Synthesis, Molecular Docking and Potential Antioxidant Activity of Di/Trisubstituted Pyridazinone Derivatives

JOURNAL OF THE CHINESE

CHEMICAL SOCIETY

Article

Synthesis, Molecular Docking and Potential Antioxidant Activity of Di/Trisubstituted

Pyridazinone Derivatives

Sukhbir Lal Khokra,^aShah Alam Khan,^bPramila Thakur,^aDeepika Chowdhary,^aAftab Ahmad^cand

Asif Husain^d*

^aInstitute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra-136119, Haryana, India

^bDepartment of Pharmacy, Oman Medical College, Muscat, Oman

^cHealth Information Technology Department, Jeddah Community College, King Abdulaziz University, Jeddah-21589,

KSA

^dDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard University, New Delhi-110062, India

(Received: Jan. 31, 2016; Accepted: Jun. 3, 2016; Published Online: ???; DOI: 10.1002/jccs.201600051)

A number of pyridazinone derivatives bearing substituted benzylidene and heterocyclic/aromatic rings at

4^thand 6^thpositions, respectively were synthesized in good to moderate yields and screened for antioxi-

dant activity. Antioxidant activity of pyridazinone derivatives was evaluated by using several in vitro radi-

cal scavenging methods such as 1,1-diphenylpicrylhydrazyl (DPPH), hydrogen peroxide (H₂O₂), nitric

oxide (NO), reducing power, and metal chelating assay etc. Molegro virtual docker software was used to

study the binding affinity of the title compounds with the xanthine oxidoreductase enzyme. Amongst the

tested compounds, 5a, 5d, 5g & 5j were found to exhibit excellent antioxidant activity at par with the posi-

tive control, ascorbic acid. The molecular docking studies of these compounds demonstrated a good selec-

tivity profile with xanthine oxidoreductase receptors. A preliminary study of the structural-activity rela-

tionship showed that the presence of electron withdrawing group and heterocyclic ring on pyridazinone

nucleus are associated with the best potency and selectivity profile. It could be proposed that xanthine

oxidoreductase receptor may be involved in observed antioxidant activity of pyridazinone derivatives

bearing aromatic ring and benzylidene substituents and thus the synthesized compounds are worthy of fur-

ther exploration.

Keywords: Antioxidant activity; DPPH; Molecular docking; Pyridazinone; Xanthine oxidoreductase.

INTRODUCTION

Free radicals are well-known destructive as well as

teins caused by the oxidation of these biomolecules.²The

nitrogenous bases (purine and pyrimidine) along with

deoxyribose backbone of Deoxyribo nucleic acid (DNA),

the genetic material of living organisms is known to be

damaged by the highly reactive hydroxyl (OH) radical.³

The results of numerous experimental and clinical

studies have indicated the involvement of oxidative stress

induced by reactive oxygen and nitrogen species in a vari-

ety of disorders including carcinogenesis⁴, atherosclero-

sis,⁵neurodegenerative disorders and aging.⁶In general,

free radicals have been implicated in the pathogenesis of

several chronic inflammatory diseases. Luckily, formation

of these free radicals is governed by a number of un-

surprisingly beneficial substances collectively referred to

as antioxidants. These natural protective compounds fight

against free radicals by neutralizing their effects produced

during the cellular metabolic processes and therefore can

play a vital role in prevention and development of several

beneficial species which usually contain reactive atoms

such as oxygen as a part of their structure. Low levels of re-

active oxygen species (ROS) in humans, produced during

normal cellular metabolism contribute to healthy functions.

Mammalian cells have a natural defense mechanism against

the damaging effects of free radicals. In a healthy state, our

cells detoxify free radicals and maintain a balance between

generation of ROS and their detoxification. However, an

imbalance between the detoxification and formation of free

radicals either due to low level of antioxidants or excessive

generation of damaging species or low level of antioxi-

dants could send a cell into a state of oxidative stress which

eventually leads to cell damage.¹A high concentration of

ROS is extremely harmful to organisms because it damages

and alters the functions of main structural components of

cell such as nucleic acids, carbohydrates, lipids and pro-

* Corresponding author. Tel: +91-11-26059681, 26059688, ext. 5647; Email: drasifhusain@yahoo.com, ahusain@jamiahamdard.ac.in

Supporting information for this article is available on the www under http://dx.doi.org/10.1002/jccs.201600051

J. Chin. Chem. Soc. 2016, 63, 000-000

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Article

Khokra et al.

life threatening diseases.⁷Our antioxidant defense system,

the first line of defense against harmful effects of free radi-

cals, comprises of several enzymes such as glutathione

reductase (GSH), superoxide dismutase (SOD), S-methyl

transferase, catalase (CAT) and glutathione peroxidase

(GPx) etc. There are several naturally occurring non enzy-

matic compounds such as carotenoids, ascorbate (Vitamin

C), tocopherol (vitamin E), polyphenols, and alkaloids etc.,

which are known to scavenge free radicals. Few synthetic

antioxidants used in food and pharmaceutical industries in-

clude butylated hydroxyl toluene (BHT), butylated hydoxy

anisole (BHA), propyl gallate etc. Antioxidants used in

food industry are substituted phenolic compounds which

donate hydrogen and stabilize free radicals by virtue of

their low activation energy. Numerous studies have sug-

gested that consumption of whole grains, fresh fruits and

vegetables as a part of regular diet promote and strengthen

the body’s antioxidant status that help to fight and slow the

progression of degenerative diseases caused by oxidative

stress.^8,9

findings, a series of substituted pyridazinones bearing the

desired structural features for antioxidant activity were de-

signed (Figure 1) and synthesized as per the protocol out-

lined in Scheme1. The title compounds were also docked

on the xanthine oxidoreductase to examine the interactions

of the active compounds with the target enzyme.

Fig. 1. Rational design of new pyridazinones deriva-

tives.

Synthesis of pyridazinone derivatives bear-

ing substituted hydrocarbon and substituted

benzylidene side chain (5a-s)

Scheme 1

Pyridazinone is nitrogen containing heterocyclic

compound along with a magical carbonyl moiety. The pre-

sence of these two structural features i.e. nitrogen atoms

and carbonyl group as a part of pyridazine ring makes it a

versatile scaffold for the development of pharmaceutical

agents.¹⁰Pyridazinone nucleus have been reported to pos-

sess an array of interesting and useful pharmacological ac-

tivities such as anticancer,¹¹anti-HIV,¹²antidepressant,¹³

anticonvulsant,¹⁰antihypertensive,¹⁴cardiotonic,¹⁵anti-

thrombotic,¹⁶diuretic,¹⁷antibacterial,¹⁸aldose reductase

inhibitors,¹⁹hepatoprotective²⁰and COX-2 inhibition.²¹

The ability of pyridazinone heterocyclic ring system to

scavenge free radicals could be one of the several mecha-

nisms by which they exhibit some of the useful actions viz.

anticancer, anti-inflammatory and cardioprotective ac-

tions. The pyridazinone nucleus is also added as an antioxi-

dant in several commercially available anti-aging formula-

tions.

RESULTS AND DISCUSSION

Chemistry

Compounds 2a-c were synthesized by Friedel Craft’s

acylation reaction of succinic anhydride with biphenyl, to-

luene and isobutyl benzene in presence of a lewis acid cata-

lyst, anhydrous aluminum chloride. The reaction leads to

succinoylation of aryl hydrocarbons which were further ni-

trated by electrophilic aromatic substitution reaction to ob-

tain 3a-c. Ring closure of 3-nitro derivative of (4-biphenyl/

tolyl/isobutylphenyl) 4-oxo-butanoic acid (3a-c) with hy-

drazine hydrate or phenyl hydrazine was carried out by

refluxing for 6 hours which afforded corresponding 2,3,

4,5-tetrahydropyridazin-3(2H)-one (4a-f) derivatives.

Condensation reaction of the pyridazinone ring (4a-f) with

aromatic aldehydes yielded the target compounds (5a-s) in

good to moderate yield (Table 1).

Minor structural modifications in the pyridazinone

skeleton lead to high variability in biological actions which

is quite beneficial in the design and development of new

safer antioxidants with improved efficacy, potency and

diminished toxicity. The structural activity relationship

(SAR) studies on pyridazinone derivatives revealed that in

general, presence of an acidic group, electron donating

group and a heterocyclic moiety enhance the antioxidant

activity of pyridazinone derivatives.^22,23Based on these

Chemical structures of newly prepared pyridazinone

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JOURNAL OF THE CHINESE

CHEMICAL SOCIETY

Antioxidant Activity of Pyridazinone Derivatives

Fe²⁺transformation was also investigated along with iron

chelating activity. Most of the screened compounds showed

comparably excellent antioxidant activity at par with posi-

tive control, ascorbic acid.

Table 1. Structure of substituted hydrocarbon and substituted

benzylidene side chain in pyridazinone derivatives (5a-

s)

*

R

*

R'

*

R'

O₂N

4''

O₂N

4

3''

2'

5

4

3'

2''

1''

4'

5

4

5

6

6'

5'

3'

2'

1'

3

6

3

4'

6

4'

O

H₃C

O

1'

H₃C

N

1

N

2

N

1

N

2

R

N

1

N

5'

5''

5' 6'

6''

3' 2'

R'

R

6'

2

DPPH free radical scavenging assay

CH₃

5a-f

5g-n

5o-s

The in vitro antioxidant activity of the target com-

pounds was evaluated in terms of hydrogen donating or

radical scavenging potential employing a modified DPPH

method. The interaction of the tested compounds resulting

in neutralization of the stable free radicals of DPPH illus-

trates their ability to scavenge free radicals in an iron-free

system. The results of % inhibition of DPPH free radical

assay by synthesized compounds are shown in Table 2. It

was observed that almost all the compounds showed good

DPPH radial scavenging activity (63.0-88.0%). Com-

pounds 5a, 5d, 5g, and 5j have shown excellent DPPH

radical scavenging activity ranging from 82.0 to 88.0% in

comparison to standard ascorbic acid (87.0%), while com-

pounds 5b, 5c, 5e, 5a, 5g, 5h, 5k-o, 5q, 5r have shown

good activity between 72.0 to 80.0%. The SAR studies in-

dicate that heterocyclic moiety is responsible for the anti-

oxidant activity but slight structural modification in the

Compound no

R

R¢

-furyl

5a

5b

5c

5d

5e

5f

5g

5h

5i

5j

5k

5l

5m

5n

5o

5p

5q

5r

-C₆H₅

-H

-C₆H₅

-H

-C₆H₅

-4-methoxy phenyl

-4-tolyl

-furyl

-4-tolyl

-4-methoxy phenyl

-furyl

-4-methoxy phenyl

-4-tolyl

-furyl

-4-tolyl

-4-methoxy phenyl

-4-nitrophenyl

-3-nitrophenyl

-4-tolyl

-4-methoxy phenyl

-3-nitrophenyl

-4-nitrophenyl

5s

Table 2. Antioxidant activity of compounds (5a-s)

derivatives were characterized by FTIR, ¹H NMR and ¹³

C

% Radial scavenging activity & metal chelating activity

NMR and mass spectral data. In ¹HNMR spectra of com-

pounds 4b, 4d, 4f presence of a singlet peak at d 8.00-8.75

ppm was assigned to NH protons while in compounds 4a,

4c, 4e this peak was missing as the hydrogen of NH was

substituted with phenyl ring and hence appearance of

multiplet at 6.95-7.60 ppm was accounted for aromatic pro-

tons of phenyl ring attached to nitrogen of pyridazinone

ring. Further, in ¹H NMR spectrum of 5a appearance of an

additional singlet peak for 1H at 6.57 ppm was observed

which was ascribed to methylidene proton. Similar pattern

of peaks were observed for other compounds of the series

also. Furthermore, structures of all the final compounds

were confirmed by their ¹³C NMR and mass spectrometry.

Antioxidant activity

(100 mg/mL)

Comp

DSA^[a]

HSA^[b]NOSA^[c]

ICA^[d]

RPA^[e]

5a

5b

5c

5d

5e

5f

5g

5h

5i

5j

5k

5l

5m

5n

5o

5p

5q

5r

86.85±2.3 80.30±3.1 78.09±2.6 80.86±1.1 0.981±0.2

75.53±1.5 70.00±2.0 69.89±2.5 77.03±1.0 0.920±0.2

72.47±2.1 66.66±1.8 65.45±1.5 66.98±1.6 0.941±0.2

88.37±2.2 81.51±2.1 81.19±1.1 82.45±1.5 0.887±0.1

81.65±1.9 71.21±1.1 71.85±1.5 54.86±1.0 0.940±0.2

60.85±1.5 64.84±1.5 69.67±1.5 68.89±1.5 0.929±0.1

84.09±2.2 82.42±2.0 82.52±1.9 81.81±1.8 0.993±0.2

78.59±1.9 69.09±1.5 64.56±1.1 64.91±1.2 0.909±0.2

62.99±1.5 65.75±1.6 62.34±1.6 59.64±1.5 0.959±0.2

82.26±2.5 84.54±1.2 84.27±1.5 83.25±1.9 0.986±0.2

79.51±1.6 62.12±2.1 58.18±2.1 54.86±1.1 0.960±0.2

78.89±1.8 60.90±1.6 68.14±1.2 66.50±2.5 0.987±0.2

80.12±2.6 55.75±1.1 71.01±2.2 60.12±2.1 0.938±0.1

77.98±1.2 53.93±1.9 65.04±1.3 78.14±2.6 0.901±0.1

72.17±2.3 57.57±1.2 60.61±1.1 54.54±2.1 0.937±0.3

69.72±2.1 65.15±1.3 59.29±1.5 70.01±2.3 0.971±0.2

78.89±1.2 55.45±1.5 63.93±2.1 76.23±2.3 0.942±0.2

80.12±2.6 52.42±1.1 61.72±1.2 78.78±2.0 0.967±0.2

76.15±1.1 57.87±1.5 72.78±2.6 68.26±1.2 0.950±0.2

The synthesized pyridazinone derivatives were

screened to assess their in vitro antioxidant activity using

five different antioxidant assay methods (Table 2). The

radical scavenging ability of the compounds was evaluated

against the 1,1-diphenyl-2-picrylhydrazyl (DPPH) stable

free radical, hydrogen peroxide & nitric oxide. Ferrous

(Fe²⁺) is known to be a most powerful pro-oxidant, there-

fore, the measurement of the reducing ability i.e. Fe³⁺to

5s

Ascorbic 87.46±2.8 83.93±2.1 83.80±1.5 83.09±1.2 0.992±0.2

acid

Values are mean ± SD, n = 3; [a] DPPH scavenging activity; [b]

H₂O₂scavenging activity; [c] Nitric oxide scavenging activity;

[d] Iron chelating activity; [e] Reducing power assay.

J. Chin. Chem. Soc. 2016, 63, 000-000

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Khokra et al.

basic nucleus alters the antioxidant activity and thus bio-

activity. In compounds 5a, 5d, 5g, and 5j, the higher anti-

oxidant activity of these pyridazinone derivatives may be

ascribed to the presence of furfuryl heterocyclic ring. Elec-

tron withdrawing group such as nitro on phenyl ring (5n,

5m, 5q, 5r and 5s) also seem to play a vital role in exhibit-

ing the antioxidant activity in comparison to the com-

pounds bearing a methyl or methoxy electron donating

group on phenyl ring. Compounds 5m and 5r showed

80.0% inhibition of DPPH radical while all the pyri-

dazinone derivatives with electron donating groups on

phenyl ring except 5e, exhibited lower antioxidant activity.

H₂O₂scavenging assay

decomposition in an aqueous solution at physiological pH

(7.2). The NO^-radicals in ideal conditions i.e. aerobic con-

ditions reacts with molecular oxygen to yield stable pro-

ducts (nitrate and nitrite), which can be quantitatively

analyzed by using Griess reagent.²⁶The potential of syn-

thesized title compounds to inhibit nitric oxide radical are

presented in Table 2. It is evident that the synthesized target

compounds possess promising nitric oxide radical scav-

enging activity. Compounds 5a, 5d, 5g, and 5j have shown

excellent nitric oxide radical scavenging activity (84.0-

78.0%) as compared to ascorbic acid (84.0%) and all the

rest of the compounds also showed good nitric oxide radi-

cal scavenging activity i.e. (72.0–59.0%).

In this method, the antioxidant activity of the target

compounds was determined in terms of anti-radial scav-

enging activity of hydroxyl radical. These radicals are

highly reactive and combine at high rate with reactant (tar-

get compounds). The results of H₂O₂free radical scaveng-

ing effect of synthesized target compounds is shown in Ta-

ble 2. It can be seen that almost all the compounds display

good H₂O₂radial scavenging activity. Four compounds viz.

5a, 5d, 5g and 5j were observed to be the most potent scav-

engers (85.0-80.0%) but 5j showed better antioxidant ac-

tivity as compared to ascorbic acid (84.0%). Compounds

5b & 5e exhibits good activity i.e. (71.0–70.0%) followed

by 5c, 5h, 5g, 5i, 5f, 5k, 5l, 5p, 5r (69.0-62.0%). Rests of

the compounds have also shown moderate hydroxyl radical

scavenging activity i.e. (58.0-52.0%).

Metal chelating assay

Ferrozine can form a red color complex with Fe²⁺.

The development of red color complex is restricted in the

presence of other chelating agents and eventually leads to a

decrease in the red color intensity of the ferrozine-Fe²⁺

complex. Measurement of the color reduction determines

the chelating activity to compete with ferrozine for the fer-

rous ions.²⁷Iron binding capacity of the synthesized com-

pounds and the reference compound EDTA were examined

as per the standard method and results are shown in Table 2.

Maximum chelation of metal ions was found to be for the

compound 5j (83.0%) and interestingly it was higher than

the reference compound (83.0%). The other synthesized

target compounds 5a, 5d, and 5g also showed excellent

chelating potency (82.0-81.0%) similar to positive control

while remaining compounds exhibited satisfactory activity

(55.0-78.0%).

Reducing power assay

The reducing power assay method is based on the

principle of increase in the absorbance of the reaction mix-

tures which indicates an increase in the antioxidant activ-

ity.²⁴The presence of reducers i.e. antioxidant causes the

conversion of the ferric (Fe³⁺) to the ferrous form (Fe²⁺).

Reduction of Fe(III) is often used as an indicator of elec-

tron donating activity. Reducing power of compounds is

shown in Table 2. Compounds 5a, 5d, 5g and 5j have

shown significant increase in absorbance when compared

to ascorbic acid. All the remaining compounds also showed

significant increase in absorbance.

Docking study

Docking is routinely used to study binding of an in-

hibitor to a receptor. It can identify the correct conforma-

tion of ligands in the binding pocket of a protein and can

correctly predict the affinity between the ligand and the

protein.^28-30Therefore, to gain better understanding on the

potency and SAR studies of the synthesized compounds,

we further examined the interactions of the active com-

pounds with target enzyme.

Compounds 5a-s were selected as ligands for docking

studies with established antioxidant target, xanthine oxido-

reductase. This receptor is the most important molecular

target in the design and discovery of successful antioxidant

drugs. In the present study, Molegro Virtual Docker was

used for automated flexible ligand docking of 5a-s with

above mentioned target and MolDock score and H-bond

properties were evaluated (Table 3).

Nitric oxide scavenging assay

Nitric oxide synthases (NOS) specifically generate

relatively unstable, diatomic nitric oxide radical (NO^-) in

biological tissues. NOS metabolize arginine to citrulline

with the production of a NO^-through a five electron oxida-

tive reaction.²⁵This in vitro method utilize sodium nitro-

prusside compound which produces NO^-after undergoing

4

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Antioxidant Activity of Pyridazinone Derivatives

Table 3. Docking result of compounds (5a-s)

5l

-116.197

8

2.99 Cys103 O of OCH₃with S

2.99 Arg212 O of NO₂with N

2.96 Arg212 N of NO₂with N

3.00 Arg212 N of NO₂with N

3.05 Arg102 N of NO₂with N

2.78 Arg105 N of NO₂with N

2.90 Arg105 N of NO₂with N

3.19 Arg105 N of pyridazinone

with N

2.71 Arg105 O of ketone with N

3.38 Arg105 N of NO₂with N

3.38 Cys103 N of NO₂with S

2.75 Cys103 O of NO₂with S

Mol

Comp Dock

Score

H-bond Amino

distance acid

(Å) involved

No. of

H-bond

Structural feature

5a

5b

5c

-132.45

-128.242

-118.01

4

6

3.25

2.60

Thr28 N of NO₂with O

Thr28 O of NO₂with O

3.33 Arg108 N of NO₂with N

2.55 Arg108 O of NO₂with N

3.16 Arg108 N of NO₂with N

2.60 Arg108 O of NO₂with N

3.53 Arg108 O of NO₂with N

3.11 Cys103 O of OCH₃with S

2.86 Arg212 O of NO₂with N

3.38 Arg212 N of NO₂with N

2.98 Arg212 O of NO₂with N

3.04 Arg212 O of NO₂with N

3.48 Arg105 N of NO₂with N

5m

5n

-121.022

5

3.31

Thr71 O of NO₂with O

-132.374 10

2.64 Cys103 N of NO₂with S

3.30 Cys103 O of NO₂with S

3.17

Thr74 O of NO₂with O

3.37 Arg212 O of NO₂with N

3.42 Arg212 O of NO₂with N

3.47 Arg105 O of NO₂with N

2.50 Arg105 O of NO₂with N

3.24 Arg105 O of NO₂with N

2.44 Arg105 O of ketone with N

2.80 Thr107 O of ketone with O

3.44 Arg105 N of ketone with N

3.39 Arg128 N of NO₂with N

2.94 Arg128 O of NO₂with N

3.08

Thr74 O of ketone with O

5d

-147.679

8

2.98 Arg121 O of furfuryl with N

2.99 Arg105 O of furfuryl with N

2.25 Arg105 O of furfuryl with N

2.60 Thr107 O of pyridazinone

2.97 Thr107 with O

2.60 Cys103 N of pyridazinone

2.88 Cys103 with O

5o

5p

5q

-119.012

-119.167

-140.13

6

5

7

2.75

Glu31 O of NO₂with S

O of NO₂with S

N of NO₂with N

2.60

3.20

Thr74 O of NO₂with O

Thr74 N of NO₂with N

5e

5f

-111.232

-106.232

2

4

2.61

Thr74 O of ketone with N

3.00 Asp205 O of pyridazinone

with O

2.60 Thr107 O of ketone with O

3.50 Arg105 O of ketone with N

2.97 Arg105 O of ketone with N

3.48 Arg128 O of NO₂with N

2.60 Arg128 N of NO₂with N

Thr74 O of NO₂with O

3.10 Asp205 N of pyridazinone

2.93 Arg128 with O

3.27 Cys103 N of pyridazinone

2.51 Cys103 with N

N of NO₂with S

3.19 Cys103 O of ketone with S

3.29 Cys103 N of NO₂with S

2.66 Cys103 O of NO₂with S

2.90 Arg103 O of NO₂with N

3.35 Arg103 N of NO₂with N

3.42 Arg103 N of NO₂with N

2.91 Arg103 O of NO₂with N

3.32 Cys103 N of NO₂with S

2.86 Cys103 O of NO₂with S

3.08 Arg105 N of NO₂with N

3.30 Arg105 O of NO₂with N

2.90 Arg212 O of NO₂with N

3.38 Arg212 N of NO₂with N

2.93 Arg212 O of ketone with N

3.41 Arg105 N of NO₂with N

2.86 Arg105 N of NO₂with N

3.46 Arg128 N of NO₂with O

3.06 Arg128 O of NO₂with N

O of NO₂with S

5g

-161.066

4

2.72 Cys103 O of NO₂with S

3.24 Cys103 N of NO₂with S

3.23

2.60

3.21

Thr74 O of NO₂with O

Thr74 O of furfuryl with O

Glu31 O of NO₂with N

5h

5i

-116.675

-115.66

3

4

2.82 Cys103 O of NO₂with S

3.33 Cys103 O of NO₂with S

3.10 Cys103 O of OCH₃with S

2.68 Thr107 O of NO₂with O

3.10 Thr107 N of NO₂with O

3.00 Arg105 O of NO₂with N

5r

-106.144

9

5j

-158.82

8

6

2.99

3.01

2.26

Thr74 O of furfuryl with O

Tyr30 O of furfuryl with O

Thr30 O of ketone with O

2.95 Arg128 N of pyridazinone

2.37 Arg105 with N

2.94 Arg105 O of NO₂with N

N of NO₂with N

2.60 Thr107 O of ketone with O

2.77 Thr107 N of pyridazinone

2.89 Trp106 with O

5s

-126.639

6

2.60

Thr74 N of NO₂with O

2.63 Arg105 O of NO₂with N

3.54 Arg105 O of NO₂with N

5k

-126.022

1.89

Thr74 O of NO₂with O

2.90 Cys103 N of pyridazinone

2.88 Cys103 with N

Compounds 5a, 5d, 5g and 5j exhibited good affinity

2.99

Thr74 N of NO₂with S

N of NO₂with S

and binding properties with xanthine oxidoreductase re-

O of NO₂with O

ceptor and showed MolDock score of -132.0, -148.0,

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Khokra et al.

-161.0, -159.0 and a total of 4, 8, 4 & 8 hydrogen bond in-

teractions respectively. Compound 5g exhibited good bind-

ing properties with xanthine oxidoreductase receptor hav-

ing MolDock score of -161.0 and 4H-bond. Based on the

docking results, it can be proposed that the synthesized

compounds would act as inhibitor of xanthine oxidore-

ductase and prevent the formation of reactive oxygen spe-

cies. The docking study results of compounds (5a-s) with

crystal structure of xanthine oxidoreductase (PDB entry

1R4U) are presented in Table 3.

COOH), 2.84 (t, 2H, -CH₂-CH₂-COOH), 7.35 (m, 3H, H-3,4,5,

phenyl), 7.46 (m, 2H, H-2,6, phenyl), 7.52 and 7.90 (d, each, 2x

A₂B₂, p-substituted phenyl); MS (m/z): 254 (M⁺; C₁₆H₁₄O₃).

Synthesis of 4-(4’-tolyl)-4-oxo-butanoic acid (2b): Ap-

proximately 1 g of succinic anhydride was dissolved in 8 mL of

freshly dried toluene in a 50 mL rb flask. To the above mixture, 3

g of fresh aluminum chloride was added while stirring and the

contents were heated to 90 °C for 45 min. The mixture was al-

lowed to stand at room temperature to cool down. Sufficient cold

water was added to the cooled mixture to hydrolyze AlCl₃fol-

lowed by addition of dilute sodium hydroxide solution to make

the solution basic in nature. The mixture was filtered and to the

filtrate, dilute hydrochloric acid was added until it became perma-

nent acidic in nature. Precipitates so formed on acidification were

filtered, washed with plenty of cold water and recrystallized from

aqueous ethanol to obtain creamish needle shaped crystals which

were dried at room temperature. Yield 72%, m.p. 142-145 °C, R_f–

0.86 (T: E: F-5:4:1); IR (KBr) v_max(cm^-1): 1682 (C=O), 3433-

3410 (b, OH).¹HNMR (DMSO): d 2.16 (s, 3H, CH₃), 2.46 (t, 2H,

-CH₂-CH₂-COOH), 2.84 (t, 2H, -CH₂-CH₂-COOH), 7.18 (d, 2H,

Ar-H-3,5), 7.67 (d, 2H, Ar- H-2,6); MS (m/z): 192 (M⁺; C₁₁H₁₂O₃)

Synthesis of 4-(4-isobutylphenyl)-4-oxo-butanoic acid

(2c): Succinic anhydride (0.1 mol) was dissolved in dried

isobutylbenzene (50 mL) under anhydrous condition in the pre-

sence of anhydrous AlCl₃(0.2 mol) and the contents were re-

fluxed for 2-3 h and then decomposed by adding sufficient quan-

tity of sodium hydroxide solution. The resulting mixture was fil-

tered and subsequently neutralized by addition of dilute hydro-

chloric acid. Finally, precipitates of 4-isobutylbenzoyl 3-pro-

panoic acid were filtered, washed with plenty of cold water and

recrystallized with ethanol to obtain colorless crystals. Yield

68%, m.p. 116-120 °C, R_f- 0.64 (T: E: F-5:4:1), IR (KBr) v_max

(cm^-1): 1728, 1682, (C=O), 3433-3418 (b, OH); ¹H NMR (CDCl₃,

in ppm) d 1.03 (d, 6H, 2xCH₃isobutyl protons), 2.05 (t, 1H, -CH

isobutyl protons), 2.21 (d, 2H, -CH₂isobutyl protons), 2.40 (t,

2H, -CH₂-CH₂-COOH), 2.80 (t, 2H, -CH₂-CH₂-COOH), 7.25 (d,

2H, Ar-H-3,5), 7.90 (d, 2H, Ar-H-2,6); MS (m/z): 234 (M⁺;

C₁₄H₁₈O₃).

EXPERIMENTAL

General: Melting points of synthesized compounds were

recorded on glass slides using Lab India MR-VIS visual melting

range apparatus and are uncorrected. Infrared spectra (IR) were

recorded on Hitachi spectrophotometer 150-20. Nuclear mag-

netic resonance (¹HNMR) and ¹³C spectra were obtained using

Bruker 400 instrument in DMSO & CDCl₃. Chemical shift values

are given in ppm (d) against tetramethylsilane (TMS) as internal

standard & J values are reported in Hz. The reactions were moni-

tored on silica gel G coated TLC plates and the purified com-

pounds showed a single spot; unless stated otherwise iodine vapor

and/or UV light were used for detection of compounds. The used

solvents were dried with sodium metal (toluene, isobutyl ben-

zene) and fused calcium chloride (dichloromethane). All the start-

ing materials & reagents used were of analytical grade.

Procedure for the synthesis of (4-Biphenyl/tolyl/

isobutylphenyl) 4-oxo-butanoic acid (2a-c)

Synthesis of 4-biphenyl-4-oxobutanoic acid (2a): In a

round bottom (rb) flask, succinic anhydride (0.1 mol) was dis-

solved in a sufficient volume of freshly dried dichloromethane

(DCM) and the reaction mixture was cooled to 0 °C using an ice

bath. Anhydrous aluminum chloride was added (0.12 mol) with

stirring in small proportion under anhydrous conditions. Sepa-

rately, a solution of biphenyl (0.1 mol) was prepared in DCM

which was added drop wise to above reaction mixture. During the

addition of biphenyl the temperature of the reaction mixture was

maintained below 15 °C, which changed its color, first to deep

yellow, then green and finally black. After complete addition of

biphenyl solution, the reaction mixture was further stirred at a

room temperature for additional 2 h on a magnetic stirrer. Finally

the reaction mixture was decomposed by adding 10% HCl (100

mL) with stirring to obtain off-white precipitates of fenbufen i.e.

{4-(biphenyl-4-yl)-4-oxobutanoic acid}³¹which were and washed

with petroleum ether. Yield 74%, m.p. 180-182 °C, R_f– 0.64

(T:E:F-5:4:1), IR (KBr) v_max(cm^-1): 1713, 1636 (C=O), 3410 -

(OH); ¹HNMR (DMSO, in ppm): d 2.40 (t, 2H, -CH₂-CH₂-

General procedure for the synthesis of 3-Nitro deriv-

ative of (4-biphenyl/tolyl/isobutylphenyl) 4-oxo-

butanoic acid (3a-c)

Synthesis of 4-(4-(3-nitrophenyl) phenyl)-4-oxo-buta-

noic acid (3a): A mixture of conc. nitric acid and sulphuric acid

(12 mL each) was stirred mechanically followed by addition of 6

g of compound 2a in small increment maintaining the mixture

temperature between 0 to 10 °C by efficient cooling. The mixture

was stirred gently in the ice water and the temperature was gradu-

6

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J. Chin. Chem. Soc. 2016, 63, 000-000

JOURNAL OF THE CHINESE

CHEMICAL SOCIETY

Antioxidant Activity of Pyridazinone Derivatives

ally allowed to rise till 15 °C over next 2 h. The precipitates ob-

tained were washed with cold water and re-crystallized from

methanol to get light yellow color product. Yield 54%, m.p. 168

°C, R_f– 0.64 (T: E: F-5:4:1); IR (KBr) v_max(cm^-1): 1713, 1682

(C=O), 1353 (NO₂), 3400-3200 (OH); ¹HNMR (DMSO in ppm):

d 2.40 (t, 2H, -CH₂-CH₂-COOH), 2.84 (t, 2H, -CH₂-CH₂-COOH),

7.50-8.25 (m, 8H, Ar-H); MS (m/z): 299 (M⁺; C₁₆H₁₃NO₅).

Synthesis of 3-Nitro-4-tolyl 4-oxobutanoic acid (3b):

Compound (3b) was synthesized from compound (2b) following

the above procedure. Yield 50%, m.p. 133 °C, R_f– 0.54 (T: E:

F-5:4:1), IR (KBr) v_max(cm^-1): 1682, 1582 (C=O), 1520 (NO₂),

3433-3410 (b, OH); ¹HNMR (DMSO): d 2.26 (t, 3H, CH₃), 2.46

(t, 2H, CH₂-CH₂-COOH), 2.86 (t, 2H, -CH₂-CH₂-COOH), 7.35

(d, 1H, H-6), 8.20 (d, 1H, H-7), 8.75 (s, 1H, H-6); MS (m/z): 237

(M⁺; C₁₁H₁₁NO₅).

Ar-H), 8.51 (s, 1H, NH pyridazinone protons); MS (m/z): 233

(M⁺; C₁₁H₁₁N₃O₃).

General procedure for the synthesis of target

compounds (5a-s)

(4Z)-4-(Furan-2-ylmethylidene)-6-[4-(3-nitrophenyl)ph-

enyl]-2-phenyl-2,3,4,5-tetrahydropyridazin-3-one (5a): The

title compounds were synthesized by condensing compound (4a)

(0.005 mol) with fufuraldehyde (0.005 mol) in glacial acetic acid

(20 mL) in presence of sodium acetate (2g). The reaction mixture

was refluxed for 6-8 h on a water bath, cooled & poured on to the

ice. The solid compound obtained was recrystallized with etha-

nol. Yield 60%, m.p. 400 °C, R_f- 0.58 (T: E: F-5:4:1); IR (KBr)

v_max(cm^-1): 1645 (C=O), 1353 (NO₂), 1090 (C-O-C) and 1580

(C=C) exo, 1335 (-C=N); ¹H NMR (DMSO, in ppm): d 2.3 (s, 2H,

–CH₂pyridazinone protons), 6.57 (s, 1H, CH*), 7.5 (d, 2H, H-2’,

6’), 7.3 (d, 2H, H-3’, 5’), 7.87 (d, 1H, H-6”), 7.55 (d, 1H, H-4”),

8.13 (d, 1H, H-5”), 8.45 (s, 1H, H-2”), 7.75 (d, 1H, H-3”’), 6.75

(s, 1H, H-4”’), 7.01 (d, 1H, H-5”’), 7.0-7.54 (m, 5H, N-phenyl

protons); ¹³C (75 MHz, DMSO, in ppm): d 159.6 (C-3), 135.2

(C-4), 32.1 (C-5), 141.3 (C-6), 132.7 (C-1’), 129.3 (C-2’), 128.0

(C-3’), 138.7 (C-4’), 128.0 (C-5’), 129.3 (C-6’), 137.2 (C-1”),

134.02 (C-2”), 130.82 (C-3”), 120.02 (C-4”), 148.82 (C-5”),

122.32 (C-6”), 138.1 (C*), 151.6 (C-2”’), 145.7 (C-3”’), 112.6

(C-4”’), 111.3 (C-5”’), 137.6 (C-1””), 121.5 (C-2””), 129.0

(C-3””), 124.2 (C-4””), 129.0 (C-5””), 121.4 (C-6””). MS (m/z):

449 (M⁺; C₂₇H₁₉N₃O₄).

Synthesis of 3-Nitro-4-isobutylphenyl 4-oxobutanoic

acid (3c): Compound (3c) was synthesized from compound (2c)

following the above procedure. Yield 66%, m.p. 104 °C, R_f– 0.55

(T: E: F-5:4:1), IR (KBr) v_max(cm^-1): -1682, 1582 (C=O), 3433-

3410 (b, OH); ¹H NMR (CDCl₃, in ppm) d: 1.03 (d, 6H, 2xCH₃

isobutyl protons), 2.25 (t, 1H, CH isobutyl protons), 2.23 (d, 2H,

CH₂isobutyl protons), 2.40 (t, 2H, CH₂-CH₂-COOH), 2.80 (t, 2H,

-CH₂-CH₂-COOH), 8.00 (d, 1H, H-5), 8.4 (d, 1H, H-6), 8.7 (s, 1H,

H-2); MS (m/z): 279 (M⁺; C₁₄H₁₇NO₅).

General procedure for the synthesis of 6-{3-Nitro(bi-

phenyl/4-tolyl/4-isobutylphenyl)}-2-phenyl-2,3,4,5-tetrahy-

dropyridazin-3(2H)-one (4a), (4c), (4e): The compound 3a-c

was refluxed on water bath for 6 hours with phenyl hydrazine

(0.01 mol) in methanol (10 mL) containing sodium acetate (50

mg). The excess of solvent was evaporated and the concentrated

reaction mixture was poured over crushed ice to obtain the final

compound which was recrystallized with ethanol. 4a: Yield 65%,

m.p. 86-87 °C, R_f– 0.52 (T: E: F-5:4:1), IR (KBr) v_max(cm^-1):

1675 (CONH₂), 1353 (NO₂), 1507 (-C=N); ¹HNMR (DMSO, in

ppm): d 1.35 (t, 2H, CH₂, pyridazinone protons C-5), 1.58 (t, 2H,

CH₂, pyridazinone protons C-4), 7.0-7.12 (m, 4H, H-2’, 3’, 5’,

6’), 7.87 (d, 1H, H-2”), 7.55 (t, 1H, H-3”), 8.13 (d, 1H, H-5”),

8.45 (s, 1H, H-6”), 7.00-7.55 (m, 5H, N-phenyl protons). MS

(m/z): 371 (M⁺; C₂₂H₁₇N₃O₃).

(4Z)-4-[(4-methoxyphenyl)methylidene]6-[4-(3-nitroph-

enyl)phenyl]-2-phenyl-2,3,4,5-tetrahydropyridazin-3-one

(5b): Compound (5b) was synthesized from (4a) and 4-methoxy

benzaldehyde following the above procedure. Yield 59%, m.p.

395 °C, R_f- 0.59 (T: E: F-5:4:1), IR (KBr) v_max(cm^-1): 1687

(C=O), 1334 (-C=N), 1350 (NO₂), 1580 (C=C), and 1115 (C-

1

O-C); H NMR (DMSO, in ppm): d 2.3 (s, 2H, pyridazinone

–CH₂), 3.90 (s, 3H, OCH₃), 6.51 (s, 1H, CH*), 6.9-7.14 (m, 5H,

N-phenyl protons), 7.2 (d, 2H, H-2’, 6’), 7.4 (d, 2H, H-3’, 5’),

7.1-7.44 (m, 4H, Ar-H-2”, 4”, 5”, 6”), 6.69-7.47 (m, 4H, Ar-H of

benzylidine); ¹³C (DMSO, d): 159.6 (C-3), 135.2 (C-4), 32.1

(C-5), 141.3 (C-6), 132.7 (C-1’), 129.3 (C-2’), 128.0 (C-3’),

138.7 (C-4’), 128.0 (C-5’), 129.3 (C-6’), 137.2 (C-1”), 134.02

(C-2”), 130.82 (C-3”), 120.02 (C-4”), 148.82 (C-5”), 122.32

(C-6”), 138.1 (C*), 127.4(C-1”’), 127.3 (C-2”’), 114.1 (C-3”’),

159.8 (C-4”’), 114.1 (C-5”’), 127.3 (C-6”’), 55.8 (OCH₃), 137.6

(C-1””), 121.5 (C-2””), 129.0 (C-3””), 124.2 (C-4””), 129.0

(C-5””), 121.4 (C-6””). MS (m/z): 489 (M⁺; C₃₀H₂₃N₃O₄).

Synthesis of 6-{3-Nitro(biphenyl/4-methylphenyl/4-iso-

butylphenyl)}-2,3,4,5-tetrahydropyridazin-3(2H)-one (4b),

(4d), (4f): Compounds (4b), (4d), (4f) were synthesized from

(3a-c), following the above procedure using hydrazine hydrate

4b: Yield 67%, m.p. 215-217 °C, R_f- 0.50 (T: E: F-5:4:1); IR

(KBr) v_max(cm^-1): 1682, 1582 (CONH₂), 1520 (NO₂); ¹H NMR

(DMSO, in ppm): d 1.3 (t, 2H, CH₂, pyridazinone protons at C-5),

1.7 (t, 2H, CH₂, pyridazinone protons at C-4)), 7.1-8.45 (m, 3H,

(4Z)-4-[(4-methylphenyl)methylidene]6-[4-(3-nitroph-

enyl)phenyl]-2-phenyl-2,3,4,5-tetrahydropyridazin-3-one

(5c): Compound (5c) was synthesized from (4a) using tolual-

J. Chin. Chem. Soc. 2016, 63, 000-000

www.jccs.wiley-vch.de

7

Article

Khokra et al.

dehyde. Yield 55%, m.p. 395 °C, R_f- 0.45 (T: E: F-5:4:1), IR

(KBr) v_max(cm^-1): 1690 (C=O), 1335 (-C=N), 1352 (NO₂), 1580

(C=C) exo; ¹H NMR (DMSO, in ppm): d 2.25 (s, 3H, CH₃), 2.3 (s,

2H, –CH₂pyridazinone protons), 6.57 (s, 1H, CH*), 7.0-7.6 (m,

4H, Ar-H 2’, 3’, 5’, 6’), 7.55-8.51 (m, 4H, Ar-H-2”, 4”, 5”, 6”),

6.69-7.25 (bm, 4H, Ar-H of benzylidine), 7.01-7.59 (m, 5H,

N-phenyl protons); ¹³C (75 MHz, DMSO, d): 159.6 (C-3), 135.2

(C-4), 32.1 (C-5), 141.3 (C-6), 132.7 (C-1’), 129.3 (C-2’), 128.0

(C-3’), 138.7 (C-4’), 128.0 (C-5’), 129.3 (C-6’), 137.2 (C-1”),

134.02 (C-2”), 130.82 (C-3”), 120.02 (C-4”), 148.82 (C-5”),

122.32 (C-6”), 138.1 (=C-benzylidene), 127.4 (C-1”’), 127.3

(C-2”’), 114.1 (C-3”’), 159.8 (C-4”’), 114.1 (C-5”’), 127.3

(C-6”’), 55.8 (OCH₃), 137.6 (C-1””), 121.5 (C-2””), 129.0

(C-3””), 124.2 (C-4””), 129.0 (C-5””), 121.4 (C-6””), 24.2 (CH₃).

MS (m/z): 473 (M⁺; C₃₀H₂₃N₃O₃).

4-methoxy benzaldehyde. Yield 57%, m.p. 387 °C, R_f- 0.55 (T:

E: F-5:4:1), IR (KBr) v_max(cm^-1): 1689 (CONH₂), 1355 (NO₂),

1

1570 (C=C), 1080 (C-O-C), 1595 (-C=N); H NMR (DMSO, in

ppm): d 2.3 (s, 2H, –CH₂pyridazinone protons), 3.92 (s, 3H,

OCH₃), 6.57 (s, 1H, CH*), 7.0 (d, 2H, H-3’, 5’), 7.9 (d, 2H, H-2’,

H-6’), 7.55-8.47 (m, 4H, Ar-H-2”, 4”, 5”, 6”), 6.41-7.21 (m, 4H,

Ar-H of benzylidine), 10.3 (s, 1H, NH); ¹³C (DMSO, in ppm): d

162.6 (C-3), 135.2 (C-4), 32.1 (C-5), 141.3 (C-6), 132.7 (C-1’),

129.3 (C-2’), 128.0 (C-3’), 138.7 (C-4’), 128.0 (C-5’), 129.3

(C-6’), 137.2 (C-1”), 134.02 (C-2”), 130.82 (C-3”), 120.02

(C-4”), 148.82 (C-5”), 122.32 (C-6”), 138.1 (C*), 127.4 (C-1”’),

127.3 (C-2”’), 114.1 (C-3”’), 159.8 (C-4”’), 114.1 (C-5”’), 127.3

(C-6”’), 55.8 (OCH₃); MS (m/z): 413 (M⁺; C₂₄H₁₉N₃O₄).

(4Z)-4-(Furan-2-ylmethylidene)-6-(4-methyl-3-nitroph-

enyl)-2-phenyl-2,3,4,5-tetrahydropyridazin-3-one (5g): Com-

pound (5g) was synthesized from (4c) using fufuraldehyde. Yield

60%, m.p. 198 °C, R_f– 0.50 (T: E: F-5:4:1), IR (KBr) v_max(cm^-1):

1682 (C=O), 1674, 1520 (NO₂), 1126 (C-O-C), 1580 (C=C), 1597

(-C=N); ¹H NMR (CDCl₃, in ppm): d 2.1 (s, 3H, CH₃), 2.3 (s, 2H,

CH₂pyridazinone protons), 6.57 (s, 1H, CH*), 7.4 (d, 1H, H-6’),

7.6 (d, 1H, H-5’), 8.2 (s, 1H, Ar-H-2’), 6.75-7.75 (m, 3H, Furyl),

7.0-7.54 (m, 5H, N-phenyl protons); ¹³C (CDCl₃, in ppm): d 159.7

(C-3), 135.2 (C-4), 32.0 (C-5), 141.3 (C-6), 131.8 (C-1’), 135.1

(C-2’), 130.2 (C-3’), 138.5 (C-4’), 149.1 (C-5’), 124.0 (C-6’),

15.6 (CH₃), 138.1 (C*), 127.3 (C-2”), 114.1 (C-3”), 159.8 (C-4”),

114.1 (C-5”), 127.3 (C-1”’), 121.5 (C-2”’), 129.0 (C-3”’), 124.2

(C-4”’), 129.0 (C-5”’), 121.4 (C-6”’); MS (m/z): 387 (M⁺;

C₂₂H₁₇N₃O₄).

(4Z)-4-(Furan-2-ylmethylidene)-6-[4-(3-nitrophenyl)ph-

enyl]-2,3,4,5-tetrahydropyridazin-3-one (5d): Compound (5d)

was synthesized from (4a) and fufuraldehyde. Yield 58%, m.p.

395 °C, R_f- 0.42 (T: E: F-5:4:1), IR (KBr) v_max(cm^-1): 1683

(CONH₂), 1360 (NO₂), 1565 (C=C), 1120 (C-O-C), 1510-(C=N);

¹H NMR (DMSO, in ppm): d 2.3 (s, 2H, –CH₂pyridazinone pro-

tons), 6.57 (s, 1H, CH*), 7.3 (d, 2H, H-2’, 6’), 7.1 (d, 2H, H-3’,

5’), 7.55-8.15 (m, 4H, Ar-H-2”, 4”, 5”, 6”), 6.4-7.40 (m, 3H,

Furyl protons), 10.9 (s, 1H, NH). ¹³C (DMSO, in ppm): d 162.6

(C-3), 135.2 (C-4), 32.0 (C-5), 141.3 (C-6), 132.7 (C-1’), 129.3

(C-2’), 128.0 (C-3’), 138.7 (C-4’), 128.0 (C-5’), 129.3 (C-6’),

137.2 (C-1”), 134.02 (C-2”), 130.82(C-3”), 120.02 (C-4”),

148.82 (C-5”), 122.32 (C-6”), 138.1 (C*), 151.6 (C-2”’), 145.7

(C-3”’), 112.6 (4”’), 111.3 (5”’). MS (m/z): 373 (M⁺; C₂₁H₁₅N₃O₄).

(4Z)-4-[(4-methylphenyl)methylidene]6-[4-(3-nitroph-

enyl)phenyl]-2,3,4,5-tetrahydropyridazin-3-one (5e): Com-

pound (5e) was synthesized from (4b) using tolualdehyde. Yield

56%, m.p. 367 °C, R_f- 0.51 (T: E: F-5:4:1), IR (KBr) v_max(cm^-1):

1682 (CONH₂), 1350 (NO₂), 1550 (C=C), 1316 (-C=N). ¹H NMR

(DMSO, in ppm): d 2.3 (s, 2H, –CH₂pyridazinone protons), 2.23

(s, 3H, CH₃), 6.57 (s, 1H, CH*), 7.2 (d, 2H, H-3’, 5’), 7.8 (d, 2H,

H-2’, 6’), 7.53-8.45 (m, 4H, Ar-H-2”, 4”, 5”, 6”), 10.5 (s, 1H,

NH), 6.39-7.21 (m, 4H, Ar-H of benzylidine); ¹³C (DMSO, in

ppm): d 159.6 (C-3), 135.2 (C-4), 32.1 (C-5), 141.3 (C-6), 132.7

(C-1’), 129.3 (C-2’), 128.0 (C-3’), 138.7 (C-4’), 128.0 (C-5’),

129.3 (C-6’), 137.2 (C-1”), 134.02 (C-2”), 130.82 (C-3”), 120.02

(C-4”), 148.82 (C-5”), 122.32 (C-6”), 138.1 (C*), 127.4 (C-1”’),

127.3 (C-2”’), 114.1 (C-3”’), 159.8 (C-4”’), 114.1 (C-5”’), 127.3

(C-6”’), 24.2 (CH₃); MS (m/z): 397 (M⁺; C₂₄H₁₉N₃H₃).

(4Z)-4-[(4-methoxyphenyl)methylidene]-6-(4-methyl-3-

nitrophenyl)2-phenyl-2,3,4,5-tetrahydropyridazin-3-one

(5h): Compound (5h) was synthesized from (4c) using 4-me-

thoxy benzaldehyde. Yield 61%, m.p. 185 °C, R_f– 0.49 (T: E:

F-5:4:1), IR (KBr) v_max(cm^-1): 1682 (C=O), 1520 (NO₂), 1072

1

(C-O-C), 1580 (C=C); H NMR (CDCl₃, in ppm): d 2.1 (s, 2H,

CH₂pyridazinone protons), 2.2 (s, 3H, CH₃), 3.90 (s, 3H, OCH₃),

6.55 (s, 1H, CH*), 7.2 (d, 1H, Ar-H-3’), 7.4 (d, 1H, Ar-H-6’), 8.4

(s, 1H, Ar-H-2’), 6.69-7.44 (m, 4H, Ar-H of benzylidine), 6.9-

7.44 (m, 5H, N-phenyl protons); ¹³C (CDCl₃, in ppm) d: 159.7

(C-3), 135.2 (C-4), 32.0 (C-5), 141.3 (C-6), 131.8 (C-1’), 135.1

(C-2’), 130.2 (C-3’), 138.5 (C-4’), 149.1 (C-5’), 124.0 (C-6’),

15.6 (CH₃), 138.1 (C*), 127.4 (C-1”), 127.3 (C-2”), 114.1 (C-3”),

159.8 (C-4”), 114.1 (C-5”), 127.3 (C-6”), 55.8 (OCH₃), (C-1”’),

121.5 (C-2”’), 129.0 (C-3”’), 124.2 (C-4”’), 129.0 (C-5”’), 121.4

(C-6”’); MS (m/z): 427 (M⁺; C₂₅H₂₁N₃O₄).

(4Z)-4-[(4-methoxyphenyl)methylidene]6-[4-(3-nitroph-

enyl)phenyl]-2-phenyl-2,3,4,5-tetrahydropyridazin-3-one

(5f): Compound (5f) was synthesized from (4b) following using

(4Z)-4-[(4-methylphenyl)methylidene]-6-(4-methyl-3-

nitrophenyl)-2,3,4,5-tetrahydropyridazin-3-one (5i): Com-

pound (5i) was synthesized from (4c) using tolualdehyde. Yield

8

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J. Chin. Chem. Soc. 2016, 63, 000-000

JOURNAL OF THE CHINESE

CHEMICAL SOCIETY

Antioxidant Activity of Pyridazinone Derivatives

63%, m.p. 201 °C, R_f– 0.53 (T: E: F-5:4:1), IR (KBr) v_max(cm^-1):

1682 (CONH₂), 1520 (NO₂), 1580 (C=C), 1620 (-C=N); ¹H NMR

(CDCl₃, in ppm): d 2.5 (s, 2H, CH₂pyridazinone protons), 2.2 (s,

3H, CH₃), 2.25 (s, 3H, CH₃), 6.45 (s, 1H, CH*), 7.0 (d, 1H,

Ar-H-3’), 7.6 (d, 1H, Ar-H-6’), 8.3 (s, 1H, Ar-H-2’), 6.69-7.59

(m, 4H, Ar-H of benzylidine), 7.01-7.59 (m, 5H, N-phenyl pro-

tons); ¹³C (CDCl₃, in ppm): d 159.7 (C-3), 135.2 (C-4), 32.0

(C-5), 141.3 (C-6), 131.8 (C-1’), 135.1 (C-2’), 130.2 (C-3’),

138.5 (C-4’), 149.1 (C-5’), 124.0 (C-6’), 15.6 (CH₃), 138.1 (C*),

127.4 (C-1”), 127.3 (C-2”), 114.1 (C-3”), 159.8 (C-4”), 114.1

(C-5”), 127.3 (C-6”), 24.2 (CH₃), (C-1”’), 121.5 (C-2”’), 129.0

(C-3”’), 124.2 (C-4”’), 129.0 (C-5”’), 121.4 (C-6”’); MS (m/z):

411 (M⁺; C₂₅H₂₁N₃O₃).

1H, Ar-H-5’), 7.7 (d, 1H, Ar-H-6’), 7.9 (s, 1H, Ar-H-2’), 6.69 (d,

2H, H-3”, 5”), 7.15 (d, 2H, H-2”, 6”), 8.3 (s, 1H, NH). ¹³C

(CDCl₃, in ppm): d 162.6 (C-3), 135.2 (C-4), 32.1 (C-5), 141.3

(C-6), 131.8 (C-1’), 135.1 (C-2’), 130.2 (C-3’), 138.5 (C-4’),

149.1 (C-5’), 124.0 (C-6’), 15.6 (CH3), 138.1 (C*), 127.4 (C-1”),

127.3 (C-2”), 114.1 (C-3”), 159.8 (C-4”), 114.1 (C-5”), 127.3

(C-6”), 55.8 (OCH₃); MS (m/z): 351 (M⁺; C₁₉H₁₇N₃O₄).

(4Z)-4-[(4-nitrophenyl)methylidene]-6-(4-methyl-3-ni-

tro-phenyl)-2,3,4,5-tetrahydropyridazin-3-one (5m): Com-

pound (5m) was synthesized from (4d) 4-nitro benzaldehyde.

Yield 57%, m.p. 205 °C, R_f– 0.58 (T: E: F-5:4:1), (KBr) v_max

1

(cm^-1): 1697 (CONH₂), 1520 (NO₂), 1605 (-C=N); H NMR

(CDCl₃, in ppm): d 2.2 (s, 3H, CH₃), 2.6 (s, 2H, CH₂pyridazinone

protons), 6.37 (s, 1H, CH*), 7.2 (d, 1H, Ar-H-5’), 7.7 (d, 1H,

Ar-H-6’), 8.2 (s, 1H, Ar-H-2’), 7.18 (d, 2H, H-2”, 6”), 8.15 (d,

2H, H-3”, 5”), 8.0 (s, 1H, NH); ¹³C (CDCl₃, in ppm): d 162.6

(C-3), 135.2 (C-4), 32.1 (C-5), 141.3 (C-6), 131.8 (C-1’), 135.1

(C-2’), 130.2 (C-3’), 138.5 (C-4’), 149.1 (C-5’), 124.0 (C-6’),

15.6 (CH₃), 138.1 (C*), 141.2 (C-1”), 127.3 (C-2”), 121.0 (C-3”),

147.5 (C-4”), 121.0 (C-5”), 127.3 (C-6”); MS (m/z): 366 (M⁺;

C₁₈H₁₄N₄O₅).

(4Z)-4-(Furan-2-ylmethylidene)-6-(4-methyl-3-nitroph-

enyl)-2,3,4,5-tetrahydropyridazin-3-one (5j): Compound (5j)

was synthesized from (4d) using fufuraldehyde. Yield 58%, m.p.

192 °C, R_f– 0.53 (T: E: F-5:4:1), IR (KBr) v_max(cm^-1): 1659

1

(CONH₂), 1528 (NO₂), 1134 (C-O-C), 1589 (-C=N); H NMR

(CDCl₃, in ppm) d: 2.1 (s, 3H, CH₃), 2.0 (s, 2H, CH₂pyridazinone

protons), 6.40 (s, 1H, CH*), 7.2 (d, 1H, Ar-H-5’), 7.9 (d, 1H,

Ar-H-6’), 8.0 (s, 1H, Ar-H-2’), 6.9-7.40 (m, 3H, Furyl), 9.0 (s,

1H, NH); ¹³C (CDCl₃, in ppm): d 162.6 (C-3), 135.2 (C-4), 32.1

(C-5), 141.3 (C-6), 131.8 (C-1’), 135.1 (C-2’), 130.2 (C-3’),

138.5 (C-4’), 149.1 (C-5’), 124.0 (C-6’), 15.6 (CH₃), 138.1 (C*),

151.6 (C-2”), 145.7 (C-3”), 112.6 (C-4”), 111.3 (C-5”); MS (m/z):

311 (M⁺; C₁₆H₁₃N₃O₄).

(4Z)-4-[(3-nitrophenyl)methylidene]-6-(4-methyl-3-ni-

trophenyl)-2,3,4,5-tetrahydropyridazin-3-one (5n): Com-

pound (5n) was synthesized from (4d) 3-nitro benzaldehyde. Mo-

lecular formula, Yield 60% m.p. 227 °C, R_f– 0.56 (T: E: F-5:4:1),

IR (KBr) v_max(cm^-1): 1673 (CONH₂), 1520 (NO₂), 1598 (-C=N);

¹H NMR (CDCl₃, in ppm): d 2.2 (s, 3H, CH₃), 2.6 (s, 2H, CH₂

pyridazinone protons), 6.37 (s, 1H, CH*), 7.2 (d, 1H, Ar-H-5’),

7.7 (d, 1H, Ar-H-6’), 8.2 (s, 1H, Ar-H-2’), 7.65 (d, 1H, Ar-H-6”),

7.44 (dd, 1H, Ar-H-5”), 8.04 (d, 1H, Ar-H-4”), 8.20 (s, 1H,

Ar-H-2”), 8.6 (s, 1H, NH); ¹³C (CDCl₃, in ppm): d 162.6 (C-3),

129.2 (C-4), 32.3 (C-5), 141.3 (C-6), 131.8 (C-1’), 135.1 (C-2’),

130.2 (C-3’), 138.5 (C-4’), 149.1 (C-5’), 124.0 (C-6’), 16.4

(CH₃), 138.1 (C*), 136.1 (C-1”), 132.4 (C-2”), 129.5 (C-3”),

120.2 (C-4”), 148.3 (C-5”), 121.2 (C-6”); MS (m/z): 366 (M⁺;

C₁₈H₁₄N₄O₅).

(4Z)-4-[(4-methylphenyl)methylidene]-6-(4-methyl-3-

nitrophenyl)-2,3,4,5-tetrahydropyridazin-3-one (5k): Com-

pound (5k) was synthesized from (4d) using tolualdehyde. Yield

55%, m.p. 197 °C, R_f– 0.56 (T: E: F-5:4:1), IR (KBr) v_max(cm^-1):

1651 (CONH₂), 1373 (NO₂), 1512 (-C=N); ¹H NMR (CDCl₃, in

ppm): d 2.2 (s, 3H, CH₃), 2.25 (s, 3H, CH₃), 2.6 (s, 2H, CH₂

pyridazinone protons), 6.37 (s, 1H, CH*), 7.2 (d, 1H, Ar-H-5’),

7.7 (d, 1H, Ar-H-6’), 8.2 (s, 1H, Ar-H-2’), 6.49 (d, 2H, H-3”, 5”),

7.25 (d, 2H, H-2”, 6”), 8.9 (s, 1H, NH). ¹³C (CDCl₃, in ppm): d

162.6 (C-3), 135.2 (C-4), 32.1 (C-5), 141.3 (C-6), 131.8 (C-1’),

135.1 (C-2’), 130.2 (C-3’), 138.5 (C-4’), 149.1 (C-5’), 124.0

(C-6’), 15.6 (CH₃), 138.1 (C*), 127.4 (C-1”), 127.3 (C-2”), 114.1

(C-3”), 159.8 (C-4”), 114.1 (C-5”), 127.3 (C-6”), 24.2 (CH₃); MS

(m/z): 335 (M⁺; C₁₉H₁₇N₃O₃).

(4Z)-4-[(4-methylphenyl)methylidene]6-[4-(2methyl-

propyl)-3-nitrophenyl]-2,3,4,5-tetrahydropyridazin-3-one

(5o): Compound (5o) was synthesized from (4f) using tolual-

dehyde. Yield 59%, m.p. 135 °C, R_f– 0.59 (T: E: F-5:4:1), IR

(KBr) v_max(cm^-1): 1682 (CONH₂), 3270 (NH), 2955, 2870 (C-H,

CH₃) str, 1342 (NO₂), 1211 (C-O-C), 1420, 1458 (C=C), 1612

(-C=N); ¹H NMR (CDCl₃, in ppm): d 1.03 (d, 6H, 2xCH₃isobutyl

protons), 2.25 (t, 1H, CH, isobutyl protons), 2.53 (d, 2H, CH₂

isobutyl protons), 2.23 (s, 3H, CH₃), 2.3 (s, 2H, CH₂pyridazinone

protons), 6.37 (s, 1H, CH*), 7.6 (d, 1H, Ar-H-5’), 8.02 (d, 1H,

Ar-H-6’), 8.7 (s, 1H, Ar-H-2’), 6.39 (d, 2H, Ar-H-3”, 5”), 7.21 (d,

(4Z)-4-[(4-methoxyphenyl)methylidene]-6-(4-methyl-3-

nitrophenyl)-2,3,4,5-tetrahydropyridazin-3-one (5l): Com-

pound (5l) was synthesized from (4d) 4-methoxy benzaldehyde.

Yield 56%, m.p. 201 °C, R_f– 0.42 (T: E: F-5:4:1), IR (KBr) v_max

(cm^-1): 1697 (CONH₂), 1520 (NO₂), 1165 (C-O-C), 1566 (-C=N);

¹H NMR (CDCl₃, in ppm): d 2.3 (s, 3H, CH₃), 3.89 (s, 3H, OCH₃),

2.1 (s, 2H, CH₂pyridazinone protons), 6.41 (s, 1H, CH*), 7.3 (d,

J. Chin. Chem. Soc. 2016, 63, 000-000

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9

Article

Khokra et al.

2H, Ar-H-2”-6”), 8.2 (s, 1H, NH); ¹³C (CDCl₃, in ppm): d 162.4

(C-3), 128.9 (C-4), 33.1 (C-5), 142.1 (C-6), 131.6 (C-1’), 134.3

(C-2’), 129.6 (C-3’), 138.4 (C-4’), 148.6 (C-5’), 123.1 (C-6’),

37.1 (CH, isobutyl), 21.0 (CH₂, isobutyl), 22.8 (CH₃), 22.8 (CH₃),

138.1 (C*), 127.4 (C-1”), 127.3 (C-2”), 114.1 (C-3”), 159.8

(C-4”), 114.1 (C-5”), 127.3 (C-6”), 24.2 (CH₃); MS (m/z): 377

(M⁺; C₂₂H₂₃N₃H₃).

2.27 (t, 1H, CH, isobutyl protons), 2.46 (d, 2H, CH₂, isobutyl pro-

tons), 2.05 (s, 2H, CH₂, pyridazinone protons), 6.32 (s, 1H, CH*),

7.0 (d, 1H, Ar-H-5’), 8.0 (s, 1H, Ar-H-2’), 8.1 (d, 1H, Ar-H-6’),

7.19 (d, 2H, Ar-H-2”, 6”), 8.14 (d, 2H, H-3”, 5”), 8.5 (s, 1H, NH).

¹³C (CDCl₃, in ppm): d 162.6 (C-3), 129.2 (C-4), 32.3 (C-5),

141.3 (C-6), 131.6 (C-1’), 134.3 (C-2’), 129.6 (C-3’), 138.4

(C-4’), 148.6 (C-5’), 123.1 (C-6’), 37.1 (CH, Isobutyl), 21.0

(CH₂, Isobutyl), 22.8 (CH₃), 22.8 (CH₃), 138.1 (C*), 141.2(C-1”),

127.3 (C-2”), 121.0 (C-3”), 147.5 (C-4”), 121.0 (C-5”), 127.3

(C-6”); MS (m/z): 408 (M⁺; C₂₁H₂₀N₄O₅).

(4Z)-4-[(4-methoxyphenyl)methylidene]-6-[4-(2methyl-

propyl)-3-nitrophenyl]-2,3,4,5-tetrahydropyridazin-3-one

(5p): Compound (5p) was synthesized from (4f) using 4-me-

thoxy benzaldehyde. Yield 60%, m.p. 132 °C, R_f– 0.49 (T: E:

F-5:4:1), IR (KBr) v_max(cm^-1): 1674 (CONH₂), 1342 (NO₂), 1026

(C-O-C), (1512); ¹H NMR (CDCl₃, in ppm): d 1.0 (d, 6H, 2xCH₃

isobutyl protons), 2.26 (t, 1H, CH, isobutyl protons), 2.52 (d, 2H,

CH₂isobutyl protons), 2.23 (s, 3H, CH₃), 2.3 (s, 2H, CH₂pyri-

dazinone protons), 3.92 (s, 3H, OCH₃), 6.27 (s, 1H, CH*), 7.1 (d,

1H, Ar-H-5’), 8.05 (d, 1H, Ar-H-6’), 8.5 (s, 1H, Ar-H-2’), 6.39 (d,

(4Z)-4-[(3-nitrophenyl)methylidene]-6-[4-(2-methylpro-

pyl)-3-nitrophenyl])-2-phenyl-2,3,4,5-tetrahydropyridazin-3-

one (5s): Compound (5s) was synthesized from (4e) using 4-

nitrobenzaldehyde. Yield 59%, m.p. 146 °C, R_f– 0.48 (T: E:

F-5:4:1), IR (KBr) v_max(cm^-1): 1674 (C=O), 1520 (NO₂), 1598

(-C=N); ¹H NMR (CDCl₃, in ppm): d 1.07 (d, 6H, 2xCH₃isobutyl

protons), 2.27 (t, 1H, CH, isobutyl protons), 2.46 (d, 2H, CH₂

isobutyl protons), 2.05 (s, 2H, CH₂, pyridazinone protons), 6.32

(s, 1H, CH*), 7.0 (d, 1H, Ar-H-5’), 8.06 (d, 1H, Ar-H-6’), 8.23 (s,

1H, Ar-H-2’), 7.19 (d, 2H, Ar-H-2”, 6”), 8.14 (d, 2H, Ar-H-3”,

5”), 7.05-7.44 (m, 4H, N-phenyl protons); ¹³C (CDCl₃, in ppm): d

162.6 (C-3), 129.2 (C-4), 32.3 (C-5), 141.3 (C-6), 131.6 (C-1’),

134.3 (C-2’), 129.6 (C-3’), 138.4 (C-4’), 148.6 (C-5’), 123.1

(C-6’), 37.1 (CH, Isobutyl), 21.0 (CH₂, Isobutyl), 22.8 (CH₃),

22.8 (CH₃), 138.1 (C*), 141.2 (C-1”), 127.3 (C-2”), 121.0 (C-3”),

147.5 (C-4”), 121.0 (C-5”), 127.3 (C-6”), 137.6 (C-1”’), 121.5

(C-2”’), 129.0 (C-3”’), 124.2 (C-4”’), 129.0 (C-5”’), 121.4 (C-

6””); MS (m/z): 484 (M⁺; C₂₇H₂₄N₄O₅).

2H, Ar-H-3”, 5”), 7.21 (d, 2H, Ar-H-2”-6”), 8.6 (s, 1H, NH); ¹³

C

(CDCl₃, in ppm): d 162.6 (C-3), 129.2 (C-4), 32.3 (C-5), 141.3

(C-6), 131.6 (C-1’), 134.3 (C-2’), 129.6 (C-3’), 138.4 (C-4’),

148.6 (C-5’), 123.1 (C-6’), 37.1 (CH, Isobutyl), 21.0 (CH₂,

Isobutyl), 22.8 (CH₃), 22.8 (CH₃), 138.1 (C*), 127.4 (C-1”),

127.3 (C-2”), 114.1 (C-3”), 159.8 (C-4”), 114.1 (C-5”), 127.3

(C-6”), 55.8 (OCH₃); MS (m/z): 393 (M⁺; C₂₂H₂₃N₃O₄).

(4Z)-4-[(3-nitrophenyl)methylidene]-6-[4-(2-methylpro-

pyl)-3-nitrophenyl])-2,3,4,5-tetrahydropyridazin-3-one (5q):

Compound (5q) was synthesized from (4f) using 3-nitrobenzal-

dehyde. Yield 61%, m.p. 140 °C, R_f– 0.44 (T: E: F-5:4:1), IR

(KBr) v_max(cm^-1): 1675 (CONH₂), 1353 (NO₂), 1426 (-C=N); ¹H

NMR (CDCl₃, in ppm): d 1.02 (d, 6H, 2xCH₃isobutyl protons),

2.21 (t, 1H, CH, isobutyl protons), 2.49 (d, 2H, CH₂isobutyl pro-

tons), 2.23 (s, 3H, CH₃), 2.02 (s, 2H, CH₂pyridazinone protons),

6.34 (s, 1H, CH*), 7.5 (d, 1H, Ar-H-5’), 7.9 (d, 1H, Ar-H-6’), 8.2

(s, 1H, Ar-H-2’), 7.49 (dd, 1H, Ar-H-5”), 7.61 (d, 2H, Ar-H-6”),

Docking study on xanthine oxidoreductase: Molegro Vir-

tual Docker (MVD) software was used for automated flexible

ligand docking of compounds (5a-s) with the molecular target i.e.

xanthine oxidoreductase. The molecular docking was performed

by inserting the compounds into the binding site crystal structure

of enzyme. The 3D structure of the xanthine oxidoreductase

(PDB entry 1R4U) was retrieved from PDB to calculate the

MolDock score (kcal/mol) and hydrogen bond interaction with

ligands.

8.07 (d, 1H, Ar-H-4”), 8.20 (s, 1H, Ar-H-2”), 9.2 (s, 1H, NH); ¹³

C

(CDCl₃, in ppm): d 162.6 (C-3), 129.2 (C-4), 32.3 (C-5), 141.3

(C-6), 131.6 (C-1’), 134.3 (C-2’), 129.6 (C-3’), 138.4 (C-4’),

148.6 (C-5’), 123.1 (C-6’), 37.1 (CH, Isobutyl), 21.0 (CH₂,

Isobutyl), 22.8 (CH₃), 22.8 (CH₃), 138.1 (C*), 138.1, 136.1

(C-1”), 132.4 (C-2”), 129.5 (C-3”), 120.2 (C-4”), 148.3 (C-5”),

121.2 (C-6”); MS (m/z): 408 (M⁺; C₂₁H₂₀N₄O₅).

In vitro antioxidant assays

Free radical scavenging activity by DPPH method: 5

mL of the freshly prepared stock solution (33 mg in 1L of meth-

anol) of DPPH was mixed with 1 mL of test solution at 100

mg/mL and was kept in dark at room temperature for 30 min.³²

The optical density of the blank prepared in a similar way omit-

ting sample and test/standard solution was measured at 517 nm

and compared with standard ascorbic acid. Percentage inhibi-

tion of DPPH radical was calculated as per the formula given

below:

(4Z)-4-[(4-nitrophenyl)methylidene]-6-[4-(2-methylpro-

pyl)-3-nitrophenyl])-2,3,4,5-tetrahydropyridazin-3-one (5r):

Compound (5r) was synthesized from (4f) using 4-nitrobenzal-

dehyde. Yield 63%, m.p. 142 °C, R_f– 0.51 (T: E: F-5:4:1), IR

(KBr) v_max(cm^-1): 1674 (CONH₂), 1342 (NO₂), 1537 (-C=N); ¹H

NMR (CDCl₃, in ppm): d 1.07 (d, 6H, 2xCH₃isobutyl protons),

10

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J. Chin. Chem. Soc. 2016, 63, 000-000

JOURNAL OF THE CHINESE

CHEMICAL SOCIETY

Antioxidant Activity of Pyridazinone Derivatives

Abs_control- Abs_sample

% anti - radical activity [DPPH] =

´100

% anti-radical activity =

´100

Abs_control

Hydrogen peroxide (H₂O₂) scavenging assay: Hydrogen

peroxide solution (1 mL of 40 mM) prepared in phosphate buffer

(50 mM pH 7.4) was added to the test tubes containing 2 mL of

test and standard solutions (100 mg/mL), respectively.³³The opti-

cal density was recorded at 230 nm after 10 min against a blank

solution of phosphate buffer omitting hydrogen peroxide. Ascro-

bic acid was used as a positive control for comparison purpose.

The percentage scavenging of hydrogen peroxide was calculated

as per the formula given below:

Metal chelating assay: Procedure of Dinis et al., was used

to measure the chelation of ferrous ions.³⁵In a test tube, 0.1 mL of

sample (100 mg/mL) was mixed with 0.5 mL of ferrous chloride

solution (0.2 mM). The reaction was initiated by the addition of

0.2 mL of ferrozine (5 mM) and the mixture was incubated at

room temperature for 10 min followed by measurement of optical

density at 562 nm. EDTA was used as a positive control.

Abs_control- Abs_sample

% anti-radical activity =

´100

Abs_control

% Scavenging [H₂O₂] = [(A₀-A₁)/A₀] ´ 100

CONCLUSIONS

where, A₀is the absorbance of the control, and A₁is the ab-

sorbance of the sample and reference compound.

A novel series of pyridazinone derivatives bearing a

substituted hydrocarbon and benzylidene side chain were

synthesized. The synthesized compounds exhibited moder-

ate to potent antioxidant activities in five different in vitro

assay methods and therefore may prove beneficial in pre-

venting the progression of various oxidative stress induced

diseases. Amongst all the synthesized pyridazinone deriva-

tives, compounds 5a, 5d, 5g and 5j have shown the most

promising antioxidant activity in DPPH free radical scav-

enging assay, H₂O₂scavenging assay, reducing power as-

say, nitric oxide scavenging and iron chelating assay. Com-

pound 5d has shown the best scavenging activity in DPPH

free radical scavenging assay, 5j emerged as the most po-

tent compound in H₂O₂scavenging assay, nitric oxide scav-

enging assay and in iron chelating assay while 5g was

found to possess the best reducing power. Thus, it is pro-

posed that overall antioxidant activity of synthesized target

compounds could be due to the presence of an electron

withdrawing group and heterocyclic moiety on pyrida-

zinone ring. The docking study data strongly support the

assumption that this receptor may be involved in observed

antioxidant activity of pyridazinone derivatives bearing ar-

omatic ring and benzylidene substituents. However, further

studies are required to ascertain the precise mechanism of

action of antioxidant activity of these molecules.

Reducing power assay (RPA): The reducing power of the

prepared compounds and reference compound, ascorbic acid was

investigated as per the reported method of Oyaizu.³⁴Briefly, to

the mixture of 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5

mL of 1% w/v K₃Fe (CN)₆, 1 mL of sample solutions (100 mg/mL)

were added and the resulting mixture was allowed to stand at 50

°C for 30 min. After the incorporation of 2.5 mL of trichloro ace-

tic acid (10% w/v) to the above mixture it was centrifuged at 3000

rpm for 10 min. The clear supernatant liquid (top layer, 2.5 mL)

was collected and mixed with an equal amount of distilled water

followed by addition of 0.5 mL of FeCl₃(0.1%, w/v) solution to

produce the colored complex. The absorbance of the colored

complex formed by the tested compounds with potassium ferri-

cyanide, trichloro acetic acid and ferric chloride was recorded at

700 nm against blank sample.

Nitric oxide scavenging assay: The ability of the synthe-

sized compounds to scavenge Nitric oxide (NO) radicals was

performed as per the standard method.²⁶Briefly, to 2 mL of so-

dium nitroprusside (10 mM) was added 0.5 mL of phosphate

buffer saline (pH 7.4) and 0.5 mL of sample (100 mg/mL). The

reaction mixture was allowed to stand at room temperature of 25

°C for approximately 2 h and 30 min. An aliquot of 0.5 mL of the

incubated solution was withdrawn and mixed with 0.5 mL of

Griess reagent [(1.0) mL sulfanilic acid reagent (0.33% in 20%)

glacial acetic acid at room temperature for 5 min with (1) mL of

naphthylethylenediamine dichloride (0.1% w/v)]. The mixture

was again incubated at room temperature for 30 min and its

absorbance was recorded at 546 nm. The amount of nitric oxide

(NO) radical inhibition is calculated following the equation

given below:

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Synthesis, Molecular Docking and Potential Antioxidant Activity of Di/Trisubstituted Pyridazinone Derivatives

DOI: 10.1002/jccs.201600051

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Article abstract of DOI:10.1002/jccs.201600051

Full text of DOI:10.1002/jccs.201600051

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