Synthesis, in-vitro, in-vivo anti-inflammatory activities and molecular docking studies of acyl and salicylic acid hydrazide derivatives

Article abstract of DOI:10.1016/j.bioorg.2020.104168

Over the course of time several drugs have been synthesized and are available in market for the treatment of inflammation. However, they were unable to cure effectively and associated with side effects. To effectively deal with such diseases, heterocycles and their derivatives have gained their special position. For this reason 1,3,4-oxadiazole (15–16), 1,2,4-triazole (17–18), Schiff base (19–24) and 3,5-disubstituted pyrazole (25) derivatives were synthesized starting from salicylic acid and acyl acid hydrazides (12–14) as COX-1 and COX-2 inhibitors. In vivo anti-inflammatory activities were also tested by carrageenan-induced mice paw edema against albino mice of any sex. Structures of all the synthesized compounds were confirmed by FT-IR and ¹H NMR analysis. Schiff base derivative of 4-amiontirazole (24) with IC₅₀ value of 1.76 ± 0.05 (COX-2) and 117.8 ± 2.59 emerged as potent COX-2 inhibitor. Furthermore, we also performed in-vivo anti-inflammatory investigations by using carrageenan induced paw edema test. From in-vivo anti-inflammatory activities, it was found that after 1 h the maximum percentage inhibition 15.8% was observed by compound 14 which is comparable with that of the standard drug followed by the compound 18 with percentage inhibition of 10.5%. After 3 h, the maximum percentage inhibition was observed by compound 18 with 22.2% and compound 14 with 16.7%. After 5 h the maximum percentage inhibition was observed by compound 18 with 29.4% followed by compound 16 with 23.5%. We further explore the mechanism of the inhibition by using docking simulations. Docking studies revealed that the selective COX-2 inhibitors established interactions with additional COX-2 enzyme pocket residues.

Full text of DOI:10.1016/j.bioorg.2020.104168

BioorganicChemistry104(2020)104168
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, in-vitro, in-vivo anti-inflammatory activities and molecular
docking studies of acyl and salicylic acid hydrazide derivatives
Abida Munira,1, Adil Khushala,1, Kiran Saeeda, Abdul Sadiqb, Rahim Ullahc, Gowhar Alic,
⁎
Zaman Ashrafd, Ehsan Ullah Mughale, Muhammad Saeed Janb, Umer Rashida, , Izhar Hussainf,
⁎
Amara Mumtaza,
a Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Pakistan
b Department of Pharmacy, Faculty of Biological Sciences, University of Malakand, Chakdara 18000 Dir (L), KP, Pakistan
c Department of Pharmacy, University of Peshawer, Peshawer, Pakistan
d Department of Chemistry, Allama Iqbal Open University Islamabad, Pakistan
e Department of Chemistry, University of Gujrat, Gujrat, Pakistan
f Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus, Pakistan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Over the course of time several drugs have been synthesized and are available in market for the treatment of
inflammation. However, they were unable to cure effectively and associated with side effects. To effectively deal
with such diseases, heterocycles and their derivatives have gained their special position. For this reason 1,3,4-
oxadiazole (15–16), 1,2,4-triazole (17–18), Schiff base (19–24) and 3,5-disubstituted pyrazole (25) derivatives
were synthesized starting from salicylic acid and acyl acid hydrazides (12–14) as COX-1 and COX-2 inhibitors. In
vivo anti-inflammatory activities were also tested by carrageenan-induced mice paw edema against albino mice
of any sex. Structures of all the synthesized compounds were confirmed by FT-IR and 1H NMR analysis. Schiff
Salicylic acid
Anti-inflammatory activities
Oxadiazole
Pyrazole
base derivative of 4-amiontirazole (24) with IC50 value of 1.76
0.05 (COX-2) and 117.8
2.59 emerged as
potent COX-2 inhibitor. Furthermore, we also performed in-vivo anti-inflammatory investigations by using
carrageenan induced paw edema test. From in-vivo anti-inflammatory activities, it was found that after 1 h the
maximum percentage inhibition 15.8% was observed by compound 14 which is comparable with that of the
standard drug followed by the compound 18 with percentage inhibition of 10.5%. After 3 h, the maximum
percentage inhibition was observed by compound 18 with 22.2% and compound 14 with 16.7%. After 5 h the
maximum percentage inhibition was observed by compound 18 with 29.4% followed by compound 16 with
23.5%. We further explore the mechanism of the inhibition by using docking simulations. Docking studies re-
vealed that the selective COX-2 inhibitors established interactions with additional COX-2 enzyme pocket re-
sidues.
1. Introduction
arachidonic acid (AA) and augment the inflammatory response.
Arachidonic acid metabolism includes
a cyclooxygenase (COX)
Inflammation is a response of the immune system caused by the
invading pathogens, toxic compounds or damaged cells. These factors
may lead to tissue damage and a vast array of disorders in brain, heart,
kidney and reproductive system. Generally, inflammation is char-
acterized by redness, swelling, warmth, pain and fever. There are sev-
eral stimuli that are responsible for inflammation. These may include
microbial invasion, UV radiation, immune respone or any physical
damage or injury. In response to the foreign stimuli, inflammatory cells
produce some bioactive mediators that are the derivatives of
pathway that converts it to inflammatory prostaglandins (PGs) [1,2].
The anti-inflammatory drugs, collectively known as nonsteroidal
anti-inflammatory drugs (NSAIDs), are considered as excellent COX
inhibitors. For the first time in 1971 Vane identified the therapeutic
target of aspirin (NSAID), the COX enzyme (COX-1). During early
1990s, several anti-inflammatory drugs were discovered and led to the
identification of a distinct second isozyme named COX-2 [3–5]. These
drugs are prescribed for relieving symptoms and signs of various kinds
of inflammation and rheumatic disease [6]. Conventional NSAIDs like
⁎ Corresponding authors.
E-mail addresses: umerrashid@cuiatd.edu.pk (U. Rashid), amaramumtaz@cuiatd.edu.pk (A. Mumtaz).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2020.104168
Received 29 January 2020; Received in revised form 26 July 2020; Accepted 29 July 2020
Availableonline28August2020
0045-2068/©2020PublishedbyElsevierInc.

A. Munir, et al.
BioorganicChemistry104(2020)104168
triazole-3(4H)-thione having vicinal aryl substitutions were also
planned. In-vitro, in-vivo anti-inflammatory activities and molecular
docking studies of these derivatives were also carried out.
Cl
O
O
CH3
OH
NH
OH
CH3
Cl
OH
2. Results & discussion
O
H3C
O
O
CH3
2.1. Chemistry
(2)
(1)
(3)
The synthetic route for the target hydrazide derivatives 12–14 is
outline in Scheme 1. In the first step, methyl esters (9–11) of the car-
boxylic acids (6–8) were synthesized by refluxing in methanol using
few drops of sulfuric acid as catalyst. Finally, the esters were then
converted to hydrazide derivatives. Freshly prepared esters were re-
acted with hydrazine monohydrate 85% in methanol and heated under
reflux to afford final hydrazides 12–14. In the FT-IR spectra stretching
signal at 3406-3329 (asymmetric), 3309-3207 (symmetric) for NH2 and
at 3012-3001 for NH groups confirmed the hydrazide formation. In
1HNMR spectra characteristic signal at 6.73 (bs, 1H, NH) and 3.23 (bs,
2H, NH2) confirmed hydrazide formation [22].
O
S
O
O
O
S
H2N
O
N
N
O
F
F
F
(4)
(5)
Fig. 1. Some common and commercially available anti-inflammatory drugs.
Synthesis of 1,3,4-oxadiazole-2(3H)-thiones 15–16 were carried out
by the reaction of hydrazide derivatives 12–13 in the first step with
potassium hydroxide and carbon disulphide in ethanol to get potassium
salt of semicarbazide. Prepared potassium salt of semicarbazide
(1.1 mmol) was taken in a 100 mL beaker and dissolved in distilled
water (20 mL). Drop wise addition of concentrated hydrochloric acid
was carried out for the precipitation of product. White colored solid
product of (15–16) was obtained which was filtered, dried and
weighed. Scheme 2 Disappearance of the signals for the hydrazide NH
groups in 1HNMR spectra confirmed that ring has been closed [23].
Synthetic route for the synthesis of 1,2,4-triazole-schif base hybrid
(scheme 3) was carried out starting from hydrazide 12–13 reacting with
carbon disulfide and potassium hydroxide followed by reaction with
hydrazine monohydrazide to get target products 17–18 as an inter-
mediate which upon condensation with substituted aldehydes furnished
triazole amine-shiff base hybrid of our interest (19–24) structures of all
the synthesized compounds were confirmed by FT-IR and 1HNMR
Ibuprofen (1), diclofenac (2) and acetylsalicylic acid (aspirin, 3) block
both isoforms i.e. COX-1 and COX-2 to a similar extent. Since long,
acetylsalicylic acid (ASA, 3) has been used as analgesic agent for mild to
moderate pain. However, by the introduction of newer NSAIDs its pre-
eminent position has been challenged. However, frequent use of these
NSAIDs leads to serious side effects, among them the most observed are
kidney problems and gastrointestinal (GI) infections. The reason for
these problems is the presence of free COOH group of NSAID (1–3,
Fig. 1) which causes irritation by the direct contact with tissues pro-
ducing prostaglandin and GI mucosal cells [7]. In order to lower the
gastrointestinal disturbances, selective COX-2 inhibitors were dis-
covered. Celecoxib (4) and rofecoxib (5) were about 100–1000 times
more selective against COX-2 than COX-1 [8].
Heterocyclic derivatives of salicylic acid are used as antimicrobial,
[13] antitumor and anti-cancer agent, show activity against various
cancer cell lines and found active [14–16]. 5-Amino salicylic acid de-
rivatives are used as drug to cure TB [17]. Other derivatives such as
salicylic anilids possess significant antifungal activities [18,19].
Hydrazides are useful building blocks to synthesize various het-
erocycles and possess biological activities. These hydrazides are very
reactive and can be used as precursors for the synthesis of heterocycles.
Heterocycles are widely distributed in nature as alkaloids, hormones,
vitamins, pharmaceutical products, dyes and agrochemicals.
Oxadiazoles [9–12], triazoles amine [13], Schiff bases [14,15] and
pyrazoles [16–18] are the known derivatives of hydrazides and possess
significant anti-inflammatory activity. Moldovan et al., also studied the
anti-inflammatory potential of some new acyl-hydrazones having 2-
aryl-thiazole core [7]. Similarly, salicylic acid and acyl substituted
heterocycles have gained much importance in the field of anti-in-
flammatory activities [19–21].
1
analysis. In the HNMR spectra appearance of characteristic signal of
Schiff base proton at 8. 38–8.68 ppm confirmed the hybrid structure.
[24] Synthesis of the 3,5-dimethyl pyrazole (25) of hydrazide 14 was
carried out by refluxing it with acetyl acetone in the presence of pi-
peridine as catalyst Scheme 4. In the 1HNMR spectra appearance of
singlet at 6.06 ppm for CH group of pyrazole ring confirmed that the
ring has been closed [25].
2.2. Anti-inflammatory activities
2.2.1. In-vitro cyclooxygenase inhibition activity
All the synthesized compounds (12–25) were subjected to COX-1
and COX-2 screening assay kits for the determination of their COX-1
and COX-2 inhibitory activity against Celecoxib used as positive con-
trols. The in-vitro enzymes inhibition results as IC50 values, (means of
three) and selectivity index (SI), IC50 (COX-2)/IC50 (COX-1) are pre-
sented in Table 1. The IC50 values of hydrazide series (12–14) are not
able to show some enzyme inhibition potential. The IC50 value of the
benzohydrazide (12, n = 0) have shown IC50 value of 61.05 μM. While,
the determined IC50 value against COX-1 is 103.26 μM. However,
acetohydrazide derivatives 13–14 (n = 1) showed comparatively better
COX-2 inhibition. Oxadiazole derives (15–16) showed IC50 values in
the range of 31.5 to 39.5 μM for COX-2 and 43.91 to 27.55 μM for COX-
The selectivity for COX-2 isoform is related with the structural
features of the drug. From the structural features of celecoxib (4) and
rofecoxib (5), it can be noted that these two marketed selective COX-2
inhibitors have a central 5-membered rings and two vicinal aryl rings
bearing sulfone group [1]. In current study, we used 4-methylsalicylic
acid and substituted acyl hydrazide (12–14) as starting material. Here,
we planned to explore the effect of the 1,3,4-oxadiazole-2(3H)-thione
and 4-amino-1,2,4-triazole-3(4H)-thione as central rings with single
aryl ring as substituent. While, Schiff base derivatives of 4-amino-1,2,4-
Scheme 1.
O
O
O
n
n
R
R
n
O
R
NHNH2
OH
9-11
12-14
6-8
2

A. Munir, et al.
BioorganicChemistry104(2020)104168
2.3. Docking studies
Docking studies on the targets were carried out using MOE
(2016.08) software [26]. Protein Data Bank (PDB code 1CX2) was used
to retrieve the crystal structure of COX-2 in complex with SC-558. All
the three series of the synthesized compounds were docked into the
binding pockets of COX-1 and COX-2 isoforms. Though, the active sites
of both isoforms are alike but in COX-2, an additional secondary side
pocket, bordered by small Val523 (isoleucine in COX-1), also exists
above the Arg120/Tyr355. Additionally, in COX-2 reserved of Arg513
(replaced with His513 in COX-1) are also presented [1]. Other im-
portant residues for COX-2 selectivity are His90, Gln192, Leu352 and
Ser353. To explore the selectivity, docking simulations on COX-2 iso-
zyme and on the reference drugs (indomethacin and celecoxib) were
performed. By analyzing the 2-D interaction plot of celecoxib and a
selective COX-2 inhibitor, it was found that it interacts with amino acid
residues (His90, Leu352, Ser353 and Arg513) situated in the additional
pocket of COX-2 pocket (Fig. 2a). Similarly, indomethacin showed in-
teractions with Ser353 and Tyr355 (Fig. 2b).
Scheme 2.
Hydrazide derivatives of series 1 (12–14) were docked into the
binding site of COX-2. Binding interaction pattern shown in Fig. 3a–c
revealed compound 12 and 13 showed binding interaction with amino
acid residues present in the additional COX-2 pocket (Fig. 3a and b).
Compound 12 forms hydrogen bond interactions with His90. While,
compound 13 showed hydrogen bond interactions with His90 and
Ser353. Phenyl ring forms π-H interactions between Tyr355. Hydrazide
derivative 14 forms hydrogen bond interactions with Ser355 (Fig. 3c).
2-D docking poses of oxadiazole derivatives 15–16 are shown in
Fig. 4a and b. Derivative 15 showed two hydrogen bond interactions.
Sulfur forms a bifurcated hydrogen bond interaction with Arg120 and
Tyr355. While, Ala527 forms π-H interactions with oxadiazole ring
(Fig. 4a). Sulfur atom of compound 16 also forms a bifurcated hydrogen
bond interaction with Arg120 and Tyr355 (Fig. 4b). 4-amino deriva-
tives 17–18 also exhibited hydrogen bond interactions with amino acid
residues. Sulfur atoms of 17 forms a bifurcated hydrogen bond inter-
action with Arg120 and Tyr355. While, Ala527 forms π-H interactions
with oxadiazole ring (Fig. 5a). However, derivative 18 forms strong
hydrogen bond interactions with the key amino acids residues present
in the additional pocket of the COX-2 enzyme. His forms hydrogen bond
with His90. While, –NH2 form hydrogen bond with Arg513. Ser353 and
important residue Val523 forms π-H interactions with oxadiazole ring
(Fig. 5b).
Scheme 3.
Scheme 4.
1. Interestingly, 4-amiontirazole derivative 18 showed selectivity to-
wards COX-2 (IC50 value against COX = 23.47 μM and for COX-1
IC50 = 68.4 μM). Contrarily, compound 17 exhibited IC50 value of
25.62 μM for COX-1 and 37.51 μM for COX-2.
Schiff base derivatives of 4-amiontirazole derivatives showed ex-
cellent selectivity for COX-2 isozyme. Compound 24 with IC50 value of
1.76 μM showed excellent COX-2 inhibition. The selectivity index
shown by compound 24 is 0.02. Other compounds of this series showed
COX-2 inhibition in the range of 12.56–24.95 μM [1].
Schiff base series of the compounds (19–24) were also docked into
the binding site of COX-2 isozyme. Three-dimensional binding or-
ientation of most active compound 24 superposed on native ligand is
shown in Fig. 6a. The ribbon diagram shown in green color represent
the additional COX-2 pocket residue. While blue indicates the Arg120/
Tyr355 residue on the border of this pocket. His90 forms hydrogen
bond with NH of triazole ring. While, Ser353 forms π-H interactions
with triazole ring (Fig. 6b) [1].
2.2.2. In-vivo anti-inflammatory activity
In vivo anti-inflammatory activities were carried out by using paw
edema carrageenan injection methods. In these methods 10 mg of
samples were dissolved in DMSO as vehicle and indomethacin was used
as standard drug. The in-vivo inhibition was monitored after 1 h, 3 h and
5 h. It was found that after 1 h the maximum percentage inhibition
15.8% was observed by compound 14 which is comparable with that of
the standard drug followed by the compound 18 with percentage in-
hibition of 10.5%. After 3 h the maximum percentage inhibition was
observed by compound 18 with 22.2% and compound 14 with 16.7%.
After 5 h the maximum percentage inhibition was observed by com-
pound 18 with 29.4% followed by compound 16 with 23.5% while
compounds 14, 15, 17 and 24 showed 17.6% percentage inhibition.
Rest of the compounds showed very little or no inhibition. Results of the
in-vivo anti-inflammatory activities are presented in Table 2. The per-
cent inhibition was calculated by using the bellow formula.
3. Conclusions:
Starting from salicylic acid and acyl acid hydrazides (12–14) 1,3,4-
oxadiazole (15–16), 1,2,4-triazole (17–18), Schiff base (19–24) and
3,5-disubstituted pyrazole (25) derivatives were synthesized. Structures
1
were confirmed by IR and HNMR analysis. In-vitro anti-inflammatory
activities of all the synthesized compounds were tested against COX-1
and COX-2. While, in- vivo anti-inflammatory activities were carried out
by using carrageenan-induced mice paw edema against albino mice of
any sex. Schiff base derivatives having vicinal aryl rings attached with
4-amiontirazole nucleus (18–24) showed good to excellent selectivity
for COX-2 isozyme. Compound 24 with IC50 value of 1.76 μM showed
excellent COX-2 inhibition. Rest of the compounds showed moderate or
no activity.
% Inhibition = (A B)/A × 100
where, A: disease control, B: tested group
3

A. Munir, et al.
BioorganicChemistry104(2020)104168
Table 1
Results of in-vitro cyclooxygenase inhibition activity.
Compounds
Structures
IC50 (μM)
COX-1
COX-2
61.04
SI
12
103.26
2.31
1.01
0.59
13
14
108.3
2.09
43.37
49.43
1.13
1.21
0.40
0.61
81.1
1.57
15
43.91
1.01
31.5
0.98
0.72
16
17
27.55
25.62
1.23
1.48
39.61
37.51
1.12
1.30
1.44
1.46
18
19
68.97
133.9
1.74
2.01
23.47
24.95
0.99
1.07
0.34
0.19
20
21
22
174.32
181.49
104.31
2.10
19.21
16.71
17.98
1.17
0.81
0.64
0.11
0.09
0.17
2.45
1.15
(continued on next page)
4

A. Munir, et al.
BioorganicChemistry104(2020)104168
Table 1 (continued)
Compounds
Structures
IC50 (μM)
COX-1
COX-2
12.56
SI
23
98.93
1.07
0.29
0.13
24
25
117.8
16.27
2.59
0.18
1.76
0.05
0.02
1.63
26.58
1.41
1.02
Diclofenac
0.48
0.25
0.01
0.01
10.05
0.07
20.93
0.28
Indomethacin
0.01
aSelectivity index = (COX-2 IC50(μM)/COX-1 IC50(μM)).
4. Material and methods
was used to check the progress of the reaction. When the reaction was
completed the potassium carbonate was filtered out from the mixture.
The solvent was evaporated on rotary evaporator and methyl 2-
methoxy-4-methylbenzoate (9) was obtained [28].
4.1. General procedure for the synthesis of methyl esters (9–11)
4.1.1. Synthesis of methyl 2-methoxy-4-methylbenzoate
To synthesize methyl ester of substituted acyl acid (7–8) (6.0 mmol)
was taken and dissolved in 35 mL of dry methanol. Few drops of H2SO4
were added into the reaction mixture as a catalyst. The reaction mixture
was refluxed for 12 h. The completion of the reaction was checked by
TLC. When the reaction was completed the solvent was evaporated on
rotary evaporator. The product so obtained was neutralized with
For the synthesis of methyl 2-methoxy-4-methylbenzoate (9), me-
thyl ester of 2-hydroxy-4-methylbenzoic acid (6) (5.4 mmol) was taken
and dissolved in 30 mL of dry acetone. Potassium carbonate
(17.4 mmol) was added which was followed by the addition of methyl
iodide (14.5 mmol). The reaction mixture was refluxed for 13 h. TLC
Table 2
Effect of the test drugs (12–25) and standard drug on carrageenan induced paw edema in mice.
S.#.
Paw edema (mL)
before carrageenan
injection
Paw edema (mL) after 1 % inhibition
Paw edema (mL) after 3 % inhibition
Paw edema (mL) after 5 % inhibition
h of carrageenan
injection
(1 h)
h of carrageenan
injection
(3 h)
h of carrageenan
injection
(5 h)
12
0.09
–
0.01
0.19
–
0.01
0
0.16
–
0.01
11.11
–
0.15
–
0.01
11.76
–
13
–
14
0.09
0.09
0.9
0.02
0.01
0.16
0.19
0.19
0.22
0.17
0.2
0.01
0.01
0.01
0.01
0.02
15.8
0
0.15
0.17
0.16
0.18
0.14
0.17
–
0.01
0.01
0.01
0.01
0.01
0.01
16.7
5.6
11.11
0
0.14
0.14
0.13
0.14
0.12
0.17
–
0.01
0.01
0.01
0.01
0.01
0.01
17.6
17.6
23.5
17.6
29.4
0
15
16
0.01
0
17
0.09
0.09
0.1
0.01
0.01
−15.7
10.53
−5.3
–
18
22.2
5.6
–
19
0.1
0.1
20
–
–
–
21
0.09
0.09
–
0.01
0.01
0.21
0.21
–
0.01
0.01
−15.8
−10.5
–
0.19
0.18
–
0.01
0.01
−5.5
0
0.18
0.15
–
0.01
0.01
−5.9
11.76
–
22
23
–
24
0.09
0.09
0.09
0.09
0.01
0.01
0.01
0.01
0.18
0.18
0.19
0.16
0.01
0.01
0.1
5.3
5.3
–
0.16
0.17
0.18
0.14
0.02
0.02
0.01
0.01
11.11
5.6
–
0.14
0.17
0.17
0.11
0.01
0.01
0.01
0.02
17.6
0
25
Disease control
Indomethacin
10 mg/kg
–
0.01
15.8
22.2
35.3
ANOVA followed by Dunnet post hoc test using Graph Pad Prism 5. *P < 0.05, **P < 0.01, ***P < 0.001 compare with disease group treated with vehicle.
5

A. Munir, et al.
BioorganicChemistry104(2020)104168
Fig. 2. The two-dimensional binding orientation of R-sulfonamides (a) Celecoxib; (b) Indomethacin generated via MOE into the binding site of 1CX2.
Fig. 3. Two-dimensional (2-D) binding interaction pattern of; (a) 12; (b) 13 and (c) 14 into the binding site of 1CX2.
solution of sodium bicarbonate in distilled water and extracted with
ethyl acetate (3x10 mL). Anhydrous sodium sulphate was used to dry
the ethyl acetate layer. The mixture was filtered to remove sodium
sulfate and concentrated on rotary to get methyl ester (10–11) [22].
hexane:EtOAc. = 8:2) 0.8; mp.(°C); oil.
4.1.1.3. Methyl 2-(3-methoxyphenyl)acetate (11). Yield: 80%; Rf = (n-
hexane:EtOAc. = 8:2) 0.7; mp (°C); oil.
4.1.1.1. Methyl 2-hydroxy-4-methylbenzoate (9). Yield: 92%; Rf = .(n-
4.2. Synthesis of hydrazide (12–14)
hexane:EtOAc. = 7:3) 0.8; mp (oil).
Freshly prepared ester (9–11) (5.2 mmol) was taken and diluted
with 35 mL of methanol. In the reaction mixture hydrazine
4.1.1.2. Methyl 2-(p-tolyl)acetate (10). Yield: 87%; Rf
=
(n-
6

A. Munir, et al.
BioorganicChemistry104(2020)104168
Fig. 4. Two-dimensional (2-D) binding interaction pattern of; (a) 15; (b) 16 into the binding site of 1CX2.
monohydrate 85% (2.0 mL) was added and refluxed for 8 h. TLC was
used to monitor the completion of reaction. Upon the completion of the
reaction methanol was evaporated on rotary evaporator and white solid
was obtained. To remove excessive hydrazine the solid was washed
with minimum amount of distilled water. The solid product (12–14)
obtained was dried and weighed [23].
4.3. Synthesis of 1,3,4-oxadiazole-2(3H)-thione derivatives (15–16)
To synthesize potassium salt of 2-(2-methoxy-4-methylbenzoyl)
hydrazine carbodithioate and methyl ester of substituted acyl hydrazide
(12–13) (2.7 mmol) was taken and dissolved in 20 mL of absolute
ethanol and stirred at room temperature. In a separate beaker ethanolic
solution of potassium hydroxide (4.0 mmol) was prepared and added
slowly into the reaction mixture and followed by addition of carbon
disulfide (4.0 mmol). After stirring for 72 h yellow precipitates of po-
tassium salt were formed. Ethanol was evaporated on rotary and yel-
lowish product was obtained which was washed with diethyl ether,
dried and weighed. Prepared potassium salt of hydrazide (13–14)
(1.1 mmol) was taken in a 100 mL beaker and dissolved in distilled
water (20 mL). Drop wise addition of concentrated hydrochloric acid
was carried out for the precipitation of product. White colored solid
product of (15–16) was obtained which was filtered, dried and weighed
[23].
4.2.1. 2-Methoxy-4-methylbenzohydrazide (12)
Yield: 92%; Rf = (n-hexane:EtOAc = 6:4) 0.2; mp. (109–110 °C);
FT-IR (¯, cm−1): 3406 (NH2 asymmetric), 3309 (NH2 symmetric), 3212,
3012, 2949, 1612 (C]O stretching), 1495, 1253, 1155; 1H NMR
(400 MHz, CDCl3) δ (ppm): 9.02 (bs, 1H, NH), 8.07 (d, J = 7.9 Hz,
1HAr), 6.89 (d, J = 7.8 Hz, 1HAr), 6.75 (s, 1HAr), 4.26 (bs, 2H, NH2),
3.95 (s, 3H, OCH3), 2.39 (s, 2H, CH3)
4.2.2. 2-(p-Tolyl)acetohydrazide (13)
Yield: 85%; Rf = . (n-hexane:EtOAc. = 6:4) 0.2; mp.(153–154 °C);
FT-IR (¯, cm−1): 3329, 3087, 3018 (aromatic CH stretching), 1618 (C]
O stretching), 1514; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.14–7.16 (m,
4HAr), 6.73 (bs, 1H, NH), 3.54 (s, 2H), 3.23 (bs, 2H, NH2), 2.35 (s, 3H,
CH3).
4.3.1. 5-(2-Methoxy-4-methylphenyl)-1,3,4-oxadiazole-2(3H)-thione
(15)
Yield: 64%; Rf = (n-hexane:EtOAc = 6:4) 0.6; mp (121–122 °C); FT-
IR (¯, cm−1): 3287 (NH Streching), 3118, 2906, 1612 (C]N
stretching), 1432, 1282, 1276, 1182; 1H NMR (400 MHz, CDCl3) δ
(ppm): 7.78 (d, J = 7.9 Hz, 1HAr), 7.37 (d, J = 7.6 Hz, 1HAr), 6.93 (d,
J = 7.5 Hz, 1HAr), 3.90 (s, 3H, OCH3), 2.45 (s, 3H, CH3).
4.2.3. 2-(3-Methoxyphenyl)acetohydrazide (14)
Yield: 82%; Rf = (n-hexane:EtOAc. = 6:4) 0.2; mp.(95–96 °C);.
Fig. 5. Two-dimensional (2-D) binding interaction pattern of; (a) 17; (b) 18 into the binding site of 1CX2.
7

A. Munir, et al.
BioorganicChemistry104(2020)104168
Fig. 6. (a) Three-dimensional binding orientation of most active compound 24 superposed on native ligand into the binding site of 1CX2; (b) Two-dimensional (2-D)
binding interaction pattern of 24.
4.3.2. 5-(4-Methylbenzyl)-1,3,4-oxadiazole-2(3H)-thione (16)
4.5.1. 4-((2-Hydroxy-4-methoxybenzylidene)amino)-3-(2-methoxy-4-
methylphenyl)-1H-1,2,4-triazole-5(4H)-thione (19)
Yield: 70%; Rf = . (n-hexane:EtOAc. = 6:4) 0.6; mp.(113–114 °C);
FT-IR (¯, cm−1): 3092 (aromatic CH stretching), 2954, 1619 (C]N
stretching), 1499, 1347 1162; 1H NMR (400 MHz, CDCl3) δ (ppm):
10.71 (bs, 1H, NH), 7.17–7.21 (m, 4HAr), 4.00 (s, 2H, 2H), 2.36 (s, 3H).
Yield: 89%; Rf = .(n-hexane:EtOAc. = 6:4) 0.5; mp (225–226 °C);
FT-IR (¯, cm−1): 3413 (OH stretching band), 3342, 3256, 2904, 1622
(C]N stretching), 1505, 1246, 1221; 1H NMR (400 MHz, CDCl3) δ
(ppm): 9.84 (bs, 1H, OH), 8.70 (s, 1H, CH), 7.38 (d, J = 7.5 Hz, 1HAr),
7.32 (d, J = 8.6 Hz, 1HAr), 6.90 (d, J = 7.5 Hz, 1HAr), 6.81 (s, 1HAr),
6.53 (dd, J = 8.5, 2.1 Hz, 1HAr), 6.46 (s, 1HAr) 3.83 (s, 3H, OCH3), 3.76
(s, 3H, OCH3), 2.43 (s, 3H, CH3).
4.4. Synthesis of 4-amino-1,2,4-triazole-3-thione derivatives (17–18)
Potassium salt of hydrazide (12–13) (1.0 mmol) was dissolved in
water/ethanol (1:1) mixture (30 mL) in a round bottomed flask and
kept on reflux until the solution became clear which was followed by
the addition of hydrazine monohydrate 64% (1 equiv.). With the evo-
lution of H2S gas the color of the solution gradually turned to green. Till
the formation of homogeneous mixture, the reaction mixture was kept
on reflux. Then the solution was transferred into the beaker and kept for
30 min to cool. The reaction mixture was neutralized by adding con-
centrated HCl dropwise. The precipitates of triazole amine (17–18) so
obtained were filtered, dried and weighed [24].
4.5.2. 4-((4-Fluoro-2-hydroxybenzylidene)amino)-3-(2-methoxy-4-
methylphenyl)-1H-1,2,4-triazole-5(4H)-thione (20)
Yield: 81%; Rf = .(n-hexane:EtOAc. = 6:4) 0.5; mp.(165–166 °C);
FT-IR (¯, cm−1): 3454 (OH stretching band), 3356 (NH stretching),
3202, 2918, 1618 (C]N stretching), 1503, 1243, 1179; 1H NMR
(400 MHz, CDCl3) δ (ppm): 10.22 (bs, 1H, OH), 8.68 (s, 1H, CH), 7.38
(d, J = 7.0 Hz, 1HAr), 7.09–7.14 (m, 3HAr), 6.91 (d, J = 6.4 Hz, 1HAr),
6.84 (s, 1HAr), 3.73 (s, 3H, OCH3), 2.45 (s, 3H, CH3).
4.4.1. 4-Amino-3-(2-methoxy-4-methylphenyl)-1H-1,2,4-triazole-5(4H)-
thione (17)
4.5.3. 4-((4-Hydroxybenzylidene)amino)-3-(2-methoxy-4-methylphenyl)-
1H-1,2,4-triazole-5(4H)-thione (21)
Yield: 65%; Rf = (n-hexane:EtOAc = 6:4) 0.3; mp (146–148 °C); FT-
IR (¯, cm−1): 3387 (NH2 asymmetric), 3233 (NH2 symmetric), 3129,
Yield: 83%; Rf = (n-hexane:EtOAc. = 6:4) 0.4; mp (163–165 °C);
1
FT-IR (¯, cm−1): 3118, 2936, 1598 (C]N stretching), 1163; H NMR
1
3129, 2969, 1614 (C]N stretching), 1577, 1277; H NMR (400 MHz,
(400 MHz, CDCl3) δ (ppm): 9.89 (bs, 1H, OH), 8.38 (s, 1H, CH), 7.76 (d,
J = 8.0 Hz, 2HAr), 7.19 (d, J = 8.6 Hz, 1HAr), 6.98 (d, J = 7.6 Hz,
2HAr), 6.89 (d, J = 8.1, 1HAr), 6.86 (s, 1HAr), 3.97 (s, 3H, OCH3), 2.44
(s, 3H, CH3).
CDCl3) δ (ppm): 9.04 (bs, 1H NH), 7.37 (d, J = 7.6 Hz, 1HAr), 6.88 (s,
1HAr), 6.56 (d, J = 7.5 Hz, 1HAr), 4.16 (bs, 2H, NH2), 3.90 (s, 3H,
OCH3), 2.46 (s, 3H, CH3).
4.4.2. 4-Amino-3-(4-methylbenzyl)-1H-1,2,4-triazole-5(4H)-thione (18)
Yield: 52%; Rf. = .(n-hexane:EtOAc. = 6:4) 0.5; mp.(90–91 °C); FT-
IR (¯, cm−1): 3295 (NH2 asymmetric), 3267 (NH2 symmetric), 3248,
3036, 2931, 1622 (C]N stretching), 1346; 1H NMR (400 MHz, CDCl3)
δ (ppm): 10.64 (bs, 1H, NH), 7.16–7.21 (m, 4HAr), 6.80 (bs, 2H, NH2),
4.01 (s, 2H, CH2), 2.35 (s, 3H, CH3).
4.5.4. 4-((4-Hydroxy-3,5-dimethoxybenzylidene)amino)-3-(2-methoxy-4-
methylphenyl)-1H-1,2,4-triazole-5(4H)-thione (22)
Yield: 72%; Rf = (n-hexane:EtOAc. = 6:4) 0.2; mp.(170–172 °C);
FT-IR (¯, cm−1): 3550 (OH stretching band), 3479 (NH stretching),
3233, 2937, 1579 (C]N stretching), 1515, 1287, 1223; 1H NMR
(400 MHz, CDCl3) δ (ppm): 9.84 (bs, 1H, OH), 8.11 (s, 1H, CH), 7.65 (d,
J = 8.0 Hz, 1HAr), 7.46 (s, 2HAr), 6.98 (d, J = 8.0 Hz, 1H), 6.75 (s,
1HAr), 4.02 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 2.43
(s, 3H, CH3).
4.5. Synthesis of Schiff bases derivatives of 4-amino-1,2,4-triazole-5(4H)-
thione (19–24)
To synthesize Schiff bases of triazoleamine (17–18), it was dissolved
in 15 mL of methanol. Methanolic solution of one equivalent of sub-
stituted aldehyde was added into the reaction mixture. As a catalyst few
drops of acetic acid were added. The progress of the reaction was ob-
served by TLC. After being refluxed for 12 h the reaction was completed
and then the reaction mixture was poured into crushed ice. Precipitates
of Schiff base (19–24) were formed which were kept in freezer for 24 h
and then filtered, dried and weighed.
4.5.5. 4-((4-Hydroxybenzylidene)amino)-3-(4-methylbenzyl)-1H-1,2,4-
triazole-5(4H)-thione (23)
Yield: 82%; Rf. = . (n-hexane:EtOAc. = 6:4) 0.6; mp.(285–286 °C) ;
FT-IR (¯, cm−1): 3123, 3087, 2939, 1656 (C]N stretching), 1499
(C]C stretching), 1283, 1112; 1H NMR (400 MHz, CDCl3) δ (ppm):
10.11 (bs, 1H, OH), 9.78 (bs, 1H, NH), 8.55 (s, 1H, CH), 7.69 (d,
J = 7.5 Hz, 2HAr), 7.16–7.18 (m, 4HAr), 6.86 (d, J = 7.2 Hz, 2HAr) 4.06
(s, 2H, CH2), 2.33 (s, 3H, CH3).
8

A. Munir, et al.
BioorganicChemistry104(2020)104168
4.5.6. 4-((2-Hydroxy-4-methoxybenzylidene)amino)-3-(4-methylbenzyl)-
1H-1,2,4-triazole-5(4H)-thione (24)
4.7.2.1. Carrageenan induced paw edema model. Mice (tested animals)
of either sex with 25–30 g body weights were selected for in-vivo anti-
inflammatory activities. The animals were dispersed into different sub
groups. Six animals (n = 6) were presented in each group. Group I & II
were designated as negative and positive control respectively. The
Yield: 82%; Rf. = .(n-hexane:EtOAc. = 6:4) 0.6; mp.(173–174 °C);
FT-IR (¯, cm−1): 3119, 3062 (CH aromatic), 2956, 1629 (C]N
stretching), 1495, 1287, 1237; 1H NMR (400 MHz, CDCl3) δ (ppm):
10.3 (bs, 1H, OH), 9.62 (bs, 1H, NH), 8.63 (s, 1H, CH), 7.21 (s, 1HAr),
7.18–7.20 (m, 4HAr), 6.56–6.58 (m, 2HAr), 4.10 (s, 2H, CH2), 3.88 (s,
3H, OCH3), 2.32 (s, 3H, CH3).
negative control Group (I) was treated with normal saline (10 mL kg−1
)
while positive control group (II) was administrated with dose of
Indomethacin (10 mg/kg) according to their body weight. Remaining
groups were subjected with tested compounds at dose of 20 mg/kg
(body weight). After thirty minutes of treatments, Carrageenan of 1%
was injected in sub-planter tissue of the hind paw (right) of each animal
(mouse). After the injection of carrageenan anti-inflammatory potential
was noted down for 5 h (1, 3 and 5) hours by using plethysmometer (LE
7500 plan lab S.L). The % inhibition of edema was measured with the
help of formula given below. [27]
4.6. General procedure for the synthesis of 3,5-dimethyl pyrazole methyl
esters (25)
Prepared hydrazide (14) was dissolved in 5 mL of methanol in a
round bottom flask. Acetylacetone (1 equiv.) was added into it. As a
catalyst few drops of hydrochloric acid were added. The reaction
mixture was refluxed for 10 h. TLC was used to check the progress of
the reaction. When the reaction was completed, the reaction mixture
was concentrated on rotary evaporator. Brown colored product was
obtained which was recrystallized with ethyl acetate [25].
Pharmacological Evaluation of Tamarix aphylla
% Inhibition = A B/B × 100
In which;
Yield: 70%; Rf. = .(n-hexane:EtOAc. = 6:4) 0.7; mp (semisolid); FT-
IR (¯, cm−1): 3031 (aromatic CH stretching), 1611 (C]O stretching),
1523, 1427, 1280, 1172; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.23 (t,
J = 7.6 Hz, 1HAr), 6.86 (d, J = 7.6 Hz, 1HAr), 6.82 (s, 1HAr), 6.81 (d,
J = 9.2 Hz, 1HAr), 6.06 (s, 1H, CHPyrazole), 3.79 (s, 3H, OCH3), 3.61 (s,
2H, CH2), 2.46 (s, 6H, CH3).
A = edema volume in negative control
B = paw edema in tested groups.
4.8. Docking studies
Molecular Operating Environment (MOE 2016.08) was used for
carrying out the Docking studies. Protein Data Bank (PDB code 1CX2)
was used for retrieving of COX-2 crystal structure in complex with SC-
558 while for COX-2, the native ligand was validated by re-docking.
Our previously reported methods were used for preparation of ligands,
enzymes downloading, energy minimization, 3D protonation and de-
termination of binding site [1,29–33]. Builder option in MOE was used
to draw the ligand structures. Ligand.mdb data base of compounds was
built. By using MMFF94X force field the compounds were then energy
minimized upto 0.01 Gradient. The enzyme structure was opened in
MOE window. The water molecules (if present) were removed. At 300 K
temperature with pH = 7, and salt concentration of 0.1 the 3D proto-
nation was done for all atoms in implicit solvated environment. The
complete structure was energy minimized using MMFF94X force field
followed by the docking of all the compounds into the binding sites of
the prepared enzymes. Default docking parameters were set and for
each compound, ten different conformations were created. Lowest
binding energy ligand enzyme complexes were analyzed by MOE ligand
interaction module while, for 3-D interaction plot, discovery studio
visualizer was used [34].
4.7. Biological activities:
4.7.1. Anti-cyclooxygenase-2 assay (COX-1/COX-2)
Cyclooxygenase-1 (COX-1) from sheep (EC 1.14.99.1) SIGMA
ALDRICH, Cyclooxygenase (COX-2) from human recombinant (CAT No.
C0858) SIGMA ALDRICH and Enzyme substrate arachidonic acid (CAT
No 150384) and lineolic acid (CAS no. 60–33-3) SIGMA ALDRICH.
Indicator and co-factor substances, glutathione (CAS 70-18-8), N, N, N-
tetramethyl-p-phenylenediamine dihydrochloride (TMPD) CAS 637–01-
4 and hematin (CAS 15489-90-4) were purchased from SIGMA
ALDRICH.
Assays were performed by following Glassman and White protocol
with little modification. The enzymes COX-1 (10 µL, 0–0.7–0.8 µg) and
COX-2 (300 units/ml) were activated on ice for 5 min. The addition of
50 µL co-factor solution containing, TMPD (N,N,N,N-tetramethyl-p-
phenylenediamine dihydrochloride) (0.24 mM), hematin (01 mM) and
glutathione (0.9 mM), was done in 0.1 M Tris HCl buffer (pH 8.0) for
the activation. 20 µL of the samples under study (with different con-
centrations ranging from 31.25 to 1000 µg/ml) and 60 µL of enzyme
solution were kept at room temperature for five minutes followed by
the addition of arachidonic acid (30 mM, 20 µL) to start the reaction.
The reaction mixture was incubated for 5 min after then the absorbance
was measured at 570 nm using UV–visible spectrophotometer. The %
inhibition of COX-1 and COX-2 was determined from absorbance value
per unit time. The IC50 values (in μM) were calculated by a plot of %
inhibition against the concentrations of sample’s solution.
Indomethacin and celecoxib were used as positive controls for COX-1
and COX-2 respectively [1].
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgment
Dr. Amara Mumtaz greatly acknowledge the Higher Education
Commission of Pakistan for funding under IPFP start-up research grant.
Dr. Umer Rashid is thankful to Higher Education Commission for fi-
nancial support for the purchase of MOE license under HEC-NRPU
project
4.7.2. In-vivo anti-inflammatory activities:
During in-vivo anti-inflammatory experiments, albino BALB/C la-
boratory mice of either sex were used throughout the study period. To
keep these animals healthy, recommended guidelines were followed
throughout the experiments [27]. The present research work was ap-
proved by departmental ethical committee and experiments were car-
ried out in PCSIR-Complex, Peshawar. A total of six animals (mice) per
group for each test dose in each experiment were used. Different ex-
perimental protocols (animal models) have been reported to study anti-
inflammatory activity by using Carrageenan induced edema models.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104168.
9

A. Munir, et al.
BioorganicChemistry104(2020)104168
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