Synthesis of novel N-substitutedphenyl-6-oxo-3-phenylpyridazine derivatives as cyclooxygenase-2 inhibitors

Article abstract of DOI:10.1002/ddr.21655

Some novel non-ulcerogenic N-substitutedphenyl-6-oxo-3-phenylpyridazines as COX-2 inhibitors have been developed (Supplementary material Appendix 1). The novel aldehyde 3 was prepared by reacting 6-phenylpyridazin-3(2H)-one with 4-fluorobenzaldehyde. The aldehyde 3 was reacted with different hydrazines and thiazolidin-4-ones to obtain the novel N-substitutedphenyl-6-oxo-3-phenylpyridazine derivatives. These were assessed for their anti-inflammatory potential and gastric ulcerogenic effects. The molecular docking investigations were also undertaken. The spectroscopic data were coherent with the allocated structures of the compounds. The compounds 4a (IC₅₀ = 17.45 nm; p <.05), 4b (IC₅₀ = 17.40 nm; p <.05), 5a (IC₅₀ = 16.76 nm; p <.05), and 10 (IC₅₀ = 17.15 nm; p <.05) displayed better COX-2 inhibitory activity than celecoxib (IC₅₀ = 17.79 nm; p <.05). These findings were consistent with the molecular docking investigations of 4a, 4b, 5a, and 10. The in vivo anti-inflammatory profile of 4a, 4b, 5a, and 10 was also superior to celecoxib and indomethacin. The compounds 4b, 5a, and 10 revealed no gastric ulcerogenic effects, wherein the compound 4a produced almost negligible gastric ulcerogenic effects than celecoxib and indomethacin. The compounds 4a, 4b, 5a, and 10 have been postulated as promising non-ulcerogenic COX-2 inhibitors.

Full text of DOI:10.1002/ddr.21655

Received: 11 December 2019
DOI: 10.1002/ddr.21655
Revised: 1 February 2020
Accepted: 22 February 2020
R E S E A R C H A R T I C L E
Synthesis of novel N-substitutedphenyl-6-oxo-
3
-phenylpyridazine derivatives as cyclooxygenase-2 inhibitors
1
1
2
3
Abida Khan
| Anupama Diwan | Hamdy K Thabet | Mohd Imran
1
School of Pharmaceutical Sciences, Apeejay
Stya University, Sohna, India
Abstract
2
Department of Chemistry, Faculty of Science,
Some novel non-ulcerogenic N-substitutedphenyl-6-oxo-3-phenylpyridazines as
COX-2 inhibitors have been developed (Supplementary material Appendix 1). The
novel aldehyde 3 was prepared by reacting 6-phenylpyridazin-3(2H)-one with
Northern Border University, Rafha, Saudi
Arabia
3
Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Northern Border
University, Rafha, Saudi Arabia
4
-fluorobenzaldehyde. The aldehyde 3 was reacted with different hydrazines and
thiazolidin-4-ones to obtain the novel N-substitutedphenyl-6-oxo-
-phenylpyridazine derivatives. These were assessed for their anti-inflammatory
Correspondence
3
Abida Khan, School of Pharmaceutical
Sciences, Apeejay Stya University,
Sohna - Palwal Road, Sohna 122103, India.
Email: aqua_abkhan@yahoo.com
potential and gastric ulcerogenic effects. The molecular docking investigations were
also undertaken. The spectroscopic data were coherent with the allocated structures
of the compounds. The compounds 4a (IC50 = 17.45 nm;
p < .05), 4b
(IC50 = 17.40 nm; p < .05), 5a (IC50 = 16.76 nm; p < .05), and 10 (IC50 = 17.15 nm;
p < .05) displayed better COX-2 inhibitory activity than celecoxib (IC50 = 17.79 nm;
p < .05). These findings were consistent with the molecular docking investigations of
4a, 4b, 5a, and 10. The in vivo anti-inflammatory profile of 4a, 4b, 5a, and 10 was also
superior to celecoxib and indomethacin. The compounds 4b, 5a, and 10 revealed no
gastric ulcerogenic effects, wherein the compound 4a produced almost negligible
gastric ulcerogenic effects than celecoxib and indomethacin. The compounds 4a, 4b,
5a, and 10 have been postulated as promising non-ulcerogenic COX-2 inhibitors.
K E Y W O R D S
anti-inflammatory, gastric ulcer, molecular docking, pyridazine, synthesis
1
|
INTRODUCTION
osteoarthritis resulting in some form of disability (Lee et al., 2013).
Chronic inflammatory diseases can be treated with NSAIDs. However,
the maximum number of NSAIDs can cause serious ulcerogenic
effects on chronic use (Radi & Khan, 2019; Sehajpal, Prasad, & Singh,
2018). The adverse effects linked with NSAIDs are contributed to the
inhibition of cyclooxygenase-1 (COX-1), wherein the inflammatory
responses are contributed to the generation of cyclooxygenase-2
(COX-2) (Korbecki, Baranowska-Bosiacka, Gutowska, & Chlubek,
2014; Tortorella, Zhang, & Talley, 2016). The COX-2 is also implicated
in the generation of atherosclerosis, pancreatitis, hepatitis, asthma,
cancer, kidney injury, irritable bowel diseases, epilepsy, depression,
and Alzheimer's disease (Chen et al., 2017; Nasef et al., 2017).
Accordingly, specific COX-2 inhibitors, like celecoxib, etoricoxib, and
rofecoxib were developed (Burnier, 2005; Kumar, Kaur, Gupta,
Inflammation is a defensive immune reaction toward injurious stimuli
Chen et al., 2017). The symptoms of acute inflammation may con-
(
tinue for many days and are characterized by swelling, redness, heat,
immobility, and pain (Varela, Mogildea, Moreno, & Lopes, 2018).
The chronic inflammatory responses are not beneficial and can lead to
the advancement of ailments like rheumatoid arthritis, osteoarthritis,
ankylosing spondylitis, gout, and ulcerative colitis (Nasef, Mehta, &
Ferguson, 2017). It is reported that 61% population of age 66 to 75 in
Saudi Arabia, 14% of the US adults, and 40% population of age > 65
in the UK have arthritis (Al Rashoud, Abboud, Wang, & Wigderowitz,
2
014). It is also reported that by the year 2030, an estimated 25% of
the adult population in the United States will be afflicted with
Drug Dev Res. 2020;1–12.
wileyonlinelibrary.com/journal/ddr
© 2020 Wiley Periodicals, Inc.
1

2
KHAN ET AL.
Gupta, & Kumar, 2013; Martina, Vesta, & Ripley, 2005) for the treat-
ment of inflammatory diseases. However, some specific COX-2 inhibi-
tors also displayed cardiac toxicity (Burnier, 2005; Fanelli, Ghisi,
Aprile, & Lapi, 2017). Accordingly, current research has been concen-
trated on the progress of novel anti-inflammatory candidates with tiny
or not at all adverse effects as an alternative to NSAIDs (Carullo,
Galligano, & Aiello, 2016). Recently, pyridazine derivatives have
emerged as a novel prototype for developing anti-inflammatory
agents (Ahmed, Kassab, El-Malah, & Hassan, 2019; Bingham et al.,
respectively. The intermediate compound 2, 6, 7, 8, 9, 14, 15, and 16
were prepared by similar methods provided in the prior publications
(Hamdy, Mustafa, & El-Feky, 2015; Lv et al., 2010; Sarkis, Tran, Lang,
Garbay, & Braud, 2014; Shakeel et al., 2019). The novelty of the
compounds 3, 4a–b, 5a–b, 10–13, and 17–19 was established by car-
rying out the exact structure search in the Scifinder database. The
chemicals, reagents, and drugs were procured from Sigma, Merck, and
Spectrochem. The synthesis of the novel compounds 3, 4a, 4b, 5a, 5b,
10–13, and 17–19 is depicted in Schemes 1, 2, and 3.
2
005; Khalil, Ahmed, Mohamed, Nissan, & Zaitone, 2014; Saeed,
Khalil, Ahmed, & Eissa, 2012; Singh, Sharma, & Bansal, 2017). Some
good pyridazine derivatives have been stated as effective COX-2
inhibitors, for example, Zomipirac was derivatized to a pyridazine
derivative as an effective inhibitor of COX-2 (selectivity index >1,500)
2.1.2
|
Synthesis of 4-(6-Oxo-3-phenylpyridazin-1
[6H]-yl)benzaldehyde (3)
(
Fabiola, Damodharan, Pattabhi, & Nagarajan, 2001; Luong et al.,
996). Centered on the literature and in the persistence of our work
A mixture of 2 (10 mmoles), p-fluorobenzaldehyde (10 mmoles),
ꢀ
1
anhydrous K
2
CO
3
(10 mmoles) in DMSO (40 ml) was heated to 100 C
on anti-inflammatory agents (El-Feky et al., 2015; El-Feky, Hamdy, &
Mustafa, 2014; El-Feky, Imran, & Nayeem, 2017; Imran & Khan,
for 5 hr, and discharged in the crushed ice. The filtered solid was
ꢀ
recrystallized from ethanol. Yield (65%); m.p: 187–189 C; IR: 1,698,
1
2
004), we decided to design and prepare novel non-ulcerogenic
1,670, 1,595; H─NMR: 7.14 (d, 1H, Ar─H), 7.43 (brs, 3H, Ar─H),
pyridazine derivative as COX-2 inhibitors.
7.88 (m, 3H, Ar─H), 7.99 (brs, 3H, Ar─H), 8.12 (d, 1H, Ar─H), 10.01 (s,
13
1
H, ─CHO); C─NMR: 126.50 (2C), 126.59, 128.13, 128.61, 129.45
(
2C), 130.26, 131.78, 132.37, 134.41, 135.56, 145.22, 146.55,
+
+
2
2
2
|
MATERIALS AND METHODS
| Chemistry
154.17, 159.11, 192.85; MS: 276 (M ), 275 (M −1, 100%); Anal.
Calcd. for C, 73.90; H, 4.38; N, 10.14. Found:
C, 73.68; H, 4.18; N, 10.05.
17 12 2 2
C H N O :
.1
.1.1
|
General
2
.1.3
|
General preparation of 4a, 4b, 5a, and 5b
The spectral data and the physical data of the compounds were
obtained using the techniques described in our previous publications
The mixture of 3 (10 mmoles), thiosemicarbazide (10 mmoles), and
ꢀ
(
El-Feky et al., 2014; El-Feky et al., 2015; El-Feky et al., 2017; Imran &
AcOH (40 ml) was heated to 125 C for 3 hr to acquire a precipitate.
Khan, 2004; Shakeel, Imran, Haq, Alshehri, & Anwer, 2019). The
uncorrected melting points, FTIR spectra (KBr), NMR spectra
The filtered precipitate was recrystallized from dioxane to get the
compound 4a.
1
13
(
H─NMR and C─NMR), Mass spectra, and the elemental analyses
The compounds 4b, 5a, and 5b were prepared by replacing the
thiosemicarbazide with phenylthiosemicarbazide, phenylhydrazine,
and cyanoacetic acid hydrazide, respectively, in the process of
Section 2.1.3.
were obtained by Gallenkamp apparatus, Shimadzu 440 spectrometer,
Varian Gemini 500 MHz spectrometer, GCMS/QP 1000 Ex mass
spectrometer (70 eV), and the VARIO El Elementer apparatus,
SCHEME 1 Preparation of
compounds 4a–b and 5a–b

KHAN ET AL.
3
1
3
8
1
1
3
6
.25 (s, 1H, azomethine-H), 11.52 (s, 1H, NH); C─NMR: 126.14,
26.54 (2C), 127.89 (2C), 128.57 (2C), 129.43 (2C), 129.65, 129.72,
31.54, 134.61, 142.94, 144.85, 159.08, 160.73, 178.61; MS:
+
+
49 (M ), 348 (M −1, 100%); Anal. Calcd. for C18
15 5
H N OS: C,
1.87; H, 4.33; N, 20.04. Found: C, 61.72; H, 4.20; N, 19.84.
2-(4-(6-Oxo-3-phenylpyridazin-1[6H]-yl)benzylidene)-N-
phenylhydrazine-1-carbothioamide (4b)
ꢀ
Yield (74%); m.p: 247–249 C; IR: 3,179, 1,671, 1,615, 1,190, 1,075;
1
H─NMR: 7.15 (d, 2H, Ar─H), 7.37, 7.46, 7.59 (3brs, 7H, Ar─H), 7.75,
7.88 (2brs, 4H, Ar─H), 8.05 (brs, 3H, Ar─H), 8.23 (s, 1H, azomethine-
13
H), 10.19 and 11.93 (2 s, 2H, 2NH); C─NMR: 125.91 (2C), 126.11
2C), 126.37, 126.50, 128.21, 128.58 (2C), 129.42 (2C), 130.12,
(
131.48, 131.57, 132.33, 134.03, 134.53, 139.44, 142.32, 143.06,
+
1
44.90, 154.26, 159.14, 176.55; MS: 425 (M , 100%); Anal. Calcd. for
24 19 5
C H N OS: C, 67.75; H, 4.50; N, 16.46. Found: C, 67.49; H,
4.26; N, 16.19.
6-Phenyl-2-(4-([2-phenylhydrazineylidene]methyl)phenyl)pyridazin-3
(
2H)-one (5a)
Yield (82%); m.p: 240–241 C; IR: 3,215, 1,660, 1,589; H─NMR: 6.78
d, 1H, Ar─H), 7.13 (t, 2H, Ar─H), 7.20 (d, 1H, Ar─H), 7.25 (t, 2H,
Ar─H), 7.49 (t, 3H, Ar─H), 7.72 (d, 2H, Ar─H), 8.78 (d, 2H, Ar─H),
ꢀ
1
(
7.94 (brs, 3H, 2 Ar─H + azomethine-H), 8.10 (d, 1H, Ar─H), 10.47 (s,
13
1
H, NH); C─NMR: 112.61, 119.46, 126.04 (2C), 126.20, 126.52
(
2C), 128.54, 129.41, 129.61, 130.04, 131.39, 131.52, 132.35,
SCHEME 2 Preparation of compounds 10–13
1
1
7
34.70, 135.86, 135.97, 136.05, 141.39, 144.69, 145.61, 159.01,
+
59.07; MS: 366 (M ), 255 (100%); Anal. Calcd. for C23
18 4
H N O: C,
5.39; H, 4.95; N, 15.29. Found: C, 75.20; H, 4.76; N, 15.10.
0
2
-Cyano-N -(4-(6-oxo-3-phenylpyridazin-1[6H]-yl)benzylidene)
acetohydrazide (5b)
ꢀ
Yield (68%); m.p: 251–253 C; IR: 3,245, 3,077, 2,959, 2,206, 1,673,
1
1
,660, 1,610; H─NMR: 3.95 (s, 2H, CH
2
), 7.25 (d, 1H, Ar─H), 7.49
(
brs, 3H, Ar─H), 7.91, (t, 4H, Ar─H), 8.10 (m, 3H, Ar─H), 8.20 (s, 1H,
13
azomethine-H), 10.75 (s, 1H, NH); C─NMR: 27.68, 114.63, 126.39,
1
1
3
1
26.56, 128.37, 128.59, 128.76, 129.19, 129.43 (2C), 129.59, 130.16,
31.63, 134.55, 135.51, 144.88, 154.56, 157.36, 159.06, 172.53; MS:
+
57 (M , 100%); Anal. Calcd. for C20
H N
15 5
O
2
: C, 67.22; H, 4.23; N,
9.60. Found: C, 67.05; H, 4.10; N, 19.45.
2
.1.4
|
General preparation of 10, 11, 12, and 13
The mixture of 3 (10 mmoles), appropriate intermediate 6, 7, 8, or
(10 mmoles) and anhydrous sodium acetate (20 mmoles) in AcOH
9
ꢀ
SCHEME 3 Preparation of compounds 17–19
(40 ml) was heated at 125 C for 5–7 hr to acquire a precipitate. The
filtered solid was recrystallized from AcOH.
2-(4-(6-Oxo-3-phenylpyridazin-1[6H]-yl)benzylidene)hydrazine-
1-carbothioamide (4a)
2-Amino-5-(4-(6-oxo-3-phenylpyridazin-1[6H]-yl)benzylidene)
ꢀ
Yield (73%); m.p: 235–237 C; IR: 3,389, 3,262, 3,170, 1,668, 1,595,
thiazol-4(5H)-one (10)
1
ꢀ
1
,240, 1,093; H─NMR: 7.18 (d, 1H, Ar─H), 7.49 (brs, 3H, Ar─H),
Yield (78%); m.p: 288–289 C; IR: 3,360, 3,242, 3,047, 1,714, 1,670,
1
7
.74 (d, 2H, Ar─H), 7.94 (m, 4H, Ar─H), 8.12 (m, 3H, Ar─H + NH
2
),
1,601; H─NMR: 7.17 (d, 1H, Ar─H), 7.49 (m, 3H, Ar─H), 7.64 (s, 1H,

4
KHAN ET AL.
benzylidene-H), 7.72 (t, 2H, Ar─H), 7.88 (m, 2H, Ar─H), 7.94 (t, 2H,
2-(4-Oxo-5-(4-(6-oxo-3-phenylpyridazin-1[6H]-yl)benzylidene)-
13
Ar─H), 8.04 (d, 1H, Ar─H), 9.28 and 9.50 (2s, 2H, 2NH); C─NMR:
26.27, 126.35 (2C), 126.55, 127.69 (2C), 128.06, 128.38, 129.43
4,5-dihydrothiazol-2-yl)acetonitrile (17)
ꢀ
1
Yield (81%); m.p: 257–259 C; IR: 3,062, 2,942, 2,180, 1,720, 1,665,
1
(
2C), 130.14, 131.63, 134.57, 143.33, 144.59, 153.93, 156.81,
1,615; H─NMR: 3.95 (s, 2H, CH
2
), 7.16 (d, 1H, Ar─H), 7.46 (brs, 5H,
+
1
57.10, 159.07, 172.72; MS: 374 (M ), 255 (100%); Anal. Calcd. for
Ar─H), 7.64 (s, 1H, Ar─H), 7.82 (brs, 3H, Ar─H), 7.93 (s, 1H,
13
C
H N
20 14 4
O
2
S: C, 64.16; H, 3.77; N, 14.96. Found: C, 63.94; H,
benzylidene-H), 8.25 (d, 1H, Ar─H);
C─NMR: 56.51, 116.78,
3
.51; N, 14.73.
125.33, 126.09, 126.66 (2C), 128.71 (2C), 128.97 (2C), 129.47,
129.70, 130.18, 131.78, 132.41, 134.85, 143.47, 145.09, 155.50,
+
5-(4-(6-Oxo-3-phenylpyridazin-1[6H]-yl)benzylidene)-3-phenyl-
159.24, 160.03, 168.52; MS: 398 (M ), 255 (100%); Anal. Calcd. for
2-thioxothiazolidin-4-one (11)
22 14 4 2
C H N O S: C, 66.32; H, 3.54; N, 14.06. Found: C, 66.20; H,
ꢀ
Yield (71%); m.p: 249–251 C; IR: 3,076, 2,969, 1,697, 1,668, 1,611,
3.42; N, 13.88.
1
1
,248, 1,090; H─NMR: 7.24 (d, 2H, Ar─H), 7.37 (brs, 2H, Ar─H),
.43 (brs, 3H, Ar─H), 7.59 (brs, 2H, Ar─H), 7.75 (brs, 2H, Ar─H), 7.82
7
2-(4-Oxo-5-(4-(6-oxo-3-phenylpyridazin-1[6H]-yl)benzylidene)-
(
brs, 2H, Ar─H), 8.05 (brs, 3H, Ar─H), 8.19 (s, 1H, benzylidene-H);
4,5-dihydrothiazol-2-yl)acetamide (18)
13
ꢀ
C─NMR: 126.52 (3C), 126.59 (3C), 128.13 (2C), 128.61, 129.45
Yield (69%); m.p: 274–275 C; IR: 3,410, 3,323, 2,954, 1,714, 1,672,
1
(
3C), 130.13 (2C), 130.35 (2C), 131.62, 132.43, 134.41, 135.56,
1,605; H─NMR: 5.23 (s, 2H, NH
2
), 5.78 (s, 1H, methylidene-H), 6.89
+
1
1
8
45.23, 146.58, 156.31, 161.13, 168.23, 193.59; MS: 467 (M ),
(d, 1H, Ar─H), 7.51 (t, 3H, Ar─H), 7.87 (brs, 4H, Ar─H), 8.09 (d, 2H,
Ar─H), 8.22 (d, 1H, Ar─H), 8.39 (s, 1H, benzylidene-H), 10.75 (s, 1H,
90 (100%); Anal. Calcd. for C26
17 3 2 2
H N O S : C, 66.79; H, 3.66; N,
13
.99. Found: C, 66.60; H, 3.70; N, 8.72.
NH); C─NMR: 66.15, 126.50, 126.59 (2C), 128.13 (2C), 128.61
(
2C), 129.45, 130.26, 130.35 (2C), 131.78, 132.37, 134.41, 140.56,
+
2-(2-Cyclohexylidenehydrazineyl)-5-(4-(6-oxo-3-phenylpyridazin-1
154.47, 154.87, 158.68, 159.11, 169.25, 175.16; MS: 416 (M ),
[
6H]-yl)benzylidene)-thiazol-4(5H)-one (12)
16 4 3
255 (100%); Anal. Calcd. for C22H N O S: C, 63.45; H, 3.87; N,
ꢀ
Yield (68%); m.p: 262–264 C; IR: 3,178, 3,072, 2,978, 1,705, 1,671,
13.45. Found: C, 63.30; H, 3.90; N, 13.23.
1
1
,605; H─NMR: 1.52–1.64 (bs, 4H, cyclohexanone), 1.76 (bs, 2H,
cyclohexanone), 2.44–2.47 (m, 2H, cyclohexanone), 2.61 (bs, 2H,
cyclohexanone), 7.09 (brs, 2H, Ar─H), 7.43 (m, 4H, Ar─H), 7.50 (s, 1H,
Ar─H), 7.72 (brs, 1H, Ar─H), 7.88 (m, 2H, Ar─H), 8.20 (s, 1H, Ar─H),
Ethyl 2-(4-oxo-5-(4-(6-oxo-3-phenylpyridazin-1[6H]-yl)benzylidene)-
4,5-dihydrothiazol-2-yl)acetate (19)
ꢀ
Yield (81%); m.p: 265–266 C; IR: 3,056, 2,968, 1,705, 1,678, 1,612;
1
3
1
8
2
1
1
.33 (s, 1H, benzylidene-H), 8.85 (s, 1H, NH); C─NMR: 22.46, 23.0,
3.73, 28.67, 30.18, 125.20, 125.83, 126.41 (2C), 126.49 (2C),
27.31, 128.62, 129.41, 129.97 (3C), 131.33, 131.47, 134.63, 141.84,
52.32, 154.81, 157.10, 159.20, 162.90; Anal. Calcd. for
3 2
H─NMR: 1.20 (t, 3H, CH ), 4.11 (q, 2H, CH ), 5.64 (s, 1H,
methylidene-H), 7.18 (d, 1H, Ar─H), 7.55 (brs, 2H, Ar─H), 7.77 (brs,
2H, Ar─H), 8.98 (m, 4H, Ar─H), 8.12 (m, 3H, 2 Ar─H + benzylidene-
13
H), 10.47 (hump, 1H, NH); C─NMR: 14.64, 61.56, 98.87, 126.19,
126.65 (2C), 128.93 (2C), 129.47, 130.19, 130.98, 131.14 (2C),
131.78, 133.39, 134.45, 134.58, 135.19, 145.10, 157.72, 159.13,
C
H N
26 23 5
O
2
S: C, 66.51; H, 4.94; N, 14.91. Found: C, 66.38; H,
4
.80; N, 14.73.
+
+
1
60.95, 168.65, 176.31; MS: 445 (M ), 444 (M −1; 100%); Anal.
Calcd. for C24 S: C, 64.71; H, 4.30; N, 9.43. Found: C,
64.54; H, 4.10; N, 9.50.
5-(4-(6-Oxo-3-phenylpyridazin-1[6H]-yl)benzylidene)-
19 3 4
H N O
2-(2-[2-oxoindolin-3-ylidene]-hydrazineyl)thiazol-4(5H)-one (13)
ꢀ
Yield (66%); m.p: 270–272 C; IR: 3,178, 3,058, 2,965, 1,795, 1,668,
1
1
,600; H─NMR: 6.88 (d, 2H, Ar─H), 7.20 (d, 2H, Ar─H), 7.56 (brs,
4
H, Ar─H), 7.85 (m, 5H, Ar─H), 8.24 (d, 2H, Ar─H), 8.60 (s, 1H,
2.2
|
Biological activity
13
benzylidene-H), 10.75 and 11.17 (2s, 2H, 2NH); C─NMR: 121.93,
22.12, 126.12 (2C), 126.66 (3C), 128.71 (2C), 129.04 (2C), 129.48
2C), 130.19, 131.78, 132.30, 132.42, 134.32, 134.58, 143.50,
1
2.2.1
|
In vitro COX-1 and COX-2 inhibition assay
(
1
5
1
45.11, 155.24, 157.51, 159.13, 159.43, 161.05, 169.02, 173.43; MS:
The compounds were subjected to the COX-1 and COX-2 inhibition
assay by 10-folds dilution technique (1–0.0000000001 μg/ml) using
DMSO (Ahmed et al., 2019; El-Feky et al., 2014; El-Feky et al., 2015).
The assay kits of the human COX-1 and COX-2 were obtained from
Cayman Chemicals (560131, Ann Arbor, MI). The components were
prepared as per the supplier's instructions. Briefly, samples (20 μl), the
COX-1 and COX-2 enzyme (10 μl), and heme (10 μl) were mixed with
the buffer solution (160 μl), which was supplied with the kits. The
+
18 (M ; 100%); Anal. Calcd. for C28
H
18
6
N O
3
S: C, 64.86; H, 3.50; N,
6.21. Found: C, 64.70; H, 3.37; N, 16.10.
2
.1.5
|
General preparation of 17, 18, and 19
The mixture of 3 (10 mmoles), appropriate intermediate 14, 15, or 16
ꢀ
(
10 mmoles) and 2–3 drops of piperidine in EtOH (40 ml) was heated
resultant combination was incubated at 37 C in a water bath for
ꢀ
at 100 C to acquire a precipitate. The filtered precipitate was rec-
rystallized from AcOH.
10 min, and arachidonic acid (10 μl) was added to initiate the COX
reaction. A saturated solution of stannous chloride (30 μl) was added

KHAN ET AL.
5
after 2 min to halt the COX reaction. The resultant mixture was incu-
bated at ambient temperature for 5 min. The PGF2α developed after
COX reaction was measured through ELISA. The samples were shifted
for preprocessing of compounds such as ChemDraw pro to design
compounds and further processed in ChemBio3D Ultra to convert
into 3D conformations. Then all the ligand molecules were converted
from PDB to PDBQTs in PyRX using the Open Babel module to facili-
tate molecular docking. The X-ray crystallographic 3D structure of
Cyclooxygenase-2 enzyme (COX-2) (PDB entry 5KIR) was retrieved
from Protein Data Bank (Jiao et al., 2019; Orlando & Malkowski,
2016; Pérez-Villanueva et al., 2017). The AutoDock tool was used to
prepare COX-2 protein, adding hydrogens and charges for docking
purposes. AutoDock Vina was used to execute molecular docking.
Discovery Studio Visualizer (BIOVIA), Maestro10.5, and PyMOL were
used for interaction analysis, binding conformation, and making
diagrams.
ꢀ
to a 96-well plate and incubated for 18 hr at 25 C. The plate was
washed to get rid of the unbound components. The Ellman's reagent
(
200 μl), containing the acetylcholine substrate, was mixed and further
ꢀ
incubated at 25 C for 1–1.5 hr till the absorbance (410 nm) of B
o
well
was in the range of 0.3 to 0.8 A.U. The plate was read through the
ELISA reader. The IC50 values of the COX-1 and COX-2 were deter-
mined by the regression analysis.
2
.2.2
|
In vitro anti-inflammatory activity
The in vivo activity of 4a, 4b, 5a, and 10 was assessed in Wistar Albino
rats (130–150 g) of either sex by carrageenan-induced edema method
2.4
|
Statistical analysis
(
El-Feky et al., 2014; El-Feky et al., 2015; Imran & Khan, 2004; Win-
ter, Risley, & Nuss, 1962). The animals were approved and kept as per
the CPCSEA guideline with approval number IAEC/KSOP/E/18/12.
The compounds (10 mg/kg), celecoxib (10 mg/kg), and indomethacin
The statistical investigations (N = 3, Mean ± SD, p-value) were per-
formed by SPSS software (version 20). The standard p-value (< .05)
reflected statistically significant results and is stated at the applicable
places of the manuscript.
(
10 mg/kg), were administered orally as 10% solution in Tween-80.
The control group was administered 1 ml of saline solution. After 1 hr,
a solution of carrageenan (0.1 ml, 1%) was injected in the right hind
paw of the rats in the control, standard, and test groups. The volume
of the rat's paw was determined after the carrageenan injection (0 hr)
and after 1, 2, 3, and 4 hr using a plethysmometer. The percentage of
edema was calculated as follows.
3
|
RESULTS AND DISCUSSION
| Chemistry
3.1
The intermediate compound 2, 6, 7, 8, 9, 14, 15, and 16 were pre-
pared by similar methods provided in the prior publications (Hamdy
et al., 2015; Lv et al., 2010; Sarkis et al., 2014; Shakeel et al., 2019).
pawdiameter after carrageenan−pawdiameter before carrageenan
%edema =
pawdiameter before carrageenan
x 100
The novel aldehyde
3 was prepared by reacting compound
2
with 4-fluorobenzaldehyde. The aldehyde 3 was condensed with
thiosemicarbazide, phenylthiosemicarbazide, phenylhydrazine, and
cyanoacetic acid hydrazide to obtain 4a–b and 5a–b (Scheme 1). The
condensation of 3 with the intermediates 6, 7, 8, and 9 provided
the compounds 10, 11, 12, and 13, respectively (Scheme 2). The con-
densation of 3 with the intermediates 14, 15, and 16 provided the
compounds 17, 18, and 19, respectively (Scheme 3).
2
.2.3
|
Gastric ulcerogenic activity
The compounds 4a, 4b, 5a, and 10 were assessed for their gastric
ulcerogenic consequences. Adult Wistar rats were kept for 18 hr
without any diet. The compounds (10 mg/kg), celecoxib (10 mg/kg),
and indomethacin (10 mg/kg), were given orally as 10% solution in
Tween-80. The control group received 1 ml of saline solution. The rats
were sacrificed after 4 hr, and their stomachs were isolated. A longitu-
dinal cut was made along the greater curvature, and the presence of
ulcers was evaluated. The scoring of the ulcers was done from
The structures of 3, 4a–b, 5a–b, 10–13, and 17–19 were con-
1
firmed based on their spectroscopical data. The H─NMR spectra and
13
the C─NMR spectra of 3, 4a–b, 5a–b, 10–13, and 17–19 displayed
signals for the protons and the carbons, respectively, at the appropri-
ate positions, which were in concurrence with their assigned struc-
tures. The mass spectral data were also in harmony with the allocated
structures. The characteristic spectroscopic peaks for these com-
pounds are summarized in Table 1.
0
(no ulcer) to 5 (≥ 3 ulcers) (Ahmed et al., 2019; El-Feky et al., 2014;
El-Feky et al., 2015).
2
.3
|
Molecular docking
3.2
|
In vitro COX-1 and COX-2 activity
Molecular docking studies of the selected compounds were carried
The derivatives 3, 4a–b, 5a–b, 10–13, and 17–19 were evaluated for
COX-1/COX-2 inhibition assay (Ahmed et al., 2019; El-Feky et al.,
2014; El-Feky et al., 2015) with respect to indomethacin and
®
®
TM
out on the HP computer system with Intel Core i5-4570 CPU @
.20GHz processor, Windows 10 operating system. Tools were used
3

6
KHAN ET AL.
TABLE 1 Summary of the characteristic spectroscopic peaks of 3, 4a–b, 5a–b, 10–13, and 17–19
1
13
H─NMR signals (500 MHz, DMSO-d
δ ppm)
6
;
6
C─NMR signals (125 MHz, DMSO-d ;
IR signals (KBr, cm−
1
)
δ ppm)
Compound
3
1,698 & 1,670 (C═O)
10.01 (─CHO)
159.11 & 192.85 (C═O)
2
3,389, 3,262 & 3,170 (NH & NH); 1,668
4
a
(C═O); 1,240 & 1,093 (C═S)
11.52 (─NH─)
160.73 (C═O); 178.61 (C═S)
3,179 (─NH─); 1,671 (C═O); 1,190 &
1,075 (C═S)
4b
5a
5b
10.19 (─NH─); 11.93 (─NH─)
10.47 (─NH─)
159.15 (C═O); 176.55 (C═S)
159.07 (C═O)
3,215 (─NH─); 1,660 (C═O)
3,245 (─NH─); 2,206 (C≡N); 1,673 &
3.95 (─CH
2
─); 10.75 (─NH─)
2
27.68 (─CH ─); 114.63 (C≡N); 159.06 &
1
,660 (C═O)
3,360 & 3,242 (─NH
C═O)
1,697 & 1,668 (C═O); 1,248 & 1,090
C═S)
,178 (─NH─); 1,705 & 1,671 (C═O)
172.53 (C═O)
1
0
1
2
); 1,714 & 1,670
7.64 (CH─benzylidene); 9.28 (─NH─);
9.50 (─NH─)
143.33 (thiazole-C5), 159.07 & 172.72
(
(C═O)
1
8.19 (benzylidene-H)
161.13 & 168.23 (C═O); 193.59 (C═S)
(
3
1.52–2.61 (10H, cyclohexanone); 8.33
(benzylidene-H); 8.85 (─NH─)
22.46, 23.0, 23.73, 28.67, 30.18
12
13
17
(cyclohexanone'C); 159.20 & 162.90
(
C═O)
3,178 (─NH─); 1,795 & 1,668 (C═O)
2,180 (C≡N); 1,720 & 1,665 (C═O)
8.60 (benzylidene-H), 10.75 (─NH─);
161.05, 169.02 & 173.43 (C═O)
1
1.17 (─NH─)
3.95 (─CH ─)
2
56.51 (─CH
2
─); 116.78 (C≡N); 160.03 &
1
68.52 (C═O)
3
,410 & 3,323 (─NH
(C═O)
2
); 1,714 & 1,672
5.23 (─NH
(─NH─)
2
); 5.78 (methylidene-H); 10.75
66.15 (─CH
(C═O)
2
─); 159.11, 169.25 & 175.16
1
8
9
1
.20 (─CH
3
), 4.11 (─CH
2
─); 5.64
14.64 (─CH
3
); 61.56 (─CH
2
─); 98.87
1
1,705 & 1,678 (C═O)
(methylidene-H); 10.47 (─NH─)
(methine-C); 160.95, 168.65 & 176.31
C═O)
(
TABLE 2 In vitro COX-1 and COX-2 inhibition (N = 3, Mean ± SD)
Compound
3
COX-1 (IC50 in nM)
350 ± 0.15
390 ± 0.10
550 ± 0.10
590 ± 0.10
480 ± 0.15
440 ± 0.16
300 ± 0.16
350 ± 0.15
460 ± 0.14
260 ± 0.32
400 ± 0.38
310 ± 0.52
320 ± 0.01
220 ± 0.01
% COX-1 inhibition
COX-2 (IC50 in nM)
23.14 ± 2.15
17.45 ± 0.10
17.40 ± 0.10
16.76 ± 0.22
20.98 ± 0.16
17.15 ± 0.11
22.0 ± 0.10
% COX-2 inhibition
76.87
SI
% SI
62.85
56.41
40
15.12
22.34
31.60
35.20
22.87
25.65
13.63
16.57
18.4
84.09
124.24
175.75
195.77
127.19
142.65
75.80
92.15
102.33
68.40
82.09
81.31
100
4
4
5
5
1
1
1
1
1
1
1
a
101.94
102.24
106.14
84.79
b
a
37.28
45.83
50
b
0
1
2
3
7
8
9
103.73
78.13
73.33
62.85
47.42
84.61
55
21.12 ± 0.14
25.0 ± 0.12
84.23
71.16
21.13 ± 0.44
27.10 ± 4.13
21.20 ± 0.40
17.79 ± 0.69
67.72 ± 2.62
84.19
12.30
14.76
14.62
17.98
3.24
65.31
70.96
68.75
100
83.91
Celecoxib
100
Indometacin
26.26
18.02
Note: All values had p < .5 (SPSS software); SI: IC50 for COX-1 in nM/IC50 for COX-2 in nm.
celecoxib. The data is provided in Table 2. For comparison, the per-
centage of COX-1 inhibition (IC50) for indomethacin has been consid-
ered as 100%, wherein the percentage of COX-2 inhibition (IC50) and
the % Selectivity Index (% SI) for celecoxib has been taken as 100%.
The IC50 values of celecoxib and indomethacin against COX-1 and
COX-2 were closer to the IC50 values of the earlier report (Ahmed
et al., 2019). All the compounds, including celecoxib (IC50 = 320 nM)
showed higher IC50 values against COX-1, in comparison to indo-
methacin (IC50 = 220 nM) (Figure 1). This observation indicates that
our pyridazine derivatives are less potent inhibitors of COX-1 than
indomethacin. This observation also indicates that our compounds
should have a lesser propensity to produce ulcerogenic effects

KHAN ET AL.
7
(
Gökçe, Utku, & Küpeli, 2009; Singh et al., 2017), wherein the hydra-
zine/hydrazone derivative of pyridazines are mentioned as potent
anti-inflammatory agents. Among the thiazolidinone derivatives the
COX-2 inhibitory potency decreased as 10 (2-Amino group; % COX-2
inhibition = 103.73%) > 12 (2-Cyclohexylidenehydrazineyl group; %
COX-2 inhibition = 84.23%) > 17 (2-Acetonitrile group; % COX-2
inhibition = 84.19%) > 19 (Ethyl 2-acetate group; % COX-2 inhibition =
8
3.91%) > 11 (3-phenyl-2-thioxothiazolidin-4-one;
inhibition = 78.13%) > 13 (2-isatinylhydrazine; % COX-2 inhibition =
1.16%) > 18 (2-acetamide; % COX-2 inhibition = 65.31%). This
%
COX-2
7
observation revealed that the substituent at C-2 of the thiazolidinone
ring plays an important role in the COX-2 inhibitory activity. The pres-
2
ence of a free -NH group at C-2 of the thiazolidinone ring produces
potent COX-2 inhibitors. The thiazolidinone ring bearing pyridazine
derivatives are novel as per the Scifinder database search. Hence, the
earlier reports (Gökçe et al., 2009; Singh et al., 2017) are silent about
the structure–activity relationship studies of the thiazolidinone ring
bearing pyridazine derivatives. However, the compounds having a
FIGURE 1 The IC50 of 3, 4a–b, 5a-b, 10–13, 17–19, celecoxib,
and indomethacin
(
Korbecki et al., 2014; Tortorella et al., 2016). All the compounds were
2
free ─NH group at the C-2 of the thiazolidinone ring are reported
better inhibitors of COX-2 than COX-1. This observation strengthens
our assumption that our compounds will produce lesser ulcerogenic
effects (Korbecki et al., 2014; Tortorella et al., 2016). However,
compound 17 displayed equal inhibition of COX-1 and COX-2. The
compounds 4a (IC50 = 17.45 nm, 101.94%), 4b (IC50 = 17.40 nm,
as potent COX-2 inhibitors (Liaras, Fesatidou, & Geronikaki, 2018).
The presence of some groups like 2-cyclohexylidenehydrazineyl,
2-acetonitrile, ethyl 2-acetate, 2-isatinylhydrazine, and 2-acetamide at
the C-2 of the thiazolidinone ring reduces the COX-2 inhibitory activ-
ity. These observations were also in concurrence with the earlier
reports (Gökçe et al., 2009; Liaras et al., 2018; Singh et al., 2017).
1
1
02.24%), 5a (IC50 = 16.76 nm, 106.14%), and 10 (IC50 = 17.15 nm,
03.73%) displayed better COX-2 inhibitory activity than celecoxib
(
IC50 = 17.79 nm, 100%). The selectivity index of the compounds 4a
SI = 22.34, 124.24%), 4b (SI = 31.60, 175.75%), 5a (SI = 35.20,
(
3.3
|
Iv vivo anti-inflammatory activity
1
95.77%), and 10 (SI = 25.65, 142.65%) were also better than the
celecoxib (SI = 17.98, 100%). The compounds 5b (% SI = 127.19%)
and 13 (% SI = 102.33%) also showed better % SI than celecoxib (%
SI = 100%). However, the % COX-2 inhibitory effect of 5b (84.79%)
and 13 (71.16%) was less than celecoxib (100%). Therefore, they were
not selected for the in vivo anti-inflammatory activity.
The derivatives 4a, 4b, 5a, and 10 were selected for the in vivo anti-
inflammatory action (El-Feky et al., 2014; El-Feky et al., 2015;
Imran & Khan, 2004; Winter et al., 1962). The results are provided in
Table 3 and are also graphically represented in Figure 2. It is evident
from the data of Table 3 and Figure 2 that the compounds 4a, 4b,
5a, and 10 have slightly more potent, superior, and consistent activ-
ity than celecoxib and indomethacin. The onset and the duration of
action of these compounds were also comparable to celecoxib
and indomethacin (Figure 2). Accordingly, it may be concluded
For the structure–activity analysis, the N-substitutedphenyl-
6
-oxo-3-phenylpyridazines can be divided into two categories.
The hydrazine derivatives consisting of compounds 4a, 4b, 5a, and 5b.
The thiazolidinone derivatives 10–13, and 17–19. Based on the
structure–activity analysis, it can be assumed that hydrazine deriva-
tives (4a, 4b, 5a, and 5b) were potent inhibitors of the COX-2 than
the thiazolidinone derivatives (10–13 and 17–19). Among the
hydrazine derivatives the COX-2 inhibitory potency decreased as
that N-substitutedphenyl-6-oxo-3-phenylpyridazines having
a
hydrazine group, phenylhydrazine group, and 2-aminothiazolidinone
ring system are efficient lead compounds to develop better COX-2
inhibitors and anti-inflammatory agents (Gökçe et al., 2009; Singh
et al., 2017).
5
a (2-phenylhydrazine group; % COX-2 inhibition = 106.14%) > 4b
N-phenylhydrazine-1-carbothioamide group; % COX-2 inhibition =
02.24%) > 4a (hydrazine-1-carbothioamide group; % COX-2 inhibi-
tion = 101.94%) > celecoxib (% COX-2 inhibition = 100%) > 5b
2-Cyano-N-acetohydrazide group; % COX-2 inhibition = 84.79%).
(
1
3.4
|
Ulcerogenic activity
(
This observation revealed that the presence of hydrazine or
phenylhydrazine group in these compounds produce potent COX-2
inhibitors. However, the introduction of the acetohydrazide group
and/or the ─CN group decrease the COX-2 inhibitory activity.
These results were consistent with the earlier reports on the
structure–activity relationship studies for the pyridazinone derivatives
Gastric ulcers and gastrointestinal erosion are the two most promi-
nent adverse events of the NSAIDs. Accordingly, the compounds 4a,
4b, 5a, and 10 were assessed for the gastric ulcerogenic effects
(Ahmed et al., 2019; El-Feky et al., 2014; El-Feky et al., 2015) in com-
parison to celecoxib and indomethacin. The data of the gastric ulcero-
genic effects are provided in Table 4.

8
KHAN ET AL.
TABLE 3 In vivo activity of compounds 4a, 4b, 5a, and 10
0
hr
1 hr
2 hr
3 hr
4 hr
Compound
Control
PD
PD
% edema
31.88
11.17
11.20
11.33
10.79
11.14
10.82
PD
% edema
37.10
9.45
PD
% edema
39.13
7.73
PD
% edema
42.02
6.87
3.45 ± 0.16
3.49 ± 0.22
3.48 ± 0.14
3.53 ± 0.26
3.52 ± 0.03
3.50 ± 0.18
3.51 ± 0.50
4.55 ± 0.20
3.88 ± 0.18
3.87 ± 0.10
3.93 ± 0.16
3.90 ± 0.08
3.89 ± 0.42
3.89 ± 0.18
4.73 ± 0.10
3.82 ± 0.30
3.80 ± 0.22
3.87 ± 0.40
3.85 ± 0.30
3.85 ± 0.10
3.86 ± 0.20
4.80 ± 0.40
3.76 ± 0.64
3.75 ± 0.40
3.83 ± 0.10
3.82 ± 0.20
3.83 ± 0.14
3.84 ± 0.32
4.90 ± 0.22
3.73 ± 0.14
3.70 ± 0.10
3.81 ± 0.50
3.79 ± 0.44
3.81 ± 0.20
3.80 ± 0.14
4
4
5
1
a
b
a
0
9.19
7.75
6.32
9.63
8.49
7.93
a
9.37
8.52
7.67
Celecoxib
10
9.42
8.85
Indomethacin
9.97
9.4
8.26
Abbreviation: PD, paw diameter (mm).
a
All values had p < .5 (SPSS software).
TABLE 5 Binding affinities (docking scores) of celecoxib, 4a, 4b,
a, and 10
5
Binding
affinity
Hydrogen bonding
interacting
Compound (kcal/Mol) residues
Celecoxib −9.4 ARG 120, MET
22,
Interacting groups
─NH of sulphonamide
and ─F of ─CF
3
5
group
4
a
−9.8
SER 353, GLY 354 Two hydrogens of the
NH group
SER 353, PRO 514, ─NH of the Ph─NH─,
─
2
4
b
−10.3
ASP 515,
HIS 90,
sulfur of the C═S,
nitrogen of the
─N═CH─Ph, and
C═O of the
TYR 355
pyridazinone ring
5
1
a
−10.5
−9.9
HIS 90, SER 353,
TYR 355
The nitrogen of the
─N═CH─Ph and the
C═O of the
FIGURE 2 The comparative illustration of the in vivo activity of
a, 4b, 5a, 10, celecoxib and indomethacin
4
pyridazinone ring
TABLE 4 The gastric ulcerative effect of 4a, 4b, 5a, and 10
0
THR 212, ASN
Two hydrogens of the
3
4
82, GLN
2
─NH , and nitrogen
of the thiazolidinone
ring
Score
54, HIS 386
a
a
Group
Number of gastric ulcers
Severity lesions
Control
0
0
4
4
5
1
a
b
a
0
0.13 ± 0.1
0.16 ± 0.10
activity (Table 4) was in concurrence with the data of Table 2 because
the ulcerogenic effects are produced by the inhibition of the COX-1
enzyme (Korbecki et al., 2014; Tortorella et al., 2016). Another possi-
bility of the non-ulcerogenic effect of the 4a, 4b, 5a, and 10 is the lack
of ─COOH group in their structure, which also contributes to the
ulcerogenic effects of the NSAIDs (Sehajpal et al., 2018).
0
0
0
0
0
0
Celecoxib
2.55 ± 0.22
8.35 ± 0.41
6.0 ± 0.10
12.39 ± 0.69
Indomethacin
a
All values had p < .5, Mean ± SE, SPSS software.
3
.5
|
Molecular modeling
The results of the gastric ulcerogenic effects revealed no ulcero-
genic effects of the compounds 4b, 5a, and 10. The compound 4a
exhibited negligible ulcerogenic effect, which was far less than the gas-
tric ulcerogenic effects produced by celecoxib, and indomethacin. The
To validate the data obtained by the in vitro COX-2 assay, molecular
docking studies of 4a, 4b, 5a, and 10 were performed in comparison
to celecoxib using COX-2 protein (PDB entry 5KIR) (Jiao et al., 2019;
Orlando & Malkowski, 2016; Pérez-Villanueva et al., 2017). The data
of the molecular docking studies are provided in Table 5.
4
a, 4b, 5a, and 10 are more potent inhibitors of COX-2 than COX-1,
which is evident from the data of Table 2. The data of the ulcerogenic

KHAN ET AL.
9
The celecoxib showed hydrogen bond interactions (green color) of
the ─NH with MET 522 and ─F atom with ARG 120; Pi-sulfur interac-
tion (yellow color) of the ─SO ─ group with TRP 387, and pyrazole ring
Mol), 4b (−10.3 kcal/Mol), 5a (−10.5 kcal/Mol; (Figure 4), and 10
(−9.9 kcal/Mol) had better binding affinity for the COX-2 enzyme than
celecoxib (−9.4 kcal/Mol) (Table 5). The better binding affinity of these
compounds resulted in the better COX-2 inhibitory potential of 4a
(IC50 = 17.45 nm), 4b (IC50 = 17.40 nm), 5a (IC50 = 16.76 nm), and 10
(IC50 = 17.15 nm) than celecoxib (IC50 = 17.79 nm). Table 5 also pro-
vides information about the hydrogen bond interactions of celecoxib,
4a, 4b, 5a, and 10 with the COX-2 protein. It can be observed that
compounds 4a, 4b, 5a, and 10 had similar or more number of H-bond
interactions with various amino acids of the COX-2 protein. We believe
2
with ARG 120; Pi-alkyl interaction (pink color) of the N1-phenyl ring
with ALA 527 and VAL 349, N-2 of pyrazole with VAL 349, carbon of
3 3
─CF with LEU 359, 5-phenyl group with VAL 523, and the ─CH
group with PHE 518; and the Pi-Pi stacked interaction (magenta color)
of the 5-phenyl group with LEU 352 (Figure 3). A similar interaction
pattern for celecoxib has been reported in the literature (Jiao et al.,
2
019; Pérez-Villanueva et al., 2017). It is apparent that 4a (−9.8 kcal/
FIGURE 3 Molecular docking studies of celecoxib with COX-2 protein (2D/3D plot)
FIGURE 4 Molecular docking studies of compound 5a with COX-2 protein (2D/3D plot)

1
0
KHAN ET AL.
FIGURE 5 Overlay of the compounds 4a (red), 4b (blue), 5a (yellow), and 10 (green) over celecoxib (violet)
that these H-bond interactions participate in the COX-2 inhibition, and
ultimately better anti-inflammatory actions of 4a, 4b, 5a, and 10. The
overlay of the docked celecoxib, 4a, 4b, and 5a exhibited super-
imposability with each other (Figure 5).
COX-2 enzyme is implicated in the advancement of ailments like ath-
erosclerosis, pancreatitis, hepatitis, asthma, cancer, kidney injury, irri-
table bowel diseases, epilepsy, depression, Alzheimer's disease,
rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gout, and
ulcerative colitis. The compounds 4a, 4b, 5a, and 10 can be beneficial
to treat diseases associated with the higher activity of the COX-2.
However, further toxicity studies are recommended for 4a, 4b, 5a,
and 10. The structure–activity analysis revealed that the hydrazine
derivatives, 4a, 4b, 5a, and 5b, were potent inhibitors of the COX-2
than the thiazolidinone derivatives (11–13 and 17–18). Accordingly,
the development of more hydrazine derivatives of N-sub-
stitutedphenyl-6-oxo-3-phenylpyridazines is also recommended.
This observation indicates that a common COX-2 inhibitory mech-
anism accounts for the anti-inflammatory activity of 4a, 4b, 5a, and
celecoxib. The compounds 10 was not superimposable over celecoxib
(
Figure 3). This indicates that compound 10 might be exerting its COX-
inhibitory potential by binding at a different site of the COX-2 pro-
2
tein, which is uncommon to celecoxib. A clear picture of this observa-
tion may be obtained by performing a detailed molecular modeling
study of 10 along with reported COX-2 inhibitors/NSAIDs with respect
to already established targets (Ahmed et al., 2019; Bingham et al.,
2
005; Jiao et al., 2019; Khalil et al., 2014; Orlando & Malkowski, 2016;
ACKNOWLEDGMENTS
Pérez-Villanueva et al., 2017; Saeed et al., 2012; Singh et al., 2017).
The authors thank Apeejay Stya University and the Northern Border
University for providing facilities and technical support to perform this
research work.
4
|
CONCLUSION
CONFLICT OF INTEREST
Four compounds, 4a, 4b, 5a, and 10, displayed better inhibition of
COX-2 as well as better selectivity index than celecoxib. The in vivo
anti-inflammatory activity evaluation of these compounds also rev-
ealed that 4a, 4b, 5a, and 10 had an almost similar onset of action and
potency concerning celecoxib. It is worthful to mention that 4a, 4b,
The authors declare no potential conflict of interest.
ORCID
Abida Khan
https://orcid.org/0000-0001-6186-461X
5
a, and 10 were free of any ulcerogenic effect in comparison to
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