1,2,4-Triazole-based benzothiazole/benzoxazole derivatives: Design, synthesis, p38α MAP kinase inhibition, anti-inflammatory activity and molecular docking studies

Article abstract of DOI:10.1016/j.bioorg.2018.09.015

Novel N-(benzothiazol/oxazol-2-yl)-2-[(5-(phenoxymethyl)-4-aryl-4H-1,2,4-triazol-3-yl)thio] acetamide derivatives (5a-n) were synthesized and investigated for in vitro anti-inflammatory activity and p38α MAP kinase inhibition. Compounds showing good in vitro activities (5a, 5b, 5d, 5e, 5i, 5k and 5l) were studied for their in vivo anti-inflammatory activity using carrageenan induced rat paw edema model. Compound 5b emerged as the most active compound with an edema inhibition of 84.43%. It also showed improved GI safety profile with lower ulcer severity index and lipid peroxidation potential. Also, p38α MAP kinase assay of 5b showed superior inhibitory potency (IC₅₀:0.031 ± 0.14 μM) than the standard SB 203580 (IC₅₀:0.043 ± 0.14 μM). To predict their binding mode compounds were also docked against p38α MAP kinase enzyme. Compound 5b and SB 203580 showed hinge region interaction with MET 109.

Full text of DOI:10.1016/j.bioorg.2018.09.015

Accepted Manuscript
1,2,4-Triazole-Based Benzothiazole/Benzoxazole Derivatives: Design, Synthe-
sis, p38α MAP Kinase Inhibition, Anti-Inflammatory Activity And Molecular
Docking Studies
Sana Tariq, Payal Kamboj, Ozair Alam, Mohd. Amir
PII:
S0045-2068(18)30536-4
DOI:
https://doi.org/10.1016/j.bioorg.2018.09.015
YBIOO 2512
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 June 2018
31 August 2018
7 September 2018
Please cite this article as: S. Tariq, P. Kamboj, O. Alam, Mohd. Amir, 1,2,4-Triazole-Based Benzothiazole/
Benzoxazole Derivatives: Design, Synthesis, p38α MAP Kinase Inhibition, Anti-Inflammatory Activity And
Molecular Docking Studies, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.09.015
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1,2,4-Triazole-Based Benzothiazole/Benzoxazole Derivatives: Design,
Synthesis, p38α MAP Kinase Inhibition, Anti-Inflammatory Activity And
Molecular Docking Studies
Sana Tariq, Payal Kamboj, Ozair Alam, Mohd. Amir*
Department of Pharmaceutical Chemistry, School of Pharmaceutical Education and Research,
Jamia Hamdard, New Delhi-110062, India.
Abstract: Novel N-(benzothiazol/oxazol-2-yl)-2-[(5-(phenoxymethyl)-4-aryl-4H-1,2,4-
triazol-3-yl)thio] acetamide derivatives(5a-n) were synthesized and investigated for in vitro
anti-inflammatory activity and p38α MAP kinase inhibition. Compounds showing good in
vitro activities (5a, 5b, 5d, 5e, 5i, 5k and 5l) were studied for their in vivo anti-inflammatory
activity using carrageenan induced rat paw edema model. Compound 5b emerged as the most
active compound with an edema inhibition of 84.43%. It also showed improved GI safety
profile with lower ulcer severity index and lipid peroxidation potential. Also, p38α MAP
kinase assay of 5b showed superior inhibitory potency (IC50:0.031 ± 0.14 µM) than the
standard SB 203580 (IC50:0.043 ± 0.14µM). To predict their binding mode compounds were
also docked against p38α MAP kinase enzyme. Compound 5b and SB 203580 showed hinge
region interaction with MET 109.
Keywords Benzoxazole ● p38α MAP kinase ● Docking ● Anti-inflammatory ●
Ulcerogenicity
___
*Corresponding author, Department of Pharmaceutical Chemistry, School of Pharmaceutical
Education and Research, Hamdard University, New Delhi-110062, India
Email address: mamir_s2003@yahoo.co.in

1. Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are the most commonly used drugs for the
treatment of inflammation, pain and fever. Inflammation is a part of body’s immune response
to noxious stimuli. Though useful to treat tissue damage, wounds and infections, chronic
inflammation can lead to many diseases including rheumatoid arthritis. Most of the NSAIDs
available in market are known to inhibit the two isoforms of cyclooxygenase (COX) enzyme:
COX-1 (constitutive form) and COX-2 (inducible form). But these are not devoid of adverse
effects. Concomitant use of non-selective NSAIDs which act on both the COX isoforms
causes increased risk of gastrointestinal (GI)complications, such as GI bleeding and ulcers
[1]. Though COX-2 selective NSAIDs offer GI advantages but these are obviated by serious
cardiovascular effects like congestive heart failure, myocardial infarction and death [2].
Inflammation is mediated by pro-inflammatory cytokines such as interleukins and TNF-α
(Tumor Necrosis Factor-α) produced predominantly by activated macrophages [3]. These
cytokines take part in the pathogenesis of inflammatory diseases such as rheumatoid arthritis,
psoriasis, psoriatic arthritis etc. [4]. Also, TNF-α blockers have displayed good efficacy in
randomized controlled clinical trials for the treatment of chronic immune-mediated or
inflammatory diseases [5, 6]. TNF-α inhibitors available for clinical use like Remicade,
Humiraand Enbrelare biological agents which come with the drawbacks of high cost,
inconvenient dosage regimen, lack of oral efficacy and short half-life.[7].
p38 MAP (Mitogen Activated Protein) kinase activation plays a crucial role in the
inflammatory response. There are four identified p38 MAP kinase isoforms (p38α, p38β,
p38γ, and p38δ), among which the α-form is a key regulator of the pro-inflammatory
cytokines biosynthesis including TNF-α and IL-1β at transcriptional and translational levels
[8-10]. Moreover, inhibition of p38α MAP kinase is also reported toblock theproduction of
COX-2 thereby preventing tissue destruction and inflammation [11-12]. Owing to its multiple
functions in modulating the inflammatory response, it is envisaged that p38α MAP kinase
inhibitors may be fruitful in treating the underlying cause of chronic inflammatory diseases
and cease their progression [13].In addition, small p38α MAP kinase inhibitors with low

molecular weight have the benefits of oral efficacy and are cost effective [14]. Thus, p38α
MAP kinase has developed as a key molecular target to treat inflammation. SB-203580 is a
prototypic p38α MAP kinase inhibitor which competitively binds with the active site of p38α
MAP kinase enzyme (Fig. 1). It was originally developed by Smith Kline Beecham and is
widely used as an ATP-competitive reference inhibitor in the literature [15]. Compounds with
bicyclic heteroaryl moieties have been reported to possess good p38α MAP kinase inhibitory
activity [16-18].
Fig. 1 SB203580 and its essential binding interactions with p38α MAP kinase binding site
(PDB ID: 3GCP)
Benzothiazoles and benzoxazoles are important bicyclic heteroaryl compounds with wide
spectrum of biological activities [19-24]. Previous studies reveal that benzothiazole and
benzoxazole moieties can be good templates for anti-inflammatory activity and p38α MAP
kinase inhibition [24-29]. Triazole derivatives have also shown good anti-inflammatory
activity alongwith potent p38α MAP kinase inhibition [30-35].
Prompted by the aforementioned findings and in the course of our research work [17, 18] we
synthesized triazolo-benzothiazole and benzoxazole derivatives (Fig. 2). The synthesized
compounds were evaluated for anti-inflammatory and p38α MAP kinase inhibitory activities
as well as ulcerogenicity. Besides, molecular docking studieswere performed to predict the
binding mechanism of newly synthesized compounds on the crystal structure of p38α MAP

kinase and in silicoADME analyses was also carried out to evaluate their drug likeliness
characters.
Fig. 2 Some p38α MAP kinase inhibitors (Structure I, PDB ID 1ZZL [36] and Structure II,
PDB ID 3CTQ [37], Structure III, PDB ID 3C5U [16]) and the designed compounds 5a-n

2. Results and discussion
2.1. Chemistry
The synthetic procedure to obtain the final compounds is depicted in Scheme-1. N-
(Benzothiazol-2-yl)-2-chloroacetamide (1a) and N-(Benzoxazol-2-yl)-2-chloroacetamide
(1b) were prepared by stirring 2-aminobenzothiazole/ 2-aminobenzoxazole respectively with
chloroacetyl chloride in dichloromethane and few drops of triethylamine. 2-
Phenoxyacetohydrazide (2) was obtained by esterification of phenoxyacetic acid to the
corresponding ethyl ester and its further treatment with hydrazine hydrate in absolute ethanol.
Treatment of compound 2 with aryl isothiocyanates in ethanol gave the corresponding 2-(2-
phenoxyacetyl)-N-phenylhydrazinecarbothioamides (3a-g). Reaction of compounds 3a-g
with 8% w/v NaOH solution resulted in 5-(phenoxymethyl)-4-phenyl-4H-1,2,4-triazole-3-
thiolderivatives (4a-g). N-(Benzothiazol/benzoxazol-2-yl)-2-[(5-(phenoxymethyl)-4-aryl-4H-
1,2,4-triazol-3-yl)thio]acetamides(5a-n) were prepared by coupling compounds 1a/ 1b with
compounds 4a-g. The formation of newly synthesized compounds was confirmed by their IR,
NMR, Mass spectral data and elemental analysis.

Scheme-1 Synthesis of N-(benzothiazol/oxazol-2-yl)-2-[(5-(phenoxymethyl)-4-aryl-4H-
1,2,4-triazol-3-yl)thio]acetamide derivatives(5a-n)
2.2. Anti-inflammatory activity
2.2.1. In vitro anti-inflammatory activity
All the synthesized compounds, 5a-n were initially screened for their in-vitro anti-
inflammatory activity using albumin denaturation assay. Percent inhibition of protein
denaturation for all the test compounds and the standard (diclofenac sodium) was calculated
with respect to the control. The percent inhibition shown by the compounds was found to be
in the range of 47.97 - 85.36 % (Table 1). Compound 5b having para substituted phenyl ring

with F atom, connected to N4 of the triazole displayed the highest activity (85.36 %) while
the standard drug (diclofenac sodium) showed 81.69 %inhibition. Activity was found to
decrease on replacement of F atomwithCl (5d) and Br (5e) on the para position of phenyl
ring (81.48 and 77.07% respectively). Compounds having electron releasing groups [4-
methyl (5f) and 2-methoxy (5g)] were the least active (59.26 and 49.38% respectively).
Also,it was observed that compounds having a benzothiazole ring (5a-g) were more active
than the compounds possessing benzoxazole ring (5h-n).
2.2.2. p38α MAPK assay
The compounds 5a–nwere further evaluated forp38α MAP Kinase inhibition using a non-
radioactive p38α MAP kinase assay kit. It provided rapid and sensitive screening of p38α
MAP kinase inhibitors using ELISA technique. Results are shown in Table 1.The
benzothiazole derivative 5b having para substituted phenyl ring with F atom showed highest
potency (IC50: 0.031 ± 0.14 µM) while the isosteric replacement of sulphur atom of
benzothiazole ring with oxygen (5i) resulted in slightly reduced activity (IC50: 0.038 ± 0.12
µM). Compounds 5b and 5i were more active than the standard SB203580 (IC50: 0.043 ±
0.14µM). Replacement of 4-F atom by 4-Cl (5d) and 4-Br (5e) atoms resulted in a decrease
in p38α MAP Kinase inhibitory activity (IC50: 0.075 ± 0.11 and 0.137 ± 0.03 µM
respectively). Their benzoxazole counterparts (5k and 5l) were found to be less active (IC50:
0.082 ± 0.09 and 0.248 ± 0.13 µM respectively). With an increase in size of halogen atom
activity was found to decrease (5b >5d >5e and 5i >5k >5l). Compounds with unsubstituted
phenyl ring (5a and 5h) showed nearly comparable inhibition (IC50: 0.497 ± 0.21 and 0.584 ±
0.21 µM). The compounds 5f and 5g bearing 4-CH3 and 2-OCH3 groups respectively showed
decreased activity (IC50: 0.928 ± 0.16 and 1.125 ± 0.14 µM respectively). It was found that
isosteric replacement of sulphur atom in benzothiazole ring with oxygen atom showed
reduced activity. In general, the compounds with electron attracting groups on the phenyl ring
demonstrated higher activity than the compounds with electron releasing groups.

Table 1
In vitro anti-inflammatory activity (albumin denaturation inhibition assay) and p38α MAP
kinase inhibitory activity
p38α MAP
kinase inhibitory
Albumin Denaturation inhibition
assay
activity
Compound
X
R
% Inhibition
of
denaturation
Mean Absorbance
± SDa
IC50 (µM) ±
SEMb
S
S
C6H5-
4-F- C6H4-
2-F- C6H4-
4-Cl- C6H4-
4-Br- C6H4-
4-CH3- C6H4-
2- OCH3-C6H4-
C6H5-
0.312 ± 0.04
0.350 ± 0.08
0.306 ± 0.04
0.343 ± 0.10
0.335 ± 0.07
0.301 ± 0.15
0.282 ± 0.17
0.313 ± 0.15
0.346 ± 0.14
0.299 ± 0.24
0.339 ± 0.10
0.317 ± 0.11
0.280 ± 0.30
0.294 ± 0.10
0.345 ± 0.12
65.08
0.497 ± 0.21
0.031 ± 0.14
0.871 ± 0.06
0.075 ± 0.11
0.137 ± 0.03
0.928 ± 0.16
1.125 ± 0.14
0.584 ± 0.21
0.038 ± 0.12
0.930 ± 0.05
0.082 ± 0.09
0.248 ± 0.13
1.273 ± 0.26
1.061 ± 0.04
---
5a
5b
5c
5d
5e
5f
85.36
61.90
81.48
77.07
59.26
49.38
65.78
82.89
58.38
79.19
67.55
47.97
55.56
82.54
S
S
S
S
S
5g
5h
5i
O
O
O
O
O
O
O
---
4-F- C6H4-
2-F- C6H4-
4-Cl- C6H4-
4-Br- C6H4-
4-CH3- C6H4-
2- OCH3-C6H4-
---
5j
5k
5l
5m
5n
Diclofenac
sodium
SB203580
---
---
---
---
0.042 ± 0.27
aMean values ± Standard Deviation (SD) of three experiments.
bMean values ± Standard Error of the Mean (SEM) of three experiments.

Seven compounds (5a, 5b, 5d, 5e, 5i, 5k and 5l) showing good in vitro activity were further
screened for their in vivo anti-inflammatory activity.
2.2.3. In vivo Anti-inflammatory activity
The seven selected compounds (5a, 5b, 5d, 5e, 5i, 5k and 5l) were evaluated for in vivo anti-
inflammatory activity using carrageenan induced rat paw edema model. The test compounds
and the standard were administered orally in equimolar doses to 10 mg/kg of the standard
(diclofenac sodium). The percentage inhibition was calculated at 3 and4 h interval and since
it was found to be more after 4 h, this was made the basis of discussion. The test compounds
displayed anti-inflammatory activity in the range of 61.07- 84.43% after 4 h, while diclofenac
sodium showed 82.48 % inhibition (Table 2). Compound 5b having para substituted phenyl
ring with F atom was the most active compound in the series showing 84.43 % inhibition. It
was observed that on replacing benzothiazole ring with benzoxazolering (5i) activity
decreased (82.24%). Activity was also found to decrease when F was replaced by Cl and Br
atoms in the phenyl ring.
2.3. Ulcerogenicity
Compounds (5a, 5b, 5d, 5e, 5i, 5k and 5l) were further evaluated for their acute ulcerogenic
potential. The compounds were administered at an equimolar oral dose to 30 mg/kg of the
standard (diclofenac sodium). The test compounds showed ulcerogenic activity (S.I: 0.917 ±
0.154- 1.417 ± 0.083) which was much lower than the standard diclofenac sodium (S.I: 1.750
± 0.171) (Table 2). Thus, the tested compounds have better GI safety profile in comparison
to the standard.
2.4. Lipid peroxidation
Lower ulcerogenic activity of compounds is combined with a reduced malondialdehyde
(MDA) content in the affected area of the gastrointestinal tract, end products of lipid
peroxidation [38]. Therefore, an attempt was made to correlate the decrease in ulcerogenic
activity of the compounds with that of lipid peroxidation. All the compounds screened for
ulcerogenic activity were also analyzed for lipid peroxidation.The testcompounds (5a, 5b, 5d,
5e, 5i, 5k and 5l) exhibited significantly reduced lipid peroxidation potential (4.060 ± 0.032 -

6.026 ± 0.177 nmolMDA/100 mg tissue) compared to the standard (6.795 ± 0.089
nmolMDA/100 mg tissue) (Table 2). The results suggest that the protection of gastric
mucosa might be related to the inhibition of lipid peroxidation.
Table 2.
Anti-inflammatory activity, ulcerogenicity and lipid peroxidation potential of compounds 5a,
5b, 5d, 5e, 5i, 5k, 5l and standard drug
Increase in
paw edema volume (ml)
± SEM
Activity
relative to
diclofenac
Mean ulcer
severity index (SI)
± SEM
Lipid peroxidation
nmolMDA/
100mg tissue ± SEM
Compounds
% Inhibition
0.267 ± 0.015 a
0.107 ± 0.011 a
0.152 ± 0.018 c
0.215 ± 0.021b
0.122 ± 0.013d
0.178 ± 0.017 a
0.237 ± 0.019 c
0.715 ± 0.048
0.120 ± 0.015
61.07
84.43
77.86
68.61
82.24
73.97
65.45
-
74.04
102.36
94.40
83.19
99.71
89.68
79.35
-
0.917 ± 0.154b
1.000 ± 0.224c
1.083 ± 0.154c
1.417 ± 0.083a
1.250 ± 0.112d
1.167 ± 0.167a
1.333 ± 0.279b
0.000 ± 0.000
1.750 ± 0.171
4.060 ± 0.032a
4.850 ± 0.036b
5.256 ± 0.007a
6.026 ± 0.177c
5.566 ± 0.189c
5.203 ± 0.347d
5.929 ± 0.296b
3.248 ± 0.133
6.795 ± 0.089
5a
5b
5d
5e
5i
5k
5l
Control
82.48
100
Diclofenac
sodium
Anti-inflammatory activity of the compounds was compared with respect to control.
Data were analyzed by unpaired student’s t-test for n=6.
a P<0.05; b P<0.01; c P<0.001; d P<0.0001

2.5. Molecular Docking:
Molecular docking study of the novel synthesized benzothiazole/benzoxazole derivatives was
carried out against p38α MAP Kinase enzyme. The docking scores of the fourteen
compounds (5a-n) along with the prototypic inhibitor against p38α MAP kinase are displayed
in Table 3. The 2D LigPlot diagramme of compound 5b is shown in Fig. 3. Due to the added
flexibility of -NHCO-CH2S- (linker) connecting the benzothiazole/benzoxazole & triazole
moieties, the synthesized molecules can be housed more favourably and effectively in p38α
MAP kinase binding site. The compound 5b makes vital hydrogen bonding with hinge region
MET 109 which provides potent inhibitory activity. The hydrogen bond with MET 109 seems
to play a role of anchor in binding of the compound 5b to p38α MAPK because amino acid
residues 107–110 are located at the entrance of the ATP-binding site. The binding interaction
of the 5b complex with p38α MAP Kinase revealed that amino group and carboxyl group
which are placed in between benzothiazole and triazole ring showed two hydrogen bond
interactions with ASP 168 and LYS 53 at distance of 1.76 Å and 2.42 Å respectively.
Furthermore, the docking interaction study of 5b also revealed that the triazole ring formed
strong hydrogen bonding with MET 109 present in the hinge region binding site at a distance
of 2.51 Å shown in Fig. 4a. Compound 5i at the binding site of p38α MAPKinase
demonstrates an interaction pattern almost similar to that of compound 5b, except ASP 168
shown in Fig. 4b.
The overall outline detected by docking displayed that electron withdrawing para
substitution on aryl moiety can yield optimum docking scores and also promising in vitro
results. The introduction of fluorine atom resulted in improvement of binding affinity to the
target protein receptor. Fluorine because of its most electronegative nature can modify
polarity as well as electronic distribution of adjacent groups. Also, fluorine due to its smaller
size than Cl & Br accommodated appropriately at p38α MAP Kinase receptor binding site
and produced slightest steric hindrance. The noteworthy in vitro pharmacological potential of
5b and 5i as well as good molecular docking outcomes suggested that derivatives with
electron attracting groups like F at para position can interact excellently in the active region
of p38αMAP kinase receptor. SB203580, the prototypic inhibitor wAS also docked at the
same active site of the receptor molecule in order to relate with the docked poses of
synthesized derivatives. The SB203580 docked pose showed hydrogen bonding associations

with MET 109 and LYS 53 and pi-pi interaction with TYR 35 is depicted in Fig. 5a. The
same residue LYS 53 also forms pi-cation interaction with SB203580. The superimposed
structures of prototypic inhibitor SB203580 with conformer 5b (most potent) were shown in
Fig. 5b and showed common hydrogen bond interactions with amino acids (MET 109 and
LYS 53) as well as proper alignment at p38α MAP Kinase binding site. Furthermore, the
molecular surface view of superimposed 5b and SB203580 at p38α MAP Kinase binding site
were shown in Fig. 6. The binding region of the MAP Kinase inhibitor is demarcated by
hydrophobic region. The benzothiazole ring engaged the hydrophobic region which marks the
one essential p38a MAP kinase binding site for hydrophobic part of the designed and
synthesized inhibitors. The appropriate hydrophobicity assists the derivatives for improved
action in the biological organisation. The XP visualization pose of 5b is displayed in Fig. 7a
where the hydrophobic groups in 5b are revealed in green colour ball-and-stick, and
hydrophobic groups on protein that encloses hydrophobic atoms in the ligand are revealed
with cyan colour in CPK demonstration. p38α MAP Kinase hydrophobic amino acids
enclosure are displayed in blue colour. The benzothiazole moiety of 5b is encircled by amino
acids (hydrophobic) such as VAL 83, ALA 51, LEU 75, ILE 84, LEU 86, LEU 104 and VAL
105 of hydrophobic region as shown in Fig. 7a, while fluoro phenyl moiety of SB203580 is
enclosed by same amino acids of hydrophobic region as shown in Fig. 7b. The overlay
representation of all compounds was also shown in Fig. 8. The compound 5n fail to produce
optimum in vitro activity in spite of having the highest docking score, this might be due to its
non-interaction with hinge region amino acids as shown in Fig. 9.
Validation of molecular docking was done by re-docking of co-crystallized ligand into active
site of p38α MAP Kinase receptor. The molecular docking of co-crystal ligand against the
generated receptor grid displayed accurately similar docking manner and interaction as
observed in 5b with 2.0 RMSD value and this validates the molecular docking protocol by
the generated receptor grid. Lastly, it was concluded that molecular docking and in vitro
study clarified that the administration of a smaller halogen atom like F at para position of the
phenyl ring leads to enhance affinity and potency when correlated with para substituted
electron releasing group on the same aryl ring. QikProp study was done in order to predict in
silico ADME properties of the synthesized derivatives which is mentioned in Table 3.

Table 3: Docking scores and ADME of compounds at the active sites of p38α-MAP Kinase
(PDB ID: 1A9U)
S. N
Comp.
R
X
Docking
Scores
Glide
aLog
Po/w
bPSA cLog S
dDHB eAHB
fRule
g% Oral
Energy
of five Absorption
1
2
5a
H
S
-5.186
-7.353
-6.564
-5.496
-5.422
-4.870
-5.544
-5.090
-6.463
-6.345
-5.428
-5.364
-4.968
-7.944
-7.151
-74.485
-79.844
-75.623
-79.544
-85.910
-83.339
-87.432
-68.663
-61.981
-78.558
-78.096
-85.385
-82.105
-83.071
-64.198
5.650
5.600
5.567
5.858
5.935
5.668
5.157
4.653
4.881
4.881
5.118
5.119
4.933
4.748
3.700
84.603 -6.615
84.603 -6.980
89.929 -7.651
84.603 -7.355
84.603 -7.470
84.603 -7.175
92.527 -6.935
99.297 -6.127
99.299 -6.454
99.824 -7.185
99.298 -6.729
99.298 -6.823
99.297 -6.484
104.85 -6.791
53.737 -4.409
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
6.75
6.75
6.75
6.75
6.75
6.75
7.50
7.25
7.25
7.25
7.25
7.25
7.25
8.00
7.00
1
1
1
2
2
1
2
0
0
0
1
2
0
0
0
100
100
4F
2F
S
5b
3
5c
S
100
4
5d
4Cl
S
91.75
92.20
100
5
5e
4Br
S
6
5f
4CH3
2OCH3
H
S
7
5g
S
80.26
100
8
5h
O
O
O
O
O
O
O
9
4F
100
5i
10
11
12
13
14
15
5j
2F
100
5k
5l
4Cl
94.02
81.49
100
4Br
5m
4CH3
2OCH3
5n
100
SB203580
92.30
b
aPredicted octanol/water partition coefficient (< 5); Van der Waals surface area of polar nitrogen and
c
oxygen atoms and carbonyl carbon atoms (Range 7 to 200); Predicted aqueous solubility, log S. S in mol
dm–3 is the concentration of the solute in a saturated solution that is in equilibrium with the crystalline solid
d
e
f
(Range –6.5 to 0.5); Donor hydrogen bond (<5); Acceptor hydrogen bond (<10); Lipinski’s violations (≤
1) and g% Oral Absorption (>80 High)

Fig.3. 2D LigPlot diagramme of Compound 5b

Fig 4: 3D Docked conformer of (a) 5b, presented as cyan color tube model (b) 5i, presented as white color
tube model in the binding site of p38α MAP Kinase receptor observing hydrogen bond interactions (dash
lines).
Fig. 5. (a) 3D Docked conformer of SB203580 prototype, presented as blue color tube model (b)
Superimposition of SB203580 (blue tube color) with 5b (cyan tube color) in the binding site of p38α MAP
Kinase receptors showing hydrogen bond interaction (dash lines).

Fig 6: Molecular surface view of superimpose of 5b with SB203580 complexes with p38α MAP Kinase protein.

Fig 7. XP visualization of (a) 5b showing hydrophobic interactions (b) SB203580 showing hydrophobic
interactions with p38α MAP Kinase receptor.

Fig 8: Overlay docked pose of all compounds, presented as thin wire model, surrounded by binding site amino
acids residue (yellow tube model) in the binding site of p38α MAP Kinase receptor.
Fig 9: 3D Docked conformer of high docking scores 5n, presented as yellow tube model in the binding site of
p38α MAP Kinase receptor.

3. Conclusion
In this work, hybrid molecules of benzothiazole/benzoxazole and triazole moiety were
synthesized and tested for p38α MAP kinase inhibitory and anti-inflammatory activities.
Among the series, compound 5b displayed the highest activity both in vitro and in vivo. It
also showed significant reduction in ulcerogenic potential and lipid peroxidation than that of
the standard drug which highlights its promising GI safety profile. Besides, molecular
docking of compound 5b exhibited good interaction with the active site of p38α MAP kinase.
4. Experimental Section
4.1. Chemistry
All the solvents, starting materials, and reagents were purchased from Merck, Sigma-Aldrich
and S.D. Fine chemicals and used without further purification. Precoated aluminium TLC
plates (Merck) were used for thin layer chromatography to monitor the reactions and
visualized using UV light (254 nm). Melting points were determined using Labtronics Digital
Auto Melting Point Apparatus (India) and are uncorrected. IR spectra were recorded on
Perkin-Elmer 1720 FTIR spectrometer (USA) using KBr discs and values were represented in
cm-1. 1H NMR spectra (400 MHz) and 13C-NMR spectra (100 MHz) were recorded on Bruker
Avance spectrometer (Switzerland) using tetramethylsilane (TMS) as the internal standard.
Chemical shifts were reported in ppm on d scale and J (coupling constant) were estimated in
hertz. Mass spectra were recorded on Jeol SX-102/DA-6000 spectrometer using electrospray
ionization (ESI) method.Elemental analyses (C, H and N) were conducted using CHNS Vario
EL III apparatus(Germany) and the results are within ± 0.4 % of theoretical values.N-
(Benzothiazol/benzoxazol-2-yl)-2-chloroacetamide(1a-b) [39],2-phenoxyacetohydrazide (2),
2-(2-phenoxyacetyl)-N-substituted-phenylhydrazinecarbothioamide
(3a-g)
and
5-
(phenoxymethyl)-4-(substituted)-4H-1,2,4-triazole-3-thiol (4a-g) were prepared by the
procedure reported in literature [40].
4.1.1. General procedure for synthesis of N-(Benzothiazol/oxazol-2-yl)-2-[(5-
(phenoxymethyl)-4-(substituted-phenyll)-4H-1,2,4-triazol-3-yl)thio]acetamide (5a-n)

Equimolar (1 mmol) mixtures of benzothiazolyl/benzoxazolyl acetamides (1a-b), appropriate
1,2,4-triazole-3-thiols (4a-g) and sodium carbonate were dissolved in small quantity of DMF
(15-20 ml). The resultant mixture was stirred for 4-6 h maintaining the temperature between
0-10 ºC throughout the reaction. The solid products thus obtained were filtered, washed with
water and recrystallized from ethanol. The completion of the reaction was ascertained by
TLC using Toluene: Ethyl acetate: Formamide (5:4:1)
4.1.1.1. N-(Benzothiazol-2-yl)-2-[(5-(phenoxymethyl)-4-phenyl-4H-1,2,4-triazol-3-
yl)thio]acetamide(5a)
Light yellow powder; yield 72%; m.p. 351-353 ºC; IR: (KBr, cm-1): 3061(NH), 1674 (C=O) ;
1H NMR (400MHz, DMSO-d6): δ = 4.25 (s, 2H, SCH2), 5.06 (s, 2H, OCH2), 6.83-7.92 (m,
14H, Ar-H), 12.64 (s, 1H, CONH, D2O exchangeable); 13C NMR (DMSO-d6): δ = 38.8
(SCH2), 61.2 (OCH2), 117.5, 118.1, 120.7, 121.5, 123.2, 125.3, 129.1, 130.2, 132.0, 132.6,
133.9, 150.4, 151.8, 153.6, 156.4, 168.2, 171.5; ESI-MS (m/z): 496 [M+Na]+; Anal. Calcd.
for C24H19N5O2S2: C, 60.87; H, 4.04; N, 14.79. Found: C, 60.73; H, 3.97; N, 14.78.
4.1.1.2. N-(Benzothiazol-2-yl)-2-[(4-(4-fluorophenyl)-5-(phenoxymethyl)-4H-1,2,4-triazol-3-
yl)thio]acetamide (5b)
Off white powder; yield 68%; m.p. 369-371 ºC; IR: (KBr, cm-1): 3055(NH), 1680 (C=O); 1H
NMR (400MHz, DMSO-d6): δ = 4.31 (s, 2H, SCH2), 5.12 (s, 2H, OCH2), 6.85-7.96 (m, 13H,
13
Ar-H), 11.09 (s, 1H, CONH, D2O exchangeable); C NMR (DMSO-d6): δ = 38.6 (SCH2),
61.7 (OCH2), 116.1, 118.4, 120.3, 121.7, 123.5, 124.6, 125.9, 129.0, 130.8, 132.3, 132.7,
134.1, 150.8, 152.1, 153.5, 156.7, 168.9, 174.5; ESI-MS (m/z): 492 [M+H]+; Anal. Calcd. for
C24H18FN5O2S2: C, 58.64; H, 3.69; N, 14.25. Found: C, 58.61; H, 3.67; N, 14.26.

4.1.1.3. N-(Benzothiazol-2-yl)-2-[(4-(2-fluorophenyl)-5-(phenoxymethyl)-4H-1,2,4-triazol-3-
yl)thio]acetamide (5c)
White powder; yield 73%; m.p. 358-360 ºC; IR: (KBr, cm-1): 3178 (NH), 1701 (C=O);1H
NMR (400MHz, DMSO-d6): δ = 4.27 (s, 2H, SCH2), 5.16 (s, 2H, OCH2), 6.73-7.89 (m, 13H,
13
Ar-H), 10.03 (s, 1H, CONH, D2O exchangeable); C NMR (DMSO-d6): δ = 38.4 (SCH2),
61.2 (OCH2), 116.8, 117.1, 118.6, 120.5, 121.8, 122.7, 124.5, 125.1, 128.3, 129.2, 130.7,
131.3, 132.5, 134.3, 150.9, 152.2, 153.6, 155.8, 169.0, 175.1; ESI-MS (m/z): 496 [M+Na]+;
Anal. Calcd. for C24H18FN5O2S2: C, 58.64; H, 3.69; N, 14.25. Found: C, 58.65; H, 3.61; N,
14.23.
4.1.5.4. N-(Benzothiazol-2-yl)-2-[(4-(4-chlorophenyl)-5-(phenoxymethyl)-4H-1,2,4-triazol-3-
yl)thio]acetamide (5d)
Yellow powder; yield 81%; m.p. 365-367 ºC; IR: (KBr, cm-1): 3167 (NH), 1691 (C=O);1H
NMR (400MHz, DMSO-d6): δ = 4.17 (s, 2H, SCH2), 5.08 (s, 2H, OCH2), 6.87-8.40 (m, 13H,
13
Ar-H), 10.39 (s, 1H, CONH, D2O exchangeable); C NMR (DMSO-d6): δ = 38.7 (SCH2),
60.0 (OCH2), 116.6, 118.4, 120.1, 120.5, 123.1, 124.0, 125.1, 129.1, 129.2, 132.1, 132.6,
132.8, 150.4, 150.7, 153.3, 156.1, 168.1, 171.1; ESI-MS (m/z): 525 [M+NH4]+; Anal. Calcd.
for C24H18ClN5O2S2: C, 56.74; H, 3.57; N, 13.79. Found: C, 56.71; H, 3.59; N, 13.75.
4.1.5.5. N-(Benzothiazol-2-yl)-2-[(4-(4-bromophenyl)-5-(phenoxymethyl)-4H-1,2,4-triazol-3-
yl)thio]acetamide (5e)
Yellow powder; yield 77%; m.p. 360-362 ºC; IR: (KBr, cm-1): 3178 (NH), 1701 (C=O) ;
1H NMR (400MHz, DMSO-d6): δ = 4.23 (s, 2H, SCH2), 5.16 (s, 2H, OCH2), 6.83-8.41 (m,
13H, Ar-H), 10.34 (s, 1H, CONH, D2O exchangeable); 13C NMR (DMSO-d6): δ = 38.8
(SCH2), 61.3 (OCH2), 114.8, 116.7, 120.5, 121.8, 123.6, 125.4, 127.4, 129.2, 130.1, 131.5,
132.6, 135.2, 149.6, 151.5, 153.2, 156.8, 167.1, 172.5; ESI-MS (m/z): 553 [M+H]+;

554[M+2]+; Anal. Calcd. for C24H18BrN5O2S2: C, 52.18; H, 3.28 N, 12.68. Found: C, 52.15;
H, 3.25; N, 12.64.
4.1.5.6.N-(Benzothiazol-2-yl)-2-[(5-(phenoxymethyl)-4-(p-tolyl)-4H-1,2,4-triazol-3-
yl)thio]acetamide (5f)
White powder; yield 79%; m.p. 343-345 ºC; IR: (KBr, cm-1): 3059 (NH), 1672 (C=O);1H
NMR (400MHz, DMSO-d6): δ = 2.38 (s, 3H, CH3), 4.19 (s, 2H, SCH2), 5.13 (s, 2H, OCH2),
6.91-7.97 (m, 13H, Ar-H), 10.42 (s, 1H, CONH, D2O exchangeable); 13C NMR (DMSO-d6):
δ = 20.8 (CH3), 38.3 (SCH2), 61.5 (OCH2), 115.2, 116.8, 120.2, 121.7, 123.5, 125.8, 127.5,
129.1, 130.4, 131.2, 132.7, 134.8, 149.5, 151.7, 153.1, 157.5, 167.6, 172.1; ESI-MS (m/z):
489 [M+H]+; Anal. Calcd. for C25H21N5O2S2: C, 61.58; H, 4.34; N, 14.36. Found: C, 61.52;
H, 4.39; N, 14.34.
4.1.5.7.N-(Benzothiazol-2-yl)-2-[(4-(2-methoxyphenyl)-5-(phenoxymethyl)-4H-1,2,4-triazol-
3-yl)thio]acetamide (5g)
White powder; yield 74%; m.p. 347-349 ºC; IR: (KBr, cm-1): 3180 (NH), 1704 (C=O);1H
NMR (400MHz, DMSO-d6): δ = 3.74 (s, 3H, OCH3), 4.13 (s, 2H, SCH2), 5.07 (s, 2H,
OCH2), 6.79-7.94 (m, 13H, Ar-H), 12.64 (s, 1H, CONH, D2O exchangeable); 13C NMR
(DMSO-d6): δ = 38.1 (SCH2), 54.8 (OCH3), 61.7 (OCH2), 115.1, 116.5, 120.4, 121.6, 123.4,
125.2, 127.3, 129.7, 130.2, 131.5, 133.9, 135.7, 149.3, 151.6, 154.2, 157.3, 159.0, 165.1,
167.8, 173.2; ESI-MS (m/z): 527 [M+Na]+; Anal. Calcd. for C25H21N5O3S2: C, 59.62; H,
4.20; N, 13.91. Found: C, 59.61; H, 4.18; N, 13.93.
4.1.5.8.N-(Benzoxazol-2-yl)-2-[(5-(phenoxymethyl)-4-phenyl-4H-1,2,4-triazol-3-
yl)thio]acetamide (5h)
Brown powder; yield 76%; m.p. 362-365 ºC; IR: (KBr, cm-1): 3059 (NH), 1674 (C=O);1H
NMR (400MHz, DMSO-d6): δ = 4.27 (s, 2H, SCH2), 5.11 (s, 2H, OCH2), 6.77-7.91 (m, 14H,

13
Ar-H), 10.39 (s, 1H, CONH, D2O exchangeable); C NMR (DMSO-d6): δ = 38.2 (SCH2),
62.5 (OCH2), 116.1, 118.4, 120.3, 121.7, 124.2, 125.2, 127.3, 129.1, 130.5, 132.8, 134.7,
150.1, 152.4, 153.2, 156.5, 168.4, 174.2; ESI-MS (m/z): 459 [M+H]+; Anal. Calcd. for
C24H19N5O3S: C, 63.01; H, 4.19; N, 15.31. Found: C, 62.98; H, 4.16; N, 15.34.
4.1.5.9.N-(Benzoxazol-2-yl)-2-[(4-(4-fluorophenyl)-5-(phenoxymethyl)-4H-1,2,4-triazol-3-
yl)thio]acetamide (5i)
Yellow powder; yield 79%; m.p. 371-373 ºC; IR: (KBr, cm-1): 3181 (NH), 1691 (C=O);1H
NMR (400MHz, DMSO-d6): δ = 4.25 (s, 2H, SCH2), 5.12 (s, 2H, OCH2), 6.87-7.96 (m, 13H,
13
Ar-H), 10.42 (s, 1H, CONH, D2O exchangeable); C NMR (DMSO-d6): δ = 38.1 (SCH2),
64.4 (OCH2), 115.4, 118.1, 120.5, 121.6, 124.5, 125.8, 127.2, 128.6, 129.5, 130.3, 132.7,
134.5, 150.6, 152.8, 154.1, 156.4, 168.5, 175.3; ESI-MS (m/z): 476 [M+H]+; Anal. Calcd. for
C24H18FN5O3S: C, 60.62; H, 3.82; N, 14.73. Found: C, 60.64; H, 3.79; N, 14.72.
4.1.5.10.N-(Benzoxazol-2-yl)-2-[(4-(2-fluorophenyl)-5-(phenoxymethyl)-4H-1,2,4-triazol-3-
yl)thio]acetamide (5j)
Light yellow powder; yield 81%; m.p. 369-371 ºC; IR: (KBr, cm-1): 3168 (NH), 1701
(C=O);1H NMR (400MHz, DMSO-d6): δ = 4.27 (s, 2H, SCH2), 5.09 (s, 2H, OCH2), 6.81-
13
7.93 (m, 13H, Ar-H), 10.76 (s, 1H, CONH, D2O exchangeable); C NMR (DMSO-d6): δ =
38.5 (SCH2), 63.2 (OCH2), 116.1, 117.2, 118.4, 120.6, 121.5, 122.4, 124.6, 125.8, 128.2,
129.1, 130.4, 131.7, 132.2, 134.8, 151.9, 152.6, 153.4, 155.1, 169.3, 174.1; ESI-MS (m/z):
476 [M+H]+; Anal. Calcd. for C24H18FN5O3S: C, 60.62; H, 3.82; N, 14.73. Found: C, 60.59;
H, 3.80; N, 14.71.
4.1.5.11.N-(Benzoxazol-2-yl)-2-[(4-(4-chlorophenyl)-5-(phenoxymethyl)-4H-1,2,4-triazol-3-
yl)thio]acetamide (5k)

White powder; yield 86%; m.p. 380-382 ºC; IR: (KBr, cm-1): 3172 (NH), 1709 (C=O) ;1H
NMR (400MHz, DMSO-d6): δ = 4.22 (s, 2H, SCH2), 5.13 (s, 2H, OCH2), 6.87-7.97 (m, 13H,
13
Ar-H), 10.49 (s, 1H, CONH, D2O exchangeable); C NMR (DMSO-d6): δ = 38.2 (SCH2),
62.4 (OCH2), 114.6, 117.2, 120.3, 121.9, 124.7, 125.6, 127.5, 128.4, 129.2, 130.5, 133.6,
135.8, 151.6, 152.8, 154.7, 156.1, 168.2, 175.4; ESI-MS (m/z): 493 [M+H]+, 494 [M+2]+,
Anal. Calcd. for C24H18ClN5O3S: C, 58.59; H, 3.69; N, 14.24. Found: C, 58.57; H, 3.67; N,
14.25.
4.1.5.12.N-(benzoxazol-2-yl)-2-((4-(4-bromophenyl)-5-(phenoxymethyl)-4H-1,2,4-triazol-3-
yl)thio)acetamide (5l)
Light yellow powder; yield 84%; m.p. 369-371 ºC; IR: (KBr, cm-1): 3178 (NH), 1704
(C=O);1H NMR (400MHz, DMSO-d6): δ = 4.00 (s, 2H, SCH2), 5.10 (s, 2H, OCH2), 6.90-
13
7.77 (m, 13H, Ar-H), 10.29 (s, 1H, CONH, D2O exchangeable); C NMR (DMSO-d6): δ =
38.2 (SCH2), 60.5 (OCH2), 115.7, 117.4, 120.1, 121.6, 123.3, 124.7, 126.8, 129.1, 130.7,
131.3, 132.5, 134.8, 148.3, 151.4, 153.7, 156.4, 168.0, 173.2; ESI-MS (m/z): 537 [M+H]+;
Anal. Calcd. for C24H18BrN5O3S: C, 53.74; H, 3.38; N, 13.06. Found: C, 53.71; H, 3.35; N,
13.08.
4.1.5.13.N-(benzoxazol-2-yl)-2-((5-(phenoxymethyl)-4-(p-tolyl)-4H-1,2,4-triazol-3-
yl)thio)acetamide (5m)
White powder; yield 79%; m.p. 343-345 ºC; IR: (KBr, cm-1): 3167 (NH), 1692 (C=O);1H
NMR (400MHz, DMSO-d6): δ = 2.35 (s, 3H, CH3), 4.11 (s, 2H, SCH2), 5.08 (s, 2H, OCH2),
6.88-7.95 (m, 13H, Ar-H), 10.12 (s, 1H, CONH, D2O exchangeable); 13C NMR (DMSO-d6):
δ = 20.5 (CH3), 38.2 (SCH2), 61.7 (OCH2), 115.1, 116.5, 120.4, 121.8, 123.6, 125.9, 127.2,

129.4, 130.6, 131.8, 132.5, 134.7, 149.3, 151.6, 153.4, 157.3, 168.4, 171.2; ESI-MS (m/z):
473 [M+H]+; Anal. Calcd. for C25H21N5O3S: C, 63.68; H, 4.49; N, 14.85. Found: C, 63.62; H,
4.47; N, 14.82.
4.1.5.14.N-(Benzoxazol-2-yl)-2-[(4-(2-methoxyphenyl)-5-(phenoxymethyl)-4H-1,2,4-triazol-
3-yl)thio]acetamide (5n)
Light brown powder; yield 79%; m.p. 352-354 ºC; IR: (KBr, cm-1): 3179 (NH), 1701 (C=O);
1H NMR (400MHz, DMSO-d6): δ = 3.75 (s, 3H, OCH3), 4.11 (s, 2H, SCH2), 5.08 (s, 2H,
OCH2), 6.87-7.93 (m, 13H, Ar-H), 10.37 (s, 1H, CONH, D2O exchangeable); 13C NMR
(DMSO-d6): δ = 38.4 (SCH2), 55.6 (OCH3), 61.9 (OCH2), 115.2, 116.6, 120.2, 121.7, 123.9,
125.3, 127.5, 129.4, 130.6, 131.3, 133.8, 135.5, 149.4, 151.7, 154.6, 157.2, 159.1, 164.7,
167.3, 173.5; ESI-MS (m/z): 489 [M+H]+; Anal. Calcd. for C25H21N5O4S: C, 61.59; H, 4.34;
N, 14.36. Found: C, 61.54 H, 4.32; N, 14.38.
Biological Screening
In vitro studies
Anti-inflammatory screening
The in vitro anti-inflammatory activity of the synthesized compounds was carried out by
BSA denaturation technique of Mizushima et al [41].
p38α MAPKinase assay
The compounds were assayed for p38α MAP Kinase inhibition using the CycLex p38 Kinase
assay kit (MBL, USA). p38α positive control was also procured from CycLex(MBL, USA).
Inhibition of p38α MAPKinase was determined using the reported method of Forrer et
al. [42].

In vivo studies
Animals
Animals used in the study were housed in the Central Animal House Facility of Jamia
Hamdard, New Delhi in compliance with the guidelines and rules laid down by the
Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA). Animal facilities and other areas in contact with the laboratory animals were
cleaned and disinfected. Wistar rats (12 weeks old) of either sex weighing 180–200 g were
used and acclimatized to room conditions for one week before the experiments. Food and
water were available ad libitum up to the time of experiments.
In-vivo Anti-inflammatory activity
Anti-inflammatory activity of the synthesized compounds was carried out using carrageenan
induced rat hind paw edema method of Winter et al [43].
Ulcerogenic activity
Compounds (5a, 5b, 5d, 5e, 5i, 5k and 5l) showing significant anti-inflammatory activity
were further screened for their ulcerogenic potential by the reported method of Cioli et al
[44].
Lipid peroxidation
Lipid peroxidation in the gastric mucosa was carried out by the earlier reported method of
Ohkawa et al [45].
Molecular Docking Methodology
Docking approach is a very essential tool in designing of compounds which guide to predict
the interactions between a ligand molecule and a receptor in order to anticipate the affinity
and the activity of the designed inhibitor. Docking study of designed derivatives equipped
with benzothiazole/benzoxazole associated triazole were performed against p38α MAP
kinase target that is essential for inflammatory process using Glide extra precision (XP)
Maestro 10.1 Schrodinger, running on Linux 64 operating system. The generated 2D
structures of synthesized derivatives were then transformed to their corresponding 3D
structures by using LigPrep module. The PDB file with PDB-ID 1A9U and resolution 2.5 Å

of p38α MAP Kinase which was firmly attached to inhibitor was claimed from RCSB Protein
Data Bank (www.rcsb.org). The desired protein was carried out using the protein preparation
wizard and receptor grid was generated for co-crystal ligand. The water molecules that were
beyond 5 Å were excluded. The targeted protein was optimized by performing energy
minimization at OPLS 2005 force field and by assigning hydrogen bonds. Finally, the docked
poses and their requisite binding interactions with concerned amino acids within the receptor
site were observed thoroughly.
Conflict of interest
The authors have declared no conflict of interest.
Acknowledgements
The author (Sana Tariq) is thankful to Hamdard National Foundation (HNF), Delhi, India,
for financial support. The co-author (Dr. Ozair Alam) is also thankful to DST-SERB
(SB/FT/LS-203-2012), New Delhi, for providing molecular modelling software (Schrodinger,
USA) under the Fast track young scientist Scheme. Authors are also thankful to Incharge
CIF, School of Pharmaceutical Education and Research, Hamdard University for spectral
analysis of compounds.
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