Synthesis, anti-inflammatory evaluation in vivo and docking studies of some new 5-(benzo[b]furan-2-ylmethyl)-6-methyl-pyridazin-3(2H)-one derivatives

Article abstract of DOI:10.1016/j.molstruc.2017.09.092

Seven novel 5-(benzo[b]furan-2-ylmethyl)-6-methyl-pyridazin-3(2H)-one derivatives (6a to 6g) have been synthesized by the condensation of appropriate 3-(benzofuran-2-ylmethylene)-4-oxopentanoic acid and hydrazine hydrate in ethanol. Structures of all compounds were elucidated by elemental analysis, IR, ¹H NMR and ¹³C NMR. These compounds were tested for their anti-inflammatory activity in carrageenan-induced rat paw edema model. In silico molecular docking study has been executed to study the binding interactions of the synthesized compounds with COX-2 protein. Compounds 6a, 6b, 6e and 6g showed a good anti-inflammatory activity at 50 mg/kg compared with the indometacin at 10 mg/kg and the aspirin at 150 mg/kg and good binding affinity with COX-2.

Full text of DOI:10.1016/j.molstruc.2017.09.092

Accepted Manuscript
Synthesis, anti-inflammatory evaluation in vivo and docking studies of some new 5-
(benzo[b]furan-2-ylmethyl)-6-methyl-pyridazin-3(2H)-one derivatives
Youness Boukharsa, Wiame Lakhlili, Jaouad El harti, Bouchra Meddah, Ramata
Yvette Tiendrebeogo, Jamal Taoufik, My El Abbes Faouzi, Azeddine Ibrahimi, M’hammed Ansar
PII:
S0022-2860(17)31291-7
10.1016/j.molstruc.2017.09.092
MOLSTR 24341
DOI:
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Revised Date:
Accepted Date:
22 April 2017
24 September 2017
25 September 2017
Please cite this article as: Youness Boukharsa, Wiame Lakhlili, Jaouad El harti, Bouchra Meddah,
Ramata Yvette Tiendrebeogo, Jamal Taoufik, My El Abbes Faouzi, Azeddine Ibrahimi, M’hammed
Ansar, Synthesis, anti-inflammatory evaluation in vivo and docking studies of some new 5-(benzo[b]
furan-2-ylmethyl)-6-methyl-pyridazin-3(2H)-one derivatives, Journal of Molecular Structure (2017),
doi: 10.1016/j.molstruc.2017.09.092
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form.
Please note that during the production process errors may be discovered which could affect the
content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Highlights
 Synthesis of a new series of pyridazin-3(2H)-ones derivatives,
 Biological evaluation of anti-inflammatory,
 Compounds 6a, 6b, 6e and 6g were found to be the most active compound,
 Molecular docking studies showed binding interactions at the active site of COX-2.

ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
1
2
Synthesis, anti-inflammatory evaluation in vivo and docking studies of some new 5-
(benzo[b]furan-2-ylmethyl)-6-methyl-pyridazin-3(2H)-one derivatives
3
4
5
Youness Boukharsa 1*, Wiame Lakhlili 2, Jaouad El harti 1, Bouchra Meddah 3, Ramata
Yvette Tiendrebeogo 3, Jamal Taoufik 1, My El Abbes Faouzi 3, Azeddine Ibrahimi 2,
M’hammed Ansar 1.
6
7
1- Laboratory of Therapeutic Chemistry, Faculty of Medicine and Pharmacy, BP 6203,
Rabat Institutes, Mohammed V University, Rabat, Morocco
8
2- Biotechnology Laboratory (MedBiotech), Faculty of Medicine and Pharmacy, Rabat
Institutes, Mohammed V University in Rabat, Morocco
9
10
11
3- Laboratory of Pharmacology and Toxicology, Pharmacokinetic Research Team, Faculty
of Medicine and Pharmacy, Rabat Institutes, Mohammed V University, Rabat, Morocco
12
*Corresponding author:
13
14
15
16
Dr. Youness BOUKHARSA
Laboratory of Therapeutic Chemistry, Faculty of Medicine and Pharmacy, Mohammed V University
BP 6203, Rabat Institutes, Rabat, Morocco
Email: boukharsay@yahoo.fr
17
E-mails:
Youness Boukharsa
Wiame Lakhlili
E-mail: boukharsay@yahoo.fr
E-mail:wlakhlili@yahoo.com
E-mail: harti.jaouad@gmail.com
E-mail: bouchra_meddah@yahoo.fr
E-mail: mfal19@yahoo.com
Jaouad El harti
Bouchra Meddah
Ramata Yvette Tiendrebeogo
Jamal Taoufik
E-mail: taoufikjamal@gmail.com
E-mail: myafaouzi@yahoo.fr
My El Abbes Faouzi
Azeddine Ibrahimi
M’hammed Ansar
E-mail: azeibrah@yahoo.com
E-mail: ansarmhammed@gmail.com
1

ACCEPTED MANUSCRIPT
19
Abstract
20
21
22
23
24
25
26
27
28
Seven novel 5-(benzo[b]furan-2-ylmethyl)-6-methyl-pyridazin-3(2H)-one derivatives (6a to
6g) have been synthesized by the condensation of appropriate 3-(benzofuran-2-ylmethylene)-
4-oxopentanoic acid and hydrazine hydrate in ethanol. Structures of all compounds were
1
13
elucidated by elemental analysis, IR, H NMR and C NMR. These compounds were tested
for their anti-inflammatory activity in carrageenan-induced rat paw edema model. In silico
molecular docking study has been executed to study the binding interactions of the
synthesized compounds with COX-2 protein. Compounds 6a, 6b, 6e and 6g showed a good
anti-inflammatory activity at 50 mg/kg compared with the indometacin at 10 mg/kg and the
aspirin at 150 mg/kg and good binding affinity with COX-2.
29
30
Keywords: Pyridazin-3(2H)-one, benzo[b]furan, anti-inflammatory activity, Molecular
docking.
2

ACCEPTED MANUSCRIPT
32
1. Introduction
33
34
35
36
37
38
39
40
41
42
43
Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely used
therapeutics for the treatment of pain and inflammation. The first NSAID discovered was
aspirin which has been used for more than 100 years. However, the molecular mechanism of
aspirin and other NSAIDs was elucidated only in the 1970s. Vane proposed that NSAIDs
produce their therapeutic and also undesirable effects by inhibition of cyclooxygenase (COX)
enzyme in prostaglandin synthetic pathway (1). Until recently, prostaglandin synthesis was
thought to result from only one form of COX enzyme however, in the early 1990s, it was
discovered that COX exists in two isoforms, COX-l (constitutive form) and COX-2 (inducible
form) (2). Thus a new hypothesis has been suggested to explain the effects of NSAIDs;
inhibition of COX-l accounts for the undesirable side effects, while inhibition of COX-2
accounts for the therapeutic benefits of NSAIDs (3).
44
45
46
47
48
49
Among these compounds, rofecoxib, celecoxib, valdecoxib and etoricoxib were approved and
marketed. However rofecoxib was taking out of the markeft in 2004 because of its cardiac
toxicity. Subsequently, some of the other coxibs have been voluntarily withdrawn from the
market. On the other hand, some studies have suggested that rofecoxib's adverse cardiac
events may not be a class effect but rather an intrinsic chemical property related to its
metabolism (4,5).
50
51
52
53
54
55
56
57
58
59
The use of pyridazinone compounds constitutes an attractive approach for the treatment of
several inflammatory disorders. Diverse pyridazinone derivatives have been reported to
exhibit a wide range of pharmacological activities (6) such as antihypertensive (7),
anticonvulsant (8), cardiotonic (9,10), antibacterial (11), saludiuretic (12), anti-HIV (13),
antihistaminic (14), antidepressant (15), phosphodiesterase (PDE) inhibitors (16) and COX-2
inhibitors (17). It has also been reported that pyridazinone derivative have remarkable
anticancer activity (18–20). Thus new pyridazinone derivatives are considerable interest in
medicinal chemistry and since inflammation is well known to be associated with many
diseases (21,22), the protective action of pyridazinone compounds against several diseases
could be associated with their anti inflammatory effects.
60
61
62
63
In this paper, we report synthesis of seven novel pyridazinone derivatives which have been
tested for their anti-inflammatory activity in vivo in comparison with the indometacine used as
reference product. On other hand and to understand the mechanism of the anti-inflammatory
activity of the synthesized compounds, the in silico approach was used by performing
3

ACCEPTED MANUSCRIPT
64
65
66
molecular modeling and docking studies on the X-ray crystal structure of COX-2 in complexe
with indometacine (PDB ID: 4COX) using the autodock vina program. These studies
provided a potential target for these compounds.
67
68
2. Experimental
2.1. Synthesis
69
70
71
72
73
74
75
Melting points of all synthesized compounds were obtained on a Büchi Melting Point SMP-
20 apparatus and were uncorrected. Infrared (IR) spectra were recorded with an IR VERTEX
1
70 FT-IR (Bruker Optics) spectrometer. H Nuclear magnetic resonance (1H NMR) spectra
were recorded on a Bruker Avance (400 MHz) spectrometer, using tetramethylsilane (TMS)
as internal standard and CDCl3 and DMSO-d6 as solvent. Mass spectra were recorded on an
API 3200 LC/MS/MS mass spectrometer using electrospray ionization (ESI) in positive
polarity.
76
General procedures for the synthesis of compounds 6a-g
77
78
79
80
81
82
To a solution of the substituted phenol 1a-g (0.5 mol) in 300 ml of 10 N NaOH (3 mol) was
heated to 65°C. Then 80 ml of CHCl3 was added in three portions over 15 min. The mixture
was heated at reflux in chloroform for 2 h. After cooling, the mixture was acidified to pH 1
with 12 N HCl, the organic layer collected and the aqueous layer 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.
83
84
85
86
87
To a solution of the substituted 2-hydroxybenzaldehydes 2a-g (0.15 mol) in N,N-
dimethylformamide (100 ml) were added bromoacetaldehyde diethyl acetal (31.5 g, 0.16 mol)
and potassium carbonate (42 g, 0.3 mol) at room temperature. The mixture was refluxed for 4
h at 150 °C. After cooling to room temperature, the precipitate was filtered off and the solvent
evaporated under reduced pressure. The oily residue was distilled.
88
89
90
A solution of compounds 3a-g (0.1 mol) in 35 ml of concentrated acetic acid was refluxed for
24 h. After cooling to room temperature, the mixture was evaporated to dryness. The crude
product was distilled or recrystallized from an appropriate solvent.
91
92
93
To a solution of appropriate aromatic aldehyde 4a-g (0.05 mol) and levulinic acid (0.06 mol)
were dissolved into 20 ml of concentrated acetic acid and saturated with dry hydrogen
chloride. Then, the mixture was stirred for 24 h at room temperature. The precipitate which
4

ACCEPTED MANUSCRIPT
94
95
was formed was filtered off and washed with ethyl ether. The crude acids were recrystallized
from an ethyl acetate.
96
97
To a solution of appropriate oxopentanoic acid 5a-g (0.02 mol) in absolute ethanol (20-30
mL) was added hydrazine hydrate (0.02 mol). The reaction mixture was refluxed for 2h. The
solid residue thus obtained was filtered and recrystallized from ethanol. The purity of the
compounds was checked by column chromatography on silica gel in the solvent system ethyl
acetate.
98
99
100
101
102
The analytical data of the products 6a-g is depicted below.
5-(Benzo[b]furan-2-ylmethyl)-6-methylpyridazin-3(2H)-one (6a).
Obtained as yellow solid, yield 69 %, mp 181–183 °C (ethanol); IR (KBr ʋmax cm-1), 1604
103
104
105
106
107
108
109
110
1
(C=N), 1662 (C=O), 2900–3000 (C–H); H NMR (DMSO-d6, 400 MHz), d = 2.22 (s, 3H, –
N=C–CH3), 4.08 (s, 2H, –CH2–), 6.97 (s, 1H, H4), 6.71 (s, 1H, H3’), 7.19-7.58 (m, 4H, H4’,
13
H5’, H6’, H7’), 12.71 (ls, 1H, NH); C NMR (DMSO-d6, 300 MHz): δ (ppm) = 18.90 (CH3),
31.31 (CH2), 105.19 (C-3’), 111.35 (C-7’), 121.31 (C-4’), 123.39 (C-5’), 124.45 (C-6’),
128.31 (C-9’), 128.62 (C-4), 143.92 (C-2’), 144.69 (C-5), 154.52 (C-8’), 154.79 (C-6), 161.17
(C=O); MS (ESI) m/z (relative abundance %): 241.2 [M-H]+ (39,20 %), 263.3 [M-Na]+ (100
%).
111
5-(5-Methylbenzo[b]furan-2-ylmethyl)-6-methylpyridazin-3(2H)-one (6b).
112
113
114
115
116
117
118
119
120
Obtained as yellow solid, yield 80 %, mp 183–184 °C (ethanol); IR (KBr ʋmax cm-1), 1603
1
(C=N), 1666 (C=O), 2900-3000 (C–H); H NMR (DMSOd6, 400 MHz), d = 2.23 (s, 3H, –
N=C–CH3), 2.37 (s, 3H, Ar–CH3), 4.07 (s, 2H, –CH2–), 6.58 (s,1H, H4), 6.64 (s, 1H, H3’),
7.07 (dd, 1H, j1 = 8.41 Hz, j2 = 1.21 Hz, H6’), 7.36 (d, 1H, j = 1.21 Hz, H4’), 7.40 (d, 1H, j =
13
8.41 Hz, H7’), 12.74 (ls, 1H, NH); C NMR (DMSO-d6, 300 MHz): δ (ppm) = 18.89 (CH3-
pyr), 21.04 (CH3-bzfr), 31.21 (CH2-pyr), 104.85 (C-3’), 111.77 (C-7’), 120.47 (C-4’), 125.30
(C-6’), 127.27 (C-9’), 128.30 (C-5’), 130.50 (C-4), 143.30 (C-2’), 143.67 (C-5), 151.85 (C-
8’), 155.90 (C-6), 161.14 (C=O); MS (ESI) m/z (relative abundance %): 255.1 [M-H]+ (55,68
%), 276.9 [M-Na]+ (100 %).
121
5-(5-Ethylbenzo[b]furan-2-ylmethyl)-6-methylpyridazin-3(2H)-one (6c).
122
123
124
Obtained as brown solid, yield 73 %, mp 180–181 °C (ethanol); IR (KBr ʋmax cm-1), 1605
1
(C=N), 1661 (C=O), 2900–3000 (C–H); H NMR (DMSOd6, 400 MHz), d = 1.18 (t, 3H, j =
7.50 Hz, –CH2–CH3), 2.21 (s, 3H, –N=C–CH3), 2.65 (q, 2H, j = 7.50 Hz, –CH2–CH3), 4.05 (s,
5

ACCEPTED MANUSCRIPT
125
126
127
128
129
130
131
2H, –CH2–), 6.56 (s, 1H, H4), 6.64 (s, 1H, H3’), 7.08 (dd, 1H, j1 = 8.40 Hz, j2 = 1.80 Hz, H6’),
13
7.37 (d, 1H, j = 1.80 Hz, H4’), 7.41 (d, 1H, j = 8.40 Hz, H7’), 12.76 (ls, 1H, NH); C NMR
(DMSO-d6, 300 MHz): δ (ppm) = 16.76 (CH3-CH2-bzfr), 18.88 (CH3-pyr), 28.56 (CH3-CH2-
bzfr), 31.37 (CH2-pyr), 105.08 (C-3’), 110.97 (C-7’), 119.87 (C-4’), 124.51 (C-6’), 128.28
(C-9’), 128.71 (C-5’), 138.94 (C-4), 143.97 (C-2’), 144.68 (C-5), 153.36 (C-8’), 154.57 (C-6),
161.18 (C=O); MS (ESI) m/z (relative abundance %): 269.5 [M-H]+ (32,38 %), 291.3 [M-
Na]+ (100 %).
132
5-(5-Isopropylbenzo[b]furan-2-ylmethyl)-6-methylpyridazin-3(2H)-one (6d).
133
134
135
136
137
138
139
140
141
Obtained as brown solid, yield 82 %, mp 185–186 °C (ethanol); IR (KBr ʋmax cm-1), 1605
(C=N), 1682 (C=O), 2900–3000 (C–H); 1H NMR (DMSOd6, 400 MHz), d = 1.24 (d, 6H, j =
6.90 Hz, H3C–CH–CH3), 2.10 (s, 3H, –N=C–CH3), 2.99 (h, 1H, j = 6.90 Hz, H3C–CH–CH3),
3.90 (s, 2H, –CH2–), 7.30–7.76 (m, 5H, H4, H3’, H4’, H6’, H7’), 12.32 (ls, 1H, NH); 13C NMR
(DMSO-d6, 300 MHz): δ (ppm) = 18.89 (CH3-pyr), 24.87 (2CH3-CH-bzfr), 31.37 (CH2-pyr),
33.84 (2CH3-CH-bzfr), 105.20 (C-3’), 110.96 (C-7’), 118.33 (C-4’), 123.15 (C-6’), 128.26
(C-9’), 128.62 (C-4), 143.68 (C-2’), 144.00 (C-5’), 144.69 (C-5), 153.38 (C-8’), 154.56 (C-6),
161.17 (C=O); MS (ESI) m/z (relative abundance %): 283.4 [M-H]+ (53,97 %), 337.4 [M-
Na]+ (100 %).
142
5-(5-Methoxybenzo[b]furan-2-ylmethyl)-6-methylpyridazin-3(2H)-one (6e).
143
144
145
146
147
148
149
150
Obtained as yellow solid, yield 78 %, mp 190–192 °C (ethanol); IR (KBr ʋmax cm-1), 1650
(C=O), 2950–3000 (C–H); 1H NMR (DMSOd6, 400 MHz), d = 2.25 (s, 3H, –N=C–CH3), 3.74
(s, 3H, –OCH3), 4.04 (s, 2H, –CH2–), 6.55 (s, 1H, H4), 6.64 (s, 1H, H3’), 6.82 (dd, 1H, j = 8.70
and 2.71 Hz, H6’), 7.08 (d, 1H, j = 2.71 Hz, H4’), 7.40 (d, 1H, j = 8.70 Hz, H7’), 12.75 (ls, 1H,
13
NH); C NMR (DMSO-d6, 300 MHz): δ (ppm) = 18.86 (CH3-pyr), 31.38 (CH2-pyr), 56.12
(OCH3-bzfr), 103.8 (C-3’), 105.22 (C-4’), 117.64 (C-7’), 117.79 (C-6’), 128.60 (C-9’), 129.90
(C-4), 143.00 (C-2’), 144.90 (C-5), 149.00 (C-8’), 154.20 (C-6), 155.60 (C-5’), 161.15
(C=O).
151
5-(5-Propoxybenzo[b]furan-2-ylmethyl)-6-methylpyridazin-3(2H)-one (6f).
152
153
154
155
156
Obtained as brown solid, yield 87 %, mp 194–196 °C (ethanol); IR (KBr ʋmax cm-1), 1652
1
(C=O), 2940–3000 (C–H); H NMR (DMSOd6, 400 MHz), d = 0.96 (t, 3H, j = 6.90 Hz, –
OCH2–CH2–CH3), 1.71 (m, 2H, –OCH2–CH2–CH3), 2.21 (s, 3H, –N=C–CH3), 3.90 (t, 2H, j =
6.90 Hz, –OCH2–CH2–CH3), 4.04 (s, 2H, –CH2–), 6.55 (s, 1H, H4), 6.62 (s, 1H, H3’), 6.81
(dd, 1H, j = 9.00 and 2.71 Hz, H6’), 7.06 (d, 1H, j = 2.71 Hz, H4’), 7.38 (d, 1H, j = 9.00 Hz,
6

ACCEPTED MANUSCRIPT
13
157
158
159
160
161
H7’), 12.74 (ls, 1H, NH); C NMR (DMSO-d6, 300 MHz): δ (ppm) = 10.91 (CH3-CH2-CH2-
bzfr), 18.89 (CH3-pyr), 22.58 (CH3-CH2-CH2-bzfr), 31.40 (CH2-pyr), 70.00 (CH3-CH2-CH2-
bzfr), 104.75 (C-3’), 105.43 (C-4’), 111.78 (C-7’), 113.23 (C-6’), 128.27 (C-9’), 129.29 (C-
4), 143.96 (C-2’), 144.68 (C-5), 149.61 (C-8’), 154.46 (C-6), 155.19 (C-5’), 161.17 (C=O);
MS (ESI) m/z (relative abundance %): 299.4 [M-H]+ (34,09 %), 321.4 [M-Na]+ (100 %).
162
5-[(5-Chlorobenzo[b]furan-2-yl)methyl]-6-methylpyridazin-3(2H)-one (6g).
163
164
165
166
167
168
169
170
Obtained as yellow solid, yield 70 %, mp 198–200 °C (ethanol); IR (KBr ʋmax cm-1), 1649
1
(C=O); H NMR (DMSOd6, 400 MHz), d = 2.23 (s, 3H, –N=C–CH3), 4.11 (s, 2H, –CH2–),
6.60 (s, 1H, H4), 6.74 (s, 1H, H3’), 7.29 (dd, 1H, j = 8.71 and 2.16 Hz, H6’), 7.58 (d, 1H, j =
13
8.71 Hz, H4’), 7.66 (d, 1H, j = 2.16 Hz, H7’), 12.76 (ls, 1H, NH); C NMR (DMSO-d6, 300
MHz): δ (ppm) = 18.91 (CH3-pyr), 31.17 (CH2-pyr), 106.12 (C-3’), 115.43 (C-7’), 120.51 (C-
4’), 124.45 (C-6’), 124.77 (C-5’), 128.32 (C-9’), 130.53 (C-4), 143.63 (C-2’), 144.65 (C-5),
150.27 (C-8’), 155.86 (C-6), 161.13 (C=O); MS (ESI) m/z (relative abundance %): 275.3 [M-
H]+ (100 %), 297.3 [M-Na]+ (17,61 %).
171
2.2. Anti-inflammatory activity
172
173
The anti-inflammatory activity of the synthesized compounds 6a-g was evaluated by
carrageenan-induced paw edema according to the method described by Winter et al. (23–25).
174
175
176
177
The carrageenan-induced rat paw edema method is highly reproducible and is the most test
widely used primary to evaluate and study anti-inflammatory effect of several compound
(26), what justifies our choice of the carrageenan test to evaluate the anti-inflammatory effect
of the synthesized compounds.
178
179
180
181
182
183
184
The adult Wistar rats of both sexes, with weights between 150-280g were used. A total of 10
lots were studied and each lot consists of 6 rats (n = 6). After 18 hours of fasting, the control
group received oral administration of orally corn oil (vehicle control, 5 ml/kg), the standard
groups received the reference drug (indomethacin at a dose of 10 mg/kg and aspirin at dose of
150 mg/kg), and the test groups received different suspensions of tested pyridazinones (6a-g)
at a dose of 50 mg/kg. Thirty minutes after feeding, each animal received in the right hind
paw, a sub-plantar injection of a suspension of carrageenan in 2.5% NaCl (0.9%), at 0.05 ml.
185
186
The volume of the treated paw was measured before and at 30 min, 1, 2, 3 and 4 hours after
injection of carrageenan using a plethysmometer LE7500 (Ugo Basile e Italy). The
7

ACCEPTED MANUSCRIPT
187
188
percentages of inhibition in inflammation were calculated for each animal using the following
formula:
189
% inhibition=(퐕퐧 ‒ 퐕퐨)퐂퐨퐧퐭퐫퐨퐥 ‒ (퐕퐧 ‒ 퐕퐨)퐓퐫퐞퐚퐭퐞퐝
× ퟏퟎퟎ
(퐕퐧 ‒ 퐕퐨)퐂퐨퐧퐭퐫퐨퐥
190
191
192
Where Vo is volume of the right hind paw measured before injection of carrageenan and Vn is
volume of the right hind paw measured at several time-points; at 30 min, 1, 2, 3 and 4 h after
carrageenan injection.
193
2.3. Molecular docking Analysis
194
195
196
197
198
199
The purpose of this section is to provide a potential target for the synthetized compounds by
an in silico approach using docking studies. The search strategy for potential target was
initiated based on several criteria. A biological target involved and validated in the
inflammatory process was the first criteria. The second one was availability of the
experimental tridimentional strucutre of the target and finally, a target which is complexed
with indometacin.
200
201
Using these criteria, we were able to propose the cyclooxygenase-2 (COX2) as one of the
target protein which was used in this study.
202
203
204
205
The three dimensional structure of COX-2 has been extensively studied and several
complexes of COX-2 are present on the PDB (protein Data Bank) (27,28). For these studies
we have chosen to work with the crystal identified by the ID PDB: 4COX (27). Which is the
complex of cyclooxygenase-2 with indometacin and that has a resolution of 2.9 Å.
206
207
208
209
210
211
The molecular docking files (ligands + protein active site) were prepared for Autodock vina
software, by using AutoDock Tools (v1.5.6) (29,30). The following parameters were adjusted
in this preparation step (31): (i) the Gastieger charges and polar hydrogens were added; (ii)
the grid-box dimensions were set at 24 Å (X), 21 Å (Y) and 7,75Å (Z); and (ii) the center of
the box was positioned at the midpoint of the active site, and the box volume covered the
entire active site area plus a significant portion of the protein’s solvent-exposed surface.
212
213
214
215
216
The following docking parameters were used in Autodock vina:(a) all bonds in the inhibitor
structures were allowed to rotate freely, except for the multiple bonds and the bonds in
aromatic entities; (b) the 3D protein structures were considered to be rigid; (c) a Lamarckian
genetic algorithm was used for searching the conformational space in the active site; (d) the
default grid spacing was set at 1 Å; (e) 100 different conformations were assessed, and the 9
8

ACCEPTED MANUSCRIPT
217
218
219
220
221
highest scored binding modes were maintained for visual inspection; (f) the maximum energy
difference between the best and the worst binding modes was set at 3 kcal/mol; (g) the scoring
function was a stochastic global optimization method inspired chiefly by X-Score (32); and
(h) visual inspection of the docking results, and image building, were done using PyMOL
software (www.pymol.org/funding.html) (33).
222
2.4. Statistical Analysis
223
224
225
226
227
After checking the homogeneity of variances by ANOVA test, statistical comparisons
between control and treated lots were performed using two-way ANOVA test on the
individual volume changes before and after injection carrageenan (Vn - Vo). A value of 푃<
0.001 was considered to be significant. Analysis was performed with GraphPad prism
Software (6.0).
228
229
3. Results and Discussion
3.1. Synthesis
230
231
232
233
234
235
236
237
238
239
240
241
The pyridazinone derivatives incorporating Benzofuran moiety studied in this work
(compounds 6a-g) were synthesized according to the general procedures outlined in Scheme
1. In the first step, salicylaldehydes 2a-g were synthesized via Reimer Thieman (34)
formylation of the appropriate substituted phenol 1a-g with CHCl3 and NaOH. Aldehydes 2a-
g has been previously synthesized with very low yield. Briefly, reaction of intermediates 2a-g
with bromacetaldehyde diethyl acetal in the presence of potassium carbonate in DMF
(dimethylformamide) yielded compounds 3a-g. Next, these compounds 3a-g were cyclized to
benzo[b]furan-2-ylcarboxaldehydes 4a-g by heating in concentrated acetic acid.
Benzo[b]furanaldehydes 4a-g were prepared according to the methods described in the
literature (35). Finally, treatment of substituted aldehydes 4a-g with levulinic acid in the
presence of HCl (chlorhydric acid) in acetic acid gave adducts 5a-g, which were heated by
hydrazine hydrate to afford the pyridazin-3(2H)-ones 6a-g (15).
242
243
244
9

R
CHO
OH
OH
I
II
OC2H5
OC2H5
O
CH2 CH
R
CHO
R
2a-g
1a-g
3a-g
III
O
COOH
CH3
NH
H2C
N
O
R
IV
C
C
R
V
CHO
C
O
CH2
CH3
R
O
H
O
6a-g
4a-g
5a-g
245
Compound
R
a
b
c
d
e
f
H
CH3
CH2CH3
CH(CH3)2
OCH3
OCH2CH2CH3
Cl
d
246
247
Scheme 1. Synthesis of target compounds 6a-g. Reagents I: CHCl3/NaOH 10N, reflux 2h; II: BrCH2CH(OC2H5)2/K2CO3/DMF, reflux 4h; III:
CH3COOH, reflux 24h; IV: H3CCOCH2CH2COOH/CH3COOH, reflux 24h; V: H2NNH2/EtOH, reflux 2h
10

ACCEPTED MANUSCRIPT
248
249
250
251
The proposed reaction mechanism is reported in Scheme 2. It is carried out according to a
condensation reaction of the derivatives of hydrazine with 1,4-dicarbonyl (appropriate
oxopentanoic acid), Intermolecular hydrazone formation followed by intramolecular
cyclization led to the formation of pyridazinones 6a-g.
252
253
254
255
The Pyridazinones are the derivatives of pyridazine which belong to an important group of
heterocyclic compounds containing two nitrogen atoms at 1 and 2 positions in a six member
ring. In pyridazinone derivatives the amine group (NH) is suitably placed with the carbonyl
group and most of the pyridazinone derivatives exhibit tautomerism.
COOH
CH3
COOH
H2C
C
H2C
C
CH3
NH2
NH2
R
R
C
C NH NH2
C
H
C
O
O
H
O
H
O
COOH
COOH
CH3
CH3
H2C
H2C
C
R
R
C
H
C
N
NH2
C
C
N
NH2
C
OH H
OH2
O
O
H
-H2O
O
HO
C
NH
N
O
CH3
H2C
C
R
R
-H2O
C
N
NH2
CH2
6a-g
CH3
C
O
O
H
256
257
258
Scheme 2. The proposed mechanism for the formation of products 6a-g
259
260
261
262
The chemical structures of all compounds were deduced from their spectral and analytical
data. Thus, IR spectrum showed absorption band at ʋ = 2833-3465 and 1661-1668 cm-1 due to
1
NH and carbonyl groups respectively. In addition, the H NMR spectrum revealed the
presence of singlet signals at δ = 2.21-2.25 ppm due to (CH3-pyridazinone), a singlet signals
11

ACCEPTED MANUSCRIPT
263
264
265
266
267
at δ = 4.04-4.11 ppm attributed to (benzofuran-CH2 –pyridazinone), a signals at δ = 6.55-7.66
ppm corresponding to aromatic protons of benzofuran-pyridazinone ring and a singlet signals
13
at δ= 12.71-12.76 ppm for NH protons. Moreover, the C NMR data exhibited the presence
of signals at δ = 18.88-18.90 ppm for (CH3-pyridazinone) and a signal at δ = 31.28-31.40 ppm
for (benzofuran-CH2-pyridazinone).
268
3.2. Anti-inflammatory activity
269
270
271
272
273
274
Inflammation is a complex phenomenon with series of interactions between cellular and
inflammatory mediators. Regardless of the triggering factor, the progression of edema is
expressed by a release of several mediators (36,37). The first phase of inflammation lasts up
to 2 h, and involves the participation of histamine, serotonin, and bradykinin; while the
second phase, is from 3 to 4 h and mainly sustained by prostaglandins, cytokines and nitric
oxide release (23,38).
275
276
277
278
279
As illustrated in Table 1, the injection of carrageenan into the sub-plantar tissue in the right
hind paw of rats in the control groups caused edema development during 30 minutes, 1, 2, 3
and 4 h after carrageenan injection with a peak volume (1.28 ml) at 4h. This result confirms
that experimental carrageenan injection into the rat paw provokes a significant local and acute
inflammatory reaction (39).
280
281
282
283
284
285
286
The single oral treatment with oily tested suspensions of pyridazinones compounds at a dose
of 50 mg on carrageenan induced rat paw edema remarkably decreased the level of paw
inflammation compared to the negative control group. The average volume of the right paw of
the treated group with 6a, 6b, 6c, 6d, 6e, 6f and 6g compounds at 30min after carrageenan
injection were 0.52, 0.57, 0.49, 0.55, 0.59, 0.58 and 0.71 ml respectively and were
progressively increased to 0.62, 0.60, 0.57, 0.68, 0.60, 0.68 and 0.64 mL at 2h and to 0.98,
0.90, 0.95, 1.20, 0.97, 1.15 and 0.68 ml (Table 1).
287
288
Table 1. Anti-inflammatory activity of the target compounds against the carrageenan-induced
paw edema in rats with different time intervals.
Average volume of the right paw (ml)*
Compound Dose (mg/kg)
30min
1h
2h
3h
4h
Control
-
0,79
0,84
0,58
0,87
0,555
0,91
0,54
1,28
0,56
Indomethacin
10
0,608
12

ACCEPTED MANUSCRIPT
Aspirin
6a
150
50
50
50
50
50
50
50
0,71
0,52
0,57
0,49
0,55
0,59
0,58
0,71
0,66
0,53
0,51
0,45
0,61
0,54
0,53
0,66
0,608
0,62
0,6
0,64
0,68
0,63
0,61
0,8
0,71
0,98
0,9
6b
6c
0,57
0,68
0,6
0,95
1,2
6d
6e
0,68
0,78
0,67
0,97
1,15
0,68
6f
0,68
0,64
6g
289
290
* Each value represents the mean of difference between of volume of paw edema before and
after injection carrageenan (Vn - Vo) for six rats (n = 6).
291
292
293
294
295
Based on percentages of inhibition values (Figures 1-7), 6a compound caused the peak effect
4h after carrageenan injection and inhibited edema by 23.43%, however the peak effect of 6b,
6c, 6d, 6e, 6f and 6g was produced at 1h after carrageenan injection with respectively 39,28,
46.42, 27.38, 35.71, 36.90 and 21.42 %. These effects were statistically insignificant (P<
0.05) compared from that produced by 10 mg/kg indomethacin and 150 mg /kg aspirin.
296
297
298
299
300
In the presence of indomethacin 10 mg/kg there was a decrease of the volume of edema with a
percent inhibition that starting at 23.04% after 30 min to get to 56.25% after 4 hours of
injection of carrageenan. Additionally, aspirin at a dose of 150 mg /kg also caused a decrease
in edema following kinetic increase and reached a value of 44.53% after 4h. p = 0.26099 (p>
0.05) compared to vehicle treated group.
301
302
303
Figure 1. Effect of 6a on edema induced by carrageenan injection at 2.5% in the right hind
paw of the rat.
13

ACCEPTED MANUSCRIPT
304
305
306
Figure 2. Effect of 6b on edema induced by carrageenan injection at 2.5% in the right hind
paw of the rat.
307
308
309
Figure 3. Effect of 6c on edema induced by carrageenan injection at 2.5% in the right hind
paw of the rat.
14

ACCEPTED MANUSCRIPT
310
311
312
Figure 4.Effect of 6d on edema induced by carrageenan injection at 2.5% in the right hind
paw of the rat.
313
314
315
Figure 5. Effect of 6e on edema induced by carrageenan injection at 2.5% in the right hind
paw of the rat.
15

ACCEPTED MANUSCRIPT
316
317
318
Figure 6. Effect of 6f on edema induced by carrageenan injection at 2.5% in the right hind
paw of the rat.
319
320
321
Figure 7. Effect of 6g on edema induced by carrageenan injection at 2.5% in the right hind
paw of the rat.
322
323
324
325
326
327
Figure 8 shows the difference between the effects of pyridazinones synthesized and reference
substances. p = 0.09 (p> 0.05) for all pyridazinones, concluding that compounds 6a, 6b, 6e
and 6g have a good anti-inflammatory activity, because there is no significant difference
between them and the effect of the indomethacin at 10 mg/kg and the aspirin at 150 mg/kg of
aspirin. The pyridazinone ring is known for its anti-inflammatory properties and confirmed by
this study (40–42).
16

ACCEPTED MANUSCRIPT
328
329
330
331
Figure 8. Area under the curve (AUC) of average percentages of inhibition corresponding to
the effect of the pyridazinones (6a-g) at 50 mg / kg on carrageenan induced edema in
comparison to the two references: Aspirin 150 mg / kg and indomethacin 10 mg / kg.
332
333
334
335
336
Considering that the production of histamine, serotonin and bradykinin are the main factors
responsible for both the first phase of the inflammatory response (lasts up to 2 h), our results
suggest that the anti-inflammatory effect of the tested pyridazinones compounds could be
related to the inhibition of the release or synthesis of arachidonic acid metabolites. The anti-
inflammatory activities of several pyridazinone derivatives have been previously reported.
337
338
339
340
341
342
Similar results were found by Mogilski et al. (43) and Abouzid et al. (44), which revealed that
several pyridazinone derivatives had an excellent inflammatory activity using the carageenan-
induced paw edema test. Ochiai et al. (45) also demonstrated that another series of
pyridazinone derivatives exhibited significant in-vivo anti-inflammatory. In another series of
pyridazinone developed by Gokce et al. (46), compound 7 emerged as a potent anti-
inflammatory agent.
17

ACCEPTED MANUSCRIPT
343
344
345
Since our findings are preliminary results; further studied need to be carried out to investigate
the other specifications such as side effect-activity profiles or toxicological studies of these
compounds.
346
3.3. Molecular docking Analysis
347
348
349
350
351
In order to execute the docking of different compounds, the binding and docking sites were
validated by the reference product (indomethacin) and Figure 9 shows the overlapping of
docked COX2 with the experimental indomethacin (Co-crystaled with COX2). The docking
scores (affinity) were between -8,9 and -9,7 kcal/mol for the seven compounds and -10,7
kcal/mol for indometacien (Table 2).
352
353
Table 2. Functional groups, Anti-inflammatory and docking score in cyclooxygenase 2 in
comparison to indometacin
H3C
R
N
CH2
NH
O
O
354
Anti-inflammatory
activity*
Docking score in cyclooxygenase 2
(ID PDB:4COX)
Compound
R
6a
6b
6c
H
23,43
29,68
25,78
6,25
-9,3
-9,7
-9,6
-9,3
-9,1
-8,9
-9,4
-10,7
CH3
CH2CH3
CH(CH3)2
OCH3
OC3H7
Cl
6d
6e
24,21
10,15
46,87
56,25
6f
6g
Ref
Indometacin
18

ACCEPTED MANUSCRIPT
355
356
* Anti-inflammatory activitiy exprimed in (% inhibition of edema) at 4h, using 50mg/kg
for compounds vs 10mg/kg for indomethacin.
357
358
359
360
361
362
Figure 9. Validation of the docking box. COX2 docked in cyan is superposed at COX2
extracted from PDB (ID PDB 4COX) margenta. Indometacine docked (in white) is
superposed at indomethacin co-crystalesed (in green) with COX2 extracted from PDB (ID
PDB 4COX) presented in margenta. Tyr355 and Tyr385, two residues of the binding site are
shown.
363
364
365
366
367
368
369
The binding site analysis of the COX2 shows that it is a closed one and do not allow the
reception of large molecule. Compound 6f of our series is the one with the larger radical (R =
OC3H7) and in looking at the docking result, we observe that this compound has the lowest
affinity (the lowest score) against the compound 6b (R=CH3) with a methyl radical has the
highest score confirms this hypothesis but compound 6a (R=H) despite its small size it has a
low score in docking, this is explained by the lack of possibility of making hydrogen bond
with hot active site residues (Figure 10).
19

ACCEPTED MANUSCRIPT
A
B
370
371
Figure 10. A : Compound (R=CH3) ; B : Compound (R=H)
372
373
374
This observation is in the same way as the biological activity. (29,6875% for CH3 compound
vs 23,4375% for H compound of inhibition. both are has less activity compared to the
reference product, as for biological activity as in silico analysis.
375
4. Conclusion
376
377
378
379
380
381
382
383
384
Pyridazinones derivatives showed anti-inflammatory activity at 50 mg /kg compared to the
reference products at 10 mg/kg (indomethacin) and at 150 mg/kg (aspirin) with a maximum
effect between 1 hour and 2 hours. Synthesized products are faster than reference ones but are
had fleeting effects. This fugacious effect may be enhanced by prolonged release dosage
forms. Comparison of in silico and biological data suggests that COX2 is a potential
therapeutic target for our compounds. However, target validation test are required to be
undertaken to confirm these results. It will be interesting to study the acute and chronic
toxicity effects of these synthesized compounds to evaluate their safety especially the more
promising ones (6a, 6b, 6e and 6g).
385
5. Aknowledgments
386
387
388
This work was carried out under National funding from the Moroccan Ministry of Higher
education & Scientific research (PPR program) to AI. This work was also supported by a
grant from the NIH for H3Africa BioNet to AI.
20

ACCEPTED MANUSCRIPT
390
References
391
392
1. Vane JR. Inhibition of Prostaglandin Synthesis as a Mechanism of Action for Aspirin-
like Drugs. Nature. 23 juin 1971;231(25):232‑5.
393
394
2. Simmons DL, Botting RM, Hla T. Cyclooxygenase Isozymes: The Biology of
Prostaglandin Synthesis and Inhibition. Pharmacol Rev. 1 sept 2004;56(3):387‑437.
395
396
397
3. Wallace JL, McKnight W, Reuter BK, Vergnolle N. NSAID-induced gastric damage in
rats: Requirement for inhibition of both cyclooxygenase 1 and 2. Gastroenterology. sept
2000;119(3):706‑14.
398
399
4. Castle RN. The Chemistry of Heterocyclic Compounds, Condensed Pyridazines
Including Cinnolines and Phthalazines. John Wiley & Sons; 2009. 1137 p.
400
401
402
5. Mason RP, Walter MF, McNulty HP, Lockwood SF, Byun J, Day CA, et al. Rofecoxib
increases susceptibility of human LDL and membrane lipids to oxidative damage: a
mechanism of cardiotoxicity. J Cardiovasc Pharmacol. 2006;47:S7‑14.
403
404
405
6. Boukharsa Y, Zaoui Y, Taoufik J, Ansar M. Pyridazin‐3 (2H)‐ones: Synthesis,
Reactivity, Applications in Pharmacology and Agriculture. J Chem Pharm Res.
2014;6(12):297‑310.
406
407
408
7. Siddiqui AA, Mishra R, Shaharyar M, Husain A, Rashid M, Pal P. Triazole incorporated
pyridazinones as a new class of antihypertensive agents: Design, synthesis and in vivo
screening. Bioorg Med Chem Lett. février 2011;21(3):1023‑6.
409
410
411
8. Sharma B, Verma A, Sharma UK, Prajapati S. Efficient synthesis, anticonvulsant and
muscle relaxant activities of new 2-((5-amino-1,3,4-thiadiazol-2-yl)methyl)-6-phenyl-
4,5-dihydropyridazin-3(2H)-one derivatives. Med Chem Res. 1 janv 2014;23(1):146‑57.
412
413
414
9. McGettigan P, Henry D. Cardiovascular Risk and Inhibition of Cyclooxygenase: A
Systematic Review of the Observational Studies of Selective and Nonselective Inhibitors
of Cyclooxygenase 2. JAMA. 4 oct 2006;296(13):1633‑44.
415
416
417
10. Wang T, Dong Y, Wang L-C, Xiang B-R, Chen Z, Qu L-B. Design, Synthesis and
Structure-Activity Relationship Studies of 6-Phenyl-4,5-dihydro-3(2H)-pyridazinone
Derivatives as Cardiotonic Agents. Arzneimittelforschung. nov 2008;58(11):569‑73.
418
419
420
11. El-Hashash MA, Guirguis DB, El-Wahed NAA, Kadhim MA. Synthesis of Novel Series
of Phthalazine Derivatives with Antibacterial and Antifungal Evaluation. J Chem Eng
Process Technol [Internet]. 30 avr 2014 [cité 17 févr 2017];5(4).
421
422
423
12. Cherkez S, Herzig J, Yellin H. Synthesis, saludiuretic, and antihypertensive activity of
6,7-disubstituted 1(2H)- and 3,4-dihydro-1(2H)-phthalazinones. J Med Chem. juin
1986;29(6):947‑59.
424
425
426
13. Livermore D, Bethell RC, Cammack N, Hancock AP, Hann MM, Green D, et al.
Synthesis and anti-HIV-1 activity of a series of imidazo [1, 5-b] pyridazines. J Med
Chem. 1993;36(24):3784‑94.
21

ACCEPTED MANUSCRIPT
427
428
429
430
14. Tao M, Aimone LD, Gruner JA, Mathiasen JR, Huang Z, Lyons J, et al. Synthesis and
structure–activity relationship of 5-pyridazin-3-one phenoxypiperidines as potent,
selective histamine H3 receptor inverse agonists. Bioorg Med Chem Lett. 15 janv
2012;22(2):1073‑7.
431
432
433
15. Boukharsa Y, Meddah B, Tiendrebeogo RY, Ibrahimi A, Taoufik J, Cherrah Y, et al.
Synthesis and antidepressant activity of 5-(benzo[b]furan-2-ylmethyl)-6-
methylpyridazin-3(2H)-one derivatives. Med Chem Res. 1 mars 2016;25(3):494‑500.
434
435
436
437
16. Ochiai K, Takita S, Eiraku T, Kojima A, Iwase K, Kishi T, et al. Phosphodiesterase
inhibitors. Part 3: Design, synthesis and structure–activity relationships of dual PDE3/4-
inhibitory fused bicyclic heteroaromatic-dihydropyridazinones with anti-inflammatory
and bronchodilatory activity. Bioorg Med Chem. 1 mars 2012;20(5):1644‑58.
438
439
440
17. Özadalı K, Özkanlı F, Jain S, Rao PPN, Velázquez-Martínez CA. Synthesis and
biological evaluation of isoxazolo[4,5-d]pyridazin-4-(5H)-one analogues as potent anti-
inflammatory agents. Bioorg Med Chem. mai 2012;20(9):2912‑22.
441
442
443
18. Jerome KD, Hepperle ME, Walker JK, Xing L, Devraj RV, Benson AG, et al. Discovery
of 5-substituted-N-arylpyridazinones as inhibitors of p38 MAP kinase. Bioorg Med
Chem Lett. mai 2010;20(10):3146‑9.
444
445
446
19. Dorsch D, Schadt O, Stieber F, Meyring M, Grädler U, Bladt F, et al. Identification and
optimization of pyridazinones as potent and selective c-Met kinase inhibitors. Bioorg
Med Chem Lett. avril 2015;25(7):1597‑602.
447
448
449
450
20. Csókás D, Zupkó I, Károlyi BI, Drahos L, Holczbauer T, Palló A, et al. Synthesis,
spectroscopy, X-ray analysis and in vitro antiproliferative effect of ferrocenylmethylene-
hydrazinylpyridazin-3(2H)-ones and related ferroceno[d]pyridazin-1(2H)-ones. J
Organomet Chem. 15 oct 2013;743:130‑8.
451
452
21. Balkwill F, Charles KA, Mantovani A. Smoldering and polarized inflammation in the
initiation and promotion of malignant disease. Cancer Cell. 1 mars 2005;7(3):211‑7.
453
454
22. Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH. Mechanisms Underlying
Inflammation in Neurodegeneration. Cell. 19 mars 2010;140(6):918‑34.
455
456
457
23. Winter CA, Risley EA, Nuss GW. Carrageenin-Induced Edema in Hind Paw of the Rat
as an Assay for Antiinflammatory Drugs. Proc Soc Exp Biol Med. décembre
1962;111(3):544‑7.
458
459
460
24. Gyires K, Budavári I, Fürst S, Molnár I. Morphine inhibits the carrageenan-induced
oedema and the chemoluminescence of leucocytes stimulated by zymosan. J Pharm
Pharmacol. février 1985;37(2):100‑4.
461
462
463
25. Bignotto L, Rocha J, Sepodes B, Eduardo-Figueira M, Pinto R, Chaud M, et al. Anti-
inflammatory effect of lycopene on carrageenan-induced paw oedema and hepatic
ischaemia–reperfusion in the rat. Br J Nutr. févr 2009;102(1):126‑33.
22

ACCEPTED MANUSCRIPT
464
465
466
26. Yonathan M, Asres K, Assefa A, Bucar F. In vivo anti-inflammatory and anti-
nociceptive activities of Cheilanthes farinosa. J Ethnopharmacol. 6 déc 2006;108(3):462
‑70.
467
468
469
27. Kurumbail RG, Stevens AM, Gierse JK, McDonald JJ. Structural basis for selective
inhibition of cyclooxygenase-2 by anti-inflammatory agents. Nature.
1996;384(6610):644.
470
471
472
28. Kiefer JR, Pawlitz JL, Moreland KT, Stegeman RA, Hood WF, Gierse JK, et al.
Structural insights into the stereochemistry of the cyclooxygenase reaction. Nature. mai
2000;405(6782):97‑101.
473
474
475
476
29. Morris GM, Huey R, Olson AJ. Using AutoDock for Ligand-Receptor Docking. In:
Current Protocols in Bioinformatics [Internet]. John Wiley & Sons, Inc.; 2002 [cité 17
févr 2017]. Disponible sur:
http://onlinelibrary.wiley.com/doi/10.1002/0471250953.bi0814s24/abstract
477
478
479
30. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, et al.
AutoDock4 and AutoDockTools4: Automated docking with selective receptor
flexibility. J Comput Chem. décembre 2009;30(16):2785‑91.
480
481
482
31. Lakhlili W, Chevé G, Yasri A, Ibrahimi A. Determination and validation of mTOR
kinase-domain 3D structure by homology modeling. OncoTargets Ther. 30 juill
2015;8:1923‑30.
483
484
485
32. Wang R, Lai L, Wang S. Further development and validation of empirical scoring
functions for structure-based binding affinity prediction. J Comput Aided Mol Des. 1
janv 2002;16(1):11‑26.
486
33. DeLano WL. The PyMOL user’s manual. DeLano Sci San Carlos CA. 2002;452.
487
488
489
34. Ansar M, Al Akoum Ebriki S, Mouhoub R, Berthelot P, Vaccher C, Vaccher M, et al. 3-
Benzo[b]furyl- and 3-benzo[b]thienylaminobutyric acids as GABAB ligands. Synthesis
and structure-activity relationship studies. Eur J Med Chem. 1 janv 1996;31(6):449‑60.
490
491
492
35. Hirota T, Fujita H, Sasaki K, Namba T. A novel synthesis of benzofuran and related
compounds. III. The vilsmeier reaction of phenoxyacetaldehyde diethyl acetals. J
Heterocycl Chem. 1 nov 1986;23(6):1715‑6.
493
494
36. Vinegar R, Schreiber W, Hugo R. Biphasic Development of Carrageenin Edema in Rats.
J Pharmacol Exp Ther. 1 mars 1969;166(1):96‑103.
495
496
497
37. Seibert K, Zhang Y, Leahy K, Hauser S, Masferrer J, Perkins W, et al. Pharmacological
and biochemical demonstration of the role of cyclooxygenase 2 in inflammation and
pain. Proc Natl Acad Sci. 12 juin 1994;91(25):12013‑7.
498
499
38. Della Loggia R, Tubaro A, Dri P, Zilli C, Del Negro P. The role of flavonoids in the
antiinflammatory activity of Chamomilla recutita. Prog Clin Biol Res. 1986;213:481‑4.
23

ACCEPTED MANUSCRIPT
500
501
502
39. Boughton-Smith N k., Deakin A m., Follenfant R l., Whittle B j. r., Garland L g. Role of
oxygen radicals and arachidonic acid metabolites in the reverse passive Arthus reaction
and carrageenin paw oedema in the rat. Br J Pharmacol. 1 oct 1993;110(2):896‑902.
503
504
40. Pieretti S, Dal Piaz V, Matucci R, Giovannoni MP, Galli A. Antinociceptive activity of a
3(2H)-pyridazinone derivative in mice. Life Sci. août 1999;65(13):1381‑94.
505
506
41. Gokçe M, Dogruer D, Sahin MF. Synthesis and antinociceptive activity of 6-substituted-
3-pyridazinone derivatives. Il Farm. avril 2001;56(3):233‑7.
507
508
509
42. Nassar E, Abdel-Aziz HA, Ibrahim HS, Mansour AM. Synthesis of Diarylpyrazoles
Containing a Phenylsulphone or Carbonitrile Moiety and their Chalcones as Possible
Anti-Inflammatory Agents. Sci Pharm. 3 juill 2011;79(3):507‑24.
510
511
512
513
43. Mogilski S, Kubacka M, Redzicka A, Kazek G, Dudek M, Malinka W, et al.
Antinociceptive, anti-inflammatory and smooth muscle relaxant activities of the
pyrrolo[3,4-d]pyridazinone derivatives: Possible mechanisms of action. Pharmacol
Biochem Behav. 1 juin 2015;133:99‑110.
514
515
44. Abouzid K, Bekhit SA. Novel anti-inflammatory agents based on pyridazinone scaffold;
design, synthesis and in vivo activity. Bioorg Med Chem. 15 mai 2008;16(10):5547‑56.
516
517
518
519
45. Ochiai K, Ando N, Iwase K, Kishi T, Fukuchi K, Ohinata A, et al. Phosphodiesterase
inhibitors. Part 2: Design, synthesis, and structure–activity relationships of dual PDE3/4-
inhibitory pyrazolo[1,5-a]pyridines with anti-inflammatory and bronchodilatory activity.
Bioorg Med Chem Lett. 15 sept 2011;21(18):5451‑6.
520
521
522
46. Gökçe M, Utku S, Küpeli E. Synthesis and analgesic and anti-inflammatory activities 6-
substituted-3(2H)-pyridazinone-2-acetyl-2-(p-substituted/nonsubstituted
benzal)hydrazone derivatives. Eur J Med Chem. 1 sept 2009;44(9):3760‑4.
24