 1680 (C=O); 1H NMR (CDCl ,
(d, 2H, J = 5.10 Hz, -CH -CH), 4.87 (t, 1H, J = 5.10 Hz,
2
-CH -CH), 6.90 (d, 1H, J = 8.20 Hz, H ), 7.40 (dd, 1H,
2
6
J = 2.11 Hz, J = 8.20 Hz, H ), 7.62 (d, 1H, J = 2.11 Hz,
max
3
1
2
5
H ), 10.50 (s, 1H, -CHO).
400 MHz; d, ppm): 7.26 – 7.74 (m, 5H, H
H H H H ),
3
3¢, 4¢, 5¢, 6¢, 7¢
(2-Formyl-4-ethylphenoxy) acetaldehyde diethylace-
9.84 (s, 1H, -CHO).
tal (3c).This compound was obtained as yellow oil; yield,
81%; bp, 198 – 200°C (5 mmHg); IR (KBr; n , cm-1):
2-Formyl-5-methylbenzo[b]furan (4b).This compound
was obtained as yellow solid; yield, 66%; mp, 26 – 27°C
(ehanol/water 9/1); IR (KBr; n , cm-1): 1680 (C=O),
max
1690 (C=O); 2900 – 3000(C-H); 1H NMR (CDCl ,
3
max
1
2900 – 3000 (C-H); H NMR (CDCl , 400 MHz; d, ppm):
400 MHz; d, ppm): 1.00 – 1.38 (m, 9H, (-OCH -CH ) ,
2
3 2
3
-CH -CH ), 2.62 (q, 2H, J = 7.61 Hz, -CH -CH ),
2.38 (s, 3H, -CH ), 7.40 – 7.77 (m, 4H, H
H
H
H ),
2
3
2
3
3
3¢,
4¢,
6¢,
7¢
3.50 – 4.00 (m, 4H, (-OCH -CH ) ), 4.08 (d, 2H,
9.87 (s, 1H, -CHO).
2
3 2
J = 4.70 Hz, -CH -CH), 4.88 (t, 1H, J = 4.70 Hz, -CH -CH),
5-Ethyl-2-formylbenzo[b]furan (4c).This compound
was obtained as yellowish oil; yield. 80%; bp, 130 – 132°C
2
2
6.88 (d, 1H, J = 8.50 Hz, H ), 7.57 (dd, 1H, J = 8.50 Hz,
6
1

898
Najat Bouchmaa et al.
2.41 (s, 3H, -OCH ), 7.43 – 7.80 (m, 4H, H
H
H H ),
3
3¢,
4¢,
6¢, 7¢
9.89 (s, 1H, -CHO).
2-Formyl-5-propoxybenzo[b]furan (4f). This com-
pound was obtained as yellow solid; yield, 85%; mp,
65 – 67°C (diisopropyl ether); IR (KBr; n , cm-1): 1700
max
1
(C=O), 2900 – 3000 (C-H); H NMR (CDCl , 400 MHz; d,
3
ppm): 0.97 (t, 3H, J = 7.20 Hz, -OCH -CH -CH ), 1.73 (m,
2
2
3
2H,
-OCH -CH -CH ),
3.94
(t,
2H,
J = 6.60,
2
2
3
-OCH -CH -CH ), 7.15 (dd, 1H, J = 9.10 Hz, J = 2.70 Hz,
2
2
3
1
2
H ), 7.31 (d, 1H, J = 2.70 Hz, H ), 7.61 (d, 1H, J = 9.10 Hz,
6¢
H ), 7.86 (s, 1H, H ), 9.79 (s, 1H, -CHO).
4¢
7¢
3¢
3.1.5. General procedures for the synthesis of substi-
tuted 3-benzo[b]furan-2-ylmethylene-levulinic acids 5a -
5f (Method D). A stirred solution of compound 4 (0.1 mol)
in 35 mL of concentrated acetic acid was refluxed for 24 h.
After cooling, the solution was evaporated to dryness and ob-
tained residue was used crude for continuation of the drug
synthesis.
Fig. 4. Cytotoxicity of compound 6f against PBMCs from healthy
donors (****p values < 0.0001 indicate significant difference).
3.1.6. General procedures for the synthesis of substi-
tuted 5-(benzo[b]furan-2-ylmethyl)-6-methylpyridazin-
3(2H)-ones 6a - 6f (Method E). The mixture of acid 5 and
hydrazine hydrate solution in ethanol was refluxed for 2h.
The precipitate was filtered and recrystallized from an appro-
priate solvent.
(3 mmHg); IR (KBr; n , cm-1): 1680 (C=O), 2900 – 3000
max
1
(C-H); H NMR (CDCl , 400 MHz; d, ppm): 1.30 (t, 3H,
3
J = 7.90 Hz, -CH -CH ), 2.76 (q, 2H, J = 7.90 Hz,
2
3
5-(Benzo[b]furan-2-ylmethyl)-6-methylpyridazin-3(2
-CH -CH ), 7.25 – 7.70 (m, 4H, H
H
H
H ), 9.86 (s,
2
3
3¢,
4¢,
6¢,
7¢
H)-one (6a). This compound was obtained as yellow solid;
yield, 69%; mp, 181 – 183°C (ethanol); IR (KBr; n , cm-1):
1H, -CHO).
max
2-Formyl-5-isopropylbenzo[b]furan (4d).This com-
1
1604 (C=N), 1662 (C=O), 2900 – 3000 (C-H); H NMR
pound was obtained as yellowish oil; yield, 56%; bp,
(DMSOd , 400 MHz; d, ppm): 2.22 (s, 3H, -N=C-CH ), 4.08
6
3
131 – 133°C (3 mmHg); IR (KBr; n , cm-1): 1700 (C=O),
max
(s, 2H, -CH -), 6.97 (s, 1H, H ), 6.71 (s, 1H, H ), 7.19 – 7.58
2
4
3¢
(m, 4H, H H , H , H ), 12.71 (ls, 1H, NH); MS (m/z):
1
2900 – 3000 (C-H); H NMR (CDCl , 400 MHz; d, ppm):
3
4¢,
5¢
6¢
7¢
241.2 [M+H]+, 238.9 [M+H]-, 263.3 [M+Na]+.
1.28 (d, 6H, J = 6.30 Hz, H C-CH-CH ), 3.00 (h, 1H,
3
3
J = 6.30 Hz, H C-CH-CH ), 7.48 – 7.63 (m, 4H, H
H H
5-(5-Methylbenzo[b]furan-2-ylmethyl)-6-methylpyri
dazin-3(2H)-one (6b). This com-pound was obtained as yel-
low solid: yield, 80%; mp, 183 – 184°C (ethanol); IR (KBr;
3
3
3¢, 4¢, 6¢,
H ), 9.85 (s, 1H, -CHO).
7¢
2-Formyl-5-methoxybenzo[b]furan (4e). This com-
, cm-1): 1603 (C=N), 1666 (C=O), 2900 – 3000 (C-H);
pound was obtained as yellow solid; yield, 70%; mp, 82 – 84
n
max
(diisopropyl ether); IR (KBr; n , cm-1): 1700 (C=O),
1H NMR (DMSOd , 400 MHz; d, ppm): 2.23 (s, 3H,
max
6
1
2900 – 3000 (C-H); H NMR (CDCl , 400 MHz; d, ppm):
-N=C-CH ), 2.37 (s, 3H, Ar-CH ), 4.07 (s, 2H, -CH -), 6.58
3
3
2
3
TABLE 2. Cytotoxic Activity of Pyridazin-3(2H)-one Derivative 6f against P815 Cells
Lysis (%)
Compound
Concentration (mM)
1,95
3,90
7,81
15,62
31,25
62,50
125
250
IC50
6a
3,80 ± 0,13 12,57 ± 0,86 21,35 ± 1,62 30,12 ± 2,36 38,89 ± 3,12 47,66 ± 3,87 56,43 ± 4,65 66,705 ± 7,49
10,50 ± 4,91 19,73 ± 3,28 29,43 ± 2,05 36,84 ± 2,19 45,41 ± 3,07 54,72 ± 2,78 61,85 ± 1,29 72,78 ± 3,08
1,92 ± 2,58 11,45 ± 2,83 21,12 ± 2,89 30,78 ± 2,96 40,45 ± 3,03 49,19 ± 1,78 59,78 ± 3,16 68,96 ± 2,52
10,62 ± 4,60 17,96 ± 2,01 25,17 ± 1,36 35,38 ± 2,14 57,08 ± 13,33 64,84 ± 10,49 63,49 ± 4,28 70,88 ± 1,93
82,5 mM
42,5 mM
66 mM
46 mM
50 mM
35 mM
7,5 mM
6b
6c
6d,
6e
74,18 2,905
7,62 ± 3,246 14,67 ± 3,79 23,84 ± 4,47 31,64 ± 2,77 42,79 ± 3,45 52,76 ± 2,68 60,63 ± 3,02
6f
8,82 ± 1,18 15,68 ± 2,12 27,19 ± 2,14 36,73 ± 2,12 48,29 ± 2,16 59,93 ± 1,92 67,63 ± 3,08 80,34 ± 2,44
24,17 ± 0,28 34,46 ± 5,36 44,463 ± 5,36 60,81 ± 1,75 77,69 ± 0,93 78,35 ± 0,93 79,56 ± 1,71 81,23 ± 3,13
MTX

In Vitro Antitumor Activity of Newly Synthesized Pyridazin-3(2H)-one Derivatives
899
(KBr; n , cm-1): 1652(C=O), 2940 – 3000(C-H); 1H NMR
(s, 1H, H ), 6.64 (s, 1H, H ), 7.07 (dd, 1H, J = 8.41 Hz,
4
3¢
J = 1.21 Hz, H ), 7.36 (d, 1H, J = 1.21 Hz, H ), 7.40 (d, 1H,
1
max
(DMSOd , 400 MHz; d, ppm): 0.96 (t, 3H, J = 6.90 Hz,
-OCH -CH -CH ), 1.71 (m, 2H, -OCH -CH -CH ), 2.21 (s,
2
6¢
J = 8.41 Hz, H ), 12.74 (ls, 1H, NH); MS (m/z): 255.1
4¢
6
7¢
2
2
3
2
2
3
[M+H]+, 253.3 [M+H]-, 276.9 [M+Na]+.
3H, -N=C-CH ), 3.90 (t, 2H, J = 6.90 Hz, -OCH -CH -CH ),
3
2
2
3
5-(5-Ethylbenzo[b]furan-2-ylmethyl)-6-methylpyrida
zin-3(2H)-one (6c). This com-pound was obtained as brown
4.04 (s, 2H, -CH -), 6.55 (s, 1H, H ), 6.62 (s, 1H, H ), 6.81
2
4
3¢
(dd, 1H, J = 9.00 and 2.71 Hz, H ), 7.06 (d, 1H, J = 2.71 Hz,
6¢
H ), 7.38 (d, 1H, J = 9.00 Hz, H ), 12.74 (ls, 1H, NH).
solid; yield, 73%; mp, 180 – 181°C (ethanol); IR (KBr; n
,
4¢
7¢
max
1
cm-1): 1605 (C=N), 1661 (C=O), 2900 – 3000 (C-H); H
3.2. Cytotoxic Activity
NMR (DMSOd , 400 MHz; d, ppm): 1.18 (t, 3H,
6
Biological evaluation of pyridazin-3(2H)-one derivatives
6a - 6f using the MTT assay showed that compounds 6b, 6e
and 6f exhibited potent cytotoxicity against both tumor cell
lines (MCF7 and P815) in the micromolar range, whereas the
other three compounds (6a, 6c, and 6d) exhibited moderate
cytotoxicity as compared to that of the former compounds
and positive control (methotrexate, MTX). Analysis of re-
sults suggested that the activity was dose- and cell-depend-
ent. Comparison of data presented in Figs. 2 and 3 allow the
six compounds to be classified on the basis of their
cytotoxicity levels as 6f > 6e > 6a > 6c > 6b > 6d against
MCF-7 cell line and as 6f > 6b > 6d > 6e > 6c > 6a against
J = 7.50 Hz, -CH -CH ), 2.21 (s, 3H, -N=C-CH ), 2.65 (q,
2
3
3
2H, J = 7.50 Hz, -CH -CH ),), 4.05 (s, 2H, -CH -), 6.56 (s,
2
3
2
1H, H ), 6.64 (s, 1H, H ), 7.08 (dd, 1H, J = 8.40 Hz,
4
3¢
J = 1.80 Hz, H ), 7.37 (d, 1H, J = 1.80 Hz, H ), 7.41 (d, 1H,
1
2
6¢
J = 8.40 Hz, H ), 12.76 (ls, 1H, NH); MS (m/z): 269.5
[M+H]+, 267.1 [M+H]-, 291.3 [M+Na]+.
4¢
7¢
5-(5-Isopropylbenzo[b]furan-2-ylmethyl)-6-methylpy
ridazin-3(2H)-one (6d). This compound was obtained as
brown solid; yield, 82%: mp, 185 – 186°C (ethanol); IR
(KBr; n , cm-1): 1605 (C=N), 1682 (C=O), 2900 – 3000
max
(C-H); 1H NMR (DMSOd , 400 MHz; d, ppm): 1.24 (d, 6H,
6
J = 6.90 Hz, H C-CH-CH ), 2.10 (s, 3H, -N=C-CH ), 2.99
3
3
3
P815 cell line. The obtained IC values (Tables 2a and 2b)
50
(h, 1H, J = 6.90 Hz, H C-CH-CH ), 3.90 (s, 2H, -CH -),
3
3
2
ranged from 14.5 to 40 mM for MCF-7 and from 35 to
82.5 mM for P815. Compound 6f bearing the propoxy
(OCH CH CH ) group in the radical position showed maxi-
7.30 – 7.76 (m, 5H, H , H H , H , H ), 12.32 (ls, 1H, NH);
4
3¢, 4¢
6¢
7¢
MS (m/z): 283.4 [M+H]+, 281.2 [M+H]-, 337.4 [M+Na]+.
5-(5-Methoxybenzo[b]furan-2-ylmethyl)-6-methylpyr
idazin-3(2H)-one (6e). This compound was obtained as yel-
low solid; yield, 78%; mp, 190 – 192°C (ethanol); IR (KBr;
2
2
3
mum activity, i.e., the lowest IC values (14.5 and 35 mM
for MCF-7 and P815, respectively).
50
3.3. Cytotoxicity of Tested Molecules with Respect to Human
Normal Cells
n
, cm-1): 1650 (C=O), 2950 – 3000 (C-H); 1H NMR
max
(DMSOd , 400 MHz; d, ppm): 2.25 (s, 3H, -N=C-CH ), 3.74
6
3
(s, 3H, -OCH ), 4.04 (s, 2H, -CH -), 6.55 (s, 1H, H ), 6.64 (s,
The majority of clinically approved anticancer drugs are
characterized by a narrow therapeutic window that results
mainly from a high systemic toxicity of drugs. Thus, we
tested the most important compounds (which were more
cytotoxic against tumor cells) with respect to human periph-
eral blood mononuclear cells (PBMCs) in order to determine
their effect against normal cells. The results presented in
Fig. 4 show the absence of any cytotoxic effect of com-
3
2
4
1H, H ), 6.82 (dd, 1H, J = 8.70 and 2.71 Hz, H ), 7.08 (d,
3¢
1H, J = 2.71 Hz, H ), 7.40 (d, 1H, J = 8.70 Hz, H ), 12.75
6¢
4¢
7¢
(ls, 1H, NH); MS (m/z): 299.4 [M+H]+, 297.1 [M+H]-, 321.4
[M+Na]+.
5-(5-Propoxybenzo[b]furan-2-ylmethyl)-6-methylpyr
idazin-3(2H)-one (6f). This compound was obtained as
brown solid; yield, 87%; mp, 194 – 196°C (ethanol); IR
TABLE 3. Cytotoxic Activity of Pyridazin-3(2H)-one Derivative 6f against MCF-7 Cells
Lysis (%)
Compound
Concentration ( mM)
0,78125
1,5625
3,125
6,25
12,5
25
50
100
IC50
6a
11,51 ± 2,33 21,26 ± 6,49 26,71 ± 1,67 37,28 ± 5,91 44,90 ± 3,52 50,65 ± 0,94 55,92 ± 5,19 62,06 ± 2,66
4,71 ± 2,56 13,37 ± 3,28 27,29 ± 2,48 38,44 ± 3,29 46,58 ± 2,76 48,9 5 ± 2,53 52,81 ± 1,21 60,94 ± 1,05
7,51 ± 2,75 14,62 ± 3,14 26,97 ± 2,32 36,14 ± 2,46 43,30 ± 4,42 49,41 ± 6,42 55,86 ± 6,88 64,73 ± 3,94
12,31 ± 1,14 18,90 ± 3,39 25,50 ± 5,82 32,09 ± 3,92 38,69 ± 3,05 45,28 ± 5,19 51,88 ± 2,14 58,47 ± 4,23
23 mM
31 mM
28 mM
40 mM
19 mM
14,5 mM
4,9 mM
6b
6c
6d
6e
6,06 ± 3,87 16,36 ± 5,71 27,86 ± 2,20 34,83 ± 2,19 43,44 ± 1,42 54,04 ± 4,80 61,35 ± 3,16
71,17 ± 2
6f
9,94 ± 1,28 17,05 ± 4,73 27,61 ± 0,65 38,68 ± 0,93 48,55 ± 1,98 59,84 ± 5,09 73,87 ± 2,24 82,14 ± 1,37
19,72 ± 7,63 26,18 ± 3,66 30,18 ± 3,50 61,38 ± 2,20 70,72 ± 5,29 76,183 ± 6,02 80,40 ± 2,92 90,21 ± 5,39
MTX

900
Najat Bouchmaa et al.
During the last decade, many interesting synthetic com-
pounds such as pyridazin-3(2H)-ones draw the attention of
medicinal chemists due to their potential bioactivity. In fact,
the handleable functional structure of pharmacophore moi-
eties in pyridazinones makes them an attractive source of
new active derivatives [21, 22]. To the best of our knowl-
edge, the combination of benzofuran and pyridazinones is of
this kind and has led to good results for the first time. Indeed,
our results demonstrated the ability of newly synthesized
pyridazin-3(2H)-ones derivatives to induce cytotoxicity
against human breast adenocarcinoma (MCF-7) and the
murine mastocytoma (P815) tumor cell lines (Figs. 2 and 3)
suggesting no specificity of organs. These results corroborate
recent findings showing a cytotoxic activity of other pyrida-
zin-3(2H)-one derivatives against lung cancer, central ner-
vous system cancer, leukemia, and pancreatic cell lines
[23, 24, 25].
More importantly, the most active compound 6f did not
show any cytotoxic effect against human PBMCs from
healthy donors. These PBMCs considered as normal cells
were used to evaluate the side effects of this product (Fig. 4).
These results suggest that this compound acts selectively
against tumor cells.
Considering the structure–activity relationship, the sub-
stitution at C in the benzofuran ring was of interest. In fact,
5
the increase in the aliphatic chain size in radical in the C of
5
benzofuran
enhanced
the
cytotoxic
effect
of
yridazin-3(2H)-one derivatives against both MCF7 and P815
cell lines. Furthermore, as shown in Fig. 2 and 3, compound
6f was the most active among all tested pyridazinones. The
interesting cytotoxic effect of 6f can probably be attributed to
the propoxy radical, which is characterized by its long
aliphatic chain associated with oxygen molecule. These re-
sults are in agreement with data demonstrating that com-
pounds substituted with this radical (propoxy) are relatively
potent as cytotoxic agents [26].
Fig. 5. Apoptosis induction analysis by annexin-V FITC test on
MCF7 tumor cells.
pounds tested on normal PBMCs, thus indicating a selective
cytotoxicity of these substances.
3.4. Annexin-V Biotin-Streptavidin FITC Analysis
for Apoptosis Assay
The search for new anti-tumor molecules employs vari-
ous approaches. The known approaches involving antimito-
tics, antimetabolites as well as alkylating and intercalating
agents are currently classified as classical. However, new de-
veloping approaches include the inducers of apoptosis/cellu-
lar senescence, molecules that control the cell cycle, those
that interfere with cell signaling, and agents controlling cel-
lular resistance, metastasis development, and angiogenesis
[27]. Among these new approaches, we are interested in that
based on the induction of apoptosis.
In order to understand the molecular mechanisms in-
volved in the observed cytotoxic activity of the most
cytotoxic compound (6f), apoptosis induction assay was per-
formed using the MCF-7 cell line and analyzed by
Annexin-V Biotin-Streptavidin staining as described in ma-
terials and methods. The results are reported in Fig. 5. As
shown in this figure, the product 6f induced a significant cell
apoptosis.
Apoptosis is the very tightly programmed cell death with
distinct biochemical and genetic pathways that play a critical
role in the development and homeostasis in normal tissues
[28]. Upon receiving specific signals instructing the cells to
undergo apoptosis, a number of distinctive changes occur in
the cell. A family of proteins known as caspases is typically
activated in the early stages of apoptosis. These proteins
cleave key cellular components that are required for normal
cellular function including structural proteins in the
cytoskeleton and nuclear proteins such as DNA repair en-
MCF-7 cells were treated with the IC of compound 6f
50
(C) after 6-h incubation. The assay was based on the ability
of annexin-V (green fluorescence) to bind to phosphatidylse-
rine exposed on the surface of cells undergoing apoptosis.
Cells cultured in a medium without serum were used as the
positive control (B). Cells were treated with DMSO alone as
negative control (A). The technique was performed as de-
scribed in materials and methods.

In Vitro Antitumor Activity of Newly Synthesized Pyridazin-3(2H)-one Derivatives
901
zymes. The caspases can also activate other degradative en-
zymes such as DNases, which begin to cleave the DNA in the
nucleus.
REFERENCES
1. S.-K. Kim (ed.) in: Handbook of Anticancer Drugs from Marine
Origin, Springer, Switzerland (2015), (p.) 1.
2. C. E. DeSantis, F. Bray, J. Ferlay, et al., Cancer Epidemiol.,
Biomarkers Prev., 24(10), 1495 – 1506 (2015).
3. K. Sato, S. Watanabe, and A. Ohtsubo, BMC Cancer, 16(1), 222
(2016).
During the apoptotic process, apoptotic cells display dis-
tinctive morphology. Typically, the cell begins to shrink fol-
lowing the cleavage of lamins and actin filaments in the
cytoskeleton. The apoptotic breakdown of chromatin in the
nucleus often leads to nuclear condensation and/or a “horse-
shoe” like appearance [28]. In order to correlate between the
cytotoxic activity of the compound 6f and induction of
apoptosis, cells were treated for 6 h with this compound and
then studied using the annexin-V/FITC test with fluores-
cence microscopy. Figure 5 shows a series of changes such
as membrane blebbing, cell shrinkage, and apoptotic body
formation. These data confirmed that cells treated with com-
pound 6f were involved in the apoptotic process. Other mo-
lecular mechanisms should also be investigated.
4. S. Zhu, N. Pabla, C. Tang, et al., Arch. Toxicol, 89(12),
2197 – 2205 (2015).
5. G. S Oh, H. J. Kim, A. Shen, et al., Electrolyte Blood Pressure,
12(2), 55 – 65 (2014).
6. S. Ovais, K. Javed, S. Yaseen, et al., Eur. J. Med. Chem, 67,
352–358 (2013).
7. M. S. R. Murty, K. R. Ram, R. V. Rao, et al., Med. Chem. Res,
20(5), 576 – 586 (2011).
8. M. B. Boxer, J. K. Jiang, M. G. Vander Heiden, et al., J. Med.
Chem., 53(3), 1048 – 1055 (2010).
9. V. Pogacic, A. N. Bullock, O. Fedorov, et al., Cancer Res.,
67(14), 6916 – 6924 (2007).
10. K. Natarajan, J. Bhullar, S. Shukla, et al., Biochem. Pharmacol.,
85(4), 514 – 524 (2013).
4. CONCLUSION
11. L. S. Chen, S. Redkar, P. Taverna, et al., Blood, 118(3),
693 – 702 (2011).
The obtained data showed an interesting cytotoxic activ-
ity of the newly synthesized pyridazin-3(2H)-one with
propoxy radical substitution. This substitution elongating the
aliphatic chain enhances the structure reactivity. Generally,
all the obtained compounds exhibited promising cytotoxic
effect that was especially pronounced for compound 6f,
which was highly cytotoxic against P815 and MFC-7 cell
12. J. M. Foulks, K. J. Carpenter, B. Luo, et al., Neoplasia, 16(5),
403 – 412 (2014).
13. L. Brault, S. Knapp, L. Brault, et al., Br. J. Cancer, 107(3),
491 – 500 (2012).
14. M. Asif, Int. J. Adv. Chem, 2(2), 148 – 161 (2014).
15. I. G. Rathish, K. Javed, S. Ahmad, et al., Eur. J. Med. Chem., 49,
304 – 309 (2012).
16. R. Bansal and S. Thota, Med. Chem. Res., 22(6), 2539 – 2552
(2013).
lines (with IC =35 and 14,5 mM, respectively). It is impor-
50
17. T. Hirota, H. Fujita, K. Sasaki, et al., J. Heterocycl. Chem.,
23(6), 1715 – 1716 (1986).
tant that this compound produces no cytotoxic action against
normal PBMCs. The interesting cytotoxic effect of 6f may
probably be attributed to the presence of propoxy radical.
Furthermore, the morphological alteration of MCF-7 cells
observed using the annexin-V/FITC test indicated that com-
pound 6f acted in part via apoptosis induction. In addition,
further studies should be conducted to explore the molecular
mechanisms underneath the cytotoxic activity, in vivo inves-
tigation and the interaction effects between this synthetizes
compounds with conventional drugs.
18. A. Benmoussa, J. El harti, M. Ansar, et al., Int. J. PharmTech
Res., 4(4), 1591 – 1594 (2012).
19. M. Tilaoui, H. A. Mouse, A. Jaafari, et al., Nat. Prod.
Bioprospect., 4(3), 189 – 196 (2014).
20. R. Sen, K. Natarajan, J. Bhullar, et al., Mol. Cancer Ther., 11(9),
2033 – 2044 (2012).
21. M. A. Ibrahim, A. H. Elmenoufy, M. Elagawany, et al., J.
Biosci. Med., 3(10), 59 (2015).
22. R. Bansal and S. Thota, Med. Chem. Res., 22(6), 2539 – 2552
(2013).
23. I. G. Rathish, K. Javed, S. Ahmad, et al., Eur. J. Med. Chem., 49,
304 – 309 (2012).
ACKNOWLEDGEMENTS
24. A. Pau, G. Murineddu, B. Asproni, et al., Molecules, 14(9),
3494 – 3508 (2009).
The authors are grateful to the University Mohammed V
and the Laboratory of Control of Drugs, Rabat-Morocco, for
measuring IR, NMR and mass spectra.
25. W. Malinka, A. Redzicka, and O. Lozach, Farmaco, 59(6),
457 – 462 (2004).
26. B. Wang, Z. W. Miao, J. Wang, et al., Amino Acids, 35(2),
463 – 468 (2008).
27. R. A. Lockshin and C. M. Williams, J. Insect Physiol., 11(2),
123 – 133 (1965).
CONFLICT OF INTEREST
28. M. Hassan, H. Watari, A. AbuAlmaaty, et al., BioMed Res. Int.,
2014, 1 – 23 (2014).
The authors declare no conflict of interest.