Synthesis and biological screening of some novel 6-substituted 2-alkylpyridazin-3(2H)-ones as anti-inflammatory and analgesic agents

Article abstract of DOI:10.1002/ardp.201900295

Some novel derivatives of 2-alkyl 6-substituted pyridazin-3(2H)-ones were synthesized by condensation of 3,6-dichloropyridazine with the sodium salt of benzyl cyanide, followed by hydrolysis and coupling with alkyl halides. The synthesized compounds were

Full text of DOI:10.1002/ardp.201900295

Received: 7 October 2019
Revised: 19 December 2019
Accepted: 21 December 2019
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DOI: 10.1002/ardp.201900295
F U L L P A P E R
Synthesis and biological screening of some novel
6‐substituted 2‐alkylpyridazin‐3(2H)‐ones as
anti‐inflammatory and analgesic agents
Yasser M. Loksha1
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Mohammad M. Abd‐Alhaseeb2
1Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Sinai University,
Al‐Arish, North Sinai, Egypt
Abstract
Some novel derivatives of 2‐alkyl 6‐substituted pyridazin‐3(2H)‐ones were synthesized by
condensation of 3,6‐dichloropyridazine with the sodium salt of benzyl cyanide, followed by
2Department of Pharmacology and
Toxicology, Faculty of Pharmacy, Damanhour
University, Damanhour, Egypt
hydrolysis and coupling with alkyl halides. The synthesized compounds were screened as
cyclooxygenase (COX)‐1/COX‐2 inhibitors and as analgesic and anti‐inflammatory agents.
Among the synthesized compounds, 6‐benzyl‐2‐methylpyridazin‐3(2H)‐one (4a), 6‐benzoyl‐
2‐propylpyridazin‐3(2H)‐one (8b), and 6‐(hydroxy(phenyl)methyl)‐2‐methylpyridazin‐3(2H)‐
one (9a) displayed the highest COX‐2 selectivity indices of 96, 99, and 98, respectively, and
analgesic efficacies of 47%, 46%, and 45% protection, respectively. Also, compounds
4a, 8b, and 9a showed anti‐inflammatory activities of 65%, 60%, and 62% inhibition of
edema, respectively, at a dose of 10 mg/kg, which is higher than that of diclofenac (58%
inhibition of edema).
Correspondence
Yasser M. Loksha, Department of
Pharmaceutical Chemistry, Faculty of
Pharmacy, Sinai University, Al‐Arish, North
Sinai, Egypt.
Email: yml@su.edu.eg
K E Y W O R D S
analgesic activity, anti‐inflammatory activity, COX‐1, COX‐2, pyridazinone
1
INTRODUCTION
and COX‐2.[4,5] The new isoform of COX was discovered in the early
1990s, providing a novel target to develop anti‐inflammatory agents
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The prostaglandins (PGs) are produced by enzymes called cycloox-
ygenases (COX). There are two types of COX enzymes, COX‐1 and
COX‐2. Both enzymes produce PGs but with different roles inside
the body. COX‐2 pathway is the main pathway that promotes
inflammation, pain, and fever. On the contrary, COX‐1 produces PGs
that activate platelets and protect the stomach and intestinal lining.
PGs are classified into many categories and are involved in many
processes in the human body.[1] PGE2 and PGI2 are important
inflammatory mediators and pain‐producing mediators. The pain and
inflammation induced by PGs affect the lifestyle of patients, and,
hence, nonsteroidal anti‐inflammatory drugs (NSAIDs) are used to
control their synthesis and relieve pain.[2,3]
with superior safety profiles compared to the older NSAIDs.[1,6]
Celecoxib is one of the drugs that are selective COX‐2 inhibitors.[1,6]
The recent drug discovery efforts have been oriented toward
developing novel small molecule ring templates as NSAIDs and
selective COX‐2 inhibitors.[7]
The pyridazin‐3‐one moiety has a diverse set of biological activities.
A wide range of biological activities was recorded for pyridazine
derivatives such as antiangiogenic,[8] anticancer,[9] antimicrobial,[10]
analgesic,[10,11] antidepressant,[12] antihypertensive,[13] antifungal, anti-
feedant,[14] phosphodiesterase‐4 (PDE4), inhibitors are effective anti‐
inflammatory, selective COX‐2 inhibitors,[15,16] and antiplatelet[17]
activities. Various drugs include pyridazinone ring as a core nucleus
like sulmazole, levosimendan, amipizone, indolidan, imazodan, pimoben-
dan, emorfazone, zardaverine, and milrinone.[18–25]
In the previous work of our research group[16] focusing on the
synthesis of 2,6‐disubstituted pyridazin‐3(2H)‐one derivatives as
analgesics, anti‐inflammatories, we derived the conclusion that the
presence the propyl group at position 2 of the pyridazinone ring
The classical types of NSAIDs block COX‐1 and COX‐2 enzymes
in different ratios. The newer COX‐2 inhibitors block only the COX‐2
enzyme. Since COX‐2 inhibitors do not block COX‐1 they do not
cause ulcers or increase the risk of bleeding as much as the older
NSAIDs. One of the older NSAIDs is diclofenac (Figure 1). It works by
decreasing the production of prostaglandin by blocking both COX‐1
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Arch Pharm Chem Life Sci. 2020;e1900295.
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© 2020 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201900295

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(a)
(c)
(b)
SCHEME 1 Synthesis of compounds 4a,b. Reagents and
conditions: (a) NaH, DMF, RT; (b) 4 M HCl, AcOH, reflux; (c) R‐X,
K2CO3, DMF, RT
FIGURE 1 Structure of diclofenac and the most active
synthesized compounds 4a, 8b, and 9a
provided better substituents than arylmethyl groups required for
good in‐vitro COX‐2 inhibition and anti‐inflammatory activity.
In the present work and in continuation of the previous work,
6‐substituted 2‐alkylpyridazin‐3(2H)‐ones were synthesized
and screened as COX‐1/COX‐2 inhibitors, analgesic, and anti‐
inflammatory agents. Alkyl groups (methyl and propyl) were
selected at position 2 with different substituents at position
6 for optimization and enhancing their COX‐2 selectivity index (SI)
and anti‐inflammatory activity.
according to the procedure described by Loksha et al.[27] Hydrolysis of
compound 2 by acetic acid furnished 2‐(6‐oxo‐1,6‐dihydropyridazin‐3‐yl)‐
2‐phenylacetonitrile (6), which was treated with sodium hydride in
dimethylformamide followed by oxidation using oxygen air to afford the
desired compound 7. Compound 7 was previously synthesized by
Heinisch et al.[28] as an intermediate to be reduced to the hydroxyl
derivative through the treatment of compound 2 with an aqueous
solution of sodium hydroxide followed by a stream of oxygen. It was
separated from the reaction and was used for the further reduction step
without identification of its melting point, yield, and spectroscopic
analyses.[28] 13C‐NMR of compound 7 showed the disappearance of CH
and CN, which were detected in compound 6 at δ 40.51 and 118.44 ppm,
respectively. CO–NH and CO–Ph were detected in the spectrum of
compound 7 at δ 160.53 and 189.49 ppm, respectively. Alkylation of
compound 7 with methyl iodide and/or n‐propyl bromide in basic medium
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
Treatment of 2,6‐dichloropyridazine (1) with the sodium salt of
benzyl cyanide in anhydrous N,N‐dimethylformamide (DMF) as a
solvent gave 2‐(6‐chloropyridazin‐3‐yl)‐2‐phenylacetonitrile (2) in
78% yield, which was hydrolyzed in a mixture of 4 M hydrochloric
acid and acetic acid to afford 6‐benzylpyridazin‐3(2H)‐one (3).
Compound 2 was previously prepared by Bemis et al.[26] in 54%
yield through a reaction of compound 1 with benzyl cyanide using
sodium amide as a base and tetrahydrofuran as a solvent at room
temperature. The melting point of compound 2 and its spectro-
scopic analysis were not published by Bemis et al.[26] The
significant CH–CN in compound 2 appeared as a singlet in 1H‐
NMR (nuclear magnetic resonance) at δ 5.70 ppm, which was
converted to the singlet CH2 in compound 3 at δ 3.88 ppm. 2‐Alkyl‐
6‐benzylpyridazin‐3(2H)‐ones 4a,b were obtained by alkylation of
compound 3 with methyl iodide or n‐propyl bromide in basic
medium using dimethylformamide as a solvent (Scheme 1).
using N,N‐dimethylformamide as
a
solvent afforded 2‐alkyl‐6‐
benzoylpyridazin‐3(2H)‐ones 8a,b. The carbonyl group of CO–Ph in
compounds 8a,b was reduced by sodium borohydride in methanol to the
corresponding hydroxyl derivatives 9a,b (Scheme 2).
Compound 2 was alkylated with methyl iodide in strong basic
medium (sodium hydride in anhydrous N,N‐dimethylformamide) to
furnish 2‐(6‐chloropyridazin‐3‐yl)‐2‐phenylpropanenitrile (10), which was
hydrolyzed in strong acidic medium of a mixture of acetic acid and 4 M
hydrochloric acid to 6‐(1‐phenylethyl)pyridazin‐3(2H)‐one (11). Hydrolysis
of compound 10 by only acetic acid gave 2‐(6‐oxo‐1,6‐dihydropyridazin‐
3‐yl)‐2‐phenylpropanenitrile (13). The significant CH–CH3 for compound
11 appeared in 1H‐NMR as a doublet at δ 1.49 ppm and a quartet at δ
4.09 ppm for CH–CH3. The signal of CH3–C–CN in compound 13
appeared as a singlet at δ 2.04 ppm. Both of the compounds 11 and 13
were alkylated in basic medium with methyl iodide or n‐propyl bromide
to afford 2‐alkyl‐6‐(1‐phenylethyl)pyridazin‐3(2H)‐ones 12a,b and 2‐
(1‐alkyl‐6‐oxo‐1,6‐dihydropyridazin‐3‐yl)‐2‐phenylpropanenitriles 14a,b
(Scheme 3).
In an attempt to get the 6‐benzoylpyridazin‐3‐one (7), a trial to
oxidize the sodium salt of compound 2 in N,N‐dimethylformamide with a
stream of oxygen bubbled through the reaction mixture did
not furnish the desired (6‐chloropyridazin‐3‐yl)(phenyl)methanone (5),

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(a)
(b)
(a)
(c)
(a)
(b)
(c)
(d)
(d)
(c)
SCHEME 2 Synthesis of compounds 9a,b. Reagents and
conditions: (a) NaH, DMF, O2, RT; (b) AcOH, reflux; (c) R‐X, K2CO3,
DMF, RT; (d) NaBH4, MeOH, RT
SCHEME 3 Synthesis of compounds 12a,b and 14a,b. Reagents
and conditions: (a) NaH, MeI, DMF, RT; (b) 4 M HCl, AcOH, reflux;
(c) R‐X, K2CO3, DMF, RT; (d) AcOH, reflux
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2.2
Biology
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2.2.1
In‐vitro inhibition assay for COX enzymes
Compounds 4a, 8b, and 9a (Figure 1) exhibited the highest analgesic
activity among the synthesized compounds of 47%, 46%, and 45%,
respectively in a dose of 10 mg/kg, which was around the same
activity of diclofenac (47%). In addition, the analgesic activity of the
mentioned compounds agreed with its anti‐inflammatory activity.
Compounds 3, 6, 12b, and 14a showed the lowest activity that can be
explained depending on different pharmacokinetic parameters of
these compounds that affect absorption and metabolism of the
compounds in an in vivo study.
The IC50 results for COX inhibition as well as the SI are shown in
Table 1. The assay results demonstrated that all compounds exhibited
potent inhibitory activity on COX‐2 enzyme with IC50 values that were
lower than those on COX‐1 in the range of 0.04–0.41 µM for COX‐2.
On the contrary, the inhibitory concentrations against COX‐1 were
found to be in the range from 4.23 to 11.64 µM for COX‐1. Among
these compounds, 8b possess the highest activity with an IC50 value of
5.33 µM for COX‐1 and 0.054 µM for COX‐2, and also showed the
highest SI of 99. Furthermore, compounds 2, 3, 4a,b, 6, 8a, 9a,b, 12b,
and 14a,b exhibited high COX‐2 SIs.
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2.2.3
Anti‐inflammatory activity
From the previous results, the selected compounds were 4a,b, 8a,b, 9a,b,
12a, 13, and 14b. Carrageenan‐induced paw edema in rats was used to
evaluate the anti‐inflammatory activity of these compounds as reported
by Puttaswamy et al.[31] All compounds exhibited significant (p < .05,
p = .01–.0001) anti‐inflammatory activity by reducing paw height and,
hence, rat paw edema was reduced after 3 hr in comparison with the
control group, as shown in Table 3. The compounds 4a, 8b, and 9a
exhibited the highest anti‐inflammatory activity among all the synthesized
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2.2.2
Analgesic activity
The analgesic activity of the compounds was screened to confirm
their in vivo activity using a 10‐mg/kg dose by applying writhing test
as reported by Gawade[29] and evaluated compared to the same dose
level of the drug diclofenac as a reference drug (Table 2). The
writhing response was counted, and %protection was calculated.[30]

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TABLE 1 IC50 and selectivity index (SI) values of the compounds
TABLE 2 Analgesic activity of the compounds using writhing test
Compound
COX‐1 IC50 (µM)a COX‐2 IC50 (µM)a COX‐2 (SI)b
No. of writhing in
Analgesic activity
(% protection)
Compound
20 min (mean SEM)
Celecoxib
16.10
4.80
6.94
11.64
7.62
10.21
8.63
10.34
7.52
5.33
4.23
6.21
11.23
5.61
9.87
7.32
9.34
0.059
0.940
0.093
0.150
0.079
0.110
0.140
0.340
0.098
0.054
0.043
0.100
0.410
0.088
0.220
0.088
0.260
273
5
Control
50 0.8
0
Diclofenac
Diclofenac
26 1.5a
40 1.6a,b
51 2.2b
27 1.6a
44 0.4a,b
50 0.5b
44 1.8a,b
44 1.9a,b
27 1.6a
28 1.8a
35 0.4a,b
34 2.5a,b
50 0.5b
34 1.1a,b
50 0.4b
45 2.2a,b
47 1.36a
2
75
78
96
93
62
30
78
99
98
62
27
64
45
83
36
2
20 1.28
3
3
3 1.96
4a
4b
6
4a
4b
6
47 1.2a
12 0.4
0
7
7
12 1.6
11 1.74
46 1.47a
45 1.62a
30 0.4
32 2.23
8a
8b
9a
9b
12a
12b
13
14a
14b
8a
8b
9a
9b
12a
12b
13
14a
14b
1
0.49
32 0.98
0
11 1.96
aThe concentration of the compound producing 50% inhibition of ovine
cyclooxygenase (COX)‐1 and COX‐2 enzymes, the result is the mean of
two determinations obtained by assay of enzyme kits obtained from
Cayman Chemicals Inc., Ann Arbor, MI.
Note: The value is expressed as mean standard error of the means (SEM;
n = 5). Data were analyzed using one‐way analysis of variance followed by
Bonferroni’s test as post hoc test. A p value is considered as significant if
it is <.05 compared to control (p = .0001).
aSignificantly different from control group.
bSignificantly different from the diclofenac group.
bThe in‐vitro COX‐2 selectivity index (COX‐1 IC50/COX‐2 IC50
)
compounds. These activities appeared from the first hour of inflammation
with an edema inhibition percentage of 65, 60, and 62 in a dose of
10 mg/kg for compounds 4a, 8b, and 9a, respectively, and were superior
to diclofenac with rapid onset of action after 1 hr. The duration sustained
until the third hour after the administration of each compound.
was decreased for the 2‐methyl derivatives with benzoyl and/or
(1‐cyano)(1‐phenyl)ethyl groups at position 6 of the pyridazine ring
(compounds 4b, 9b, and 12b). Meanwhile, the activity was increased
with benzoyl and group at position 6 when the substituent at position
2 was n‐propyl group (compound 8b) and decreased when the
substituent at position 2 was the methyl group (compound 8a).
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2.2.4
Serum level of interleukin‐1β (IL‐1β)
At the end of the paw edema test, serum was prepared for enzyme‐
linked immunosorbent assay (ELISA) determination of IL‐1β concentra-
tion in the most significant groups; the selected compounds were 4a, 8b,
and 9a. As shown in Table 4, compound 4a exhibited the most
significant reduction in the IL‐1β concentration, which confirmed the in‐
vitro inhibition of COX activity and also inhibition of the inflammatory
mediators from this pathway. The relation between COX activity and
ILs was studied and illustrated in many studies.[32–34] ILs were produced
from macrophages during inflammation process to induce the produc-
tion of PGs.[34] COX pathway was reported in many studies to be
upregulated and over expressed by many proinflammatory mediators
such as IL‐1β in inflammatory and cancer diseases.[35]
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3
CONCLUSION
It could be concluded that when the group at position 6 of the pyridazine
ring is flexible, the alkyl substituent at position 2 is preferred to be short
as a methyl group (4a, 9a, and 12a) but it showed high activity when the
alkyl group was long as a n‐propyl group with the rigid substituent at
position 6 as the benzoyl group (8b).
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4
EXPERIMENTAL
Chemistry
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Form the data of in‐vitro COX‐1, COX‐2 inhibition
(Table 1), analgesic activity (Table 2), and anti‐inflammatory activity
(Table 3), the activity was increased with the derivatives that
included methyl group at position 2 of the pyridazine ring with
flexible methylene derivatives (benzyl, 1‐phenylethyl, hydroxyl(phe-
nyl)methyl) at position 6 (compound 4a, 9a, and 12a). The activity
4.1
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4.1.1
General
NMR spectra were recorded on Bruker NMR spectrometer at
400 MHz for 1H and 100 MHz for 13C, with tetramethylsilane

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TABLE 3 Percentage changes in rat paw height after carrageenan‐induced paw edema in rats
Anti‐inflammatory activity
(% inhibition of edema)
% Change in paw height (mean SEM)
Compound
1 hr
2 hr
3 hr
3 hr
–
Control
Diclofenac
4a
0.38 0.02
0.17 0.04a
0.21 0.01a
0.34 0.02b
0.38 0.02b
0.24 0.02a
0.24 0.02a
0.36 0.02b
0.36 0.02b
0.38 0.02b
0.42 0.02b
0.46 0.02
0.15 0.03a
0.16 0.02a
0.32 0.02a,b
0.34 0.02b
0.22 0.02a
0.18 0.02a
0.32 0.020a,b
0.30 0.03a,b
0.34 0.02b
0.38 0.02b
0.52 0.02
0.22 0.02a
0.18 0.02a
0.34 0.02a,b
0.38 0.02a,b
0.21 0.02a
0.20 0.03a
0.38 0.02a,b
0.28 0.02a
0.34 0.02a,b
0.38 0.02a,b
58
65
35
27
60
62
27
46
35
27
4b
8a
8b
9a
9b
12a
13
14a
Note: The value is expressed as mean standard error of the means (SEM; n = 5). Data were analyzed using one‐way analysis of variance followed by
Bonferroni’s test as a post hoc test. A p value considered significant if it is <.05 compared to control (p = .01–.0001).
aSignificantly different from the control group.
bSignificantly different from the diclofenac group.
(TMS) as an internal standard for all compounds except 1H‐NMR of
The InChI codes of the investigated compounds together
with some biological activity data are provided as supporting
compound 11 was recorded on Mercury‐300BB NMR spectrometer
information.
at 300 MHz with TMS as an internal standard. Mass spectra were
recorded on Shimadzu Qp‐2010 plus spectrometer for compounds 7,
8a,b, 9a,b, 11, 13, and 14a, Thermo Scientific Trace GC Ultra/ISQ
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Procedure for the synthesis of
2‐(6‐chloropyridazin‐3‐yl)‐2‐phenylacetonitrile 2
spectrometer for compounds 4a, 6, and 10, and Advion compact mass
spectrometer (CMS) NY using atmospheric pressure chemical
ionization (APCI) as ion source technique for compounds 4b, 12a,b,
and 14b. Melting points (m.p.) were determined on a Mel‐Temp
melting point apparatus and are uncorrected. Elemental analyses
were performed at Micro Analytical Center, Cairo University, Cairo,
Egypt. Reactions were monitored by thin‐layer chromatography
(TLC) and performed on silica gel TLC plate 60 F254 that was
purchased from Merck. The starting material 3,6‐dichloropyridazine
(1) is commercially available and was purchased from Sigma‐Aldrich.
4.1.2
To a stirred solution of 3,6‐dichloropyridazine (5, 1.49 g, 0.01 mol)
and benzyl cyanide (1.2 ml, 0.01 mol) in anhydrous DMF (25 ml),
sodium hydride (0.92 g, 0.021 mol, 55% suspension in paraffin oil)
was added portionwise at 0°C. The reaction mixture was left to reach
room temperature gradually with stirring, then poured on ice‐cold
water (100 ml) with stirring, and the precipitated material was
collected by filtration and dried to give 1.8 g of compound 2 as a pure
white solid; yield: 78%; m.p.: 120–122°C; 1H‐NMR (400 MHz, CDCl3)
δ (ppm): 5.70 (s, 1H, CH), 7.37–7.50 (m, 5H, Harom), 7.57 (s, 2H, H4 &
H5). 13C‐NMR (100 MHz, CDCl3) δ (ppm): 43.08 (CH), 117.54 (CN),
127.42 (C4), 127.69, 129.10, 129.59 (Carom), 129.63 (C5), 132.73
(Carom), 156.92 (C3), and 157.83 (C6); electron ionization mass
spectrometry (EI‐MS): m/z = 228 (100%), 229 (M+, 37%). Anal. calcd.
for C12H8ClN3 (229.67): C, 62.76; H, 3.51; N, 18.30. Found: C, 63.05;
H, 3.61; N, 18.11.
TABLE 4 Serum level of IL‐1β after paw edema in rats
Groups
IL‐1β concentration (pg/ml)
22 1.53
Normal animals
Paw edema‐bearing animals
41 0.58a
Diclofenac
30 0.67b
4a
8b
9a
31 0.67b
34 1.33b
35 0.58b
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4.1.3
General procedure for the synthesis of
Note: The value is expressed as mean standard error of the means (SEM;
n = 5). Data were analyzed using one‐way analysis of variance followed by
Bonferroni’s test as post hoc test. A p value is considered significant if it is
<.05 compared to control (p = .0001). Normal animals mean control
animals that did not take any medications during the experiment and
exhibit no edema.
6‐benzylpyridazin‐3(2H)‐one (3) and 6‐(1‐phenylethyl)
pyridazin‐3(2H)‐one (11)
Compound 2 and/or 10 (5 mmol) was refluxed in a mixture of
concentrated hydrochloric acid (20 ml), acetic acid (10 ml), and water
(10 ml) for 30 hr. The solvents were evaporated under reduced
aSignificant from normal animals.
bSignificant from paw edema‐bearing animals.

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pressure, then water (20 ml) was added to the residual material and
neutralized with saturated solution of sodium bicarbonate. The solid
product formed was collected by filtration and dried to give compounds
3 and 11 as pure white solids.
6‐Benzyl‐2‐propylpyridazin‐3(2H)‐one 4b
Yield: 43%, as an oil; 1H‐NMR (400 MHz, CDCl3) δ (ppm): 0.91 (t, 3H,
J = 7.5 Hz, CH2CH3), 1.78 (sxt, 2H, J = 7.5 Hz, CH2CH2CH3), 3.84
(s, 2H, CH2–Ph), 4.05 (t, 2H, J = 7.5 Hz, CH2CH2CH3), 6.75 (d, 1H,
J = 9.7 Hz, H5), and 7.12–7.27 (m, 5H, Harom); 13C‐NMR (100 MHz,
CDCl3) δ (ppm): 11.11 (CH3CH2CH2), 21.73 (CH3CH2CH2), 40.96
(CH2–Ph), 53.23 (CH2–N), 126.98, 128.80, 128.84 (C5), 130.04
(Carom), 132.18 (C4), 137.48 (Carom), 146.60 (C6), and 159.78 (C3);
APCI‐MS (C14H16N2O): m/z = 229.5 (M+ + 1, 100%).
6‐Benzylpyridazin‐3(2H)‐one 3
Yield: 79%; m.p. 76–78°C; 1H‐NMR (400 MHz, dimethyl sulfoxide
[DMSO]‐d6) δ (ppm): 3.88 (s, 2H, CH2), 6.82 (d, 1H, J = 9.8Hz, H5),
7.21–7.34 (m, 6H, H4 and Harom), and 12.88 (s, 1H, NH); 13C‐NMR
(100 MHz, DMSO‐d6) δ (ppm): 38.78 (CH2), 126.68, 128.72 (Carom),
128.89 (C5), 130.12 (Carom), 134.21 (C4) 138.19 (Carom), 146.91 (C6),
and 160.29 (C3); EI‐MS: m/z = 186 (M+, 100%). Anal. calcd. for
C11H10N2O (186.21): C, 70.95; H, 5.41; N, 15.04. Found: C, 70.70;
H, 5.58; N, 14.98.
6‐Benzoyl‐2‐methylpyridazin‐3(2H)‐one 8a
Yellow solid; yield: 84%; m.p.: 130–132°C; 1H‐NMR (400 MHz,
CDCl3) δ (ppm): 3.84 (s, 3H, CH3), 7.01 (d, H, J = 9.7 Hz, H5),
7.47–7.50 (m, 2H, Harom), 7.59–7.62 (m, 1H, Harom), 7.97 (d, 1H,
J = 9.7 Hz, H4), and 8.03 (d, 2H, J = 7.6 Hz, Harom); 13C‐NMR
(100 MHz, CDCl3) δ (ppm): 40.89 (CH3), 128.06 (Carom), 128.83
(C5), 130.41, 131.41, 133.00 (Carom), 134.33 (C4), 142.06 (C6),
160.04 (C3), and 188.57 (Ph–C═O); EI‐MS: m/z = 105 (100%) and 214
(M+, 39%). Anal. calcd. for C12H10N2O2 (214.22): C, 67.28; H, 4.71;
N, 13.08. Found: C, 67.00; H, 5.03; N, 13.09.
6‐(1‐Phenylethyl)pyridazin‐3(2H)‐one 11
Yield: 86%; m.p.: 118–120°C; 1H‐NMR (300 MHz, DMSO‐d6) δ (ppm):
1.49 (d, 3H, J = 7.0 Hz, CH3–CH), 4.09 (q, 1H, J = 7.0 Hz, CH3–CH),
6.77 (d, 1H, J = 9.6 Hz, H5), 7.20–7.33 (m, 5H, H4 & Harom), and 12.86
(bs, 1H, NH); 13C‐NMR (100 MHz, DMSO‐d6) δ (ppm): 19.39 (CH3),
43.26 (CH3–CH), 126.67, 127.42, 128.66 (Carom), 129.92 (C5), 133.58
(C4), 143.60 (Carom), 149.93 (C6), and 160.26 (C3); EI‐MS: m/z = 200
(M+, 100%). Anal. calcd. for C12H12N2O (200.24): C, 71.98; H, 6.04;
N, 13.99. Found: C, 72.06; H, 6.27; N, 13.82.
6‐Benzoyl‐2‐propylpyridazin‐3(2H)‐one 8b
Yellow crystals; yield: 46%; m.p.: 192–194°C; 1H‐NMR (400 MHz,
CDCl3) δ (ppm): 0.88 (t, 3H, J = 7.4 Hz, CH3CH2CH2), 1.78 (sxt, 2H,
J = 7.4 Hz, CH3CH2CH2), 4.09 (t, 2H, J = 7.4 Hz, CH3CH2CH2), 6.91
(d, 1H, J = 9.7 Hz, H5), 7.38–7.41 (m, 2H, Harom), 7.50–7.53 (m, 1H,
Harom), 7.86 (d, 1H, J = 9.7 Hz, H4), and 7.94 (d, 2H, J = 7.6 Hz, Harom);
13C‐NMR (100 MHz, CDCl3) δ (ppm): 10.96 (CH3CH2CH2–N), 21.48
(CH3CH2CH2–N), 53.80 (CH3CH2CH2–N), 127.99 (Carom), 129.09
(C5), 130.57, 130.97, 132.92 (Carom), 135.41 (C4), 141.96 (C6),
159.77 (C3), and 188.71 (Ph–C═O); EI‐MS: m/z = 105 (100%) and 242
(M+, 37%). Anal. calcd. for C14H14N2O2 (242.28): C, 69.41; H, 5.82;
N, 11.56. Found: C, 69.47; H, 5.58; N, 11.90.
|
4.1.4
General procedure for the synthesis of the
2‐alkylpyridazin‐3(2H)‐one derivatives 4a,b, 8a,b,
12a,b and 14a,b
A mixture of 3, 6, 8, and/or 12 (1 mmol), methyl iodide and/or
n‐propyl bromide (1.1 mmol), and anhydrous potassium carbonate
(0.41 g, 3 mmol) in anhydrous DMF (10 ml) was stirred at room
temperature for 6 hr. The reaction mixture was poured on ice‐cold
water (25 ml) with continuous stirring. To obtain compounds 4a,b,
12a,b and 14a,b, the mixture was extracted with ether (3 × 15 ml).
The combined ethereal extracts were dried over sodium sulfate,
filtered, and the solvent was removed under reduced pressure. The
2‐Methyl‐6‐(1‐phenylethyl)pyridazin‐3(2H)‐one 12a
White solid; yield: 55%; m.p.: 60–62°C; 1H‐NMR (400 MHz, CDCl3)
δ (ppm): 1.52 (d, 3H, J = 7.4 Hz, CH3–CH), 3.72 (s, 3H, CH3–N), 3.98 (q,
1H, J = 7.4 Hz, CH3–CH); 6.72 (d, 1H, J = 9.5, H5), 6.90 (d, 1H,
J = 9.5 Hz, H4), and 7.15–7.34 (m, 5H, Harom); 13C‐NMR (100 MHz,
CDCl3) δ (ppm): 19.44 (CH3–CH), 40.15 (CH3–N), 44.24 (CH3–CH),
126.95, 127.43, 128.77 (Carom), 129.46 (C5) 132.13 (Carom), 142.97
(C4), 150.21 (C6), and 160.16 (C3); APCI‐MS: m/z = 215.5 (M+ + 1,
100%). Anal. calcd. for C13H14N2O (214.27): C, 72.87; H, 6.59;
N, 13.07. Found: C, 73:00; H, 6.66; N, 12.88.
residual material was chromatographed on
a column of silica
gel using petroleum ether/ethyl acetate (2:1, v/v). To obtain
compounds 8a,b, the solid product formed was filtered off, washed
with water followed by petroleum ether, and dried to give a pure
white solid.
6‐Benzyl‐2‐methylpyridazin‐3(2H)‐one 4a
White crystals; yield: 45%; m.p.: 63–65°C; 1H‐NMR (400 MHz,
CDCl3) δ (ppm): 3.78 (s, 3H, CH3), 3.90 (s, 2H, CH2), 6.82 (d, 1H,
J = 9.5 Hz, H5); 7.02 (d, 1H, J = 9.5 Hz, H4), and 7.20–7.33 (m, 5H,
Harom); 13C‐NMR (100 MHz, CDCl3) δ (ppm): 39.91 (CH2), 40.71
(CH3), 126.89, 128.70, 128.72 (Carom), 129.58 (C5) 132.48 (Carom),
137.23 (C4), 159.93 (C6), and 164.64 (C3); EI‐MS: m/z = 200
(M+, 100%). Anal. calcd. for C12H12N2O (200.24): C, 71.98; H, 6.04;
N, 13.99. Found: C, 72.20; H, 6.18; N, 13.68.
6‐(1‐Phenylethyl)‐2‐propylpyridazin‐3(2H)‐one 12b
Yield: 41%; as an oil; 1H‐NMR (400 MHz, CDCl3) δ (ppm): 0.92 (t, 3H,
J = 7.5 Hz, CH3CH2), 1.52 (d, 3H, J = 7.0 Hz, CH3CH), 1.80 (sxt, 2H,
J = 7.5 Hz, CH3CH2CH2), 3.98 (q, 1H, J = 7.0 Hz, CHCH3), 4.07 (t, 2H,
J = 7.5 Hz, CH3CH2CH2–N), 6.71 (d, 1H, J = 9.6 Hz, H5), and 6.87
(d, 1H, J = 9.6 Hz, H4); 13C‐NMR (100 MHz, CDCl3) δ (ppm) 11.15
(CH3CH2CH2–N), 19.51 (CH3CH), 21.69 (CH3CH2CH2–N), 44.35
(CH3CH), 53.19 (CH3CH2CH2–N), 126.92, 127.43 (Carom), 128.78

LOKSHA AND ABD‐ALHASEEB
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(C5), 129.77, 131.75 (Carom), 143.15 (C4), 149.94 (C6), and 159.89
(C3); APCI‐MS (C15H18N2O): m/z = 243.2 (M+ + 1, 100%).
reaction mixture was neutralized with saturated solution of
sodium bicarbonate. The solid product formed was filtered off,
washed with water followed by petroleum ether and dried to
afford compounds 6 and/or 13 as pure white solids.
2‐(1‐Methyl‐6‐oxo‐1,6‐dihydropyridazin‐3‐yl)‐2‐phenylpropanenitrile
14a
White solid; yield: 72%, m.p.: 98–100°C; 1H‐NMR (400 MHz, CDCl3)
δ (ppm): 2.03 (s, 3H, CH3–C), 3.82 (s, 3H, CH3–N), 6.84 (d, 1H,
J = 9.7 Hz, H5), 7.08 (d, 1H, J = 9.7 Hz, H4), and 7.32–7.42 (m, 5H,
Harom); 13C‐NMR (100 MHz, CDCl3) δ (ppm): 25.83 (CH3–C), 40.31
(CH3–N), 46.39 (CH3–C), 120.83 (CN), 125.81, 128.52 (Carom), 129.23
(C5), 130.22, 130.96 (Carom), 138.31 (C4), 144.38 (C6), and 159.40
(C3). EI‐MS: m/z = 239 (M+, 100%). Anal. calcd. for C14H13N3O
(239.28): C, 70.28; H, 5.48; N, 17.56. Found: C, 70.55; H, 5.70;
N, 17.29.
2‐(6‐Oxo‐1,6‐dihydropyridazin‐3‐yl)‐2‐phenylpropanenitrile 13
White solid; yield: 89%; m.p.: 53–55°C; 1H‐NMR (400 MHz,
DMSO‐d6) δ (ppm): 2.04 (s, 3H, CH3), 6.90 (d, 1H, J = 9.9 Hz,
H5), 7.31 (d, 1H, J = 9.9 Hz, H4), 7.37–7.48 (m, 5H, Harom), and
13.23 (s, 1H, NH); 13C‐NMR (100 MHz, DMSO‐d6) δ (ppm): 24.85
(CH3), 46.15 (C–CN), 121.39 (CN), 126.23 (Carom), 128.60 (C5),
129.37, 131.25, 131.34 (Carom), 138.44 (C4), 145.40 (C6), and
159.89 (C3); EI‐MS: m/z = 225 (M+, 100%). Anal. calcd.
for C13H11N3O (225.25): C, 69.32; H, 4.92; N, 18.66. Found:
C, 69.23; H, 5.25; N, 18.53.
2‐(6‐Oxo‐1‐propyl‐1,6‐dihydropyridazin‐3‐yl)‐2‐phenylpropanenitrile
14b
2‐(6‐Oxo‐1,6‐dihydropyridazin‐3‐yl)‐2‐phenylacetonitrile 6
White solid; yield: 95%; m.p. 183–185°C; 1H‐NMR (400 MHz,
DMSO‐d6) δ (ppm): 5.84 (s, 1H, CH–CN), 6.91 (d, 1H, J = 9.8 Hz,
H5), 7.33 (d, 1H, J = 9.8 Hz, H4), 7.38–7.48 (m, 5H, Harom), and
13.23 (s, 1H, NH); 13C‐NMR (100 MHz, DMSO‐d6) δ (ppm): 40.51
(CH–CN), 118.44 (CN), 127.97 (Carom), 128.82 (C5), 129.56,
131.18, 132.49 (Carom), 134.05 (C4); 142.55 (C6), and 160.03
(C3); EI‐MS: m/z = 210 (100%) and 211 (M+, 85%). Anal. calcd. for
C12H9N3O (211.22): C, 68.24; H, 4.29; N, 19.89. Found: C, 68.08;
H, 4.53; N, 20.00.
Yield: 44%; as an oil; 1H‐NMR (400 MHz, CDCl3) δ (ppm): 0.93 (t, 3H,
J = 7.5 Hz, CH3CH2), 1.82 (sxt, 2H, J = 7.5 Hz, CH3CH2CH2), 4.10 (t,
2H, J = 7.5 Hz, CH3CH2CH2), 6.79 (d, 1H, J = 9.6 Hz, H5), 7.00 (d, 1H,
J = 9.6 Hz, H4), and 7.19–7.36 (m, 5H, Harom); 13C‐NMR (100 MHz,
CDCl3) δ (ppm): 11.13 (CH3CH2CH2–N), 21.61 (CH3CH2CH2–N),
26.06 (CH3CH), 46.65 (CH3CH), 53.45 (CH3CH2CH2–N), 121.05 (CN),
125.89, 128.65 (Carom), 129.39 (C5), 130.66, 130.69 (Carom), 138.56
(C4), 144.32 (C6), and 159.32 (C3); APCI‐MS (C16H17N3O):
m/z = 268.2 (M+ + 1, 100%).
|
|
4.1.5
Synthesis of 2‐(6‐chloropyridazin‐3‐yl)‐2‐
4.1.7
Synthesis of 6‐benzoylpyridazin‐3(2H)‐one 7
phenylpropanenitrile 10
Sodium hydride (1.44 g, 55% suspension in paraffin oil, 60 mmol) was
added to a solution of compound 6 (4.22 g, 20 mmol) in anhydrous
DMF (20 ml). The reaction mixture was stirred in open air for 5 hr.
The reaction mixture was poured on ice‐cold water and the solid
product formed was filtered off, washed with water followed by
petroleum ether and dried to afford 3.1 g of the pure white solid of
compound 7; yield: 78%; m.p.: 194–196°C; 1H‐NMR (400 MHz,
DMSO‐d6) δ (ppm): 7.04 (d, 1H, J = 9.8 Hz, H5), 7.51–7.55 (m, 2H,
Harom), 7.64–7.68 (m, 1H, Harom), 7.93–7.96 (m, 3H, H4 & Harom), and
13.54 (s, 1H, NH); 13C‐NMR (100 MHz, DMSO‐d6) δ (ppm): 128.17
(Carom), 129.68 (C5), 130.38, 131.97, 132.95 (Carom), 135.63 (C4),
142.53 (C6), 160.53 (C3), and 189.49 (Ph–C═O); EI‐MS: m/z = 106.05
(100%) and 200 (M+, 34%). Anal. calcd. for C11H8N2O2 (200.20):
C, 66.00; H, 4.03; N, 13.99. Found: C, 66.15; H, 4.18; N, 13.95.
Sodium hydride (1.2 g, 55% suspension in paraffin oil, 36 mmol) was
added portion‐wise to a solution of compound 2 (6.87 g, 30 mmol) in
anhydrous DMF (15 ml) with stirring for 15 min at room temperature.
Methyl iodide (2.28 ml, 36 mmol) was added in one portion to the
reaction mixture and stirred for an additional 3 hr. The reaction was
quenched with ethanol (1 ml) followed by the addition of cold water
(50 ml). The solid product formed was filtered off, washed with water
and dried to afford 6.5 g of compound 10 as pure white crystals; yield:
71%; m.p.: 115–117°C; 1H‐NMR (400 MHz, CDCl3) δ (ppm): 2.33 (s, 1H,
CH3) and 7.35–7.57 (m, 7H, Harom, H4 & H5); 13C‐NMR (100 MHz,
CDCl3) δ (ppm): 26.70 (CH3), 47.81 (C–CN), 121.46 (CN), 126.12 (C4),
128.34, 128.68, 129.12 (Carom), 129.40 (C5), 138.85 (Carom), 156.87
(C3), and 160.43 (C6); EI‐MS: m/z = 242 (100%), 243 (M+, 75%), and 244
(44%). Anal. calcd. for C13H11N3O (243.69): C, 64.07; H, 4.14; N, 17.24.
Found: C, 63.93; H, 3.83; N, 17.04.
|
4.1.8
Synthesis of 2‐alkyl‐6‐(hydroxy(phenyl)
methyl)‐pyridazin‐3(2H)‐ones 9a,b
|
4.1.6
General procedure for the synthesis of
compounds 6 and 13
Under an ice cooling bath, sodium borohydride (0.21 g, 5.5 mmol)
was added portionwise to a stirred solution of 8a,b (5 mmol) in
methanol (10 ml). The reaction mixture was left to reach room
temperature gradually with stirring for 0.5 hr. Acetic acid (1 ml)
Compound 2 and/or 5 (3 mmol) was refluxed in acetic acid (20 ml)
for 6 hr. The solvent was concentrated to 10 ml and cooled. The

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LOKSHA AND ABD‐ALHASEEB
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was added followed by addition of water (20 ml). The mixture was
extracted with ethyl acetate (3 × 15 ml). The combined organic
layers were dried over sodium sulfate and filtered. Then,
petroleum ether (20 ml) was added with scratching by glass rod.
The crystalized product formed was filtered off and dried to
afford 9a,b as pure white solids.
4.2.3
In‐vitro COX assay
Compounds 2, 3, 4a,b, 6,7, 8a,b, 9a,b, 12a,b, 13, and 14a,b were screened
for in‐vitro COX‐inhibiting activity[16] using an enzyme immunoassay
(EIA) kit (Cayman Chemical Company, Ann Arbor, MI). The IC50 values for
the compounds were determined using diclofenac and celecoxib as the
reference drug, which are considered as non‐selective and selective COX‐
inhibiting medicine, respectively. Furthermore, the SI for COX‐2 (COX‐1
IC50/COX‐2 IC50) was calculated for the compounds. The inhibition ability
of the compounds to inhibit ovine COX‐1 and the human recombinant
COX‐2 form was determined and evaluated using a COX inhibitor
screening assay kit (catalog no. 560131; Cayman Chemical Company) in
agreement with the instructions recommended and mentioned by the
supplier. An inhibitor‐screening assay kit was used in this assay. The kit
contained two types of COX‐1 and 2 enzymes. The inhibitory activity of
compounds was measured using the immune assay technique.
6‐(Hydroxy(phenyl)methyl)‐2‐methylpyridazin‐3(2H)‐one 9a
White solid; yield: 88%; m.p.: 102–104°C; 1H‐NMR (400 MHz, CDCl3)
δ (ppm): 2.97 (bs, 1H, OH), 3.81 (s, 3H, CH3), 5.69 (s, 1H, CH), 6.97 (d,
1H, J = 9.5 Hz, H5), 7.18 (d, 1H, J = 9.5 Hz, H4), and 7.34–7.40 (m, 5H,
Harom); 13C‐NMR (100 MHz, CDCl3) δ (ppm): 40.17 (CH3), 74.03 (CH‐
OH), 126.39, 128.39 (Carom), 128.82 (C5), 130.09, 130.48 (Carom),
140.55 (C4), 148.24 (C6), and 160.30 (C3); EI‐MS: m/z = 111 (100%)
and 216 (M+, 31%). Anal. calcd. for C12H12N2O2 (216.24): C, 66.65; H,
5.59; N, 12.96. Found: C, 66.86; H, 5.44; N, 12.66.
|
Screening of analgesic activity
6‐(Hydroxy(phenyl)methyl)‐2‐propylpyridazin‐3(2H)‐one 9b
White solid; yield: 66%; m.p.: 66–68°C; 1H‐NMR (400 MHz, CDCl3) δ
(ppm): 1.00 (t, 3H, J = 7.5 Hz, CH3CH2CH2), 1.87 (sxt, 2H, J = 7.5 Hz,
CH3CH2CH2), 4.15 (m, 2H, CH3CH2CH2), 5.69 (s, 1H, CH‐OH), 6.86 (d,
1H, J = 9.5 Hz, H5), 7.14 (d, 1H, J = 9.5 Hz, H4), and 7.34–7.40 (m, 5H,
Harom); 13C‐NMR (100 MHz, CDCl3) δ (ppm): 11.10 (CH3CH2CH2), 21.65
(CH3CH2CH2), 53.34 (CH3CH2CH2), 74.04 (CH‐OH), 126.38, 128.35
(Carom), 128.82 (C5), 130.06, 139.36 (Carom), 140.67 (C4), 148.06 (C6),
and 159.99 (C3); EI‐MS: m/z = 57 (100%) and 244 (M+, 3%). Anal. calcd.
for C14H16N2O2 (244.29): C, 68.83; H, 6.60; N, 11.47. Found: C, 68.59; H,
6.31; N, 11.85.
4.2.4
The analgesic activity of the compounds was performed using the
writhing test as described by Gawade.[29] Mice weight ranged from 25 to
30 g. The mice were then divided into 17 different groups, each
containing five animals. The animals were fasted for 8 hr before the
experiment. The administration of the compounds (10 mg/kg, p.o.) was
performed orally. Equal dose of diclofenac as the reference drug and
saline as negative control were also administrated orally. Writhing was
induced after 1 hr from compounds administration by using 0.1 ml of 1%
glacial acetic acid in a volume of 0.1 ml/10 g body weight. The number of
writhing responses (stretching of the abdomen, extension of hind limbs,
twisting of the trunk, and elongation of the body) observed were counted
for 20 min. The protection percentile against acetic acid‐induced writhing
|
4.2
Biological screening
Chemicals and drugs
was calculated according to the following formula[16]
:
|
4.2.1
n − n′
n
% Analgesic activity =
× 100,
Carrageenan, celecoxib, and diclofenac sodium were purchased from
Sigma‐Aldrich. IL‐1β ELISA Kit was purchased from Reddot Biotech.,
Inc. (Canada).
where n is the mean of writhes of the control group and n′ is the
mean of writhes of the tested compound group.
|
4.2.5
Evaluation of the anti‐inflammatory activity
|
4.2.2
Animals
The method of paw edema, reported by Puttaswamy et al.,[31] was used
for the evaluation and screening of the activity of the compounds that
exhibited analgesic activity against inflammation‐induced. Albino rats
(150–180 g) were used. The rats were divided into 11 experimental
groups of five rats each. One hour after oral administration of the
compounds (10 mg/kg), 0.1 ml of 1% carrageenan solution was injected in
the left hind paw of each animal. Rat paw volumes were measured using a
digital caliper (Germany) used to measure inflammation height at 0, 1, 2,
and 3 hr, after carrageenan injection. The percentage increase in left hind
paw, in comparison to the uninjected right hind paw, was determined,
calculated, and expressed as the amount of inflammation. All the tested
compounds were orally administered at equivalent doses of the reference
drug diclofenac (10 mg/kg, p.o.).
Male albino rats and mice were used for the evaluation of anti‐
inflammatory and analgesic activities, respectively. The animals
were purchased from the animal house at The Holding Company
for Biological products; vaccines and drugs (VACSERA, Giza,
Egypt) were acclimatized for 2 weeks before the experiments at
the animal house of Faculty of Pharmacy, Damanhour University.
The animals were divided to different separated groups, five
animals in each group. The food and water were supplied ad
libitum to the animals. All the protocols used in this study were
approved and agreed by the Ethical Committee at the Faculty of
Pharmacy, Damanhour University, Damanhour, Egypt (with
ethical code number 120PO18).

LOKSHA AND ABD‐ALHASEEB
9 of 9
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The anti‐inflammatory activity of the selected analgesic com-
[10] M. Asif, Eur. J. Biol. Sci. 2016, 8, 142.
[11] M. Asif, D. Singh, A. Singh, Global J. Pharmacol. 2011, 5, 18.
pounds after 3 hr was expressed as percentage inhibition of edema
[12] C. Rubat, P. Coudert, P. Bastide, P. Tronche, Chem. Pharm. Bull. 1988, 36,
5000.
that was calculated according to the following equation[16]
:
[13] J. Il‐Longom, M. D. R. Laguna, I. Verde, M. E. Castro, F. Oralloj,
J. A. Fontenla, J. M. Calleja, E. Ravina, C. Teran, J. Pharm. Sci. 1993,
82, 286.
V
− V
cont
test
% Inhibition of edema =
× 100,
V
cont
[14] M. Asif, Mini Rev. Org. Chem. 2013, 10, 113.
[15] M. Asif, J. Chem. 2014, ID 703238. https://doi.org/10.1155/2014/
703238
where Vcont is the edema volume in the control group and Vtest is the
edema volume in the group of the screened compound.
[16] T. H. Ibrahim, Y. M. Loksha, H. A. Elshihawy, D. M. Khodeer,
M. M. Said, Arch. Pharm. Chem. Life Sci. 2017, 350, e1700093.
[17] A. Ya Kots, M. A. Grafov, Y. V. Khropov, V. L. Betin, N. N. Belushkina,
O. G. Busygina, M. Yu Yazykova, I. V. Ovchinnikov, A. S. Kulikov,
N. N. Makhova, N. A. Medvedeva, T. V. Bulargina, I. S. Severina,
Br. J. Pharmacol. 2000, 129, 1163.
|
4.2.6
ELISA determination of IL‐1
IL‐1 is one of the important inflammatory mediators used to confirm
the anti‐inflammatory activity of new anti‐inflammatory compounds.
At the end of the paw edema test, blood samples were collected from
the orbital axis of the eye under light ether anesthesia of the rats.
Sera were separated after centrifugation of blood at 5,000 rpm for
15 min; the sera were stored at −80°C until the ELISA assay was
performed according to the manufacturer’s instructions.
[18] M. Gokce, G. Bakir, M. F. Sahin, E. Kupeli, E. Yesilada, Arzneimittel-
forschung 2005, 55, 318.
[19] L. P. Guan, X. Sui, X. Q. Deng, Y. C. Quan, Z. S. Quan, Eur. J. Med.
Chem. 2010, 45, 1746.
[20] F. Z. Hu, G. F. Zhang, Y. Q. Zhu, X. M. Zou, B. Liu, H. Z. Yang, Chin.
J. Org. Chem. 2007, 27, 758.
[21] K. Javed, S. Yaseen, R. Bashirand, P. Rathore, Eur. J. Med. Chem. 2013,
67, 352.
[22] Y. Kawano, H. Nagayaand, M. Gyoten. WO 2000020417. 2000 [Chem.
Abstr. 2000, 132, 265202].
|
4.2.7
Statistical analysis
[23] R. D. Li, X. Zhai, Y. F. Zhao, S. Yu, P. Gong, Arch. Pharm. 2007, 340, 424.
[24] W. D. Liu, Z. W. Li, Z. Y. Li, X. G. Wang, B. D. Gao, Chin. J. Org. Chem.
2005, 25, 445.
Data were expressed as mean standard error of the means. One‐way
ANOVA followed by Bonferroni’s post hoc test was used to analyze the
data using the Statistical Package for Social Sciences, version 19 (SPSS,
Inc., Chicago, IL). p < .05 was considered to be statistically significant.
[25] W. Malinka, A. Redzicka, O. Lozach, Farmaco 2004, 59, 457.
[26] G. W. Bemis, F. G. Salituro, J. P. Duffy, E. M. Harrington. US 6147080
A1 2000 [Chem. Abstr. 2000, 133, 350242].
[27] Y. M. Loksha, D. Globisch, R. Loddo, G. Collu, P. La Colla,
E. B. Pedersen, Chem. Med. Chem. 2010, 5, 1847.
[28] G. Heinisch, T. Langer, J. Heterocycl. Chem. 1993, 30, 1685.
[29] S. P. Gawade, J. Pharmacol. Pharmacother. 2012, 3, 348.
[30] E. Shawky, N. M. El Newehy, A. M. Beltagy, M. M. Abd‐Alhaseeb,
G. A. Omran, F. M. Harraz., J. Chromatogr. B 2018, 1087‐1088, 108.
[31] N. Puttaswamy, V. H. Malojiao, Y. H. E. Mohammed, A. Sherapura,
B. T. Prabhakar, S. A. Khanum, Biomed. Pharmacother. 2018, 103, 1446.
[32] L. A. J. O’Neill, M. L. Barrett, G. P. Lewis, Fed. Eur. Biochem. Soc. 1987, 212,
35.
CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.
ORCID
Yasser M. Loksha
http://orcid.org/0000-0001-5731-0315
[33] J.‐H. Lin, M.‐T. Lin, Neurosci. Lett. 1996, 208, 155.
[34] Z. Yang, W. Ji, M. Li, Z. Qi, R. Huang, J. Qu, H. Wang, H. Wang, Am. J.
Transl. Res. 2019, 11, 6024.
Mohammad M. Abd‐Alhaseeb
http://orcid.org/0000-0001-5388-
5250
[35] W. Liu, N. Reinmuth, O. Stoeltzing, A. A. Parikh, C. Tellez, S. Williams,
Y. D. Jung, F. Fan, A. Takeda, M. Akagi, M. Bar‐Eli, G. E. Gallick,
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