Design, Synthesis and Biological Evaluation of 5-amino-3-aryl-1-(6′-chloropyridazin-3′-yl)pyrazoles and their Derivatives as Analgesic Agents

Article abstract of DOI:10.1055/a-1202-9959

An efficient and environmental benign solvent-free synthesis of 5-amino-3-aryl-1-(6 ^'-chloropyridazin-3'-yl)pyrazoles (4a-e) was accomplished by grinding 3-chloro-6-hydrazinopyridazine (2) and β-ketonitriles (3a-e) in the presence of p -toulenesulfonic acid as a catalyst. Subsequently, 6'-chloro group in 4a-e was replaced with cyclic 2° amine derivatives viz. pyrrolidine 5a, piperidine 5b and morpholine 5c to obtain 6a-e, 7a-e, 8a-e respectively. The newly synthesized compounds were characterized by using IR, NMR (¹H and ¹³C), mass spectral studies, elemental analyses. All the synthesized compounds were studied for their docking interaction with target protein 6COX and screened for their in vivo analgesic mode of action against swiss albino mice (animal model) using acetic-acid induced writhing test. Consequently, docking simulations data justifies the potential of synthesized series as an analgesic and very well correlated with in vivo study. Preliminary results revealed that most of the synthesized compounds exhibited moderate to good analgesic activity as compared to reference/standard drug (s) sodium diclofenac and candidates 4d and 7c protrude out as a promising lead for further investigation.

Full text of DOI:10.1055/a-1202-9959

Published online: 2020-07-24
Thieme
Original Article
Design, Synthesis and Biological Evaluation of 5-amino-3-aryl-1-
(6'-chloropyridazin-3'-yl)pyrazoles and their Derivatives as
Analgesic Agents
Authors
Swati1, Ranjana Aggarwal1, 2, Pawan Kaushik3, Ajay Kumar3, Deepika Saini3
Affiliations
Supporting Information for this article is available
1
2
3
Department of Chemistry, Kurukshetra University,
online https://doi.org/10.1055/a-1202-9959
Kurukshetra, India
Department of Scientific and Industrial Research
NISTADS, New Delhi, India
AbStRAct
An efficient and environmental benign solvent-free synthesis
of 5-amino-3-aryl-1-(6'-chloropyridazin-3'-yl)pyrazoles (4a-e)
was accomplished by grinding 3-chloro-6-hydrazinopyridazine
(2) and β-ketonitriles (3a-e) in the presence of p-toulenesul-
fonic acid as a catalyst. Subsequently, 6'-chloro group in 4a-e
was replaced with cyclic 2 ° amine derivatives viz. pyrrolidine
5a, piperidine 5b and morpholine 5c to obtain 6a-e, 7a-e,
8a-e respectively. The newly synthesized compounds were
characterized by using IR, NMR (1H and 13C), mass spectral
studies, elemental analyses. All the synthesized compounds
were studied for their docking interaction with target protein
6COX and screened for their in vivo analgesic mode of action
against swiss albino mice (animal model) using acetic-acid in-
duced writhing test. Consequently, docking simulations data
justifies the potential of synthesized series as an analgesic and
very well correlated with in vivo study. Preliminary results re-
vealed that most of the synthesized compounds exhibited
moderate to good analgesic activity as compared to reference/
standard drug (s) sodium diclofenac and candidates 4d and 7c
protrude out as a promising lead for further investigation.
Department of Pharmaceutical Sciences, Kurukshetra
University, Kurukshetra, India
Key words
analgesic drugs, anti-inflammatory drugs, biopharmaceuti-
cals, drug research
received
accepted
24.03.2020
18.05.2020
Bibliography
DOI https://doi.org/10.1055/a-1202-9959
Published online: 2020
Drug Res
© Georg Thieme Verlag KG Stuttgart · New York
ISSN 2194-9379
Correspondence
Ranjana Aggarwal
National Institute of Science Technology and Development
Studies
110012 New Delhi
India
Tel.: (+91-11) 25846064, 25843227
director@nistads.res.in
Introduction
analgesic action by inhibition of the cyclooxygenase enzyme (COX),
known for reducing the level of prostaglandins within the hypotha-
lamic region, e. g. PGE2, which sensitizes nociceptors at nerve fibre
terminals [5]. Moreover, 5-lipoxygenase (5-LOX) catalyses the con-
version of arachidonic acid to leukotriene B4 which contributes to
the hyperalgesia seen during inflammation [6, 7]. Neutrophils are
the first responders of inflammatory cells which migrate to the site
of inflammation following chemical signal, leukotriene, during the
beginning phase of inflammation. For these reasons, compounds
that achieve the dual inhibition of enzymes COX and 5-LOX become
an important therapeutic strategy in the combat of the pain toler-
able trauma.
Pain is an unpleasant sensory and emotional event that works as a
piece of physiological advice to convey discomfort signals to the
brain by sensory neurons and thereby alerting the body for poten-
tially tissue-damaging situations which are triggered by aseptic
stimuli [1, 2]. Body inflammation is a key element to pain induction
which is an outcome of tissue damage developed by a sequence of
pain, fever or microbial infection [3]. Several drugs are available in
the market which nullifies and prevent the sensitization of nocice-
ptors, e. g., dipyrone and indomethacin respectively, also, known
as peripheral analgesic agents for their mode of action to regulate
the inflammatory hyperalgesia [4]. Most of the NSAID exert their
Sharma S et al. Analgesics COX-2 Inhibitor Drug Research… Drug Res

Thieme
Original Article
▶Fig. 1 Marketed drugs containing pyridazine ring collaborated with cyclic secondary amines.
Out of the three isomeric forms of diazines: pyridazine, pyrimi-
dine and pyrazine, pyridazine moiety does not show its abundant
existence in any natural product probably due to difficulty in for-
mation of N-N bond by living organisms. However, pyridazine nu-
cleus has occupied an influential place in the therapeutic arena be-
cause of its bioavailability to the cell membrane, especially to CNS,
due to its water soluble nature. Pyridazine scaffold participates as
hydrogen bond receptor and has the highest capacity to complex
with targets due to its highest dipole moment 3.9 D, as compared
to pyrimidine 2.42 D and pyrazine 0.6 D [8].
Pyrrolidine, piperidine, morpholine are few cyclic secondary (2°)
amines have received considerable attention in the pharmaceuti-
cal field due to their existence as an essential part in a wide range
of drugs such as procyclidine, bepridil, droperidol, risperidone,
melperone, meperidine, edronax, fenpropimorph, phendimetra-
zine etc. Also, these are present in many natural alkaloids: pyrroli-
dine in nicotine, hygrine, piperidine in solenopsins, piperine (black
pepper), anabasine, lobeline, coniline and morpholine in polyg-
onapholine, chelonine, acortatarins etc.
Evidence for the potent activity of pyridazine entity endowed
with cyclic 2 ° amines in biological system has been highlighted by
its remarkable presence in a number of pharmaceutical drugs such
as minaprine (brantur), emorfazone (pentoil or nandron), carbaz-
eran, endralazine, irdabisant and AG-246 etc (▶Fig. 1). Consider-
ing our matter of interest, pyridazine moiety has emerged as an
important target in pain therapy aiming at improved nonsteroidal
analgesics which are effective pain killers without any gastric and
renal ulcerations. Emorphazone is one of the best drug known
which shows better gastric profile, marketed in Japan as an analge-
sic and anti-inflammatory drug [9, 10] and AG 246 is also known
for its good analgesic action [11] etc.
The medicinal value of 5-aminopyrazoles is widespread and
achieved a remarkable position in the biological system as antimi-
crobial, anticancer, antimalarial, antiprotozoal, anti-HIV agents and
receptor antagonist [12–17] etc. This privileged scaffold has fasci-
nated the attention of most of the researchers in the field of me-
dicinally active candidates. The rapid growth of 5-aminopyrazole
motif has been witnessed in a wide range of compounds. Fipronil,
a member of 5-aminopyrazole family, is the most effective pesti-
cide as it blocks the GABA-gated, glutamate-gate chloride chan-
nels and disrupts the central nervous system of insects. Interest-
ingly, ethyl 5-amino-1-(2-(6-methyl-1-phenyl-1H-pyrazolo[3,4-d]
pyrimidin-4-yloxy)acetyl)-1H-pyrazole-4-carboxylate [18] and
5-amino-1-(2-fluorophenyl)-N-(5-(isoxazol-3-ylcarbamoyl)-
2-methylphenyl-1H-pyrazole-4-carboxamide [19] are reported in
the literature as an anti-inflammatory agent.
Aim: Designing of Target molecules
The above-mentioned applications envisaged us to combine these
three active pharmacophores (pyridazine, cyclic 2 ° amines and
5-aminopyrazole) to generate a small library of novel compounds
which are supposed to exhibit interesting biological properties in
animal models. In literature, 5-aryl-1-(4-substitutedpyridazinyl)
pyrazol-3-propanoic acids (A) have been reported to inhibit LTB4
biosynthesis in human neutrophils with IC50 values of 12–14 µM
[20, 21]. Keeping this in view and in continuation of our interest in
5-aminopyrazoles as precursors for the synthesis of heterocyclic
rings and as a promising biological active entity [22–29], target
molecules have been designed by replacing acidic chain of pyra-
zole ring with aryl ring, aryl ring at position-5 of pyrazole ring with
amino group and widening the scope of substituents such as R
group at phenyl ring present at position-3 of pyrazole ring, and sub-
stitution of Cl group by pyrrolidine, piperidine and morpholine of
pyridazine ring (▶Fig. 2).
Materials and Methods
Experimental protocol
Melting points were determined in open capillaries on digital in-
strument Make. Infrared spectra were recorded on a spectropho-
tometer (IR M-500, Buck Scientific Inc, Norwalk, CT) in KBr pellets
(νmax in cm-1). 1H and 13C NMR spectra for the analytical purpose
were recorded in CDCl3 on a Bruker instrument (Billerica, MA) at
400 and 100 MHz respectively. Tetramethylsilane was taken as an
integral standard. Chemical shifts (δ) were measured in ppm. Cou-
pling constants (J) are given in Hertz (Hz). Mass and elemental anal-
yses were performed on Q-TOF micro mass spectrometer and
CHNS-O elemental analyser at Sophisticated Analytical Instrumen-
tation Facility, Panjab University, Chandigarh.
Step-wise solvent-mediated procedures
Synthesis of 1-(6'-Chloropyridazin-3'-yl)-3-phenyl-1H-pyrazole-
5-amine (4a)
Ethanol medium
3-Chloro-6-hydrazinopyridazine (0.144g, 1mmol) was added to a
solution β-ketonitrile (3a) (0.145g, 1mmol) in 15 ml ethanol fol-
lowed by the addition of a catalytic amount of conc. HCl (4–5
Sharma S et al. Analgesics COX-2 Inhibitor Drug Research… Drug Res

▶Fig. 2 Designing of target molecules as analgesic agent w.r.t. model compound A.
1-(6'-Chloropyridazin-3'-yl)-3-(4''-fluorophenyl)-1H-
pyrazol-5-amine (4c), Cas No.: 1154198–00–1
drops) after 5 min. of refluxing the contents. Completion of the re-
action was observed within 30 min. of refluxing. The reaction mix-
ture was concentrated, cooled and left overnight. The solid sepa-
rated was filtered, washed and crystallised with ethanol. The TLC
(ethyl acetate/petroleum ether in 1:4) and 1H NMR spectra showed
the formation of a single product.
Yield 75.0%; Rf =0.66; M.Pt. 205.5°C; IR (KBr, cm-1): 3294 (symm.)
and 3418 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 5.83
(s, 1H, 4-H); 6.01 (s, 2H, -NH2); 7.15 (t, 2H, J = 8.7 Hz, 3'', 5''-H);
7.60 (d, 1H, J = 9.3 Hz, 5'-H); 7.79 (m, 2H, 2'', 6''-H); 8.33 (d, 1H, J=
9.3 Hz, 4'-H); MS: m/z [M + 1] + 290/292 (3:1). Anal. Cal. For
C13H9ClFN5: C, 53.90; H, 3.13; N, 24.17. Found: C, 53.00; H, 2.98;
N, 23.98.
DMF medium
3-Chloro-6-hydrazinopyridazine (0.144g, 1mmol) and β-ketonitrile
(3a) (0.145g, 1mmol) were refluxed in 6 ml DMF followed by the
addition of a catalytic amount of Cs2CO3. Completion of the reac-
tion was observed in 5–10 min. of refluxing and the excess of sol-
vent was distilled off to about 1/3 of the volume i.e 2ml, cooled,
water poured into it, sticky solid separated was filtered, washed
with hot water repeatedly and crystallised with ethanol. The TLC
(ethyl acetate/petroleum ether in 1:4) and 1H NMR spectra showed
the formation of a single product.
3-(4''-Chlorophenyl)-1-(6'-chloropyridazin-3'-yl)-1H-
pyrazole-5-amine (4d), Cas No.: 1154568–20–3
Yield 88.1 %; Rf = 0.67; M.Pt. 203 °C; IR (KBr, cm-1): 3294 (symm.)
and 3418 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 5.92
(s, 1H, 4-H); 6.96 (s, 2H, -NH2); 7.43 (d, 2H, J = 8.4 Hz, 3'',5''-H);
7.84 (d, 2H, J = 8.6 Hz, 2'', 6''-H); 7.97 (d, 1H, J= 9.4 Hz, 5'-H); 8.33
(d, 1H, J = 9.4 Hz, 4'-H); Ms: m/z [M]+ 305/307/309 (9:6:1). Anal.
Cal. For C13H9Cl2N5: C, 51.00; H, 2.96; N, 22.88. Found: C, 49.96;
H, 2.90; N, 22.80.
Similarly, compounds 4b-e were synthesized using the proce-
dure mentioned above.
Percentage yield reported in the experimental protocol is of the
ethanolic medium.
1-(6'-Chloropyridazin-3'-yl)-3-(4''-bromophenyl)-1H-
pyrazol-5-amine (4e), Cas No.: 1154319–76–2
Yield 74.6 %; Rf = 0.66; M.Pt 193.5 °C; IR (KBr, cm-1): 3394 (symm.)
and 3402 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 5.85
(s, 1H, 4-H); 6.01 (s, 2H, -NH2); 7.54 (d, 2H, J = 8.5 Hz, 3'', 5''-H);
7.61 (d, 1H, J = 9.3 Hz, 5'-H); 7.68 (d, 2H, J= 8.6 Hz, 2'', 6''-H); 8.32
(d, 1H, J = 9.4 Hz, 4'-H); Ms: m/z [M]+ 350/352/354 (3:4:1). Anal.
Cal. For C13H9ClBrN5: C, 44.53; H, 2.59; N, 19.98. Found: C, 44.40;
H, 2.50; N, 19.80.
1-(6'-Chloropyridazin-3'-yl)-3-phenyl-1H-pyrazole-5-amine
(4a) [30], Cas No.: 122651–96–1
Yield 62.4 %; Rf = 0.66; M.Pt. 185 °C; IR (KBr, cm-1): 3317 (symm.)
and 3487 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 5.87
(s, 1H, 4-H); 5.99 (s, 2H, -NH2); 7.39 (m, 3H, 3'', 4'', 5''-H); 7.58 (d,
1H, J = 9.4 Hz, 5'-H); 7.82 (m, 2H, 2'', 6''-H); 8.34 (d, 1H, J = 9.4 Hz,
4'-H); Ms: m/z [M + 1]+ 272/274 (3:1). Anal. Cal. for C13H10ClN5: C,
57.47; H, 3.71; N, 25.78. Found: C, 57.00; H, 3.00; N, 25.58.
Step-wise solvent-mediated procedure
1-(6'-Chloropyridazin-3'-yl)-3-p-tolyl-1H-pyrazol-5-amine
(4b), Cas No.: 1155511–89–9
Synthesis of 3-phenyl-1-(6'-(pyrrolidin-1'''-yl)pyridazin-3'-yl)-1H-
pyrazol-5-amine (6a)
Yield 66.9 %; Rf = 0.65; M.Pt. 173 °C; IR (KBr, cm-1): 3325 (symm.)
and 3433 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 2.39
(s, 3H, -CH3); 5.86 (s, 1H, 4-H); 5.97 (s, 2H, -NH2); 7.23 (d, 2H, J =
8.0 Hz, 3'',5''-H); 7.59 (d, 1H, J = 9.3 Hz, 5'-H); 7.71 (d, 2H, J = 8.0
Hz, 2'',6''-H); 8.35 (d, 1H, J= 9.3 Hz, 4'-H); MS: m/z [M+1]+ 286/288
(3:1). Anal. Cal. for C14H12ClN5: C, 58.85; H, 4.23; N, 24.51. Found:
C, 57.81; H, 3.76; N, 26.50.
Ethanol medium
1-(6'-Chloropyridazin-3'-yl)-3-phenyl-1H-pyrazole-5-amine (4a)
(0.271g,1mmol) was treated with pyrrolidine (0.070g, 1mmol) in
15ml ethanol and refluxed for about 28hrs, then concentrated to
about 1/5th of the volume, cooled, poured into water, solid sepa-
rated, filtered, washed with water and crystallised from ethanol.
The TLC (ethyl acetate/petroleum ether in 1:4) and 1H NMR spec-
tra showed the formation of a single product.
Sharma S et al. Analgesics COX-2 Inhibitor Drug Research… Drug Res

Thieme
Original Article
J = 9.7 Hz, 4'-H); MS: m/z [M + 1]+ 325. Anal. Cal. for C17H17FN6: C,
DMF medium
1-(6'-Chloropyridazin-3'-yl)-3-phenyl-1H-pyrazole-5-amine (4a)
(0.271g, 1mmol) was treated with pyrrolidine (0.070g, 1mmol) in
5 ml DMF and refluxed for 30 min, then excess of solvent was dis-
tilled off to about ½ of the volume, cooled, poured into water, sticky
solid separated, filtered, washed with water and crystallised from
ethanol. The TLC (ethyl acetate/petroleum ether in 1:4) and1H NMR
spectra showed the formation of a single product.
62.95; H, 5.28; N, 25.91. Found: C, 62.80; H, 5.05; N, 25.80.
3-(4''-Chlorophenyl)-1-(6'-(pyrrolidin-1'''-yl)pyridazin-3'-yl-
1H-pyrazol-5-amine (6d)
Yield 92.8 %; Rf = 0.15; M.Pt. 210 °C; IR (KBr, cm-1): 3325 (symm.)
and 3433 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 2.00
(s, 4H, 3''', 4'''-H); 3.49 (s, 4H, 1''', 2'''-H); 5.75 (s, 1H, 4-H); 5.83 (s,
2H, -NH2); 6.79 (s, 1H, 5'-H); 7.24 (s, 2H, 3'', 5''-H); 7.59 (s, 2H, 2'',
6''-H); 8.01 (s,1H, 4'-H), MS: m/z [M + 1]+ 341/343 (3:1). Anal. Cal.
for C17H17ClN6: C, 59.91; H, 5.03, N, 24.66. Found: C, 59.80; H,
4.95; N, 24.50.
One-pot solvent-free procedure
Synthesis of 3-phenyl-1-(6'-(pyrrolidin-1'''-yl)pyridazin-3'-yl)-1H-
pyrazol-5-amine (6a)
Equimolar amounts of 3-chloro-6-hydrazinopyridazine (0.144g,
1mmol) and 3-oxo-3-phenylpropanenitrile (0.145g, 1mmol) were
ground with p-toulenesulfonic acid (PTSA) (0.344g, 2mmol) in mor-
tar and pestle. The reaction was monitored on TLC (ethyl acetate/
petroleum ether in 1:4) at regular intervals and indicated that re-
action is complete in 5 min. Once the synthesis of 4a accomplished,
then in situ addition of pyrrolidine (0.070, 1mmol) was undertak-
en. The reaction mixture ground for another 15–20 min. and the
spot corresponding 4a disappeared and a new spot corresponding
to 6a appeared. Then ethanol is added to the reaction mixture, left
overnight, solid separated, filtered through suction, washed with
water and crystallised in alcohol. 1H NMR spectra showed the for-
mation of a single product.
Similarly, compounds 6b-e and 7,8 a-e with secondary amines
piperidine 5b and morpholine 5c respectively, have been synthe-
sized following the above procedure.
3-Phenyl-1-(6'-(pyrrolidin-1'''-yl)pyridazin-3'-yl)-1H-pyrazol-5-
amine (6a)
Yield 88.8%; Rf =0.15; M.Pt. 187°C; IR (KBr, cm-1): 3317 (symm.)
and 3425 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 2.08
(quintet, 4H, J= 3.1 Hz, 3''', 4'''-H); 3.57 (t, 4H, J= 6.6 Hz, 2''', 5'''-H);
5.86 (s, 1H, 4-H); 5.88 (s, 2H, -NH2); 6.86 (d, 1H, J = 9.7 Hz, 5'-H);
7.32 (m, 1H, 4''-H); 7.40 (t, 2H, J = 7.2 Hz, 3'', 5''-H); 7.82 (d, 2H, J=
7.1 Hz,2'', 6''-H); 8.13 (d, 1H, J= 9.7 Hz, 4'-H); Ms: m/z [M+1]+ 307.
Anal. Cal. For C17H18N6: C, 66.65; H, 5.92; N, 27.43. Found: C,
66.50; H, 5.85; N, 27.30.
3-(4''-Bromophenyl)-1-(6'-(pyrrolidin-1'''-yl)pyridazin-3'-yl-
1H-pyrazol-5-amine (6e)
Yield 93.9%; Rf =0.17; M.Pt. >315°C; IR (KBr, cm-1): 3325 (symm.)
and 3433 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 2.08
(quintet, 4H, J= 3.8 Hz, 3''', 4'''-H); 3.56 (t, 4H, J= 6.4 Hz, 1''', 2'''-H);
5.82 (s, 1H, 4-H); 5.90 (s, 2H, -NH2); 6.85 (d, 1H, J = 9.6 Hz, 5'-H);
7.51 (d, 2H, J = 8.4 Hz, 3'', 5''-H); 7.69 (d, 2H, J= 8.4 Hz, 2'', 6''-H);
8.08 (d,2H, J=9.6 Hz, 4'-H), MS: m/z [M+1]+ 341/343. C17H17BrN6:
C, 53.00; H, 4.45; N, 21.81. Found: C, 52.95; H, 4.30; N, 21.75.
3-Phenyl-1-(6'-(piperidin-1'''-yl)pyridazin-3'-yl)-1H-pyrazol-
5-amine (7a)
Yield 90.9%; Rf =0.44; M.Pt. >315°C; IR (KBr, cm-1): 3325 (symm.)
and 3425 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 1.70
(s, 6H, 3''', 4'''-H); 3.62 (s, 4H, 2''', 6'''-H) 5.86 (s, 1H, 4-H); 5.89 (s,
2H, -NH2); 7.14 (d, 1H, J= 9.8 Hz, 5'-H); 7.32 (m,1H, 4''-H); 7.40 (t,
2H, J = 7.2 Hz, 3'', 5''-H); 7.82 (d, 2H, J = 7.1 Hz, 2'', 6''-H); 8.12 (d,
1H, J = 9.8 Hz, 4'-H); MS: m/z [M + 1]+ 321. Anal. Cal. for C18H20N6:
C, 67.48; H, 6.29; N, 26.23. Found: C, 67.30; H, 6.00; N, 26.10.
1-(6'-(Piperidin-1'''-yl)pyridazin-3'-yl)-3-p-tolyl-1H-pyrazol-
5-amine (7b)
Yield 97.1 %; Rf = 0.45; M.Pt. 221 °C; IR (KBr, cm-1): 3317 (symm.)
and 3425 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 1.70
(s, 6H, 3''', 4''', 5'''-H); 2.37 (s, 3H, -CH3); 3.62 (s, 4H, 2''',6'''-H); 5.83
(s, 1H, 4-H); 5.87 (s, 2H, -NH2); 7.14 (d, 1H, J = 9.9 Hz, 5'-H); 7.20
(d, 2H, J = 8.0 Hz, 3'', 5''-H); 7.70 (d, 2H, J = 8.1 Hz, 2'', 6''-Hz); 8.11
(d, 1H, J=9.9 Hz, 4'-H); MS: m/z [M+1]+ 335. Anal. Cal. for C19H22N6:
C, 68.24; H, 6.63; N, 25.13. Found: C, 55.22; H, 4.40; N, 20.06.
1-(6'-(Pyrrolidin-1'''-yl)pyridazin-3'-yl)-3-p-tolyl-1H-
pyrazol-5-amine (6b)
Yield 89.3%; Rf =0.14; M.Pt. >315°C; IR (KBr, cm-1): 3279 (symm.)
and 3379 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 2.07
(quintet, 4H, 3''', 4'''-H); 2.37 (s, 3H, -CH3); 3.56 (t, 4H, J = 6.6 Hz,
J = 3.0 Hz, 2''', 5'''-H); 5.83 (s, 1H, 4-H); 5.87 (s, 2H, -NH2); 6.85 (d,
1H, J = 9.7 Hz, 5'-H); 7.20 (d, 2H, J= 8.0 Hz, 3'', 5''-H); 7.71 (d, 2H,
J = 8.1 Hz, 2'', 6''-H); 8.12 (d, 1H, J = 9.7 Hz, 4'-H); MS: m/z
[M + 1]+ 321. Anal. Cal. for C18H20N6: C, 67.48; H, 6.29; N, 26.23.
Found: C, 51.25; H, 4.53; N, 18.09.
3-(4''-Fluorophenyl)-1-(6'-(piperidin-1'''-yl)pyridazin-3'-yl-
1H-pyrazol-5-amine (7c)
Yield 87.4%; Rf =0.43; M.Pt. >315°C; IR (KBr, cm-1): 3256 (symm.)
and 3364 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 1.70
(s, 6H, 3''', 4''', 5'''-H); 3.62 (s, 4H, 2''', 6'''-H); 5.80 (s, 1H, 4-H); 5.91
(s, 2H, -NH2); 7.08 (t, 2H, J= 8.5 Hz, 3'', 5''-H);7.15 (d, 1H, J= 9.9Hz,
5'-H); 7.79 (m, 2H, 2'', 6''-H); 8.09 (d, 1H, J= 9.5 Hz, 4'-H); MS: m/z
[M + 1]+ 339. Anal. Cal. for C18H19FN6: C, 63.89; H, 5.66; N, 24.84.
Found: C, 63.75; H, 5.50; N, 24.50.
3-(4''-Fluorophenyl)-1-(6'-(pyrrolidin-1'''-yl)pyridazin-3'-yl-
1H-pyrazol-5-amine (6c)
Yield 89.8 %; Rf = 0.16; M.Pt. 218 °C; IR (KBr, cm-1): 3310 (symm.)
and 3425 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 2.08
(quintet, 4H, J= 3.1 Hz, 3''', 4'''-H); 3.56 (t, 4H, J= 6.5 Hz, 2''', 5'''-H);
5.81 (s, 1H, 4-H); 5.90 (s, 2H, -NH2); 6.86 (d, 1H, J = 9.7 Hz, 5'-H);
7.08 (t, 2H, J= 6.7 Hz, 3'', 5''-H); 7.79 (m, 2H, 2'', 6''-H); 8.10 (d, 1H,
3-(4''-Chlorophenyl)-1-(6'-(piperidin-1'''-yl)pyridazin-3'-yl-
1H-pyrazol-5-amine (7d)
Yield 91.9 %; Rf = 0.44; M.Pt. 207 °C; IR (KBr, cm-1): 3286 (symm.)
and 3387 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 1.70
Sharma S et al. Analgesics COX-2 Inhibitor Drug Research… Drug Res

(s, 6H, 3''', 4''', 5'''-H); 3.63 (s, 4H, 2''', 6'''-H); 5.82 (s, 1H, 4-H); 5.89
(s, 2H, -NH2); 7.13 (d, 1H, J = 9.8 Hz, 5'-H); 7.37 (d, 2H, J= 6.7 Hz,
3'', 5''-H); 7.74 (d, 2H, J = 6.6 Hz, 2'', 6''-H); 8.08 (d, 1H, J= 9.8 Hz,
4'-H); MS: m/z [M + 1]+ 355/357 (3:1). Anal. Cal. for C18H19ClN6: C,
60.93; H, 5.40; N, 23.68. Found: C, 60.50; H, 5.30; N, 23.50.
for C17H17ClN6O: C, 57.22; H, 4.80; N, 23.55. Found: C, 57.00; H,
4.58; N, 23.40.
1-(6'-morpholinopyridazin-3'-yl)-3-(4''-bromophenyl)-1H-
pyrazol-5-amine (8e)
Yield 80.8%; Rf =0.18; M.Pt. >315°C; IR (KBr, cm-1): 3286 (symm.)
and 3418 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 3.61
(t, 4H, J = 4.7 Hz, 3''',5'''-H); 3.87 (t, 4H, J = 5.0 Hz, 2''',6'''-H); 5.83
(s,1H, 4-H); 5.91 (s, 2H, -NH2); 7.13 (d, 1H, J = 9.8 Hz, 5'-H); 7.52
(d, 2H, J = 8.4 Hz, 3'', 5''-H); 7.69 (d, 2H, J= 8.4 Hz, 2'', 6''-H); 8.16
(d, 1H, J = 9.8 Hz, 4'-H); MS: m/z [M + 1]+ 402/404 (1:1). Anal. Cal.
for C17H17ClN6O: C, 50.89; H, 4.27; N, 20.94. Found: C, 50.45; H,
4.12; N, 20.85.
3-(4''-Bromophenyl)-1-(6'-(piperidin-1'''-yl)pyridazin-3'-yl-
1H-pyrazol-5-amine (7e)
Yield 86.5%; Rf =0.42; M.Pt. 224.5°C; IR (KBr, cm-1): 3402 (symm.)
and 3294 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 1.70
(s, 6H, 3''', 4''', 5'''-H); 3.63 (s, 4H, 2''', 6'''-H); 5.82 (s, 1H, 4-H); 5.90
(s, 2H, -NH2); 7.14 (d, 1H, J= 9.8Hz, 5'-H); 7.52 (d, 2H, J= 8.4 Hz,
3'', 5''-H); 7.60 (d, 2H, J = 8.4 Hz, 2'', 6''-H); 8.08 (d, 1H, J= 9.7 Hz,
4'-H); MS: m/z [M + 1]+ 400/402 (1:1). Anal. Cal. for C18H19BrN6: C,
54.14; H, 4.80; N, 21.05. Found: C, 54.00; H, 4.58; N, 21.00.
Molecular docking methodology
Analgesic effect of titled compounds can be understood by study-
ing the interaction between the ligand and the protein receptor in-
volved. A docking simulation study is the best way for understand-
ing drug-receptor interaction that helps in drug discovery. Molec-
ular docking has been performed using Algorithm genetic method
of the AutoDock 4.0 [31]. Openbabel software tool was employed
for converting all the mol, 2D structures of ligand molecules were
converted into pdb, 3D format. To get an insight into the active res-
idues having a vital role in an analgesic activity, the newly synthe-
sized compounds were docked against target protein (PDB ID:
6COX) [32]. The protein, Cyclooxygenase-2 complexed with a se-
lective inhibitor; SC-558, (PDB 6COX) was obtained from protein
data bank (www.rcsb.org). The water of crystallization was re-
moved while polar hydrogens and Gasteigere partial charges were
added. All the parameters were set by default during docking anal-
ysis. The grid box was set to get the region of interest, active site,
in the macromolecule under study. A grid box size of 40.0, 40.0,
40.0 Å was generated having x, y and z coordinates of 47.06, 25.441
and 36.961 respectively with a spacing of 0.375 Å for the crystal
structure of COX-2. Docking results were analysed and chimera was
used to get the pictorial presentation of drug-receptor interaction
[33].
1-(6'-Morpholinopyridazin-3'-yl)-3-phenyl-1H-pyrazol-5-
amine (8a)
Yield 84.7%; Rf =0.19; M.Pt. 170.5°C; IR (KBr, cm-1): 3279 (symm.)
and 3410 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 3.60
(t, 4H, J = 4.7 Hz, 3''',5'''-H); 3.87 (t, 4H, J = 5.0 Hz,2''',6'''-H); 5.87
(s, 1H, 4-H); 5.90 (s, 2H, -NH2); 7.13 (d, 1H, J = 9.8 Hz, 5'-H); 7.33
(m, 1H, 4''-H); 7.40 (t, 2H, J= 7.7 Hz, 3'', 5''-H); 7.82 (d, 2H, J= 7.2
Hz, 2'', 6''-H); 8.19 (d, 1H, J = 9.8 Hz, 4'-H); MS: m/z [M + 1]+ 323.
Anal. Cal. for C17H18N6O: C, 63.34; H, 5.63; N, 26.07. Found: C,
63.20; H, 5.30; N, 25.95.
1-(6'-Morpholinopyridazin-3'-yl)-3-p-tolyl-1H-pyrazol-5-
amine (8b)
Yield 79.5 %; Rf = 0.18; M.Pt. 214 °C; IR (KBr, cm-1): 3295 (symm.)
and 3401 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 2.39
(s, 3H, -CH3); 3.60 (t, 4H, J = 4.7 Hz, 3''',5'''-H); 3.87 (t, 4H, J= 5.1
Hz, 2''', 6'''-H); 5.85 (s, 1H, 4-H); 5.88 (s, 2H, -NH2); 7.13 (d, 1H, J =
9.8 Hz, 5'-H); 7.20 (d, 2H, J= 7.9 Hz, 3'', 5''-H); 7.71 (d, 2H, J= 8.0
Hz, 2'', 6''-H); 8.19 (d, 1H, J = 9.8 Hz, 4'-H); MS: m/z [M + 1]+ 337.
Anal. Cal. for C18H20N6O: C, 64.27; H, 5.99; N, 24.98. Found: C,
59.61; H, 4.71; N, 22.12.
Pharmacology
3-(4''-Fluorophenyl)-1-(6'-morpholinopyridazin-3'-yl)-1H-
pyrazol-5-amine (8c)
Acetic-acid induced writhing test
Yield 80.7 %; Rf = 0.17; M.Pt 220 °C; IR (KBr, cm-1): 3402 (symm.)
and 3294 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 3.60
(t, 4H, J = 4.7 Hz, 3''',5'''-H); 3.87 (t, 4H, J = 5.1 Hz, 2''',6'''-H); 5.81
(s,1H, 4-H); 5.92 (s, 2H, -NH2); 7.08 (t, 2H, J= 6.7 Hz, 2'', 6''-H); 7.14
(d, 1H, J = 9.8 Hz, 5'-H); 7.79 (m, 2H, 3'', 5''-H); 8.17 (d, 1H, J = 9.8
Hz, 4'-H); MS: m/z [M+1]+ 341. Anal. Cal. for C17H17FN6O: C, 59.99;
H, 5.03; N, 24.69. Found: C, 59.69; H, 4.98; N, 24.55.
The total number of writhings following intraperitoneal adminis-
tration of acetic acid solution was (1%, 10mL/kg) was recorded over
for 15min, starting 5min after acetic acid injection. The mice were
treated with the test compound (50 mg/kg) or vehicle (tween 80
(5 %)) or standard drug (diclofenac sodium, 50 mg/kg), 30 min be-
fore administration of acetic acid and number of writhings and
stretchings were recorded and permitted to express the percent-
age of protection. Calculation of percentage protection was un-
dertaken by considering the following formula:% protection= (Mc-
Mt/Mc) × 100; where Mt = Mean writhing of test group and Mc =
Mean writhing of a control group and reported values were the av-
erage of six determinations SEM of n (6) animals per group. Results
were expressed as mean + SEM differences between control and
treatment group and were tested using one-way ANOVA followed
by least significant differences. The * p < 0.05, * * p < 0.01 values
are considered as statistically significant or highly significant, re-
spectively [34].
3-(4''-Chlorophenyl)-1-(6'-morpholinopyridazin-3'-yl)-1H-
pyrazol-5-amine (8d)
Yield 84.0%; Rf =0.19; M.Pt. >315°C; IR (KBr, cm-1): 3294 (symm.)
and 3402 (asymm.) NH strech; 1H NMR (400 MHz, CDCl3) δ: 3.53
(t, 4H, J = 4.7 Hz, 3''',5'''-H); 3.80 (t, 4H, J = 5.0 Hz, 2''',6'''-H); 5.75
(s,1H, 4-H); 5.84 (s, 2H, -NH2); 7.05 (d, 1H, J = 9.8 Hz, 5'-H); 7.30
(d, 2H, J = 8.5 Hz, 3'', 5''-H); 7.67 (d, 2H, J = 8.4 Hz, 2'', 6''-H); 8.08
(d, 1H, J = 9.8 Hz, 4'-H); MS: m/z [M + 1]+ 357/359 (3:1). Anal. Cal.
Sharma S et al. Analgesics COX-2 Inhibitor Drug Research… Drug Res

Thieme
Original Article
▶Fig. 3 Synthesis of 5-amino-3-aryl-1-(6'-chloro/heterocyclicpyridazin-3'-yl)pyrazoles.
Results
▶table 1 Comparative analysis of reactions under different reaction
conditions.
The synthesis of 5-amino-3-aryl-1-(6'-chloropyridazin-3'-yl)pyra-
zoles and their derivatives 4, 6–8 (a-e) is outlined in ▶Fig 3. The
key precursor, 3-chloro-6-hydrazinopyridazine 2, synthesized by
treating 3,6-dichloropyridazine 1 with hydrazine hydrate following
the literature procedure [35]. Initially, the reaction of 3-chloro-
6-hydrazinopyridazine 2 with β-ketonitriles 3 (a-e), obtained by re-
action of α-bromoacetophenones with potassium cyanide, was car-
ried out under refluxing ethanol for 30 min. with 2–3 drops of con-
centrated HCl, which yielded single product 5-amino-3-aryl-
1-(6'-chloropyridazin-3'-yl)pyrazoles 4 (a-e) on ethyl acetate/pe-
troleum ether (1:4) TLC system. Further, the synthesis of 5-amino-
3-aryl-1-(6'-heterocyclicpyridazin-3'-yl)pyrazoles (6–8) was under-
taken by amino-de-halogenation of 4 (a-e) with cyclic secondary
amines, viz. pyrrolidine 5a, piperidine 5b, morpholine 5c, under re-
fluxing ethanol for 24–36 hrs. Interestingly, another reaction con-
dition was studied utilising DMF as a solvent with a catalytic amount
of Cs2CO3 (high polarizability and size than Na2CO3 and K2CO3), the
reaction time decreased drastically; 4 (a-e) could be accomplished
in 5–10 min. and 6–8 (a-e) in 30 min., however, with low yields and
tedious work-up. Hence, to overcome these drawbacks, an eco-
friendly approach was applied resulting in minimum reaction time,
high yield and easy work-up. Grinding of 3-chloro-6-hydrazinopyri-
dazine (2) and β-ketonitrile (3a-e) in the presence of p-toulenesul-
fonic acid (PTSA) as a solid catalyst led to the formation of 5-ami-
nopyrazoles (4a-e), which were eventually converted to their
6'-pyrrolidine (6a-e), 6'-piperidine (7a-e) and 6'-morpholine (8a-
e) derivatives after in situ addition of cyclic 2° amines, pyrrolidine/
piperidine/morpholine 5(a-c) respectively. A comparative analysis
of time for the completion of reaction under different reaction con-
ditions is depicted in ▶table 1. The structure and purity of all the
synthesized compounds were confirmed by TLC and correspond-
ing spectral data (IR, MS, 1H, 13C) and elemental analysis.
Sr.
No
Medium
Reaction
time (4a–e)
Reaction time
(6,7,8a–e)
Overall
Yeild%
1
2
3
EtOH
30 min
24–36 hrs
30 min
55–81
35–40
82–84
DMR
5–10 min
5 min (in-situ)
Solvent-free
15–20 min
The 1H NMR spectra also helped in recognising the NH2 group
in 4,6,7,8 (a-e) as it displayed a characteristic singlet integrating
for two protons at δ 5.84–6.96 ppm. Besides two doublets appear-
ing for pyridazine protons 4'-H at δ 8.32–8.35 ppm and 5'-H at δ
7.58–7.97 ppm with coupling constant 3J~ 9.3 Hz, a characteristic
sharp singlet for 4-H pyrazole appeared at δ 5.83–5.92 ppm of com-
pounds 4 (a-e). Nucleophilic substitution of chlorine by cyclic 2 °
amine resulted into an upfield shift in 6,7,8 (a-e) and the signal ap-
peared at δ 5.75–5.87, 6.79–7.15 ppm and 8.01–8.19 ppm for 4,
5' and 4'-H respectively. Besides, 1H NMR of 6, 7 and 8 exhibited
the characteristic splitting pattern of pyrrolidine, piperidine, mor-
pholine in the aliphatic region.
Moreover, the structure of the compounds was supported by
13C NMR spectroscopy and the signals appeared at δ 150, 85–87
and 151–152 ppm corresponding to C-3, C-4 and C-5 of the pyra-
zole ring, respectively. These values are following the literature
[36]. The complete assignment of the signals in the 13C NMR spec-
tra of compounds 4,6,7,8 (a-e) is given in ▶tables 2 and ▶3.
Discussion
Molecular docking study was performed to understand the inter-
action of potential sites of receptor protein with synthesized li-
gands. Aiming at the goal, 6COX was used to get an insight into the
interaction pattern of ligands with COX-2 receptor. It was observed
that N-atom present in synthesized series forms H-bond with vari-
ous amino acid residues. Most common interacting residues were
ASN375.A, ASN375.B and ASN537.B. Candidates 4d and 7c were
docked into the active site of COX-2 and showed the same orien-
The presence of amino group in compounds 4,6,7,8 (a-e) was
confirmed by IR spectra, which showed two sharp absorption bands
in the range of 3252–3560 cm-1 (symm.) and 3356–3610 cm-1
(asymm.) due to NH2 stretching.
Sharma S et al. Analgesics COX-2 Inhibitor Drug Research… Drug Res

▶table 2 C-13 NMR of compounds 4 (a-e).
carbon
C-3
4a
4b
4c
4d
4e
149.9
87.5
149.8
87.4
150.0
87.3
150.8
85.2
150.0
87.3
C-4
C-5
152.7
157.2
130.6
121.6
154.2
132.6
125.9
128.6
128.8
152.6
157.2
130.5
121.7
154.3
138.7
125.9
129.3
129.7
21.4
152.8
164.5
121.6
115.5
153.3
115.7
127.7
127.8
130.6
152.0
156.9
131.3
121.8
152.2
131.2
127.1
128.4
133.2
152.9
157.2
130.6
121.6
153.1
122.8
127.5
131.8
131.5
C-3′
C-4′
C-5′
C-6′
C-1″
C-2″/6″
C-3″/5″
C-4″
CH3
tation and binding mode to that of SC-588 (selective COX-2 inhib-
itor). In the binding mode, amino N of compound 4d and pyridazine
N of compound 7c was potently bound to the active binding site of
6COX forming hydrogen bond with ASN375.B with dock score -8.61
and -8.23 kCal/mol as shown in ▶Figs. 4 and ▶5 respectively. Sim-
ilarly, internal ligand, SC-558 was bound to the binding pocket of
COX-2 with dock score -8.67 kCal/mol, also formed a hydrogen
bond with ASN375.A as displayed in ▶Fig. 6. The data of dock score
and interactive amino acids of all the compounds of the series is
provided in ▶table 4. Results from docking simulations justify the
potential of synthesized series as an analgesic and very well corre-
lated with in vivo study.
A screening protocol was used to select the structures from all
the synthesized compounds possessing analgesic action and eval-
uation was undertaken by acetic-acid induced writhing test on
swiss albino mice. The visceral pain induced by acetic-acid is due
to the release of arachidonic acid via cyclooxygenase and prosta-
glandin (PG). A careful examination of the data in ▶table 5 shows
that all compounds, except 6b, 8b, 6d, 8d exhibited moderate-to-
good analgesic action.
It is inferred from ▶table 5 that analgesic potency estimated
by classical acetic-acid induced constrictions/writhings for certain
compounds (8a, 4b, 4,7,8c, 4d) is comparatively equal or highly
related to reference/standard drug (s) sodium diclofenac. Accord-
ing to SAR study, considering fixed group present on 6’-position of
pyridazine ring and variation is made around the R group present
on p-position of phenyl framework (shown in ▶Fig. 7). Antinocic-
eptive action of 5-amino-3-aryl-1-(6'-chloropyridazin-3'-yl)pyra-
zoles (4a-4e) follows the sequence 4d > 4b > 4c > 4a > 4e, which
shows the enhancement of analgesic activity when Me, F, Cl group
is present at p-position of phenyl group whereas Br group cause a
decline. Candidates 4b and 4d, registered a noteworthy analgesic
profile which is closest to standard reference drug (sodium di-
clofenac). Further, the sequence followed by 3-aryl-1-(6'-
(pyrrolidin-1'''-yl)pyridazin-3'-yl)-1H-pyrazol-5-amines (6a-6e) for
antinociceptive action is 6a>6e>6d>6c>6b, which exhibit a de-
cline in analgesic activity when phenyl group is substituted by Me,
F, Cl, Br groups. Moreover, antinociceptive profile of 3-aryl-1-(6'-
(piperidin-1'''-yl)pyridazin-3'-yl)-1H-pyrazol-5-amines (7a-7e) and
3-aryl-1-(6'-(morpholin-1'''-yl)pyridazin-3'-yl)-1H-pyrazol-5-amines
Sharma S et al. Analgesics COX-2 Inhibitor Drug Research… Drug Res

Thieme
Original Article
▶table 4 Binding energy of synthesized compounds against COX-2 recep-
tor (PDB ID: 6COX).
S.No
com-
pound
Docking
score
Hydrogen
bond
Amio acid
1
2
4a
4b
4c
4d
4e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
8b
8c
− 6.47
− 8.01
− 7.26
−ꢀ8.61
− 6.76
− 6.23
− 6.11
− 6.67
− 6.23
− 6.42
− 6.27
− 6.68
−ꢀ8.23
− 6.77
− 6.99
− 7.11
− 5.36
− 7.94
–
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
–
2
ASN375.B
ASN375.B
ASN375.b
ASN375.B
ASN375.B
ASN375.B
ASN375.A
ASN375.A
ASN375.B
ASN375.A
ASN375.A
ASN375.b
ASN375.B
ASN375.B
ASN375.B
–
3
4
5
6
▶Fig. 4 Docking pose of compound 4d with COX-2 receptor (PDB
ID: 6COX) showing hydrogen bond.
7
8
9
10
11
12
13
14
15
16
17
18
ASN375.B
ASN375.B
ASN375.B
ASN375.B
ASN375.A
19
20
21
8d
− 5.46
− 6.69
−ꢀ8.67
1
1
1
▶Fig. 5 Docking pose of compound 7c with COX-2 receptor (PDB
ID: 6COX) showing hydrogen bond.
8e
Sc-558
▶table 5 Analgesic action (percentage protection) of compounds using
writhing test.
S.No
1
Groups
control
No of wriths
5.56±0.81
% Protection
–
2
3
Standard(s)
13.1±0.10**
33.00 ± 2.44 *
24.00 ± 0.60 * *
26.66 ± 0.57 * *
19.8±0.40**
38.66 ± 0.17
30.33 ± 0.91 *
44.00 ± 0.7
76.5
4a
4b
4c
4d
4e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
41.69
57.59
52.89
65.01
31.69
46.41
22.26
34.34
25.51
36.69
44.94
34.62
59.08
32.57
33.46
51.13
13.42
53.78
18.44
43.18
4
5
6
7
▶Fig. 6 Docking pose of Internal ligand, SC-558 with COX-2 recep-
tor (PDB ID: 6COX) showing hydrogen bond.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
37.16 ± 0.90 *
42.16 ± 1.4
35.83 ± 1.5
(8a-8e) follows the order: 7c > 7a > 7b > 7e > 7d and
8c>8a>8e>8d>8b respectively, exhibiting decrease in analgesic
action by Me, Cl, Br groups present at p-position of phenyl frame-
work and 8c increment in analgesic action is comparable to refer-
ence drug.
31.16±0.30*
37.00 ± 0.8
23.16±0.23**
38.16 ± 0.31
37.66 ± 0.45
27.66 ± 0.45 * *
49.00 ± 0.5
▶Fig. 7 Graph representing% protection versus number of com-
pounds and % protection obtained for compounds 4d, 7c compa-
rable to reference/standard drug (s) sodium diclofenac.
Hence, analgesic study showed that candidates (8a=51.13%,
4b = 57.59 %, 4c = 52.89 %, 7c = 59.08 %, 8c = 53.78 %, 4d =
65.01%) exhibited their best analgesic action by exerting their ef-
fect of action by inhibiting the enzymatic activity of COX and con-
sequently reducing the level of prostaglandin (PGE2) within the hy-
26.16 ± 0.15 * *
46.16 ± 0.40
32.16 ± 0.55 *
n=6, All values are expressed as mean±SEM of six swiss albino mice in each
group. Statistically significant **p<0.01, *p<0.05 compared to control.
Sharma S et al. Analgesics COX-2 Inhibitor Drug Research… Drug Res

Conflict of Interest
The relationship among the authors (Dr. Ranjana Aggarwal, Swati
(research scholar), Dr. Pawan Kaushik, Dr. Ajay Kumar, Dr. Deepika
Saini) is purely a scientific collaboration.
References
[1] Purnima T, Avinash CT, Viney C et al. Syntheses, characterization and
evaluation of novel 2,6-diarylpiperidin-4-ones as potential analgesic-
antipyretic agents. Eur J Med Chem 2014; 82: 439–448
[2] Vinoth PV, Nalini G, Chidambaranathan N et al. Evaluation of anti- in-
flammatory and analgesic activity of tridax procumbenslinnagainst
formalin, acetic acid and cfa induced pain models. Int J Pharm & Pharm
Sci 2011; 3: 126–130
▶Fig. 7 Graph representing% protection versus number of com-
pounds and% protection obtained for compounds 4d, 7c compara-
ble to reference/standard drug (s) sodium diclofenac.
[3] Rangappa SK, Kallappa MH, Ramya VS et al. Analgesic, anti-pyretic and
DNA cleavage studies of novel pyrimidine derivatives of coumarin
moiety. Eur J Med Chem 2010; 45: 2597–2605
[4] Ferreira SH, Lorenzetti BH, Devissaguet M et al. S14080, a peripheral
analgesic acting by release of anendogenous circulating opioid-like
substance. Br J Pharmacol 1995; 114: 303–308
pothalamic region. Moreover, amination of 5-aminopyrazoles at
6’-position with pyrrolidine, piperidine, morpholine increases the
analgesic mode of action in case of Ph, p-FPh, p-BrPh framework
whereas reverse effects were obtained in case of p-MePh, p-ClPh.
[5] Syeda AZ, Syed MT, Afzal Q et al. Nonsteroidal anti-inflammatory
drugs. Professional Med J 2016; 23: 460–466
[6] Krystyna C, Ewa M, Magdalena BZ. Inhibitors of leukotrienes synthesis:
novel agents and their implementation. Drug Research 2016; 73:
843–849
[7] Sebastian B, Carmen BR, Andreas L et al. Synthesis and biological
evaluation of a class of 5-benzylidene-2-phenyl-thiazolinones as
potent 5-lipoxygenase inhibitors. Bioorg & Med Chem 2012; 20:
3575–3583
Conclusion
A new series of biologically active compounds: 5-amino-3-aryl-
1-(6'-chloro/heterocyclicpyridazin-3'-yl)pyrazoles 4,6,7,8 (a-e)
were synthesised and analgesic profile of synthesized compounds
showed moderate-to-good results. Results from docking simula-
tion suggested that compound 4d and 7c was the most potent
among the series when docked against COX-2 receptor. The dock-
ing study investigated a binding mode for synthetic derivatives and
provided insights for further inhibitor design. This study drives us
to the path towards the development of pyridazine derivatives as
lead molecules for further structural optimization as potential an-
algesic agents. Docking data was very well correlated to the results
of in vivo study, also, candidates 4d and7c exhibited comparable
results regarding standard drug sodium diclofenac when tested
with swiss albino mice using the acetic-acid induced writhing assay.
Hence, compound 4d and 7c could be probed further as analgesic
agents with application in the pharmaceutical industry, after test-
ing its toxicity in humans.
[8] Chambon JP, Feltz P, Heaulme M et al. An arylaminopyridazine
derivative of gamma-aminobutyric acid (GABA) is a selective and
competitive antagonist at the GABAA receptor site. Proc Natl Acad Sci
1985; 82: 1832–1836
[9] Takaya M, Sato M, Terashima K et al. A new nonsteroidal analgesic-
anti-inflammatory agent. Synthesis and activityof 4-Ethoxy-2-methyl-
5-morpholino-3(2H)-pyridazinone and related compounds. J Med
Chem 1979; 22: 53–58
[10] Asif M, Singh A., Lakshmayya A brief review on triazin-pyridazinones:
Synthesis and biological activities. Brazil. J Pharm Sci 2013; 49:
903–909
[11] Asif M. Overview on emorfazone and other related 3(2H) pyridazinone
Analogues displaying analgesic and anti-Inflammatory activity. Ann
Med Chem Res 2014; 1: 1004
[12] Tkachenko PV, Tkachenko OV, Netosova KY et al. Products of
interaction of substituted 5-aminopyrazoles and α-haloketones as
potential pharmaceutical agents. Visnik Farmacii 2017; 91: 3–9
[13] Mark CB, Terence D, Matthew CD et al. Microwave-assisted synthesis
of 5-aminopyrazol-4-yl ketones and the p38MAPK inhibitor
RO3201195 for study in Werner syndrome cells. Bioorg Med Chem
Lett 2008; 18: 3745–3748
Acknowledgment
We are thankful to the University Grants Commission, New Delhi,
India, for providing financial assistance to Swati, in the form of Sen-
ior Research Fellowship. Thanks are also extended to the Sophisti-
cated Analytical Instrument Facility, Panjab University, for use of
NMR, mass spectrometer facilities and elemental analyses.
[14] Bhatt A, Singh RK, Kant R. Discovery of novel pyrazoles as potent
antimicrobial and antimalarial agents. Der Pharm Lett 2016; 8:
182–187
[15] dos Santos FV, Leon LL, Canto-Cavalheiro MM et al. Effectiveness of
novel 5-(5-amino-1-aryl-1H-tetrazole derivatives against promastig-
otes and amastigotes of leishmania amanzonensis. Chem Biol Drug
Des 2014; 83: 272–277
[16] Mizuhara T, Kato T, Hirari A et al. Structure-activity relationship study
of phenylpyrazole derivatives as a novel class of anti-HIV agents.
Bioorg Med Chem Lett 2013; 23: 4557
Sharma S et al. Analgesics COX-2 Inhibitor Drug Research… Drug Res

Thieme
Original Article
[17] Kordik CP, Luo C, Zanoni BC et al. Pyrazolecarboxamide human
neuropeptide Y5 receptor ligands with in vivo antifeedant activity.
Bioorg Med Chem Lett 2001; 11: 2287–2290
[27] Aggarwal R, Singh G, Kumar S et al. Preparation of 4,7-Dihydro-1H
-pyrazolo[3,4-b]pyridine-5-nitriles in a multicomponent domino
process. Chem Select 2016; 1: 5990–5994
[18] Bakr RB, Azouz AA, Adellatif KRA. Synthesis, cyclooxygenase
inhibition, anti-inflammatory evaluation and ulcerogenic liability of
new 1-phenylpyrazolo[3,4-d]pyrimidine derivatives. J Enzyme Inhib
Med Chem 2016; 31: 6–12
[28] Aggarwal R, Singh G, Kaushik P et al. Molecular docking design and
one-pot expeditious synthesis of novel 2,5-diarylpyrazolo[1,5-a]
pyrimidin-7-amines as anti-inflammatory agents. Eur J Med Chem
2015; 101: 326–333
[19] Jagdamba D, Moquin RV, Dyckman AJ et al. 5-aminopyrazoles as
potent and selective p38α inhibitors. Bioorg Med Chem Lett 2010; 20:
6886–6889
[29] Aggarwal R, Kumar V, Singh G et al. An NMR and computational study
of azolo[a]pyrimidines with special emphasis on pyrazolo[1,5-a]
pyrimidines. J Het Chem doi: 10.1002/jhet.1955
[20] Burcu CE, Maria TN, Luis L et al. Synthesis of 1,5-diarylpyrazol-3-propa-
noic acids towards inhibition of cyclooxygenase-1/2 activity and
5-lipoxygenase-mediated LTB4 formation. Arzneimittelforschung
2010; 60: 497–505
[30] Geza S, Peter D. Studies on pyridazine compounds, X1X[1] Mesylation
of 5-aminopyrazoles and their related compounds. Monatshefte Fur
Chemie 1989; 120: 131–137
[31] Mohd S, Rizvi D, Shakil S et al. A simple click by click protocol to
perform docking: AutoDock 4.2 made easy for non-bioinformaticians.
Excli 2013; 12: 831–857
[21] Burcu C, Susann L, Yagmur O et al. Pyrazol-3-propanoic acid
derivatives as novel inhibitors of leukotriene biosynthesis in human
neutrophils. Eur J Med Chem 2011; 46: 5021–5033
[32] Lacerda BR, de Lima FKC, da Silva LL et al. Discovery of novel analgesic
and anti-inflammatory3-arylamine-imidazo[1,2-a]pyridine symbiotic
prototypes. Bioorg Med Chem 2009; 17: 74–84
[22] Aggarwal R, Bansal A, Rozas I et al. p-Toluenesulfonic acid-catalyzed
solvent-free synthesis and biological evaluation of new
1-(4′,6′-dimethylpyrimidin-2′-yl)-5-amino-4H-3-arylpyrazole
derivatives. Med Chem Res 2014; 23: 1454–1464
[33] Petersen EF, Goddard TD, Huang CC et al. UCSF-Chimera-a visualiza-
tion system for exploratory research and analysis. J Comput Chem
2004; 25: 1605–1612
[23] Mamta, Aggarwal R, Jessie S, Sadana R. Synthesis and cytotoxic
evaluation of some new 1-(6-chloropyridazin-3-yl)-3-H/alkyl-4-phenyl-
1H-pyrazol-5-amines and their derivatives. Indian J Het Chem 2016;
26: 59–68
[34] Anupam GB, Nirupam D, Sushant AS et al. Synthesis, characterization,
evaluation and molecular dynamics studies of 5, 6-diphenyl-1,2,4-
triazin–3(2H)-one derivatives bearing 5-substituted 1,3,4-oxadiazole
as potential anti-inflammatory and analgesic agents. Eur J Med Chem
2015; 101: 81–95
[24] Aggarwal R, Kumar V, Gupta KG et al. Synthesis of some new
3,5-diamino-4-(4'-fluorophenylazo)-1-aryl/heteroarylpyrazoles as
antimicrobial agents. Med Chem Res 2013; 22: 3566–3573
[35] Elvidge JA, Pickett JA. Heterocyclic imines and amines. Part X111.1
3,6-Dihydrazinopyridazine and the nature of the reaction between
3,6-dimethoxypyridazine and hydrazine. J Chem Soc Perkin 1972; 1:
1483–1488
[25] Aggarwal R, Kumar V, Bansal A et al. Multi-component solvent-free
versus stepwise solvent mediated reactions: Regiospecific formation of
6-trifluoromethyl and4-trifluoromethyl-1H-pyrazolo[3,4-b]pyridines. J
Fluor Chem 2012; 140: 31–37
[36] Vinod K, Aggarwal R, Tyagi P et al. Synthesis and antibacterial activity
of some new 1-heteroaryl-5-amino-3H/methyl-4-phenylpyrazoles.
Bioorg Med Chem 2006; 14: 1785–1791
[26] Aggarwal R, Rani C, Kumar R et al. Synthesis of new bi(pyrazolo[1,5-a]
pyrimidinyl)-7-one derivatives from dehydroacetic acid and its
analogues as antibacterial agents. Arkivoc 2014; 2: 120–134
Sharma S et al. Analgesics COX-2 Inhibitor Drug Research… Drug Res