Indole derivatives as cyclooxygenase inhibitors: Synthesis, biological evaluation and docking studies

Article abstract of DOI:10.3390/molecules23061250

A new series of 2-(5-methoxy-2-methyl-1H-indol-3-yl)-N-[(E)-(substituted phenyl) methylidene] acetohydrazide derivatives (S1–S18) were synthesized and evaluated for their anti-inflammatory activity, analgesic activity, ulcerogenic activity, lipid peroxidation, ulcer index and cyclooxygenase expression activities. All the synthesized compounds were in good agreement with spectral and elemental analysis. Three synthesized compounds (S3, S7 and S14) have shown significant anti-inflammatory activity as compared to the reference drug indomethacin. Compound S3 was further tested for ulcerogenic index and cyclooxygenase (COX) expression activity. It was selectively inhibiting COX-2 expression and providing the gastric sparing activity. Docking studies have revealed the potential of these compounds to bind with COX-2 enzyme. Compound S3 formed a hydrogen bond between OH of Tyr 355 and NH₂ of Arg 120 with carbonyl group and this hydrogen bond was similar to that formed by indomethacin. This study provides insight for compound S3, as a new lead compound as anti-inflammatory agent and selective COX-2 inhibitor.

Full text of DOI:10.3390/molecules23061250

molecules
Article
Indole Derivatives as Cyclooxygenase Inhibitors:
Synthesis, Biological Evaluation and Docking Studies
Mashooq Ahmad Bhat 1, ID , Mohamed A. Al-Omar 1 ID , Mohammad Raish 2,
*
Mushtaq Ahmad Ansari 3, Hatem A. Abuelizz 1 ID , Ahmed H. Bakheit 1,4 and
Ahmed M. Naglah 5,6
1
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451,
Saudi Arabia; malomar1@ksu.edu.sa (M.A.A.-O.); habuelizz@ksu.edu.sa (H.A.A.);
abujazz@gmail.com (A.H.B.)
Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia;
2
mraish@ksu.edu.sa
3
Department of Pharmacology, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia;
muansari@ksu.edu.sa
4
Department of Chemistry, Faculty of Science and Technology, El-Neelain University, P.O. Box 12702,
Khartoum 11121, Sudan
Department of Pharmaceutical Chemistry, Drug Exploration and Development Chair (DEDC),
5
College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia; amnaglah@gmail.com
Peptide Chemistry Department, Chemical Industries Research Division, National Research, Centre,
6
12622-Dokki, Cairo, Egypt
*
Correspondence: mabhat@ksu.edu.sa; Tel.: +966-55-8164-097
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 25 April 2018; Accepted: 19 May 2018; Published: 24 May 2018
Abstract: A new series of 2-(5-methoxy-2-methyl-1H-indol-3-yl)-N0-[(E)-(substituted phenyl)
methylidene] acetohydrazide derivatives (S1 S18) were synthesized and evaluated for their
–
anti-inflammatory activity, analgesic activity, ulcerogenic activity, lipid peroxidation, ulcer index
and cyclooxygenase expression activities. All the synthesized compounds were in good agreement
with spectral and elemental analysis. Three synthesized compounds (S3, S7 and S14) have shown
significant anti-inflammatory activity as compared to the reference drug indomethacin. Compound
S3 was further tested for ulcerogenic index and cyclooxygenase (COX) expression activity. It was
selectively inhibiting COX-2 expression and providing the gastric sparing activity. Docking studies
have revealed the potential of these compounds to bind with COX-2 enzyme. Compound S3 formed
a hydrogen bond between OH of Tyr 355 and NH2 of Arg 120 with carbonyl group and this hydrogen
bond was similar to that formed by indomethacin. This study provides insight for compound S3, as a
new lead compound as anti-inflammatory agent and selective COX-2 inhibitor.
Keywords: indole derivatives; docking; anti-inflammatory activity; analgesic activity; ulcerogenic
activity; cyclooxygenase expression
1. Introduction
Inflammation is an important mechanism to defend the body against infection or any physical
or chemical offense. This protection mechanism is involved in common life-threatening diseases,
including autoimmune diseases such as rheumatoid arthritis, Crohn’s disease or inflammatory bowel
syndrome [1]. Prostaglandin is the essential moderator in the inflammation process. Cyclooxygenase
enzyme (COX) catalyzes the conversion of arachidonic acid into the prostaglandin E2, because of
the instability of prostaglandin E2, an isomerase enzyme specific to this intermediate, converts it
to many prostanoids [2]. Anti-platelet activity and the protection of the gastro-intestinal tract are
Molecules 2018, 23, 1250; doi:10.3390/molecules23061250
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1250
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the beneficial effect of the prostaglandin. Fever and pain associated with the inflammation are the
unwanted effects of prostaglandin [ ]. Two isoforms of COX have been identified, COX-1 and COX-2.
3
The gastrointestinal (GI) tract cytoprotection effect is provided by COX-1 while the inflammation is
mediated by COX-2 [4].
Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used and are effective analgesics
in palliative care. However, use of these medicines is limited, because of adverse side effects.
The gastrointestinal toxicity is a common side effect, which is sometimes concomitant with gastro
duodenal perforation, bleeding or ulcer complications. This toxicity is caused by the inhibition of
cyclooxygenase-1 (COX-1). Highly risked patients with NSAIDs were prescribed with gastro protective
drugs, such as the proton pump inhibitors. Therefore, selective inhibitors of the cyclooxygenase-2
(COX-2) is attempted to be developed in order to reduce the side effects. Studies have confirmed
the reduction of the gastrointestinal toxicity incidences by the COX selective drugs [
commonly used NSAIDs have high selectivity to COX-1 more than COX-2. Therefore, their use for
long time will cause gastric irritation, ulcer or bleeding [ ]. The selective inhibition of COX-2 will have
5]. Most of the
6
the same anti-inflammatory effect as the non-selective inhibitors but with less gastrointestinal adverse
effect incidence. However, it allows the cytoprotective prostaglandin synthesis; reduce the bleeding
and ulceration [7]. Drugs such as celecoxib, rofecoxib, etoricoxib and valdecoxib have been approved
as selective COX-2 inhibitors. These drugs were having fewer gastrointestinal side effects compared to
traditional NSAIDs. Long term uses of COX-2 inhibitors have been reported with cardiovascular side
effects except celecoxib.
Indomethacin is a NSAID and one of the indole acetic acid derivative, which is known to cause
ulcers for its users. However, its safety profile has been improved by chemical modifications [8]. This
has shown, that modification by synthesis has high possibility to provide derivatives with significant
anti-inflammatory activity and fewer side effects. This will be provided by the COX-2 inhibitors as a
solution of the aforementioned criteria [
chemistry. Indole derivatives have been reported to possess significant pharmacological activities.
Derivatives of indole have been used as anti-inflammatory, analgesic and antipyretic agents [11 13].
9,10]. Indole is an important scaffold in the field of medicinal
–
Schiff bases have been reported to possess various pharmacological activities e.g., anti-inflammatory
activity [14], anti-tubercular activity [15] and anticonvulsant activity [16,17].
The aim of the present study was to synthesize novel indole derivatives and to evaluate their
potential as anti-inflammatory and analgesic agents with gastric sparing activity.
2. Results and Discussion
Indole hydrazide, 2-(5-methoxy-2-methyl-1H-indol-3-yl) acetohydrazide (
starting material for the synthesis of various substituted indole derivatives [18]. Schiff bases were
obtained by refluxing indole hydrazide ( ) with differently substituted benzaldehydes in ethanol
1), was used as a
1
with Glacial acetic acid as catalyst. The synthesis of these compounds was achieved through an
efficient synthetic route (Scheme 1). The 1H-NMR and 13C-NMR was used to assign the structures
of synthesized compounds. Moreover, the structures were characterized by elemental analysis, mass
spectrometry and FT IR. The structure of the 2-(5-methoxy-2-methyl-1H-indol-3-yl) acetohydrazide (1)
has shown similar NMR splitting pattern and δ-values (δH & δC) as that of indomethacin, including the
pattern of three aromatic protons (H-3, H-5 and H-6) and their 13C-signals. The structures of the indole
hydrazide derivatives were established on the basis of 1H-NMR analysis, which was confirmed by the
disappearance of –NH2 protons at 4.26 ppm. The detailed results of 1H-NMR and 13C-NMR and MS
are provided in the experimental part. The presence of all carbon atoms for compounds (S1–S18) were
confirmed by 13C-NMR spectra. Molecular weights of compounds were confirmed by mass spectra.
Molecular ion peak were observed in all the compounds.

Molecules 2018, 23, 1250
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Cl
O
N
CH3
OH
O
CH3
Indomethacin
O
CH3OH, H2SO4, Reflux 3 hr
Cl
O
N
CH3
H3C
O
OCH 3
O
NH2NH2.H2O, EtOH, Reflux 30 h
Cl
H
N
CH3
Compound Code
S 1
R
H3C
O
NHNH 2
Phenyl
S 2
4-Nitrophenyl
O
NH
(1)
O
3-Nitrophenyl
S 3
(2)
NH2
2-Nitrophenyl
S 4
RCHO/CH3COOH
S5
4-Chlorophenyl
H
N
S 6
2, 4-Dichlorophenyl
3,4-Dimethoxyphenyl
2-Methoxyphenyl
S 7
CH3
H3C
S 8
R
O
NH
N
S 9
4-Hydroxyphenyl
(S1-18)
S 10
S 11
S 12
S 13
S 14
S 15
S 16
S 17
S 18
3-Hydroxyphenyl
O
4- Dimethylaminophenyl
3-methoxyphenyl
4-Ethoxyphenyl
2, 4, 5-Trimethoxyphenyl
2, 3, 4-Trimethoxyphenyl
3, 4, 5-Trimethoxyphenyl
2, 4, 6-Trimethoxyphenyl
2, 4-Dimethoxyphenyl
Scheme 1. Synthetic route of compounds (S1–S18).
2.1. Anti-Inflammatory Activity
The synthesized compounds (S1–S18) were screened by the carrageenan-induced paw edema
method for anti-inflammatory activity [19]. The anti-inflammatory activity of the tested compounds after
2 h ranges between 7.35% to 62.69% inhibition. After 3 h, the inhibition was 7.06% to 63.69%, compared
to the reference drug indomethacin that showed 77.23% inhibition after 2 h and 76.89% inhibition after
3 h. The compounds (S3, S7 and S14) were found to be most potent compounds of the series compared to
the reference drug indomethacin (Table 1). A significant anti-inflammatory activity of these compounds
was observed because of their hydrazide substitution. However, the substitution of the hydrazide with
3-nitrophenyl as in compound S3 has shown 61.99% inhibition after 2 h and 61.20% inhibition after
3 h. Whereas, the substitution with 3,4-dimethoxyphenyl as in compound S7 was observed to have
61.47% and 62.24% inhibition after 2 h and 3 h, respectively. The inhibition by compound S14 with
2,4,5-trimethoxyphenyl substitution was 62.69% after 2 h and 63.69% after 3 h. These observations

Molecules 2018, 23, 1250
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provided the base for further testing and development of these compounds. The indole Schiff
base derivatives (S1 S18) having 3-nitrophenyl; 3,4 dimethoxyphenyl and 2,4,5 trimethoxyphenyl
–
substitutions showed the maximum anti-inflammatory activity. Minimum anti-inflammatory activity
was shown by the compound containing 2-methoxyphenyl substitution. The compounds containing
2-nitrophenyl; 2,4-dichlorophenyl and 4-dimethylaminophenyl substitution showed more than
50% inhibition.
Table 1. Anti-inflammatory activity of compounds (S1–S18).
Increase in Paw Volume (mm)
% Inhibition
Potency
Treatments
After 2 h
After 3 h
After 2 h
After 3 h
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
S13
S14
S15
S16
S17
0.53 ± 0.04
0.81 ± 0.03
0.53 ± 0.04 ***
0.79 ± 0.02 ***
0.37 ±0.04 ***
0.43 ± 0.02 ***
0.85 ± 0.06
43.95
14.71
61.99
54.46
13.48
51.31
61.47
4.20
39.40
28.19
52.18
14.71
19.96
62.69
7.35
44.31
17.93
61.20
55
11.89
51.89
62.24
0
0.57
0.21
0.79
0.71
0.16
0.66
0.80
0.02
0.55
0.36
0.69
0.21
0.22
0.82
0.09
0.22
0.63
0.40
1.00
-
0.36 ± 0.04 ***
0.43 ± 0.01 ***
0.82 ± 0.07
0.46 ± 0.04 ***
0.36 ± 0.02 ***
0.91 ± 0.04
0.46 ± 0.05 ***
0.36 ± 02 ***
0.95 ± 0.03
0.57 ± 0.02 ***
0.68 ± 0.02 ***
0.45 ± 0.02 ***
0.81 ± 0.03 *
0.76 ± 0.33 **
0.35 ± 0.02 ***
0.88 ± 0.01
0.51 ± 0.03 ***
0.69 ± 0.01 ***
0.43 ± 0.03 ***
0.79 ± 0.03 **
0.82 ± 0.04 *
0.34 ± 0.02 ***
0.89 ± 0.09
46.37
27.75
55
18.27
14.65
63.69
7.06
17.41
50.34
35.68
76.89
-
0.78 ± 0.04 *
0.49 ± 0.02 ***
0.69 ± 0.12
0.79 ± 0.04 *
0.48 ± 0.02 ***
0.62 ± 0.05 ***
0.22 ± 0.02 ***
0.96 ± 0.02
17.68
47.81
27.49
77.23
-
S18
indomethacin
Control
0.20 ± 0.02 ***
0.95 ± 0.02
All values represent mean
comparison test (n = 6) All data were compare with control group.
±
SEM.* p < 0.05, ** p < 0.01, *** p < 0.001; ANOVA, followed by Dunnett’s multiple
2.2. Analgesic Activity
The compounds that exhibited significant anti-inflammatory activity were selected for the
analgesic activity (Table 2). Hot-plate method was used for testing the analgesic activity [20].
The compound S14 (R = 2,4,5-trimethoxyphenyl) expressed significant analgesic activity of 70.27%
inhibition, compared with the reference drug indomethacin with 84.09% inhibition. The analgesic
activity of compound S3 (R = 3-nitrophenyl) was found to be 61.36%. The exchange of the nitro group
at 3 position of the substituted phenyl with hydroxyl group has shown analgesic activity of 62.50%
in compound S10. The compound S17 (R = 2,4,6-trimethoxyphenyl) was found to have significant
analgesic activity with 61.53% inhibition. The aforementioned analgesic results have proven that
compounds S14, S10, S17, S3 and S9 have a significant analgesic activity.
Table 2. Analgesic activities of selected compounds.
Pretreatment (0 h)
Post Treatment (3 h)
Potency
Treatments
% Inhibition
Mean ± SE (Second)
Mean ± SE (Second)
S1
S3
S4
S6
S7
8.33 ± 0.49
7.33 ± 0.42
6.83 ± 0.30
7.33 ± 0.33
7.33 ± 0.42
10.83 ± 0.79 *
11.83 ± 0.65 ***
7.33 ± 0.40
30
61.36
7.31
13.63
47.72
0.35
0.72
0.08
0.16
0.56
8.33 ± 0.49
10.83 ± 0.47 ***

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Table 2. Cont.
Post Treatment (3 h)
Pretreatment (0 h)
Potency
Treatments
% Inhibition
Mean ± SE (Second)
Mean ± SE (Second)
S9
S10
S11
S14
S17
7.16 ± 0.30
6.66 ± 0.33
7.16 ± 0.47
6.16 ± 0.30
6.50 ± 0.22
6.16 ± 0.30
7.33 ± 0.42
11.16 ± 0.60 ***
10.83 ± 0.47 ***
10.16 ± 0.65 **
10.50 ± 0.42 ***
10.50 ± 0.42 ***
7.66 ± 0.33 **
55.81
62.50
41.86
70.27
61.53
24.32
84.09
0.66
0.74
0.49
0.83
0.73
0.28
1.00
S18
indomethacin
13.50 ± 0.42 ***
All values represent mean
±
SEM.* p < 0.05, ** p < 0.01, *** p < 0.001; ANOVA, followed by Dunnett’s multiple
comparison test, (n = 6). All data were compared with control group.
2.3. Ulcerogenic Activity
All the compounds were further evaluated for their ulcerogenic and lipid peroxidation activity
(Table 3). The mucosal damage after oral administration of the compounds was examined [21].
Equimolar concentrations of each compound were administered as oral doses to the examined animals.
The examined compounds demonstrated a significant ulcerogenic reduction activity ranging between
0 to 0.966
±
0.16. The maximum ulcerogenic reduction activities were observed in compounds S4, S6,
S11 S15 and S17. The reduction of the ulcerogenic activity for compound S3 (R = 3-nitrophenyl) and
,
S7 (R = 3,4-dimethoxyphenyl) were 0.116 and 0.432, respectively. However, the ulcerogenic reduction
activity of compound S14 (R = 2,4,5-trimethoxyphenyl) was 0.616. These results of the reduction of the
ulcerogenic activity were compared with the standard drug indomethacin (0.948
±
0.21). It was noticed
that the highest activity was observed with the 2-nitrophenyl substitution as in compound S4 compared
with the 3-nitrophenyl as in compound S3. The methoxy group substitution on the phenyl presented
a significant ulcerogenic reduction. However, the 3,4-dimethoxyphenyl substitution has shown 54%
reduction in the ulcerogenic activity in compound S7, compared to S15 with an added third methoxy
group at position 2 to be 2,3,4-trimethoxyphenyl substitution. Similar observation was in compound
S17 with (R = 2,4,6-trimethoxyphenyl) that showed 0 ulcerogenic activity. This was compared with
compound S14 with (R = 2,4,5-trimethoxyphenyl) substitution with 35% of the ulcerogenic reduction
activity. It has been known that the reduction of malondialdehyde (MDA) content in liver and
kidney tissues is concomitant with the reduction of the ulcerogenic activity [22]. The gastric mucosa
was scrapped after the ulcerogenic activity screening for lipid peroxidation determination in the
gastric mucosa. The reference drug indomethacin has shown highest lipid peroxidation in liver as
8.16 nmoL/100 mg and in kidney as 6.70 nmoL/100 mg. However, compounds with less ulcerogenic
activity have also shown reduction in the lipid peroxidation values.
Table 3. Ulcerogenic and lipid peroxidation activity of compounds (S1
–
S18) in liver and kidney tissue.
Nanomoles of MDA Content
(Liver tissue)
Nanomoles of MDA Content
(Kidney Tissue)
Ulcerogenic Activity (Index)
Treatments
Mean ± SEM/
% Change
Mean ± SEM/
% Change
Mean ± SE
% Inhibition
100 mg Tissue
100 mg Tissue
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
0.582 ± 0.17
0.362 ± 0.17
0.116 ± 0.07 **
0.00
38.6
61.81
87.76
100
43.88
100
54.30
86.07
93.24
0
7.00 ± 0.25 *
5.94 ± 0.25 ***
5.55 ± 0.18 ***
4.70 ± 5.75 ***
6.08 ± 0.18 ***
4.35 ± 0.16 ***
6.32 ± 0.14 ***
6.88 ± 0.37 *
7.82 ± 0.28
14.13
27.22
31.93
40.32
18.84
45.59
22.51
15.70
4.18
4.78 ± 0.14 ***
5.89 ±0.14 **
6.83 ± 0.17
28.66
12.10
1.91
40.12
40.12
43.31
15.92
11.46
10.19
7.00
4.01 ± 0.14 ***
4.40 ± 0.27 ***
3.80 ± 0.12 ***
5.64 ± 0.20 **
5.94 ± 0.18 *
6.02 ± 0.19 *
7.17 ± 0.20
0.532 ± 0.08
0.00
0.432 ± 0.04 *
0.132 ± 0.08 **
0.064 ± 0.03 **
0.948 ± 0.17
8.58 ± 0.38
5.23

Molecules 2018, 23, 1250
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Table 3. Cont.
Nanomoles of MDA Content
(Liver tissue)
Nanomoles of MDA Content
(Kidney Tissue)
Ulcerogenic Activity (Index)
Treatments
Mean ± SEM/
% Change
Mean ± SEM/
Mean ± SE
% Inhibition
% Change
100 mg Tissue
100 mg Tissue
S11
S12
S13
S14
S15
S16
S17
S18
0.00
0.316 ± 0.09 *
0.696 ± 0.09
0.616 ± 0.11
0.00
100
66.66
26.58
35.2
100
0
100
36.91
4.74 ±0.28 ***
5.85 ± 0.18 ***
5.29 ± 0.17 ***
5.59 ± 0.19 ***
4.78 ± 0.06 ***
7.56 ± 0.43
41.88
28.27
35.07
41.36
41.36
7.32
42.93
28.79
114.60
-
4.14 ± 0.18 ***
5.29 ± 0.28 **
5.12 ± 0.16 ***
5.68 ± 0.12 **
4.01 ± 0.23 ***
7.17 ± 0.22
38.21
21.09
23.56
15.28
40.12
7.00
33.75
15.92
98.73
0.966 ± 0.16
0.00
4.65 ± 0.21 ***
5.81 ± 0.41 ***
8.16 ± 0.28 ***
3.80 ± 0.18
4.44 ± 0.17 ***
5.64 ± 0.18 **
6.70 ± 0.20 ***
3.37 ± 0.12
0.598 ± 0.11
0.948 ± 0.21
0.00
indomethacin
Control
100
Values represent mean
comparison test (n = 6). All data were compared with Indomethacin group.
±
SEM.* p < 0.05, ** p < 0.01, *** p < 0.001; ANOVA, followed by Dunnett’s multiple
2.4. Compound S3 Biological Characterization
Three compounds were found to have a significant anti-inflammatory and analgesic activity. These
compounds can be ordered according to their anti-inflammatory and analgesic activity as S14 > S3 > S7
.
However, these compounds were also evaluated for their less ulcerogenic effect in comparison to the
reference drug indomethacin as S3
> S7 > S14. Compound S3 has shown a maximum reduction in the
lipid peroxidation and gastric ulceration. Therefore, Compound S3 with 3-nitrophenyl substitution
was found to be most the potent anti-inflammatory and analgesic derivative, as well as, a significant
gastric sparing activity.
2.5. Gastro-Protective Effect of Compound S3
The gastro-protective activity was evaluated using indomethacin with ethanol-induced and the
ulcer control rat, ethanol-induced rats [23]. It was compared with pre-treated rats using compound
S3. It was found that 12.34 ulcer index for the indomethacin with ethanol-induced rat and 7.83 for
the ethanol-induced rat. However, a significant decrease in the ulcer index of the pre-treated rat
with compound S3 was found to be 2.83 (Table 4). The gastric mucosa was increased in the rats
pre-treated with compound S3. The gastric mucus has a significant role in the gastric protection.
The pre-treatment of the rats with compound S3, plays a crucial role by enhancing the free mucus in
augmenting the gastro-protection activity in comparison to the ulcer control mucosa. The mucus is
adhered to the gastric surface because it is formed from mucin-type glycoprotein. This mucus protects
the underneath epithelium against pepsin, acid, necrotizing agents such as, indomethacin and ethanol.
Moreover, it is involved in repairing the gastric epithelium damage and defending the mucosa from the
aggregation of mechanical factors and chemical [24]. Thus, the gastro-protective effect of compound
S3 against indomethacin and ethanol is demonstrated by improving the mucosal content (Figure 1).
The glycogen level of the control and the pre-treated animal was also checked using the Periodic
Acid-Schiff (PAS). The ulcers induced by ethanol or indomethacin causes extensive gastric mucosal
injury. Moreover, they exhibit hemorrhagic and necrotic lesions which infiltrate into the mucosa and
causes edema and leukocyte infiltration. However, the pre-treatment with compound S3 resulting
in expansion of mucus gel layer that with continuous PAS-positive that lines the gastric mucosal
surface (Figures 2 and 3). The magenta staining color is exhibited with the compound S3 pre-treated
group. The tissue has a normal glandular pattern and mild leucocyte infiltration (Figures 2C and 3C).
On the other hand, the gastric specimen from the control and the indomethacin pre-treatment did
not exhibit the magenta staining color. As it can be seen in (Figures 2B and 3B), the ethanol-induced
ulcer exhibits pervasive injury to the gastric mucosa. The pre-treatment with indomethacin causes
severe ulcer and injury (Figures 2D and 3D). In gastro-protective experiments, the ulcer control rats,
ethanol-induced and indomethacin with ethanol-induced rats, revealed severe mucosal damage with

Molecules 2018, 23, 1250
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an ulcer index of 7.83
exhibited a significant decrease in ulcer index (2.83
±
0.33 and 12.34
±
0.73, respectively. The compound S3 pre-treated rats
±
0.87), and less mucosal damage. These results
clearly indicate that compound S3 has gastro-protective activity. Mucus production by gastric mucosa
increased gradually in the experimental rats pre-treated with compound S3. Gastric mucus plays
crucial role in gastro-protection. The pre-treatment with compound S3 significantly augmented the
gastro-protective activity, with enhancement of the free mucus when compared to the mucus of ulcer
control animals. Thus, compound S3 has gastro-protective activity against ethanol and indomethacin
ethanol induced gastric ulcer by improving mucosal content.
Table 4. Ulcer index of compound S3 compared with standard drug indomethacin.
Ulcer Index (mm2)
Animal Groups
Treatment (5 mL/kg Dose)
Mean ± SEM
1
2
3
4
normal control
ethanol group
S3 (25.6 mg/kg)
0
7.83 ± 0.33
2.83 ± 0.87
12.34 ± 0.73
Indomethacin (25 mg/kg)
Figure 1.
(
A
) Gastric sections of control rats showing normal mucosa. (B) Gastric section of untreated
rats showing mucosal ulceration. (
C) Gastric section of compound S3 treated rats (25.6 mg/kg) showing
intact mucosa with mild ulceration (
focal mucosal ulceration.
D
) Gastric section of indomethacin-treated rats (25 mg/kg) showing
Figure 2. Cont.

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Figure 2. The ethanol-induced gastric ulcers after treatment with (
) compound S3 (25.6 mg/kg) with ethanol and ( ) indomethacin (25 mg/kg) with ethanol. PAS
staining indicates the mucus secretion in the gastric glands.
A) normal control, (B) ethanol,
(
C
D
Figure 3.
showing gastric mucosal ulceration consisting of necrosis, cellular debris neutrophils and degenerated
epithelial cells of ethanol treated rats. ( ) Gastric section showing focal mucosal ulceration consisting
of predominantly neutrophils and degenerated epithelial cells of compound S3 (25.6 mg/kg) and
ethanol treated rats. ( ) Gastric section showing intense mucosal ulceration consisting of necrosis,
(A) Gastric section showing intact gastric mucosa of control rats. (B) Gastric section
C
D
cellular debris, neutrophils and degenerated epithelial cells of indomethacin (25 mg/kg) and ethanol
treated rats.
2.6. Toxicity of Compound S3
Karber method was used to determine the LD50 of compound S3 [25]. A 24 h’s observation was
made for the toxicity symptoms and mortality. The dead animals were counted at the end of the study
and the LD100 was calculated. The LD50 of compound S3 was found to be 35 mg/kg.
2.7. COX-1 and COX-2 Protein Expression
A Western blot to assess the protein expression was applied in the pre-treatment with ethanol,
indomethacin or compound S3 [26]. The indomethacin pre-treatment has shown gastro-toxicity
because of the potent COX-1 expression inhibition in the ethanol-induced ulcer (Figure 4B). However,
compound S3 has shown potent COX-2 expression inhibition with low COX-1 expression (Figure 4A).

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This demonstrates that the gastro-protection activity of compound S3 caused by attenuating the
ethanol lesions induces the gastric mucus and decrease the production of COX-2.
Figure 4. Western blot analysis of COX-2 (A), and COX-1 (B) expression in the rat gastric mucosa
damaged by ethanol. The ethanol-administered rats showed marked increases in COX-2, and COX-1
expression in the gastric mucosa (a representative blot is shown). These increases were significantly
suppressed by pre-treatment with compound S3 (25.6 mg/kg) or indomethacin (25 mg/kg), which
were given 1 h before ethanol administration.
2.8. Docking Studies of Compounds (S1–S18) to the COX-1/COX-2
Molecular docking was performed on the synthetic compounds (S1–S18) into human
COX-1/COX-2 enzyme using MOE 2015 software-package and compared with the docking results of
indomethacin that have been reported to have COX-2 inhibitory activity [27]. In case of docking studies
of compounds to the COX-1, all the compounds fitted in the appropriate amino acids by hydrogen
bonds. In addition, hydrophobic interactions were found with most of the important lipophilic amino
acids: Leu 531, Leu 352, Ala 527, Ile 523, Val 349, Ser 530, Ser 353, Gly 526, and Tyr 355. From docking
parameters table, we observed that the interaction of these compound form pi-H bond with energy
cut-off of
−
0.3 kcal/mol for COX-1 and 0.5 kcal/mol for COX-2. 3D crystal structure of human COX-2
(PDB ID: 4COX) was used to interpret the differences in the binding interactions at the molecular
level as inhibitors of human COX-2. (Table 5) shows the values of docking scores for compounds
(S1–S18) that were docked into the site of COX-2 (PDB ID: 4COX). Docking studies have shown the
potential of this series of compounds (S1
–
S18) to bind with COX-2. All the compounds fitted in the
appropriate amino acids by hydrogen bonds. In addition, hydrophobic interactions were found with
most of the important lipophilic amino acids: Leu 352, Leu 93, Tyr 355, Tyr 115, Val 116, Val 523, Val 349,
Val 89, Ala 527 and Arg 120. It was found that Tyr 355 formed hydrogen bond acceptors with the
carboxamide groups of all compounds except S11. Arg 120 also formed hydrogen bond acceptors
with carboxamide groups of all compounds except S7, S13, S17. Leu 93 formed Pi-hydrogen bonds
with the centroid of nitrophenyl of compound S3 and trimethoxyphenyl of compound S15. Val 523
formed Pi-hydrogen bond with the centroid of 4-(dimethylamino) phenyl of compounds S11 and

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4-ethoxyphenyl of compound S13. Compound S3 formed a hydrogen bond between OH of Tyr 355
and NH2 of Arg 120 with carbonyl group in compound S3 and this hydrogen bond was similar to
that formed by indomethacin. Moreover, hydrogen bond between Leu 93 with nitro-phenyl ring in
compound S3 was also formed (Figure 5, Figure 6). In 2D, green dashed line represents hydrogen
bonding while; the green dashed line with ring represents pi-H bonding and the residues, which are
colored light green, represents hydrophobic interactions. In 3D, black dashed line represents hydrogen
bonding while; blue dashed line represents pi-H bonding. The biological activity of the compound S3
was found to be higher than that which was expected based on data from molecular docking.
Table 5. Docking of compounds (S1–S18) into COX-2 with reference drug indomethacin.
Total Binding
Energy (kcal·mol
Compd. No.
Amino Acid Residues
Interaction Type
RMSD
Distance (Å)
−1
)
SER 530
ARG 120
TYR 355
ARG 120
ARG 120
H-acceptor
H-acceptor
H-acceptor
ionic
2.92
2.84
2.84
2.42
3.04
−8.86
0.748
indomethacin
ionic
ARG 120
TYR 355
H-acceptor
H-acceptor
2.87
2.87
−7.12
−7.73
S1
S2
2.008
2.008
ARG 120
TYR 355
H-acceptor
H-acceptor
2.78
3.04
ARG 120
TYR 355
LEU 93
H-acceptor
H-acceptor
Pi-H
2.80
3.08
4.46
−7.80
S3
2.0
ARG 120
TYR 355
H-acceptor
H-acceptor
2.82
2.97
−7.86
−7.79
S4
S5
1.322
1.480
ARG 120
TYR 355
H-acceptor
H-acceptor
2.87
2.88
ARG 120
TYR 355
H-acceptor
H-acceptor
2.83
2.94
−7.80
−7.47
−7.997
S6
S7
1.645
1.058
TYR 355
H-acceptor
2.75
ARG 120
TYR 355
H-acceptor
H-acceptor
2.88
3.06
S8
1.365
1.606
1.851
0.941
2.050
1.474
1.357
ARG 120
TYR 355
H-acceptor
H-acceptor
2.88
2.99
−7.31
−7.52
−7.16
−7.73
−7.48
−8.38
S9
ARG 120
TYR 355
H-acceptor
H-acceptor
2.85
2.96
S10
S11
S12
S13
S14
ARG 120
VAL 523
H-acceptor
pi-H
2.79
4.61
ARG 120
TYR 355
H-acceptor
H-acceptor
2.87
2.87
TYR 355
VAL 523
pi-H
pi-H
3.46
4.77
ARG 120
TYR 355
H-acceptor
H-acceptor
2.83
2.92
ARG 120
TYR 355
LEU 93
H-acceptor
H-acceptor
pi-H
2.87
2.87
4.56
−8.49
S15
2.301
ARG 120
TYR 355
H-acceptor
H-acceptor
2.82
2.90
−8.22
S16
2.279
S17
S18
TYR 355
ARG 120
H-acceptor
H-acceptor
3.12
3.35
−7.71
−7.60
2.563
1.363

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(A) (B)
Figure 5. Compound S3 in the binding site of COX-2 (PDB: 4COX). (A,B) represents the 2D and
3D docking view of compound S3 with binding site of COX-2. In 2D, green dashed line represents
hydrogen bonding; green dashed line represents pi-H bonding; residues which are colored light green,
represents hydrophobic interactions. In 3D, black dashed line represents hydrogen bonding; blue
dashed line represents pi-H bonding.
(A)
(B)
Figure 6. Reference compound indomethacin in the binding site of COX-2 (PDB: 4COX).
) represents the 2D and 3D docking view of indomethacin binding site of COX-2. In 2D, green
(A,B
dashed line represents hydrogen bonding; green dashed line represents pi-H bonding; residues which
are colored light green, represents hydrophobic interactions. In 3D, black dashed line represents
hydrogen bonding; blue dashed line represents pi-H bonding.
3. Material and Methods
3.1. Experimental Section
The solvents were procured from Merck. Purity of the synthesized compounds was confirmed by
thin layer chromatography (TLC), performed on Silica gel 60 F254 coated plates (Merck, Kenilworth,
NJ, USA). UV light was used for the visualization of TLC spots. Spectrum BX, Perkin Elmer FT-IR

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spectrophotometer (Perkin Elmer, Hopkinton, MA, USA) was used for performing FTIR. Gallenkamp
melting point apparatus (Gallenkamp, Loughborough, UK) was used for performing melting points.
Bruker NMR 500 MHz and 125 MHz spectrophotometer (Bruker, Billerica, MA, USA) were used for 1H-
and 13C-NMR. All the samples were processed in DMSO-d6 with tetramethylsilane (TMS) as internal
standard. Mass spectroscopy was used for the measurement of molecular masses of compounds.
The elemental analysis of the compounds was performed on the CHN Elementar (Analysensysteme
GmbH, Langenselbold, Germany). The elemental analyses were within the limit.
3.2. Synthesis of 2-(6-Methoxy-2-methyl-1H-indol-3-yl) Acetohydrazide (1)
The methyl ester of indomethacin (0.01 mol) and hydrazine hydrate (99%) (0.2 mol) in presence
of absolute ethanol (50 mL) were refluxed for 30 h. The reaction mixture was concentrated by using
rota vapor and poured in a beaker containing ice while stirring and kept for 4 h at room temperature.
The solid was separated out by filtration. The product was dried and recrystallized from ethanol.
The product was carefully checked by thin layer chromatography. Two compounds were isolated by
column chromatography by using different fractions of n-hexane and ethyl acetate. The first compound
was 2-(6-methoxy-2-methyl-1H-indol-3-yl) acetohydrazide compound (1) and was obtained as the
major product. The second compound, 4-chlorobenzohydrazide (
2
) was obtained as minor product.
Both the compounds were fully characterized by the spectral data.
◦
2-(6-methoxy-2-methyl-1H-indol-3-yl) acetohydrazide (
1
). Color: white; Yield: 70%; m.p.: 168–170 C;
1
UV
λmax (Methanol) = 280 nm; H-NMR (500 MHz, DMSO-d6):
δ
= 2.38 (3H, s, CH3), 3.54 (2H, s,
CH2), 3.80 (3H, s, OCH3), 4.26 (2H, s, NH2, D2O exchg.), 6.67 (1H, d, J = 8.5 Hz, Ar–H), 7.16 (2H, d,
J = 7.5 Hz, Ar–H), 9.16 (1H, s, NH, D2O exchg.), 10.62 (1H, s, CONH, D2O exchg.); 13C-NMR (125 MHz,
DMSO-d6):
δ = 12.0 (CH3), 30.2 (CH2), 55.8 (OCH3), 101.1, 105.1, 109.8, 110.0, 111.7, 128.0, 129.3, 129.7,
130.6, 134.3, 153.4, 170.8 (C=O); ms: m/z = 233.11 [M]+, 234.07 [M + 1]+; Analysis: C12H15N3O2 for,
calcd. C 61.79, H 6.48, N 18.01%; found C 61.58, H 6.46, N 18.05%.
4-Chlorobenzohydrazide (
2
). Color: white; Yield: 20%; m.p.: 148–150 ◦C; UV
= 4.53 (2H, s, NH2, D2O exchg.), 7.52 (2H, d, J = 8.5 Hz, Ar–H), 7.84
λmax (Methanol) = 230 nm;
1H-NMR (500 MHz, DMSO-d6):
δ
(2H, d, J = 8.5 Hz, Ar–H), 9.87 (1H, s, CONH, D2O exchg.); 13C-NMR (125 MHz, DMSO-d6):
δ = 128.86,
129.32, 132.50, 136.25, 165.29; MS: m/z = 170.45 [M]+; Analysis: C7H7N2OCl for, calcd. C 49.28, H 4.14,
N 16.42%; found C 49.37, H 4.12, N 16.46%.
3.3. General Procedure for the Synthesis of 2-(5-Methoxy-2-methyl-1-indol-3yl)-N-[(E)-Substituted Phenyl
methylidine] Aceto Hydrazide Derivatives (S1–S18).
A solution of indole hydrazide (1) (371 mg, 1.0 mmol) in EtOH (15 mL) containing an appropriate
substituted benzaldehyde (1.1 mmol) and a catalytic amount of Glacial acetic acid was heated under
reflux for 3 h. After cooling, 5 mL of water was added to the mixture and kept in a refrigerator for 12 h.
The product was obtained by filtration. The compound was washed several times with cold water.
Ethanol was used for the recrystallization of the compound.
2-(5-Methoxy-2-methyl-1H-indol-3-yl)-N0-[(E)-phenylmethylidene] acetohydrazide (S1): Yield: 70%; m.p.:
◦
1
170–172 C; IR (KBr) cm−1: 3412 (NH), 3024 (C–H), 1654 (C=O), 1637 (C=N); H-NMR (500 MHz,
DMSO-d6):
δ = 2.37 (3H, s, –CH3), 3.55 (2H, s, CH2), 3.74 (3H, s, –OCH3), 6.65–8.00 (8H, m, Ar–H), 10.62
(1H, s, =CH), 11.26 (1H, s, –NH, D2O exchg.), 11.9 (1H, s, CONH, D2O exchg.); 13C-NMR (125 MHz,
DMSO-d6):
δ = 12.2 (CH3), 28.2 (CH2), 55.5 (OCH3), 100.7, 104.7, 110.0, 111.3, 127.2, 127.4, 127.6, 129.0,
129.2, 129.3, 130.0, 134.3, 134.8, 153.3, 167.7 (C=O); MS: m/z = 321.37 [M]+; Analysis: for C19H19N3O2,
calcd. C 71.01, H 5.96, N 13.08%; found C 71.25, H 5.94, N 13.11%.
2-(5-Methoxy-2-methyl-1H-indol-3-yl)-N0-[(E)-(4-nitrophenyl)methylidene] acetohydrazide (S2): Yield: 75%;
◦
m.p.: 220–222 C; IR (KBr) cm−1: 3411 (NH), 3000 (C–H), 1654 (C=O), 1617 (C=N); 1H-NMR (500 MHz,
DMSO-d6):
δ = 2.37 (3H, s, –CH3), 3.58 (2H, s, CH2), 3.74 (3H, s, –OCH3), 6.97–8.26 (7H, m, Ar–H), 10.63

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(1H, s, =CH), 11.70 (1H, s, –NH, D2O exchg.), 12.2 (1H, s, –CONH, D2O exchg.); 13C-NMR (125 MHz,
DMSO-d6):
δ = 11.6 (CH3), 28.2 (CH2), 55.0 (OCH3), 100.2, 110.8, 123.9, 124.0, 127.6, 128.0, 128.6, 129.6,
140.4, 142.0, 143.0, 144.0, 145.5, 151.0, 163.0, 175.0; MS: m/z = 366.37 [M]+; Analysis: for C19H18N4O4,
calcd. C 62.29, H 4.95, N 15.29%; found C 62.14, H 4.97, N 15.25%.
2-(5-Methoxy-2-methyl-1H-indol-3-yl)-N0-[(E)-(3-nitrophenyl)methylidene] acetohydrazide (S3): Yield: 68%;
◦
m.p.: 200–202 C; IR (KBr) cm−1: 3412 (NH), 3237 (C–H), 1654 (C=O), 1617 (C=N); 1H-NMR (500 MHz,
DMSO-d6):
δ = 2.37 (3H, s, –CH3), 3.58 (2H, s, CH2), 3.74 (3H, s, –OCH3), 6.99–8.57 (7H, m, Ar–H), 10.63
(1H, s, =CH), 11.5 (1H, s, NH, D2O exchg.), 12.18 (1H, s, –CONH, D2O exchg.); 13C-NMR (125 MHz,
DMSO-d6):
δ = 11.5 (CH3), 27.9 (CH2), 55.0, 100.2, 103.9, 109.4, 123.8, 124.2, 128.5, 130.3, 131.7, 133.3,
136.0, 140.33, 145.5, 148.1, 152.8, 162.2, 170.0; MS: m/z = 366.37 [M]+; Analysis: for C19H18N4O4, calcd.
C 62.29, H 4.95, N 15.29%; found C 62.36, H 4.93, N 15.24%.
2-(5-Methoxy-2-methyl-1H-indol-3-yl)-N0-[(E)-(2-nitrophenyl)methylidene] acetohydrazide (S4): Yield: 70%;
◦
m.p.: 210–212 C; IR (KBr) cm−1: 3407 (NH), 3063 (C–H), 1654 (C=O), 1617 (C=N); 1H-NMR (500 MHz,
DMSO-d6):
δ = 2.37 (3H, s, –CH3), 3.58 (2H, s, CH2), 3.74 (3H, s, –OCH3), 6.98–8.25 (7H, m, Ar–H),
10.63 (1H, s, =CH), 11.90 (1H, s, –NH, D2O exchg.), 12.10 (1H, s, –CONH, D2O exchg.); 13C-NMR
(125 MHz, DMSO-d6):
δ = 11.60 (CH3), 27.8 (CH2), 54.9 (OCH3), 100.0, 103.8, 109.4, 110.6, 124.5, 127.8,
129.6, 133.4, 134.0, 141.4, 143.2, 147.9, 148.2, 152.0; 167.0, 170.0; MS: m/z = 366.37 [M]+; Analysis: for
C19H18N4O4, calcd. C 62.29, H 4.95, N 15.29%; found C 62.15, H 4.97, N 15.24%
N0-[(E)-(4-chlorophenyl)methylidene]-2-(5-methoxy-2-methyl-1H-indol-3-yl)acetohydrazide (S5): Yield: 80%;
◦
m.p.: 180–182 C; IR (KBr) cm−1: 3411 (NH), 3071 (C–H), 1654 (C=O), 1597 (C=N); 1H-NMR (500 MHz,
DMSO-d6):
δ = 2.36 (3H, s, –CH3), 3.55 (2H, s, CH2), 3.74 (3H, s, –OCH3), 6.60–8.45 (7H, m, Ar–H), 10.63
(1H, s, =CH), 11.3 (1H, s, NH, D2O exchg.), 12.00 (1H, s, –CONH, D2O exchg.); 13C-NMR (125 MHz,
DMSO-d6):
δ = 11.6 (CH3), 28.2 (CH2), 55.1 (OCH3), 101.0, 128.5, 128.7, 128.8, 128.9, 129.5, 136.6, 146.7,
148.0, 149.0, 150.0, 151.0, 152.1,162.7, 172.0; MS: m/z = 355.81 [M]+; Analysis: for C19H18N3O2Cl, calcd.
C 64.13, H 5.10, N 11.81%; found C 64.33, H 5.12, N 11.83%.
N0-[(E)-(2,4-dichlorop◦henyl)methylidene]-2-(5-methoxy-2-methyl-1H-indol-3-yl)acetohydrazide (S6): Yield:
65%; m.p.: 238–240 C; IR (KBr) cm−1: 3411 (NH), 2940 (C–H), 1654 (C=O), 1617 (C=N); 1H-NMR
(500 MHz, DMSO-d6):
δ = 2.36 (3H, s, –CH3), 3.59 (2H, s, CH2), 3.74 (3H, s, –OCH3), 6.59–8.61 (6H, m,
Ar–H), 10.62 (1H, s, =CH), 11.51 (1H, s, –NH, D2O exchg.); 11.7 (1H, s, –CONH, D2O exchg.); 13C-NMR
(125 MHz, DMSO-d6):
δ = 11.5 (CH3), 27.8 (CH2), 55.0 (OCH3), 100.2, 103.7, 109.4, 110.8, 127.8, 128.7,
129.2, 130.0, 133.4, 133.6, 134.0, 137.5, 140.9, 154.0, 168.0, 172.0S; MS: m/z = 390.26 [M]+; Analysis: for
C19H17N3O2Cl2, calcd. C 58.47, H 4.35, N 10.77%; found C 58.25, H 4.33, N 10.74%.
0
2-(5-Methoxy-2-methyl-1H-indol-3-yl)-N -[(E)-(3,4-dimethoxyphenyl)methylidene] acetohydrazide (S7
):
Yield:
70%; m.p.: 210–212 C; IR (KBr) cm−1: 3299 (NH), 3011 (C–H), 1654 (C=O), 1599 (C=N); 1H-NMR
(500 MHz, DMSO-d6): = 2.40 (3H, s, –CH3), 3.70 (2H, s, CH2), 3.84 (3H, s, –OCH3), 6.50–8.40 (6H, m,
Ar–H), 10.50 (1H, s, =CH), 11.20 (1H, s, –NH, D2O exchg.), 11.5 (1H, s, –CONH, D2O exchg.); 13C-NMR
◦
δ
(125 MHz, DMSO-d6):
δ = 12.1 (CH3), 12.2 (CH2), 55.6 (OCH3), 55.8 (OCH3), 56.0 (OCH3), 101.0, 121.4,
122.0, 127.5, 143.1, 151.0, 175.0; MS: m/z = 381.42 [M]+; Analysis: for C21H23N3O4, calcd. C 66.13,
H 6.08, N 11.02%; found C 66.31, H 6.10, N 11.05%.
2-(5-Methoxy-2-methyl-1H-indol-3-yl)-N0-[(E)-(2-methoxyphenyl)methylidene] acetohydrazide (S8): Yield:
◦
60%; m.p.: 220–222 C; IR (KBr) cm−1: 3315 (NH), 3017 (C–H), 1664 (C=O), 1601 (C=N); 1H-NMR
(500 MHz, DMSO-d6):
δ = 2.36 (3H, s, –CH3), 3.56 (2H, s, CH2), 3.87 (3H, s, –OCH3), 7.00–8.82 (11H, m,
Ar–H), 10.61 (1H, s, =CH), 11.23(1H, s, NH, D2O exchg.), 11.92 (1H, s, –CONH, D2O exchg.); 13C-NMR
(125 MHz, DMSO-d6):
δ = 12.1 (CH3), 28.1 (CH2), 56.1 (OCH3), 63.4 (OCH3), 111.2, 112.2, 112.3, 121.2,
122.7, 125.8, 126.0, 129.0, 130.0, 132.1, 137.0, 144.0, 158.2, 162.3; MS: m/z = 351.39 [M]+; Analysis: for
C20H21N3O3, calcd. C 68.36, H 6.02, N 11.96%; found C 68.50, H 6.00, N 11.93%.

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N0-[(E)-(4-hydroxyphenyl)methylidene]-2-(5-methoxy-2-methyl-1H-indol-3-yl)acetohydrazide (S9
)
:
Yield:
70%; m.p.: 230–232 ◦C; IR (KBr) cm−1: 3411 (OH), 3411 (NH), 3300 (C–H), 1654 (C=O), 1609 (C=N);
1H-NMR (500 MHz, DMSO-d6):
(7H, m, Ar–H), 9.88 (1H, s, OH, D2O exchg.), 10.60 (1H, s, =CH), 11.0 (1H, s, –CONH, D2O exchg.),
11.73 (1H, s, –CONH, D2O exchg.); 13C-NMR (125 MHz, DMSO-d6):
= 12.1 (CH3), 28.2 (CH2), 55.5
δ = 2.37 (3H, s, –CH3), 3.58 (2H, s, CH2), 3.75 (3H, s, –OCH3), 6.59–8.36
δ
(OCH3), 100.8, 104.9, 109.8, 110.0, 116.1, 128.9, 129.4, 130.5, 134.2, 143.4, 153.3, 159.5, 162.2, 167.4, 170.7,
172.9; MS: m/z = 337.37 [M]+; Analysis: for C19H19N3O3, calcd. C 67.64, H 5.68, N 12.46%; found
C 67.43, H 5.70, N 12.43%.
N0-[(E)-(3-hydroxyphenyl)methylidene]-2-(5-methoxy-2-methyl-1H-indol-3-yl)acetohydrazide (S10): Yield:
60%; m.p.: 145–147 ◦C; IR (KBr) cm−1: 3500 (OH), 3413 (NH), 3023 (C–H), 1654 (C=O), 1617 (C=N);
1H-NMR (500 MHz, DMSO-d6):
δ
= 2.37 (3H, s, –CH3), 3.57 (2H, s, CH2), 3.74 (3H, s, –OCH3), 6.58–8.17
(7H, m, Ar–H), 9.59 (1H, s, OH, D2O exchg.), 10.62 (1H, s, =CH), 11.21 (1H, s, –NH, D2O exchg.), 11.39
(1H, s, –CONH, D2O exchg.); 13C NMR (125 MHz, DMSO-d6):
= 11.6 (CH3), 27.6 (CH2), 54.9 (OCH3),
δ
100.1, 104.0, 109.3, 110.8, 112.4, 117.2, 118.2, 128.5, 129.5, 130.0, 133.8, 135.5, 146.2, 152.8, 157.5, 175.0;
MS: m/z = 337.37 [M]+; Analysis: for C19H19N3O3, calcd. C 67.64, H 5.68, N 12.46%; found C 67.41,
H 5.70, N 12.42%.
2-(5-Methoxy-2-methyl-1H-indol-3-yl)-N0-{(E)-[4-(dimethylamino) phenyl]methylidene}acetohydrazide (S11):
◦
Yield: 65%; m.p.: 200–202 C; IR (KBr) cm−1: 3351 (NH), 2909 (C–H), 1654 (C=O), 1609 (C=N); 1H-NMR
(500 MHz, DMSO-d6):
–OCH3), 6.59–8.32 (7H, m, Ar–H), 10.60 (1H, s, =CH), 10.97 (1H, s, –NH, D2O exchg.), 11.62 (1H, s,
–CONH, D2O exchg.); 13C-NMR (125 MHz, DMSO-d6):
= 12.1 (CH3), 28.0 (CH2), 30.0 (NCH3), 31.0
δ
= 2.37 (3H, s, –CH3), 3.00 (6H, s, 2
×
NCH3) 3.59 (2H, s, CH2), 3.74 (3H, s,
δ
(NCH3), 55.5 (OCH3), 100.9, 105.0, 110.0, 111.2, 112.27, 112.3, 122.2, 128.4, 128.7, 128.9, 129.8, 130.5,
143.9, 151.7, 153.3, 172.7; ms: m/z = 364.44 [M]+; Analysis: for C21H24N4O2, calcd. C 69.21, H 6.64,
N 15.37%; found C 69.37, H 6.66, N 15.33%.
2-(5-methoxy-2-methyl-1H-indol-3-yl)-N0-[(E)-(3-methoxyphenyl)methylidene] acetohydrazide (S12): Yield:
◦
65%; m.p.: 195–197 C; IR (KBr) cm−1: 3412 (NH), 3000 (C–H), 1654 (C=O), 1636 (C=N); 1H-NMR
(500 MHz, DMSO-d6):
(7H, m, Ar–H), 10.67 (1H, s, =CH), 11.28 (1H, s, –NH, D2O exchg.), 11.46 (1H, s, –CONH, D2O exchg.);
13C-NMR (125 MHz, DMSO-d6):
= 12.1 (CH3), 28.3 (CH2), 55.5 (OCH3), 55.35 (OCH3), 100.8, 104.5,
δ = 2.37 (3H, s, –CH3), 3.58 (2H, s, CH2), 3.80 (3H, s, –OCH3), 6.59–8.44
δ
104.7, 109.8, 110.0, 111.6, 120.3, 129.3, 130.3, 134.3, 134.3, 136.2, 142.9, 146.5, 153.3, 159.9, 173.3; MS:
m/z = 351.39 [M]+; Analysis: for C20H21N3O3, calcd. C 68.36, H 6.02, N 11.96%; found C 68.15, H 6.00,
N 11.99%.
0
N -[(E)-(4-ethoxyphenyl)methylidene]-2-(5-methoxy-2-methyl-1H-indol-3-yl)acetohydrazide (S13): Yield: 75%;
m.p.: 213–215 C; IR (KBr) cm−1: 3322 (NH), 3042 (C–H), 1654 (C=O), 1571 (C=N); 1H-NMR (500 MHz,
◦
DMSO-d6):
–OCH3), 4.06 (2H, q, J = 7.5 Hz, OCH2), 6.58–8.20 (7H, m, Ar–H), 10.61 (1H, s, =CH), 11.12 (1H, s, –NH,
D2O exchg.), 11.30 (1H, s, –CONH, D2O exchg.); 13C-NMR (125 MHz, DMSO-d6):
= 12.2 (CH3), 15.0
δ = 1.33 (3H, t, J = 7.5 Hz, CH2CH3), 2.37 (3H, s, –CH3), 3.57 (2H, s, CH2), 3.74 (3H, s,
δ
(CH2), 28.2 (CH3), 55.5 (OCH3), 55.88 (OCH2), 63.7, 100.8, 104.8, 109.8, 111.2, 115.1, 127.2, 129.2, 129.3,
130.5, 134.3, 143.0, 146.5, 153.3, 160.2, 167.5, 173.0; ms: m/z = 365.42 [M]+; Analysis: for C21H23N3O3,
calcd. C 69.02, H 6.34, N 11.50%; found C 69.22, H 6.36, N 11.53%.
2-(5-Methoxy-2-methyl-1H-indol-3-yl)-N0-[(E)-(2,4,5-trimethoxyphenyl) methylidene]acetohydrazide (S14):
◦
Yield: 60%; m.p.: 238–240 C; IR (KBr) cm−1: 3412 (NH), 2943 (C–H), 1654 (C=O), 1617 (C=N); 1H-NMR
(500 MHz, DMSO-d6):
(5H, m, Ar–H), 10.61 (1H, s, =CH), 11.14 (1H, s, –NH, D2O exchg.), 11.42 (1H, s, –CONH, D2O exchg.);
13C-NMR (125 MHz, DMSO-d6):
= 12.1 (CH3), 30.1 (CH2), 55.5 (OCH3), 56.4 (OCH3), 60.9 (OCH3),
δ
= 2.36 (3H, s, –CH3), 3.51 (2H, s, CH2), 3.78 (12H, s, 4
×
–OCH3), 6.91–8.42
δ
62.1 (OCH3), 100.8, 104.8, 109.1, 109.8, 110.0, 111.2, 120.8, 130.5, 134.4, 138.9, 142.0, 152.8, 153.3, 155.2,
172.9; ms: m/z = 411.45 [M]+; Analysis: for C22H25N3O5, calcd. C 64.22, H 6.12, N 10.21%; found
C 64.35, H 6.14, N 10.24%.

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2-(5-Methoxy-2-methyl-1H-indol-3-yl)-N0-[(E)-(2,3,4-trimethoxyphenyl) methylidene]acetohydrazide (S15):
◦
Yield: 55%; m.p.: 250–252 C; IR (KBr) cm−1: 3310 (NH), 3048 (C–H), 1654 (C=O), 1595 (C=N); 1H-NMR
(500 MHz, DMSO-d6): = 2.37 (3H, s, –CH3), 3.59 (2H, s, CH2), 3.84 (12H, s, 4 –OCH3), 6.59–8.74
(5H, m, Ar–H), 10.60 (1H, s, =CH), 11.08 (1H, s, –NH, D2O exchg.), 11.33 (1H, s, –CONH, D2O exchg.);
13C-NMR (125 MHz, DMSO-d6):
= 12.1 (CH3), 28.5 (CH2), 55.6 (OCH3), 56.2 (OCH3), 56.4 (OCH3),
δ
×
δ
56.9 (OCH3), 98.3, 101.1, 104.8, 108.4, 109.8, 111.2, 114.1, 129.2, 130.6, 134.4, 138.8, 143.6, 152.1, 153.6,
167.3, 172.9; ms: m/z = 411.45 [M]+; Analysis: for C22H25N3O5, calcd. C 64.22, H 6.12, N 10.21%; found
C 64.36, H 6.10, N 10.23%.
2-(5-Methoxy-2-methyl-1H-indol-3-yl)-N0-[(E)-(3,4,5-trimethoxyphenyl) methylidene]acetohydrazide (S16):
◦
Yield: 58%; m.p.: 233–235 C; IR (KBr) cm−1: 3309 (NH), 3015 (C–H), 1654 (C=O), 1577 (C=N); 1H-NMR
(500 MHz, DMSO-d6):
(5H, m, Ar–H), 10.61 (1H, s, =CH), 11.28 (1H, s, –NH, D2O exchg.), 11.40 (1H, s, –CONH, D2O exchg.);
13C-NMR (125 MHz, DMSO-d6):
= 12.1 (CH3), 28.4 (CH2), 55.6 (OCH3), 55.8 (OCH3), 56.3 (OCH3),
δ
= 2.37 (3H, s, –CH3), 3.59 (2H, s, CH2), 3.83 (12H, s, 4
×
–OCH3), 6.97–8.20
δ
60.5 (OCH3), 60.5, 101.1, 104.4, 104.6, 104.7, 109.8, 111.2, 130.3, 130.6, 134.2, 139.3, 142.9, 153.3, 153.6,
173.2; ms: m/z = 411.45 [M]+; Analysis: for C22H25N3O5, calcd. C 64.22, H 6.12, N 10.21%; found
C 64.37, H 6.10, N 10.24%.
2-(5-Methoxy-2-methyl-1H-indol-3-yl)-N0-[(E)-(2,4,6-trimethoxyphenyl) methylidene]acetohydrazide (S17):
◦
Yield: 55%; m.p.: 230–232 C; IR (KBr) cm−1: 3412 (NH), 3056 (C–H), 1654 (C=O), 1612 (C=N); 1H-NMR
(500 MHz, DMSO-d6):
(5H, m, Ar–H), 10.61 (1H, s, =CH), 11.10 (1H, s, NH, D2O exchg.), 11.80 (1H, s, –CONH, D2O exchg.);
13C NMR (125 MHz, DMSO-d6):
= 12.1 (CH3), 28.4 (CH2), 55.5 (OCH3), 55.7 (OCH3), 56.3 (OCH3),
δ
= 2.37 (3H, s, –CH3), 3.59 (2H, s, CH2), 3.82 (12H, s, 4
×
–OCH3), 6.57–8.74
δ
60.5 (OCH3), 98.6, 106.7, 111.2, 115.6, 129.9, 142.2, 144.1, 153.3, 159.3, 159.6 162.1, 167.3, 172.9; ms:
m/z = 411.45 [M]+; Analysis: for C22H25N3O5, calcd. C 64.22, H 6.12, N 10.21%; found C 64.38, H 6.13,
N 10.17%.
2-(5-Methoxy-2-methyl-1H-in◦dol-3-yl)-N0-[(E)-(2,4-dimethoxyphenyl) methylidene]acetohydrazide (S18):
Yield: 60%; m.p.: 170–172 C; IR (KBr) cm−1: 3413 (NH), 3000 (C–H), 1654 (C=O), 1638 (C=N);
1H-NMR (500 MHz, DMSO-d6):
δ
= 2.40 (3H, s, –CH3), 3.60 (2H, s, CH2), 3.82 (3H, s, –OCH3), 6.50–8.70
(6H, m, Ar–H), 10.61 (1H, s, =CH), 11.20 (1H, s, NH, D2O exchg.); 11.80 (1H, s, –CONH, D2O exchg.);
13C-NMR (125 MHz, DMSO-d6):
= 11.5 (CH3), 28.4 (CH2), 55.0 (OCH3), 55.6 (OCH3), 55.68 (OCH3),
δ
98.2, 104.4, 106.2, 109.5, 110.7, 115.2, 128.8, 130.0, 132.0, 135.8, 138.3, 143.6, 152.8, 158.9, 162.0, 162.4,
164.7, 172.0; ms: m/z = 381.42 [M]+; Analysis: for C21H23N3O4, calcd. C 66.13, H 6.08, N 11.02%; found
C 66.34, H 6.10, N 11.05%.
3.4. Anti-Inflammatory Activity
Anti-inflammatory activity was evaluated by reported carrageenan-induced rat paw edema
method. Male rats weighing 200 g were housed in a room with a controlled temperature and
12 h light/12 h dark cycle. Each group consisted of six randomly assigned rats. Compounds were
administered p.o. as suspension prepared in 1% methyl cellulose. Intradermal injection of 50 µL of
1% carrageenan induced into the sub plantar region of the right hind paw was used for paw edema,
after one hour of test compound administration. Activity was evaluated after p.o. administration of
indomethacin or test compounds at the dose of (10 mg/kg) each. Immediately after dosing and after
2 h and 3 h, paw volume was measured by using a plethysmometer (UGO 7140 plethysmometer).
The control group was given vehicle (1% methyl cellulose) only. Percent inhibition was calculated
taking the values in the control group as 0% inhibition.
3.5. Analgesic Activity
Male albino Swiss mice (25 g) used in the study were distributed into various groups (n = 6).
Each mouse was initially placed on a hot plate (hot plate analgesia meter, Harvard apparatus Ltd.)
thermostatically maintained at 58 ◦C. The mouse was monitored carefully for the time in seconds in

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which it displayed nociceptive responses, considered as the control reaction time. To avoid damage
to the paws, a cut off time of 60 s was used. Activity was evaluated after p.o. administration of
indomethacin or test compounds at the dose of (20 mg/kg) each. The reaction time was then retested
at 0 h and 120 min after injection (each animal acted as its own control). The percentage changes in the
reaction were then calculated.
3.6. Ulcerogenic Activity
For acute ulcerogenesis testing, albino rats, six rats in each group were used (Experimental animal
ethical approval number: 7570). Activity was evaluated after p.o. administration of indomethacin or
test compounds at the dose of (20 mg/kg) each. All of the test compounds were given as equimolar oral
doses. Suspension of 1% methyl cellulose p.o. was given to control group. The stomach was opened
along the greater curvature, washed with distilled water. Magnifying glass was used to examine the
mucosal damage. For each stomach, the mucosal damage was assessed according to the reported
scoring system.
3.7. Lipid Peroxidation
For determination of lipid peroxidation, the gastric mucosa was scraped with two glass slides,
weighed (100 mg) and homogenized in 1.8 mL of 1.15% ice cold KCl solution. The reported procedure
was followed for processing the samples. The supernatant organic layer was separated out and
absorbance was measured by UV spectrophotometer at 532 nm. The results were expressed as nmol
MDA/100 mg tissue.
3.8. Ethanol Induced Ulcer Model
The ethanol induced ulcer model was used to study gastro-protective activity of compound S3
.
The rats were grouped into five groups (n = 6). Group I and II received saline solution and served as
negative-control and ulcer-control, respectively. Group III received compound S3 (25.6 mg/kg) orally
and served as the experimental drug group. Rats in groups IV received the indomethacin (25 mg/kg
bodyweight). All of the groups received (20 mL/kg) of ethanol 95% except group I. One h alter, the rats
were sacrificed under anesthesia and their stomachs were removed for further experimental studies.
Periodic Acid-Schiff (PAS) staining was used to detect the glycogen level in control and pretreated
rats. With regard to the gross gastric lesion evaluation, the stomach of each rat was opened along
the greater arc and washed with saline water to remove gastric contents. Gastric ulcers appear as
elongated bands on the gastric mucosa. A digital planimeter was used to measure the ulcers area
(hemorrhagic lesions). The length and width of each lesion were measured in (mm2). To determine
mucus production of the gastric mucosa, the gastric mucosa of each rat was obtained and weighed
using a high precision electronic balance. For the histologic studies, gastric tissues were fixed in 4%
formalin solution after sacrifice. Then, each tissue sample was embedded in paraffin and cut into 5 µm
thick slices for histopathological evaluation. Hematoxylin and eosin (H & E) were used for tissue
section staining.
3.9. Determination of LD50
The LD50 (lethal dose 50%) of compound S3 was calculated by Karber method. For determination
of LD50, an observation was made for 24 h and symptoms of toxicity and rate of mortality
were noted. Expired rats were counted at the end of the study period for the calculation of
LD50. LD50 = LD100
−
× (a × b)/n, where “n” is the total number of rats in a group, “a” is the
∑
difference between two successive doses of administered substance, “b” is the average number of dead
rats in two successive doses, and LD100 is the lethal dose causing 100% death of all tested rats.

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3.10. Western Blot
For the Western blot analysis, 20
µg of protein was shifted to PVDF membranes, restricted in
5% skim milk in TBS buffer 1% Tween 20, and then incubated overnight with the COX-1, COX-2 and
-actin, followed by HRP-conjugated anti-rat/rabbit/goat antibodies for 2 h at 25 ◦C. Bands were
β
visualized with the Luminata Western Chemiluminescent HRP Substrates and densitometry analysis
of bands was assessed using LI-COR C-DiGit blot scanners.
3.11. Docking Studies of Compounds
Docking for the synthesized compounds (S1–S18) and reference compound indomethacin was
performed via their 3D structures and energy was minimized using MMFF94x of Molecular Operating
Environment energy minimization module (MOE, Version 2015, Chemical Computing Group Inc.,
Montreal, Quebec, Canada).3D crystal structure of COX-2 enzyme (PDB ID: 4COX) was selected from
Protein Data Bank database (http://www.rcsb.org). The reported protocol was followed for preparing
selected 3D structure of COX-2 enzyme for docking. Docking procedure was also followed using the
standard protocol implemented in MOE 2015 and the geometry of resulting complexes was studied
using the MOE’s Pose Viewer utility.
4. Conclusions
In conclusion, novel indole hydrazide derivatives were synthesized in good yield. All the
compounds were fully characterized by spectral data and elemental analysis. Applying the chemical
modifications to the indole hydrazide scaffold resulted in eighteen derivatives with significant
anti-inflammatory activity. Three compounds namely S3
, S7 and S14 exhibited considerable
anti-inflammatory activities compared to the reference drug indomethacin. These compounds were
found to be highly significant anti-inflammatory and analgesic agents in the order of S14 > S3 > S7
The compounds were less ulcerogenic than the reference drug indomethacin in the order of
S3 > S7 > S14. Compound S3 (R = 3-nitro phenyl) was found to be a potent anti-inflammatory and
analgesic agent with significant gastric sparing activity. Lead compound S3, presented maximum
reduction in gastric ulceration and lipid peroxidation. The toxicity of compound S3 was found to be
very low; with significant gastro-protective activity. It was observed that 3-nitrophenyl substitution
to the indole hydrazide has significant effect on the anti-inflammatory, analgesic and ulcerogenic
reduction activities. It was found to have all the potential specification as a lead compound, which was
subjected for further biological assay. It was also found to be a potent inhibitor of the COX-2 expression.
.
Docking studies have shown the potential of this series of compounds (S1–S18) to bind with COX-2.
It formed a hydrogen bond between OH of Tyr 355 and NH2 of Arg 120 with carbonyl group and
this hydrogen bond was similar to that formed by indomethacin. These results provide the essential
information for developing new anti-inflammatory and analgesic agent with gastro-protective effect.
Supplementary Materials: The following are available online.
Author Contributions: Investigation, M.R. and M.A.A.; Methodology, A.M.N.; Project administration, M.A.A.-O.;
Resources, H.A.A.; Software, A.H.B.; Supervision, M.A.B.
Acknowledgments: The authors would like to extend their sincere appreciation to the Deanship of Scientific
Research at King Saud University for funding this research group no. (RG 1435–006).
Conflicts of Interest: The authors declare no conflict of interests.
References
1.
Shuttleworth, S.J.; Bailey, S.G.; Townsend, P.A. Histone deacetylase inhibitors: New promise in the treatment
of immune and inflammatory diseases. Curr. Drug Targets 2010, 11, 1430–1438. [CrossRef] [PubMed]
Serhan, C.N.; Levy, B. Success of prostaglandin E2 in structure-function is a challenge for structure-based
therapeutics. Proc. Natl. Acad. Sci. USA 2003, 100, 8609–8611. [CrossRef] [PubMed]
2.

Molecules 2018, 23, 1250
18 of 19
3.
4.
Amin, A.R.; Vyas, P.; Attur, M.; Leszczynska-Piziak, J.; Patel, I.R.; Weissmann, G.; Abramson, S.B. The mode
of action of aspirin-like drugs: Effect on inducible nitric oxide synthase. Proc. Natl. Acad. Sci. USA 1995, 92,
7926–7930. [CrossRef] [PubMed]
Chattopadhyay, M.; Kodela, R.; Duvalsaint, P.L.; Kashfi, K. Gastrointestinal safety, chemotherapeutic
potential, and classic pharmacological profile of NOSH-naproxen (AVT-219) a dual NO- and H2S-releasing
hybrid. Pharmacol. Res. Perspect. 2016, 4, e00224. [CrossRef] [PubMed]
5.
6.
Russell, R.I. Non-steroidal anti-inflammatory drugs and gastrointestinal damage-problems and solutions.
Postgrad. Med. J. 2001, 77, 82–88. [CrossRef] [PubMed]
Gierse, J.; Kurumbail, R.; Walker, M.; Hood, B.; Monahan, J.; Pawlitz, J.; Stegeman, R.; Stevens, A.; Kiefer, J.;
Koboldt, C.; et al. Mechanism of inhibition of novel COX-2 inhibitors. Adv. Exp. Med. Biol. 2002, 507, 365–369.
[PubMed]
7.
Almansa, C.; Alfon, J.; de Arriba, A.F.; Cavalcanti, F.L.; Escamilla, I.; Gómez, L.A.; Miralles, A.; Soliva, R.;
Bartrolí, J.; Carceller, E.; et al. Synthesis and structure-activity relationship of a new series of COX-2 selective
inhibitors: 1,5-diarylimidazoles. J. Med. Chem. 2003, 46, 3463–3475. [CrossRef] [PubMed]
Amir, M.; Kumar, S. Anti-inflammatory and gastro sparing activity of some new indomethacin derivatives.
Arch. Pharm. 2005, 338, 24–31. [CrossRef] [PubMed]
Abuelizz, H.A.; Al-Salahi, R.; Al-Asri, J.; Mortier, J.; Marzouk, M.; Ezzeldin, E.; Ali, A.A.; Khalil, M.G.;
Wolber, G.; Ghabbour, H.A.; et al. Synthesis, crystallographic characterization, molecular docking and
biological activity of isoquinoline derivatives. Chem. Cent. J. 2017, 11, 103–121. [CrossRef] [PubMed]
8.
9.
10. Abuelizz, H.A.; Hassane, A.E.; Marzouk, M.; Ezzeldin, E.; Ali, A.A.; Al-Salahi, R. Molecular modeling,
enzyme activity, anti-inflammatory and antiarthritic activities of newly synthesized quinazoline derivatives.
Future Med. Chem. 2017, 9, 1995–2009. [CrossRef] [PubMed]
11. Lamie, P.F.; Ali, W.A.M.; Bazgier, V.; Rárová, L. Novel N-substituted indole Schiff bases as dual inhibitors of
cyclooxygenase-2 and 5-lipoxygenase enzymes: Synthesis, biological activities in vitro and docking study.
Eur. J. Med. Chem. 2016, 123, 803–813. [CrossRef] [PubMed]
12. Abdellatif, K.R.; Lamie, P.F.; Omar, H.A. 3-methyl-2-phenyl-1-substituted-indole derivatives as indomethacin
analogs: Design, synthesis and biological evaluation as potential anti-inflammatory and analgesic agents.
J. Enzyme Inhib. Med Chem. 2016, 31, 318–324. [CrossRef] [PubMed]
13. Rani, P.; Srivastava, V.K.; Kumar, A. Synthesis and antiinflammatory activity of heterocyclic indole
derivatives. Eur. J. Med. Chem. 2004, 39, 449–452. [CrossRef] [PubMed]
14. Bhat, M.A.; Al-Omar, M.A.; Ansari, M.A.; Zoheir, K.M.A.; Imam, F.; Attia, S.M.; Bakheet, S.A.; Nadeem, A.;
Korashy, H.M.; Voronkov, A.; et al. Design and synthesis of N-aryl-phthalimides as inhibitors of
glucocorticoid-induced TNF receptor-related protein, pro-inflammatory mediators and cytokines in
carrageenan-induced lung inflammation. J. Med. Chem. 2015, 58, 8850–8867. [CrossRef] [PubMed]
15. Bhat, M.A.; Al-Omar, M.A. Synthesis, characterization and in vitro anti Mycobacterium tuberculosis activity
of terpene schiff bases. Med. Chem. Res. 2013, 22, 4522–4528. [CrossRef]
16. Bhat, M.A.; Al-Omar, M.A. Synthesis, characterization and in vivo anticonvulsant screening of schiff bases
of phthalimide. Acta Pol. Pharm. Drug Res. 2011, 68, 375–380.
17. Siddiqui, N.; Bhat, M.A.; Khan, S.A.; Ahsan, W.; Alam, M.S. Synthesis and in vivo anticonvulsant screening
of coumarin incorporated Schiff bases of 1,3,4-oxadiazoles. J. Chin. Chem. Soc. 2008, 55, 1326–1331. [CrossRef]
18. Bhat, M.A.; Al-Omar, M.A.; Raish, M.; Ansari, M.A.; Abuelizz, H.A. Cyclooxygenase Inhibitors. U.S. patent
9,808,443 B1, 7 November 2017.
19. Winter, C.A.; Risley, E.A.; Nuss, G.W. Carrageenin-induced edema in hind paw of the rat as an assay for
antiiflammatory drugs. Proc. Soc. Exp. Biol. Med. 1962, 111, 544–547. [CrossRef] [PubMed]
20. Eddy, N.B.; Leimbach, D.J. Synthetic analgesics. II. Dithie-nylbutenyl- and dithienylbutylamines. J. Pharmacol.
Exper. Therap. 1953, 107, 385–393.
21. Cioli, V.; Putzolu, S.; Rossi, V.; Scorza Barcellona, P.; Corradino, C. The role of direct tissue contact in the
production of gastrointestinal ulcers by anti-inflammatory drugs in rats. Toxicol. Appl. Pharmacol. 1979, 50,
283–289. [CrossRef]
22. Ohkawa, H.; Ohishi, N.; Yagi, K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction.
Anal. Biochem. 1979, 95, 351–358. [CrossRef]
23. Rafatullah, S.; Tariq, M.; Al-Yahya, M.A.; Mossa, J.S.; Ageel, A.M. Evaluation of turmeric (Curcuma longa) for
gastric and duodenal antiulcer activity in rats. J. Ethnopharmacol. 1990, 29, 25–34. [CrossRef]

Molecules 2018, 23, 1250
19 of 19
24. Kauffman, G.L., Jr.; Grossman, M.I. Prostaglandin and cimetidine inhibit the formation of ulcers produced
by parenteral salicylates. Gastroenterology 1978, 75, 1099–1102. [PubMed]
25. Karber, G. Beitrag zur kollektiven behandlung pharmakologischer reihenversuche. Arch. Exptl. Pathol. Pharmakol.
1931, 162, 480–483. [CrossRef]
26. Towbin, H.; Staehelin, T.; Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to
nitrocellulose sheets: Procedure and some applications. Biotechnology 1992, 24, 145–149. [CrossRef] [PubMed]
27. Kurumbail, R.G.; Stevens, A.M.; Gierse, J.K.; McDonald, J.J.; Stegeman, A.R.; Pak, Y.G.; Gildehaus, D.;
Iyashiro, M.J.; Penning, T.D.; Seibert, K.; et al. Structural basis for selective inhibition of cyclooxygenase-2 by
anti-inflammatory agents. Nature 1996, 384, 644–648. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds (S1–S18) in pure form are available from authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).