Click chemistry synthesis, biological evaluation and docking study of some novel 2′-hydroxychalcone-triazole hybrids as potent anti-inflammatory agents

Article abstract of DOI:10.1016/j.bioorg.2019.103505

A hybrid pharmacophore approach is used to design and synthesize two novel series of 2′-hydroxychalcone-triazole hybrid molecules 6a-j and 8a-j. These compounds were fully characterized by spectral and elemental analyses. They were evaluated in vitro and in vivo for anti-inflammatory activity. Most of compounds were selective inhibitors for COX-2. Among them, compounds 6d, 6f, 6i, 8c, 8e and 8h demonstrated highly potent dual inhibition of COX-2 (IC₅₀ = 0.037–0.041 μM) and 15-LOX (IC₅₀ = 1.41–1.80 μM). Compounds 6i, 8c and 8h showed 116%, 113% and 109% of the in vivo anti-inflammatory activity of celecoxib. Therefore, compounds 6d, 6f, 6i, 8c, 8e and 8h-j are potent dual inhibitors of COX-2 and 15-LOX. Docking study over COX-2 and 15-LOX active sites ensures the binding affinity and selectivity. These compounds are promising candidates for further development as anti-inflammatory drugs.

Full text of DOI:10.1016/j.bioorg.2019.103505

Journal Pre-proofs
Click chemistry synthesis, biological evaluation and docking study of some
novel 2’-hydroxychalcone-triazole hybrids as potent anti-inflammatory agents
Andrew N. Boshra, Hajjaj H.M. Abdu-Allah, Anber F. Mohammed, Alaa A.
Hayallah
PII:
S0045-2068(19)31829-2
DOI:
https://doi.org/10.1016/j.bioorg.2019.103505
YBIOO 103505
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Bioorganic Chemistry
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Accepted Date:
29 October 2019
26 November 2019
10 December 2019
Please cite this article as: A.N. Boshra, H.H.M. Abdu-Allah, A.F. Mohammed, A.A. Hayallah, Click chemistry
synthesis, biological evaluation and docking study of some novel 2’-hydroxychalcone-triazole hybrids as potent
anti-inflammatory agents, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103505
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Click chemistry synthesis, biological evaluation and docking study of some
novel 2’-hydroxychalcone-triazole hybrids as potent anti-inflammatory
agents
Andrew N. Boshraa, Hajjaj H. M. Abdu-Allaha*, Anber F. Mohammeda, Alaa A. Hayallahaⱶ*
a Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Assiut University,
Assiut 71526, Egypt
Prof. Dr. Alaa Hayallah
Tel. 002-088-2411312 (office.) Fax. 002-08088-332776. alaa_hayalah@yahoo.com
Dr. Hajjaj Abdu-Allah
Work: (+2088)240-1521
Mobile: +201010211566
hajjaj@aun.edu.eg
ORCID: 0000-0002-4474-9086
ⱶPresent address: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Deraya
University, El-Minia, Egypt.
1

Abstract
A hybrid pharmacophore approach is used to design and synthesize two novel series of 2’-
hydroxychalcone-triazole hybrid molecules 6a-j and 8a-j. These compounds were fully
characterized by spectral and elemental analyses. They were evaluated in vitro and in vivo for
anti-inflammatory activity. Most of compounds were selective inhibitors for COX-2. Among
them, compounds 6d, 6f, 6i, 8c, 8e and 8h demonstrated highly potent dual inhibition of
COX-2 (IC50 = 0.037-0.041 µM) and 15-LOX (IC50 = 1.41-1.80 µM). Compounds 6i, 8c and
8h showed 116%, 113% and 109% of the in vivo anti-inflammatory activity of celecoxib.
Therefore, compounds 6d, 6f, 6i, 8c, 8e and 8h-j are potent dual inhibitor of COX-2 and 15-
LOX. Docking study over COX-2 and 15-LOX active sites ensures the binding affinity and
selectivity. These compounds are promising candidates for further development as anti-
inflammatory drugs.
Keywords: Chalcone; Fries Rearrangement; Click; Triazole; Isatin; COX-2; 15-LOX; Anti-
inflammatory
2

1. Introduction
Inflammation not only mitigates infections, initiates tissue repair and regeneration but it also
causes many chronic and degenerative diseases [1]. It occurs due to stimulation of various
cyclooxygenase (COX) and lipoxygenase (LOX) isozymes to produce inflammatory
mediators like prostaglandins (PGs) and leukotrienes. The various symptoms of inflammation
include heat, swelling, redness, pain, and loss of function of body parts such as imbalances in
the movements of limbs. Other signs result in an increase in the number of white blood cells,
fibrinogen and serum amyloid A protein (SAA), presence of C-reactive proteins (CRP), and
fever [2]. Non-steroidal anti-inflammatory drugs (NSAIDs) are the most frequently used
treatment for inflammatory symptoms. Their activities result from inhibition of
cyclooxygenase (COX) mediated production of pro-inflammatory prostaglandins and
thromboxanes [3]. However, long-term therapy with NSAIDs is usually accompanied by
undesirable side effects on the gastrointestinal, renal, hepatic, and cardiovascular systems [4].
Therefore, the discovery of new NSAIDs with better safety profiles remains an active area of
research. Chalcone moiety has found to be such a privileged pharmacophore in many
biologically active compounds and continue to be of great interest to researchers owing to its
promising applications in medicinal chemistry [5]. Several natural and (semi-) synthetic
chalcones have demonstrated anti- inflammatory activity through inhibition of COX,
prostaglandin E2, inducible NO synthase, nuclear factor κB activities and tumor necrosis
factor-α (TNF- α) [6]. For example, 2’-hydroxylated chalcones showed anti-inflammatory
activity through LOX inhibition [7], while 3,4-dihydroxychalcones showed dual inhibition of
COX-2 and 5-LOX [8]. 3-(pyridine-4-yl)-1H-pyrazole-5-carboxamide chalcones exhibited
both anti-inflammatory and antioxidant activity [9]. On the other hand, many isatin
derivatives have been synthesized and tested for their anti-inflammatory activity and showed
promising results. 3-Thiosemicarbazino isatin [10], phenylhydrazone of isatins [11] exhibited
good anti-inflammatory activity, also, ring substituted isatin showed good inhibitory activity
to COX-2 [12]. Furthermore, numerous evidences in scientific literature advocate the 1,2,3-
triazole efficacy in context of inflammation and analgesia [13-15]. Their ability to intervene
in the selective COX-2 inhibition and inhibiting the 5-LOX enzyme is said to be the cardinal
factor associated with their anti-inflammatory potential [13-15]. Moreover, 1,2,3-triazole
tethered indole-3-glyoxamide derivatives have been designed as dual inhibitors of COX-2
and 5-LOX for cancer chemotherapy [16]. Combining bioactive pharmacophores to design a
hybrid molecule is a useful tool to generate lead molecules with synergetic biological
activities and superior efficacy [17]. Recently, increasing evidence indicates that
3

simultaneous modulation of multiple targets may improve both therapeutic safety and
efficacy, compared with single-target drugs [18]. Taking into consideration the
aforementioned information and as a continuation of our project on searching for new
biologically active hybrids [19-22], we envisaged that chalcone, 1,2,3-triazole and isatin
pharmacophores if linked together would generate hybrid molecules with good and broad-
spectrum anti-inflammatory activities through selective inhibition of COX-2 and LOX.
Fig. 1. The design strategy for chalcone–triazole hybrids.
4

Moreover, varied substituents were introduced to C-4 of phenyl of chalcone in order to
explore their electronic and/or steric effects and contribution to the activity. This strategy
could afford new molecules with multitarget and improved anti-inflammatory activity
profiles. The design strategy for chalcone-triazole hybrids is shown in Fig. 1.
2. Results and discussion
2. 1. Chemistry
The target compounds 6a-j and 8a-j were synthesized as depicted in Schemes (1 and 2)
starting from the key intermediate 4. p-Aminophenol was considered a proper starting
material for the synthesis of 4 by azidation, phenolic acetylation followed by Fries
rearrangement. However, product from azidation was semisolid and difficult to purify.
Alternatively, p-anisidine was used. It was N-acetylated by reaction with acetic anhydride.
The acetylated compound was subjected to Friedel-Crafts acetylation by reaction with acetyl
chloride in presence of AlCl3. As expected, under these condition methoxy ether bond was
cleaved to give phenolic OH which directly acetylated to give phenyl acetate ester that
undergone Fries rearrangement to give compound 2, since the p-position is occupied. Acidic
hydrolysis of 2 gave 5-amino 3. Diazotization of 3 followed by azide substitution furnished
5-azido-2-hydroxyacetophenone 4. The structure of 4 was verified by the disappearance of
NH2 hump at δ 3.30 in 1H NMR and confirmed by the azide peak at 2113 cm-1 in IR. The
synthesis of 4 starting from p-anisidine afforded high yield (90%), it was scalable, and all
produced intermediates are crystallizable. Synthesis of this compound was reported recently
starting from 3 [23]. In this study the work-up was modified to a simple procedure, the
reaction mixture was gradually cooled without neutralization and the product recrystallizes in
analytically pure form in excellent yield. Thus, this is a completely green step devoid of any
organic solvents in the reaction as well as work up. Aldol condensation of 4 with
un/substituted benzaldehydes and cinnamaldehyde provided ten azido chalcone derivatives
5a-j (Scheme 1). Click reaction between 5a-j and phenyl acetylene gave chalcone–
phenyltriazole hybrids 6a-j (Scheme 2). On the other hand, click reaction of 5a-j with N-
propargylisatin 7 [24] provided the second series 8a-j (chalcone–triazole–isatin hybrids)
(Scheme 2). All compounds were characterized by NMR and IR. All final compounds (6a-j
and 8a-j) were confirmed further by elemental analyses. ESI-MS was measured for
compounds 6a and 6b.
The chemical structures of compounds 5a-j were verified by the appearance of C=O band at
1644 cm-1 in IR, while 1H NMR showed two protons of alkene appear in the aromatic region
5

at δ 7.7 and δ 7.9 ppm. 1H NMR spectra of all final compounds (6a-j and 8a-j) showed the
triazole proton resonated as a singlet at δ around 8.4 ppm.
O
O
OH
O
OH
a
R
R
N3
N3
4
5a-c, 5e-j
O
OH
O
OH
O
b
N3
N3
4
5d
Scheme 1. Reagents and conditions: (a) NaOH-ethanol, 0.5-12 h, 50-70% yield; (b) NaOH-
ethanol, 1h, 60% yield.
OH
O
n
R
N
N
N
a
OH
N3
O
6a-j
n
R
O
OH
N
5a-j
n
b
R
N
N
O
N
O
8a-j
Scheme 2. Reagents and conditions: (a) phenylacetylene, CuSO4, sod. ascorbate, t-butanol-
water, 70-80 °C, 12-24 h. (b) N-propargylisatin, CuSO4, sod. ascorbate, t-butanol-water, 70-
80°C, 12-24 h.
Compd. no.
5a, 6a, 8a
5b, 6b, 8b
R
H
n Compd. no.
R
n
1
1
1
1
5f, 6f, 8f
4-Cl
4-OH
4-Me
5g, 6g, 8g
6

5c, 6c, 8c 4-DMA 1 5h, 6h, 8h
4-OMe
1
1
1
5d, 6d, 8d
5e, 6e, 8e
H
2
1
5i, 6i, 8i
5j, 6j, 8j
3,4-(OMe)2
3,4,5-(OMe)3
4-Br
2.2. Biology
2.2.1. In vitro Cyclooxygenase inhibition.
All final compounds (6a-j, 8a-j) were tested for their inhibitory potencies against ovine
COX-1 and human recombinant COX-2. Celecoxib and Indomethacin (INM) were taken as
standards. The results are shown in (Table 1). All tested compounds displayed higher
inhibitory potency against COX-2 (IC50 = 0.037- 0.23 µM) than COX-1 (IC50 = 7.4-13.7 µM)
with COX-2 selectivity indexes (COX-1 IC50 /COX-2 IC50) in the range of 32.17-360.53.
Table 1.
COX and 15-LOX inhibitory potencies and selectivity indexes of compounds 6a-j, 8a-j and
reference drugs
COX-1 COX-2 Selectivity
15-LOX
IC50
(µM)
-
a
a
a
Compd. No.
R
IC50
IC50
indexb
(SI)
(µM)
14.7
0.041
-
(µM)
0.045
0.51
Celecoxib
Indomethacin
-
-
-
326.67
0.08
-
Zileuton
6a
-
-
15.6
4.12
5.11
4.52
2.97
3.12
1.78
2.97
3.98
1.68
2.11
2.4
Ph
7.4
9.8
0.23
0.11
32.173
89.09
141.89
185.25
300
319.51
89.09
65.83
238.46
160.29
291.33
305.13
311.63
6b
6c
6d
6e
6f
6g
6h
6i
6j
8a
8b
8c
4-CH3Ph
4-(CH3)2NPh
-CH=CHPh
4-BrPh
4-ClPh
4-HOPh
4-CH3OPh
3,4-(CH3O)2Ph
10.5
11.3
12.3
13.1
9.8
0.074
0.061
0.041
0.041
0.11
7.9
12.4
0.12
0.052
0.068
0.045
0.039
0.043
3,4,5-(CH3O)3Ph 10.9
Ph
13.11
11.9
13.4
4-CH3-Ph
4-(CH3)2N-Ph
2.67
1.58
7

8d
8e
8f
8g
8h
8i
-CH=CHPh
4-BrPh
4-ClPh
4-HOPh
11.5
13.7
10.9
9.9
13.3
12.8
0.087
0.038
0.11
0.091
0.037
0.047
0.048
132.18
360.53
99.09
108.79
359.46
272.34
258.33
2.14
1.41
3.97
3.45
1.95
1.67
1.56
4-CH3OPh
3,4-(CH3O)2Ph
3,4,5-(CH3O)3Ph 12.4
8j
a IC50 value represents the concentration of the compound that produce 50% inhibition of
COX-1, COX-2 or 15-LOX which is the mean value of two determinations where the
deviation from the mean is < 10% of the mean value.b Selectivity index (COX-1 IC50 /COX-2
IC50).
Results for COX-1 inhibition indicated that all tested compounds of phenyltriazole series (6a-
j) exhibited comparable inhibitory potency (IC50 = 7.4–13.3 µM) to celecoxib (IC50 = 14.7 µM).
On the other hand, all tested compounds showed high inhibitory profiles (IC50 = 0.037- 0.23
µM) against COX-2. Six compounds of this series, 6c, 6d-f, 6i and 6j exhibited high potency
and selectivity comparable to the standard drug Celecoxib. Particularly, compounds 6e, 6f and
6i are equipotent to Celecoxib (IC50 = 0.045 µM) and more potent than INM (IC50 = 0.51 µM).
The results showed that introducing electron donating group such as 3,4-dimethoxy (6i; IC50 =
0.052 µM) or electron withdrawing group such as 4-Br (6e; IC50 = 0.041 µM)) to the phenyl
derivative (6a; IC50 = 0.23 µM) improved the potency. Particularly, introducing lipophilic
moiety as Cl or Br increases inhibitory potency (IC50 = 0.041 µM)) as well as selectivity (SI =
319.5) against COX-2. In order to investigate the effect of phenyltriazole moiety on activity,
phenyl group was replaced by isatin to give compounds (8a-j). Interestingly, some compounds
(8a, 8b and 8h) of this series showed higher potency and selectivity for COX-2 than the
corresponding 6a, 6b an 6h. While compounds 8c, 8e, 8i and 8j are slightly more potent and
selective for COX-2 than the corresponding 6c, 6e, 6i and 6j as shown in Table 1. This
improvement in potency of 8a-j series may be attributed to the synergistic effect of isatin
moiety. In contrast, compound 8f showed dramatic decrease in potency and selectivity if
compared to 6f.
2.2.2. In vitro lipoxygenase inhibition
All final compounds (6a-j, 8a-j) were evaluated for their ability to inhibit 15-LOX enzyme.
Zileuton was taken as standard. The results are shown in Table 1. The compounds exhibit a
range of potency IC50 1.41-5.11 µM higher than Zileuton (IC50 = 15.6 µM). Several
compounds (6d, 6e, 6f, 6g, 6i and 6j) of phenyltriazole series showed high potency.
Moreover, compounds 6f and 6i displayed promising potency with IC50 1.78 and 1.68 µM
respectively. Accordingly, compound 6f and 6i showed excellent dual inhibition of COX-
8

2/15-LOX and high selectivity. Similarly, isatin series (8a-j) showed higher potency than
Zileuton. Among these compounds, compounds 8e and 8h has promising dual inhibition of
both COX-2 and 15-LOX.
2.2.3. In vivo anti-inflammatory activity
The most potent compounds in in vitro studies (6e, 6f, 6i, 8b, 8e and 8h) were screened for in
vivo anti-inflammatory study by carrageenan induced paw edema bioassay in rats using
celecoxib as a reference drug. The test compound was administrated i.p. at a dose of 28
µM/kg and the % of edema inhibition was calculated. The results (Table 2) revealed that
most of the tested compounds showed a gradual increase of the anti-inflammatory activity up
to its maximum effect after 3 h, while compounds 6e and 8e maintain this activity up to 5 h.
Table 2.
In vivo anti-inflammatory in rats using celecoxib as reference drugs
% of edema inhibition ± SE
Compd
a
No.
1h
2 h
3 h
4 h
5 h
34.7 ± 7.53
54.5 ± 5.25 62.8 ± 2.85
Celecoxib 44.0 ± 7.99
61.2 ± 11.36
60.5 ± 2.8d
59.5 ± 2.10c
59.0 ± 3.11d
6e
6f
38.6 ± 2. 12c 40.1 ± 2.72c
38.0 ± 4.00c 42.4 ± 3.25c
40.0 ± 6.93b 58.7 ± 7.84d
62.5 ± 3.18c 61.1 ± 4.16d 58.1 ± 2.1d
71.4 ± 4.08d 69.7 ± 3.03d 52.8 ± 2.47d
60.3 ± 1.92d 59.1 ± 2.19c
6i
8b
8e
8h
39.8 ± 3.24b 52.6 ± 2.33b 59.3 ± 3.70a
38.0 ± 3.10c 45.1 ± 3.34d 69.2 ± 3.27d
40.2 ± 3.24a 41.14 ± 2.34c 66.5 ± 2.01c
68.2 ± 2.60a 64.2± 2.11b
64.1 ± 3.22d 60.4 ± 2.16b
Significant difference at P < 0.05, b Significant difference at P < 0.01, c Significant difference
at P < 0.001, d Significant difference at P < 0.0001.
The results are going well with that from in vitro studies. The structure activity relationship
studies for the anti-inflammatory effects of triazole derivatives 6a-j and 8a-j shown in Tables
1 and 2 illustrated that the compounds that showed anti-inflammatory activities are not only
contained electron-withdrawing substituents (6e, 6f, 8e and 8f), but also electron-donating
substituents on the phenyl moiety (6i). Of compounds with electron-withdrawing with
halogen atom substituent groups, the introduction of Cl (6e and 8e), or Br (6f and 8f) at the p-
position of the phenyl ring, resulted in the best anti-inflammatory activity, with the best
9

compounds being 8e and 8h. Both, in addition to 6i, were the most active compounds with
activity higher than celecoxib after 3 h. They showed 116%, 113% and 109% of the anti-
inflammatory activity of celecoxib at 3 h interval. To rationalize the biological results at the
molecular level docking studies were undertaken.
2.2.4. Docking study
Docking simulations of the new compounds (6a-j and 8a-j) were performed using Molecular
Operating Environment (MOE) software [25] and the crystal structures of rofecoxib bound to
human cyclooxygenase-2 (PDB: 5KIR) [26] as well as human 15-Lipoxygenase-2 bound to a
competitive inhibitor C8E4 (PDB: 4NRE) [27].
2.2.4.1. Docking to COX-2
Docking was performed and compared with three COX-2 inhibitors; indomethacin, celecoxib
and rofecoxib to rationalize the biological results of the new compounds. Visualization of the
active site revealed that COX-2 pocket is predominately lipophilic with several regions of
hydrophilicity. Moreover, docking results revealed that celecoxib and indomethacin probe the
space of the enzyme active site showing different binding modes (Table S1) that were found to
be compatible with literature [28]. Coxibs such as celecoxib and rofecoxib adopt a binding
pose within the COX-2 channel with their sulfone moiety inserted into the side pocket near
Arg513. On the other hand, indomethacin forms a salt bridge between the ligand carboxylate
moiety and the guanidinium moiety of Arg120 at the hydrophilic mouth of the active pocket.
The remaining contacts for both selective and non-selective COX-2 inhibitors are hydrophobic
in nature with Leu352, Ser353, Val349, Tyr355, Val523, Ala527.
Among the first series 6a-j, the highly potent halogen substituted derivatives 6e and 6f were
the best docked within the active site although they showed different orientations. They showed
collective binding interactions for the known COX-2 inhibitors within the active site. The bulky
bromo substituted phenyl of compound 6e occupies the side pocket forming hydrophobic
interactions with Val523, Leu352 and Ser353 as well as a CH-π interaction between the ligand
triazole and Ala527 of the hydrophobic pocket (Table S1), while its carbonyl accepts a
hydrogen bond interaction from Arg120 (Fig. 2A). Whilst compound 6f is being upturned with
its phenyl triazole moiety inserted in the side pocket forming hydrophobic interactions with
Val523 and Leu352 as well as a hydrogen bond interaction with Ser353. In addition, the chloro
group accepts a hydrogen bond interaction from Arg120 (Fig. 2B).
10

A
B
Fig. 2. Docking of compounds 6e (purple) (A) aligned with celecoxib (cyan) and 6f (B) into
the COX-2 active site showing their orientations and binding modes.
The methoxy substituted derivatives 6g-6j were found to exhibit an orientation similar to that
of the chloro substituted compound. However, the position and/or number of the methoxy
groups have an impact on the ring slope near Arg120. Compounds 6h and 6j showed weaker
binding with their distant p-methoxy or bulky di m-methoxy groups. However, the moderately
active compound 6i showed better binding with its single m-methoxy group accepting a
hydrogen bond interaction from Arg120. Also, the size of the hydrogen bond forming group
affected the binding of the compound, the small hydroxyl group of 6g failed to form a hydrogen
bond interaction with Arg120 compared with the methoxy substituted compound 6i.
The compound 6c substituted with the basic dimethylamino group prefers to get away from the
Arg120 and shows an orientation that resembles that of the bromo derivative 6e however
projection of the phenyl triazole moiety towards the hydrophilic pocket weakened the binding.
Other derivatives carrying lipophilic groups rather than hydrogen bond forming ones (6a, 6b
and 6d) showed hydrophobic interactions with Val523 and failed to form hydrogen bond
interaction with Arg120. However, the bi-vinyl containing compound 6d showed stronger
hydrophobic interactions within the active site with its triazole moiety forming a bi CH-π
interaction with Leu352 and Val523.
Analysis of the docked 8a-j compounds revealed that most derivatives bind with their isatin
moiety located in the side pocket of COX-2 active site showing a hydrogen bond interaction
between the ligand 2-oxo group and Arg513. In addition, the triazole moiety is good fitted
between Leu352 and Val523 forming bi CH-π interactions along with the optimized
hydrophobic interactions of these compounds to the hydrophobic cleft of the enzyme.
11

The mono- and dimethoxy substituted derivatives 8h and 8i showed almost similar binding
modes, although the dimethoxy substituent was better docked within the enzyme pocket.
Replacement of methoxy by more polar hydroxy group in compound 8g decreases the extent
of hydrophobic interactions of the whole compound. The methyl substituted derivative 8b is
better bound to the enzyme active site and forms stronger hydrophobic interactions than the
unsubstituted ones 8a and 8d. The bulky bi-vinyl moiety in compound 8d induces steric effect
resulting in observed shift for the isatin and triazole moieties from their best binding locations
within the enzyme pocket (Fig. 3).
Fig. 3. Docking of compounds 8a (purple), 8b (cyan) and 8d (yellow) into the COX-2 active
site showing the effect of the bi-vinyl group on the binding of 8d.
The same effect was observed with the bulky trimethoxy substituted compound 8j compared
with compounds 8h and 8j. The most potent bromo substituted compound 8e showed better
binding interactions and fitting within the active site than the chloro substituted one 8f which
failed to interact with its cholophenyl moiety to Arg513 in the side pocket. Similar to the first
series 6a-j, the moderately active compound 8c displays different binding mode with its
dimethyl amino group located in the side pocket while the 2-oxo of the isatin moiety forms
hydrogen bond interaction with Arg120.
2.2.4.2. Docking to 15-LOX
Docking simulations of the new compounds was done and compared with two reference
compounds; Quercetin and Zileuton in order to rationalize the biological results of the new
compounds. Visualization of the 15-LOX-2 active site revealed that the cavity composed of a
hydrophobic hairpin structure where the amino acid residues can adopt an equivalent U-
shaped conformation to that of the bound detergent. Also, the crystal structure contains a
12

catalytic iron Fe++ and an apparent channel for invading oxygen lying on opposite sides of the
cavity tunnel. This catalytic iron-binding site was found to be coordinated by the imidazole
nitrogens of His373, His378, and His553. Mechanistic studies have been demonstrated that
iron metal abstracts the hydrogen from the central carbon of the pentadiene and further
oxidation also occurs on opposite faces of the amino acid residues [27]. Therefore, the
distance between the main phenolic OH of the new compounds and Fe++ was measured to
give an idea about the proper orientation of the ligand within the active site and the
possibility for the compound phenolic OH to be attacked by the enzyme (Table S2).Results
indicated that the new compounds 6a-j and 8a-j display distances in the range of 5.09 -5.88 Å
compared with Quercetin (5.04 Å) and Zileuton (5.12 Å). Overall docking results revealed
that all the new compounds showed binding modes within the active site better than that of
Quercetin as well as Zileuton (Table S2). The new compounds display binding orientation in
the cavity in a U-shaped conformation and are surrounded primarily by the side chains of
hydrophobic amino acids. This conformation was adopted as the phenolic OH was found to
be capable to form an intramolecular hydrogen bond with its adjacent carbonyl group (Fig.
4).
13

Fig. 4. Docking of compounds 8e (purple) aligned with Quercetin (cyan) within 15-LOX-2
active site.
Among each series, although being inadequate to find successful correlation between the
obtained biological results and the estimated docking within the active site, the analysis could
determine some important binding interactions for the new compounds inside the cavity.
The main phenolic OH was found to be in the vicinity of the iron metal in a way comparable
to that of Quercetin forming hydrogen bond interaction with Ile412 or Asn413. The chalcone
moiety occupies the smaller hydrophobic pocket and forms CH-π interaction with the amino
acid Glu369 or Leu420. On the other side, the docking analysis revealed that the isatin
containing compounds (8a-j) showed better binding interactions and filled the cavity of the
active site in away better than that displayed by their phenyl congeners (6a-j). Docking of
compound 6d revealed that the compound completely filled the cavity forming strong
hydrophobic interactions with the surrounding amino acid residues as well as two hydrogen
bond interactions with Ile412 compared with the other lipophilic containing compounds 6a
and 6b. On the other hand, their congeners 8a, 8b, 8d showed better fitting within the active
site owing to the complete fitting of their bulky istain moiety into the larger hydrophobic
pocket. For the same reason, the dimethylamino substituted compound 8c was found to be
better oriented within the cavity than its congener 6c and got stabilized by forming two
hydrogen bond interaction with Ile412 rather than Asn413 in addition to the CH-π
interactions with Glu369 and Phe184. The halogen containing compounds 6e, 6f and their
isatin carrying congeners 8e, 8f showed almost similar binding modes inside the cavity,
however, the chloro substituted compound 8f displays longer distance from the catalytic Fe++
(5.82 Å). Replacing the hydroxyl by methoxy group and even increasing the number of
methoxy groups in compounds 6g-j and 8g-j has a good effect on the activity. The di- and
trimethoxy substituted derivatives 6i and 6j showed better binding modes and stronger
hydrophobic interactions with the surrounding hydrophobic amino acid residues than the
hydroxy and monomethoxy substituted compounds 6g and 6h owing to their increased
bulkiness within the active site. The same effect could also explain the enhanced potency of
14

compounds 8g-j in the order of hydroxy 8g< monomethoxy 8h< dimethoxy8i< trimethoxy
substituted 8j with their increasing bulkiness inside the cavity hydroxy 8g< monomethoxy
8h< dimethoxy8i< trimethoxy substituted 8j (Fig. 5).
Fig. 5. Docking of compounds 6j (yellow) aligned with 8j (cyan) within 15-LOX-2 active
site.
3. Conclusion
This study proposed a new rational design of potent anti-inflammatory compounds acting by
dual inhibition of COX-2 and 15-LOX. The design is based on combining two or more
hybrids having different anti-inflammatory mechanisms; as chalcone – triazole hybrids and
chalcone – triazole – isatin hybrids. The obtained results revealed that compounds 6e, 6f, 6i,
8b, 8e and 8h showed anti-inflammatory activity equivalent to or even higher than that of
celecoxib. In vitro COX-1/COX-2 inhibition study revealed that among the synthesized
compounds, compound 8h showed the highest inhibitory activity against COX-2 with an IC50
values of 0.037 µM and selectivity index 359.46. Additionally, most compounds showed
significant in vitro 15-LOX inhibitory activity higher than that of zileuton. Therefore,
compounds 6d, 6f, 6i, 8c, 8e and 8h-j are potent dual inhibitor of COX-2 and 15-LOX. The
docking experiments attempted to investigate the binding mode of the final compounds in the
binding site of COX-2 and 15-LOX confirmed the high binding selectivity towards COX-2 as
well as the difference in activity between different derivatives. Consequently, the in vitro and
15

in vivo anti-inflammatory profiles revealed that these compounds can be a lead for
developing new anti-inflammatory drugs.
4. Experimental
4.1. Chemistry
All solvents and reagents used for the synthesis of target compounds were of commercial
grade without further purification before use. Melting points were determined on an
electrothermal melting point apparatus [Cole-Parmer - Electrothermal IA9100, UK], and
were uncorrected. Pre-coated silica gel plates (TLC) (kieselgel 0.25 mm, 60G F254, Merck,
Germany) were used for monitoring of the chemical reactions. Spots were detected by using
ultraviolet lamp at 254 nm wavelength (Spectroline, model CM-10, USA). (IR) spectra (KBr
discs) were recorded on thermo scientific nicolet IS10 FT IR spectrometer (thermo Fischer
scientific, USA) at Faculty of science, Assiut University, Assiut, Egypt. 1H-NMR (400 MHz)
and 13C-NMR (100 MHz) Spectra were scanned on AVANCE-III High Performance FT-
NMR spectrometer, (Brucker Biospin International AG, Swtizerland) at Faculty of Science,
Sohag University, Sohag, Egypt except for compounds (5a-d, 5f, 5h) which were performed
on a Varian EM-390 NMR spectrometer (90 MHz, Varian, CA, USA) at Faculty of Science,
Assiut University, Assiut, Egypt, and compounds 6a and 6b on AVANCE 500 spectrometer
(Bruker Biospin K.K.; Yokohama, Japan). Chemical shifts are expressed in δ-values (ppm)
relative to TMS as an internal standard in (90 MHz instrument), using the appropriate solvent
as specified. Deuterium oxide was used for the detection of exchangeable protons. Coupling
constants (J) are reported in Hertz (Hz). ESI-MS were determined using Bruker Daltonics
K.K., Yokohama, Japan equipped with electrospray ionization (ESI) interface. Elemental
microanalyses were performed on elemental analyzer model flash 2000 thermo fisher at the
Regional Center for Mycology and Biotechnology (RCMB), Faculty of Science, Al-Azhar
University, Nasr city, Cairo, Egypt.
4.1.1. N-(4-Methoxyphenyl)acetamide (1)
To a stirred suspension of p-anisidine (6.04 g, 49 mmol) in dichloromethane (20 mL), acetic
anhydride (5 mL, 53 mmol) was added over 1 hour. The reaction was stirred for 1 hour more
then poured into hexane (60 mL) and stirred for 1 h. The pale grey crystals were filtered,
washed with hexane and dried; Yield 7.72 g (95%); mp 128-130 ºC as reported [29].
4.1.2. N-(3-Acetyl-4-hydroxyphenyl)acetamide (2):
To a stirred suspension of N-(4-methoxyphenyl)acetamide 1 (5.25 g, 32 mmol) and acetyl
chloride (6.6 mL, 93 mmol) in dichloromethane (55 mL), aluminum trichloride (14.55 g, 109
16

mmol) was added portion-wise over 90 min. The reaction was then heated under reflux for
4.5 h and cooled overnight. The reaction mixture was poured into ice then extracted with
dichloromethane (5 x 50 mL), dried over MgSO4, filtered and concentrated in vacuo to give
the product as green solid that was recrystallized from ethyl acetate - diethyl ether as green
crystals. Yield 5.34 g (87%); mp 167-168 ºC as reported [30].
4.1.3. 1-(5-Amino-2-hydroxyphenyl)ethan-1-one (3):
A suspension of N-(3-acetyl-4-hydroxy-phenyl)-acetamide 2 (1.03 g, 5.3 mmol) in 15% HCl
(1.5 mL, 6.2 mmol) was heated under reflux for 40 min, then cooled and neutralized with
10% aqueous ammonia. The green solid was filtered, washed with water and dried in
desiccator. Yield 0.68 g (84%); mp 107-110 ºC as reported [30].
4.1.4. 1-(5-Azido-2-hydroxyphenyl)ethan-1-one (4):
A solution of sodium nitrite (1.46 g, 21.2 mmol) in water (20 mL) was added dropwise to an
ice-cold solution of 1-(5-amino-2-hydroxyphenyl)ethan-1-one 3 (2.00 g, 13.2 mmol) in water
(100 mL) and conc. H2SO4 (15 mL). The reaction mixture was stirred at 0 °C for 30 min. A
solution of sodium azide (2.32 g, 35.7 mmol) in water (20 mL) was added dropwise to the
mixture. The reaction mixture was further stirred at room temperature for 1h, then gradually
cooled to give green crystals, filtered and washed with cooled water and dried in desiccator.
Yield 2.25 g (91%); mp 60-61 ºC as reported [23].
4.1.5. (E)-1-(5-Azido-2-hydroxyphenyl)-3- substituted prop-2-en-1-ones (5a-j):
Aqueous NaOH (2 mL of 30 g/100 mL) was added to a mixture of 4 (0.5 g, 2.8 mmol) and
the appropriate (un)substituted benzaldehyde (3.1 mmol) in ethanol (6 mL), then the reaction
mixture was stirred at room temperature for 0.5–12 h. The mixture was neutralized with 15%
hydrochloric acid in ice bath. The product was filtered, washed with ethanol, then water, and
dried. The formed compounds were crystallized from ethanol–water.
4.1.5.1. (E)-1-(5-Azido-2-hydroxyphenyl)-3-phenylprop-2-en-1-one (5a):
Yield 70.1%; mp 121-122 ºC as reported [21]; IR (cm-1, KBr): 3440 (O-H), 3062 (Ar-H),
2114 (N3) 1646, (C=O), 1576, 1487 (C=C), 692 (=C-H). 1H NMR (90 MHz, δppm CDCl3):
7.0-7.4 (m, 3H, Ar-H), 7.4-7.8 (m, 6H, alkene-H & Ar-H), 8.0 (d, J = 16 Hz, 1H, alkene-H),
13.0 (s, 1H, OH).
4.1.5.2. (E)-1-(5-Azido-2-hydroxyphenyl)-3-(p-tolyl)prop-2-en-1-one (5b):
Yield 66.8%; mp 100-102 ºC; IR (ύ cm-1, KBr): 3400 (O-H), 3060 (Ar-H), 2920 (C-H
aliphatic), 2112 (N3), 1637 (C=O), 1479, 1561 (C=C), 703 (=C-H). 1H NMR (90 MHz, δ ppm
CDCl3): 2.4 (s, 3H, -CH3), 7.1-7.4 (m, 4H, Ar-H), 7.4-7.6 (m, 4H, alkene-H & Ar-H), 7.9 (d,
J = 17 Hz, 1H, alkene-H), 13.0 (s, 1H, OH).
17

4.1.5.3. (E)-1-(5-Azido-2-hydroxyphenyl)-3-(4-(dimethylamino)phenyl)prop-2-en-1-one
(5c): Yield 63.3%; mp 128-130 ºC; IR (ύ cm-1, KBr): 3439 (O-H), 3062 (Ar-H), 2911 (C-H
aliphatic), 2111 (N3), 1630 (C=O), 1601 (C=C), 678 (=C-H). 1H NMR (90 MHz, δ ppm
CDCl3): 3.98 (s, 6H, N-(CH3)2), 6.95-7.35 (m, 5H, Ar-H), 7.4-8.1 (m, 4H, alkene-H & Ar-H),
12.95 (s, 1H, OH).
4.1.5.4. (2E,4E)-1-(5-Azido-2-hydroxyphenyl)-5-phenylpenta-2,4-dien-1-one (5d):
Yield 65.4%; mp: >300 ºC; IR (ύ cm-1, KBr): 3423 (O-H); 3027 (Ar-H), 2123 (N3), 1637
(C=O), 1563, 1483 (C=C), 690 (=C-H). 1H NMR (90 MHz, δ ppm CDCl3): 6.85–7.20 (m,
3H, alkene-H & Ar-H), 7.31-7.67 (m, 6H, alkene-H & Ar-H), 7.73–7.85 (m, 3H, alkene-H &
Ar-H), 12.09 (s, 1H, OH).
4.1.5.5. (E)-1-(5-Azido-2-hydroxyphenyl)-3-(4-bromophenyl)prop-2-en-1-one (5e):
Yield 69.8%; mp 100-102 ºC; IR (ύ cm-1, KBr): 3410 (O-H), 3105 (Ar-H), 2125 (N3), 1679
(C=O), 1574, 1484 (C=C), 717 (=C-H). 1H NMR (400 MHz, δ ppm CDCl3):7.08 (d, J = 8.8
Hz, 1H, 6’Ar-H), 7.34–7.39 (dd, J = 8.9, 2.7 Hz, 1H, 5’Ar-H), 7.67–7.77 (m, 3H, alkene-H &
Ar-H), 7.79–7.89 (m, 3H, alkene-H & Ar-H), 8.00 (d, J = 15.6 Hz, 1H, alkene-H), 12.04 (s,
1H, OH)
4.1.5.6. (E)-1-(5-Azido-2-hydroxyphenyl)-3-(4-chlorophenyl)prop-2-en-1-one (5f):
Yield 71.0%; mp 113-114 ºC; IR (ύ cm-1, KBr): 3300 (O-H), 3030 (Ar-H), 2115 (N3), 1644
(C=O), 1582, 1482 (C=C), 718 (=C-H). 1H NMR (90 MHz, δ ppm CDCl3): 7.0-7.35 (m, 3H,
Ar-H), 7.4-7.7 (m, 5H, alkene-H & Ar-H), 7.9 (d, J = 16 Hz, 1H, alkene-H), 13.00 (s, 1H,
OH).
4.1.5.7. (E)-1-(5-Azido-2-hydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one (5g):
Yield 50.4%; mp >300 ºC; IR (ύ cm-1, KBr): 3450 (O-H), 3079 (Ar-H), 2115 (N3), 1644
(C=O), 1517, 1482 (C=C), 718 (=C-H). 1H NMR (400 MHz, δ ppm CDCl3): 6.94–7.50 (m,
3H, Ar-H), 7.63–7.94 (m, 2H, alkene-H & Ar-H), 8.03–8.43 (m, 4H, alkene-H & Ar-H),
11.62 (s, 1H, OH), 11.87 (s, 1H, OH).
4.1.5.8. (E)-1-(5-Azido-2-hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (5h):
Yield 54.3%; mp 130-132 ºC; IR (ύ cm-1, KBr): 3432 (O-H), 3010 (Ar-H), 2837 (C-H
aliphatic), 2110 (N3), 1639 (C=O), 1567, 1512 (C=C), 679 (=C-H). 1H NMR (90 MHz, δ ppm
CDCl3): 3.9 (s, 3H, O-CH3), 6.95-7.2 (m, 3H, Ar-H), 7.25-7.5 (m, 3H, alkene-H & Ar-H),
7.6-8.0 (m, 3H, alkene-H & Ar-H), 13.11 (s, 1H, OH).
4.1.5.9. (E)-1-(5-Azido-2-hydroxyphenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (5i):
Yield 50.1%; mp 140-142 ºC; IR (ύ cm-1, KBr): 3400 (O-H), 3060 (Ar-H), 2115 (N3), 1644
(C=O), 1567, 1512 (C=C), 718 (=C-H). 1H NMR (400 MHz, δ ppm CDCl3): 3.84 (s, 3H, O-
18

CH3), 3.87 (s, 3H, O-CH3), 7.03–7.09 (dd, J = 8.5, 6.2 Hz, 2H, Ar-H), 7.36–7.40 (dd, J = 8.9,
2.7 Hz, 1H, Ar-H), 7.46 (d, J = 8.3 Hz, 1H, Ar-H), 7.55 (s, 1H, Ar-H), 7.77–7.89 (m, 3H,
alkene-H & Ar-H), 12.36 (s, 1H, OH).
4.1.5.10. (E)-1-(5-Azido-2-hydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one
(5j): Yield 50.2%; mp >300 ºC; IR (ύ cm-1, KBr): 3510 (O-H), 3052 (Ar-H), 2920, 2117
(N3), 1683 (C=O), 1593, 1486 (C=C), 682 (=C-H). 1H NMR (90 MHz, δ ppm CDCl3): 3.55
(s, 3H, 4-(OCH3)), 3.64 (s, 6H, 3,5-(OCH3)), 6.92–7.25 (m, 3H, Ar-H), 7.41–7.96 (m, 4H,
alkene-H & Ar-H), 13.51 (s, 1H, OH).
4.1.6. (E)-1-(2-Hydroxy-5-(4-phenyl-1H-1,2,3-triazol-1-yl)phenyl)-3- substituted prop-2-
en-1-ones (6a-j): To a stirred suspension of appropriate derivative of 5a-j (0.84 mmol) in a
mixture of tertiary butanol-water (3:1, 7 mL), phenyl acetylene (0.15 mL, 1.35 mmol) was
added. Solutions of sodium ascorbate (33 mg, 0.17 mmol) in water (0.4 mL) and CuSO4. (21
mg, 0.08 mmol) in water (0.3 mL) were added to the stirred mixture at 70 ºC and further
stirred overnight. Additional solution of sodium ascorbate (33 mg, 0.17 mmol) in water (0.4
mL) and a solution of CuSO4 (21 mg, 0.08 mmol) in water (0.3 mL) were added and the
mixture was further stirred. The reaction progress was monitored by TLC. Water (15 mL)
was added and the resultant suspension was stirred for 30 min. the formed precipitate was
filtered, washed with water and dried. The products were recrystallized from ethyl acetate-
hexane.
4.1.6.1. (E)-1-(2-Hydroxy-5-(4-phenyl-1H-1,2,3-triazol-1-yl)phenyl)-3-phenylprop-2-en-
1-one (6a): Yield 59.9%; yellow solid; mp 202-204 ºC; IR (ύ cm-1, KBr): 3450 (O-H), 3130
(Ar-H), 1644 (C=O), 1575, 1493 (C=C); 692 (Ar-H). 1H NMR (500 MHz, δ ppm CDCl3):
7.19 (d, J = 8.9 Hz, 1H, Ar-H), 7.35–7.40 (t, J = 7.4 Hz, 1H, Ar-H), 7.43–7.49 (m, 6H, Ar-
H), 7.66–7.72 (m, 2H, alkene-H & Ar-H), 7.76-7.80 (dd, J = 8.9, 2.5 Hz, 1H, Ar-H), 7.89–
7.93 (m, 2H, Ar-H), 8.00 (d, J = 15.4 Hz, 1H, alkene-H), 8.39 (s, 1H, triazole-H), 8.40 (d, J =
2.5 Hz, 1H, Ar-H), 12.38 (s, 1H, OH). 13C NMR (125 MHz): 117.97, 119.24, 120.00, 120.02,
122.26, 125.88, 128.43, 128.58, 128.70, 129.00, 129.03, 129.16, 130.09, 131.50, 134.17,
147.17, 148.61, 163.66, 193.13 (C=O). ESI-MS (m/z): calcd. (M++1) 368.1, Found (M++1)
368.3. Anal. Calcd. for C23H17N3O2 (367.40): Calculated/Found: 75.19/75.42 (%C), 4.66/4.80
(%H), 11.44/11.69 (%N).
4.1.6.2. (E)-1-(2-Hydroxy-5-(4-phenyl-1H-1,2,3-triazol-1-yl)phenyl)-3-(p-tolyl)prop-2-en-
1-one (6b): Yield 57.2%; ; yellow solid; mp 226-228 ºC; IR (ύ cm-1, KBr): 3440 (O-H),
3132 (Ar-H), 2900 (C-H aliphatic), 1644 (C=O), 1577, 1493 (C=C), 691 (Ar-H). 1H NMR
19

(500 MHz, δ ppm CDCl3): 2.42 (s, 3H, -CH3), 7.19 (d, J = 8.9 Hz, 1H, 6’Ar-H), 7.25–7.27 (t,
J = 3.9 Hz, 2H, 2’’,6’’Ar-H), 7.37–7.40 (t, J = 7.4 Hz, 1H, 4’’Ar-H), 7.46–7.49 (t, J = 7.6 Hz,
2H, 3’’,5’’Ar-H), 7.61 (d, J = 8.0 Hz, 2H, 3,5Ar-H), 7.65 (d, J = 15.4 Hz, 1H, Cb-H), 7.77–
7.79 (dd, J = 8.9, 2.5 Hz, 1H, 5’Ar-H), 7.93 (d, J = 7.2 Hz, 2H, 2,6 Ar-H) 8.00 (d, J = 15.3
Hz, 1H, Ca-H) 8.40 (s, 1H, triazole-H) 8.41 (d, J = 2.5 Hz, 1H, 3’Ar-H), 13.08 (s, 1H, OH).
13C NMR (125 MHz): 21.67 (-CH3), 117.96, 118.08, 119.69, 120.07, 122.25, 125.87, 128.28,
128.56, 128.64, 128.98, 129.10, 129.90, 130.09, 131.46, 142.32, 147.29, 148.59, 163.65,
193.13 (C=O). ESI-MS (m/z): calcd. (M++1) 382.1, Found (M++1) 382.2. Anal. Calcd. for
C24H19N3O2 (381.43): Calculated/Found: 75.57/75.80 (%C), 5.02/5.18 (%H), 11.02/11.34
(%N).
4.1.6.3. (E)-3-(4-(Dimethylamino)phenyl)-1-(2-hydroxy-5-(4-phenyl-1H-1,2,3-triazol-1-
yl)phenyl)prop-2-en-1-one (6c): Yield 55.8%; orange solid; mp 241-243 ºC; IR (ύ cm-1,
KBr): 3439 (O-H), 3119 (Ar-H), 2920 (C-H aliphatic), 1634 (C=O), 1550, 1526 (C=C), 695
(Ar-H). 1H NMR (400 MHz, δ ppm DMSO-d6): 3.05 (s, 6H, -N(CH3)2), 6.79 (d, J = 8.8 Hz,
2H, 3,5 Ar-H), 7.21 (d, J = 8.9 Hz, 1H, 6’Ar-H), 7.39–7.43 (t, J = 5.8 Hz, 1H, 4’’Ar-H),
7.48–7.56 (t, J = 7.6 Hz, 2H, 2’’,6’’Ar-H), 7.77–7.92 (m, 4H, Ca-H & Cb-H & 3’’,5’’Ar-H),
7.97 (d, J = 7.4 Hz, 2H, 2,6 Ar-H), 8.04–8.09 (dd, J = 8.9, 2.5 Hz, 1H, 5’Ar-H), 8.58 (d, J =
2.5 Hz, 1H, 3’Ar-H), 9.27 (s, 1H, triazole-H), 13.11 (s, 1H, OH). Anal. Calcd. for
C25H22N4O2 (410.47): Calculated/Found: 73.15/73.49 (%C), 5.40/5.47 (%H), 13.65/13.83
(%N).
4.1.6.4. (2E,4E)-1-(2-Hydroxy-5-(4-phenyl-1H-1,2,3-triazol-1-yl)phenyl)-5-phenylpenta-
2,4-dien-1-one (6d): Yield 55.7%.; yellow solid; mp 160-162 ºC; IR (ύ cm-1, KBr): 3400 (O-
H), 3119 (Ar-H), 1655 (C=O), 1567, 1493 (C=C), 694 (Ar-H). 1H NMR (400 MHz, δ ppm
DMSO-d6): 6.80–7.25 (m, 4H, alkene-H & Ar-H), 7.32-7.64 (m, 8H, alkene-H & Ar-H),
7.70–7.95 (m, 4H, alkene-H & Ar-H), 8.54 (s, 1H, Ar-H), 9.15 (s, 1H, triazole-H), 12.23 (s,
1H, OH). Anal. Calcd. for C25H19N3O2 (393.44): Calculated/Found: 76.32/76.15 (%C),
4.87/4.98 (%H), 10.68/10.97 (%N).
4.1.6.5. (E)-3-(4-bromophenyl)-1-(2-hydroxy-5-(4-phenyl-1H-1,2,3-triazol-1-yl)phenyl)-
prop-2-en-1-one (6e): Yield 51%.; yellow solid; mp 243-244 ºC; IR (ύ cm-1, KBr): 3448 (O-
H), 3133 (Ar-H), 1646 (C=O), 1577, 1489 (C=C), 691 (Ar-H). 1H NMR (400 MHz, δ ppm
DMSO-d6): 7.25 (d, J = 7.1 Hz, 1H, Ar-H), 7.41 (d, J = 5.2 Hz, 1H, Ar-H), 7.48-7.56 (m, 2H,
Ar-H), 7.70 (d, J = 6.2 Hz, 2H, Ar-H), 7.81–7.90 (m, 3H, alkene-H & Ar-H), 7.97 (d, J = 6.0
Hz, 2H, Ar-H), 8.02–8.12 (m, 2H, alkene-H & Ar-H), 8.53 (s, 1H, Ar-H), 9.25 (s, 1H,
20

triazole-H), 12.29 (s, 1H, OH). Anal. Calcd. for C23H16BrN3O2 (446.30): Calculated/Found:
61.90/62.14 (%C), 3.61/3.74 (%H), 9.42/9.73 (%N).
4.1.6.6. (E)-3-(4-Chlorophenyl)-1-(2-hydroxy-5-(4-phenyl-1H-1,2,3-triazol-1-yl)phenyl)-
prop-2-en-1-one (6f): Yield 52.2%; brown solid; mp 230-232 ºC; IR (ύ cm-1, KBr): 3490 (O-
H), 3136 (Ar-H), 1639 (C=O), 1582, 1501 (C=C), 691 (Ar-H). 1H NMR (400 MHz, δ ppm
DMSO- d6): 7.24–7.61 (m, 9H, alkene-H & Ar-H), 7.89–7.96 (m, 5H, alkene-H & Ar-H),
9.27 (s, 1H, triazole-H). Microanalysis for C23H16ClN3O2 (401.85): Calculated/Found:
68.74/69.01 (%C), 4.01/4.13 (%H), 10.46/10.68 (%N).
4.1.6.7. (E)-1-(2-Hydroxy-5-(4-phenyl-1H-1,2,3-triazol-1-yl)phenyl)-3-(4-hydroxyphenyl)
prop-2-en-1-one (6g): Yield 58.6%; yellowish green solid; mp 242-244 ºC; IR (ύ cm-1, KBr):
3400 (O-H), 3133 (Ar-H), 1646 (C=O), 1577, 1494 (C=C), 689 (Ar-H). 1H NMR (400 MHz,
δ ppm DMSO-d6): 7.23 (d, J = 9.7 Hz, 2H, Ar-H), 7.35–7.55 (m, 4H, Ar-H), 7.80-8.35 (m, 7H,
alkene-H & Ar-H), 8.45 (s, 1H, Ar-H), 9.28 (s, 1H, triazole-H), 11.93 (s, 1H, OH), 12.13 (s,
1H, OH). Anal. Calcd. for C23H17N3O3 (383.40): Calculated/Found: 72.05/72.32 (%C),
4.47/4.53 (%H), 10.96/11.12 (%N).
4.1.6.8. (E)-1-(2-Hydroxy-5-(4-phenyl-1H-1,2,3-triazol-1-yl)phenyl)-3-(4-ethoxyphenyl)-
prop-2-en-1-one (6h): Yield 51.2%; yellowish green solid; mp 202-204 ºC; IR (ύ cm-1, KBr):
3400 (O-H), 3130 (Ar-H), 2936 (C-H aliphatic), 1644 (C=O), 1567, 1512 (C=C), 692 (Ar-H).
1H NMR (400 MHz, δ ppm DMSO-d6): 3.84 (s, 3H, O-CH3), 7.06 (d, J = 7.6 Hz, 2H, Ar-H),
7.25 (d, J = 7.8 Hz, 1H, Ar-H), 7.38–7.42 (m, 1H, Ar-H), 7.49-7.56 (m, 2H, Ar-H), 7.86-8.00
(m, 6H, alkene-H & Ar-H), 8.09 (d, J = 7.6 Hz, 1H, Ar-H), 8.58 (s, 1H, Ar-H), 9.28 (s, 1H,
triazole-H), 12.65 (s, 1H, OH). Anal. Calcd. For C24H19N3O3 (397.43): Calculated/Found:
72.53/72.80 (%C), 4.82/4.89 (%H), 10.57/10.43 (%N).
4.1.6.9. (E)-3-(3,4-Dimethoxyphenyl)-1-(2-hydroxy-5-(4-phenyl-1H-1,2,3-triazol-1-yl)
phenyl)prop-2-en-1-one (6i): Yield 52.6%; yellow solid; mp 257-259 ºC; IR (ύ cm-1, KBr):
3432 (O-H), 3118 (Ar-H), 2931 (C-H aliphatic), 1640 (C=O), 1568, 1509 (C=C), 695 (Ar-H).
1H NMR (400 MHz, δ ppm DMSO-d6): 3.84 (s, 3H, O-CH3), 3.86 (s, 3H, O-CH3), 7.07 (d, J =
8.3 Hz, 1H, Ar-H), 7.25 (d, J = 8.9 Hz, 1H, Ar-H), 7.37-7.42 (t, J = 7.4 Hz, 1H, Ar-H), 7.49–
7.55 (m, 4H, alkene-H & Ar-H), 7.83–7.94 (m, 2H, alkene-H & Ar-H), 7.96 (d, J = 7.7 Hz, 2H,
Ar-H), 8.08 (d, J = 8.8 Hz, 1H, Ar-H), 8.54 (s, 1H, Ar-H), 9.28 (s, 1H, triazole-H), 12.56 (s,
1H, OH). Anal. Calcd. for C25H21N3O4 (427.45): Calculated/Found: 70.25/70.41 (%C),
4.95/4.87 (%H), 9.83/9.98 (%N).
4.1.6.10. (E)-1-(2-Hydroxy-5-(4-phenyl-1H-1,2,3-triazol-1-yl)phenyl)-3-(3,4,5-trimethoxy
21

phenyl)prop-2-en-1-one (6j): Yield 49.5%; grey solid; mp 281-283 ºC; IR (ύ cm-1, KBr):
3392 (O-H), 3144 (Ar-H), 2900 (C-H aliphatic), 1731 (C=O), 1495 (C=C), 689 (Ar-H). 1H
NMR (400 MHz, δ ppm DMSO- d6): 3.74 (s, 3H, 4-(OCH3)), 3.87 (s, 6H, 3,5-(OCH3)), 7.26–
7.51 (m, 6H, Ar-H) 7.80–8.48 (m, 6H, alkene-H & Ar-H), 9.28 (s, 1H, triazole-H), 12.36 (s,
1H, OH). Microanalysis for C26H23N3O5 (457.48): Calculated/Found: 68.26/68.43 (%C),
5.07/5.14 (%H), 9.19/9.37 (%N).
4.1.7. (E)-1-((1-(3-(3-Substituted acryloyl)-4-hydroxyphenyl)-1H-1,2,3-triazol-4-
yl)methyl)indoline-2,3-diones (8a-j)
To a stirred suspension of appropriate derivative of 5a-j (1.13 mmol) in a mixture of tertiary
butanol-water (3:1, 10 mL), 1-(prop-2-yn-1-yl)-1H-indole-2,3-dione 7 [22] (200 mg, 1.08
mmol) was added. Solutions of sodium ascorbate (43 mg, 0.22 mmol) in water (0.4 mL) and
CuSO4.5H2O (27 mg, 0.11 mmol) in water (0.3 mL) were added and the mixture was stirred at
70 ºC overnight. Additional solution of sodium ascorbate (43 mg, 0.22 mmol) in water (0.4
mL) and a solution of CuSO4.5H2O (27 mg, 0.11 mmol) in water (0.3 mL) were added and the
mixture was further stirred. The reaction progress was monitored using TLC. Water (20 mL)
was added and the resultant suspension was stirred for 30 min. the precipitate formed was
filtered, washed with water and dried. Crystallization was performed from dioxane-water.
4.1.7.1. (E)-1-((1-(3-Cinnamoyl-4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)indoline-
2,3-dione (8a): Yield 70.2%; yellow solid; mp 245-246 ºC; IR (ύ cm-1, KBr): 3442 (O-H),
3156 (Ar-H), 2990 (C-H aliphatic), 1745, 1644 (C=O), 1497, 1471 (C=C), 696 (Ar-H). 1H
NMR (400 MHz, δ ppm DMSO-d6): 5.09 (s, 2H, CH2), 7.13–7.26 (m, 3H, Ar-H), 7.35 (d, J =
8.8 Hz, 1H, Ar-H), 7.40–7.52 (m, 3H, Ar-H), 7.56–7.68 (m, 2H, alkene-H & Ar-H), 7.81–
8.01 (m, 3H, alkene-H & Ar-H), 8.04–8.15 (m, 1H, Ar-H), 8.40 (s, 1H, Ar-H), 8.87 (s, 1H,
triazole-H), 12.27 (s, 1H, OH). Anal. Calcd. For C26H18N4O4 (450.45): Calculated/Found:
69.33/69.15 (%C), 4.03/4.17 (%H), 12.44/12.80 (%N).
4.1.7.2. (E)-1-((1-(4-Hydroxy-3-(3-(p-tolyl)acryloyl)phenyl)-1H-1,2,3-triazol-4-
yl)methyl)indoline-2,3-dione (8b): Yield 71.7 %; yellowish brown solid; mp 253-255 ºC; IR
(ύ cm-1, KBr): 3445 (O-H), 3159 (Ar-H), 2921 (C-H aliphatic), 1744, 1643 (C=O), 1579,
1469 (C=C), 682 (Ar-H). 1H NMR (400 MHz, δ ppm DMSO-d6): 2.38 (s, 3H, -CH3), 5.09 (s,
2H, CH2), 7.13 –7.21 (m, 2H, Ar-H), 7.25 (d, J = 8.2 Hz, 1H, Ar-H), 7.31 (d, J = 7.8 Hz, 2H,
Ar-H), 7.58–7.70 (m, 2H, alkene-H & Ar-H), 7.78–7.85 (m, 3H, Ar-H), 7.92–8.00 (m, 2H,
alkene-H &Ar-H), 8.41 (s, 1H, Ar-H), 8.86 (s, 1H, triazole-H), 12.37 (s, 1H, OH). 13C NMR
(100 MHz): 21.63 (-CH3), 35.53 (CH2), 111.71, 118.13, 119.47, 122.01, 122.56, 122.74,
122.85, 123.92, 124.98, 128.39, 129.15, 129.78, 130.11, 132.15, 138.63, 138.68, 141.88,
22

145.88, 150.59, 158.32, 161.16 (C=O), 183.54 (C=O), 193.04 (C=O). Anal. Calcd. for
C27H20N4O4 (464.47): Calculated/Found: 69.82/69.95 (%C), 4.34/4.46 (%H), 12.06/12.31
(%N).
4.1.7.3. (E)-1-((1-(3-(3-(4-(Dimethylamino)phenyl)acryloyl)-4-hydroxyphenyl)-1H-1,2,3-
triazol-4-yl)methyl)indoline-2,3-dione (8c): Yield 69.9%; orange red solid; mp 277-278 ºC;
IR (ύ cm-1, KBr): 3448 (O-H), 3144 (Ar-H), 2914 (C-H aliphatic), 1739, 1633 (C=O), 1551,
1494 (C=C), 699 (Ar-H). 1H NMR (400 MHz, δ ppm DMSO-d6): 3.05 (s, 6H, N-(CH3)2), 5.10
(s, 2H s, 2H, CH2), 6.78 (d, J = 8.2 Hz, 2H, Ar-H), 7.13-7.19 (m, 2H, Ar-H), 7.26 (d, J = 7.7
Hz, 1H, Ar-H), 7.60 (d, J = 7.6 Hz, 1H, Ar-H), 7.64–7.90 (m, 6H, alkene-H & Ar-H), 7.96 (d,
J = 7.5 Hz, 1H, Ar-H), 8.48 (s, 1H, Ar-H), 8.88 (s, 1H, triazole-H), 13.09 (s, 1H, OH). Anal.
Calcd. for C28H23N5O4 (493.51): Calculated/Found: 68.14/68.39 (%C), 4.70/4.82 (%H),
14.19/14.37 (%N).
4.1.7.4.1-((1-(4-Hydroxy-3-((2E,4E)-5-phenylpenta-2,4-dienoyl)phenyl)-1H-1,2,3-triazol-
4-yl)methyl)indoline-2,3-dione (8d): Yield 53.4%; brick red solid; mp 232-234 ºC; IR (ύ cm-1,
KBr): 3443 (O-H), 3154 (Ar-H), 2852 (C-H aliphatic), 1745, 1638 (C=O), 1570, 1468 (C=C),
1
682 (Ar-H). H NMR (400 MHz, δ ppm DMSO-d6): 5.08 (s, 2H, CH2), 7.13–7.24 (m, 3H,
alkene-H &Ar-H), 7.29 (d, J = 5.4 Hz, 2H, Ar-H), 7.36–7.51 (m, 4H, alkene-H & Ar-H), 7.58-
7.69 (m, 5H, alkene-H & Ar-H), 7.97 (d, J = 8.7 Hz, 1H, Ar-H), 8.24 (s, 1H, Ar-H), 8.86 (s,
1H, triazole-H), 12.15 (s, 1H, OH). Anal. Calcd. For C28H20N4O4 (476.48): Calculated/Found:
70.58/70.76 (%C), 4.23/4.36 (%H), 11.76/11.98 (%N).
4.1.7.5. (E)-1-((1-(3-(3-(4-Bromophenyl)acryloyl)-4-hydroxyphenyl)-1H-1,2,3-triazol-4-
yl)methyl)indoline-2,3-dione (8e): Yield 69.3%; yellowish green solid; mp 257-259 ºC; IR
(ύ cm-1, KBr): 3380 (O-H); 3158 (Ar-H), 2900 (C-H aliphatic), 1743, 1645 (C=O), 1579,
1489 (C=C), 682 (Ar-H). 1H NMR (400 MHz, δ ppm DMSO-d6): 5.09 (s, 2H, CH2), 7.13-
7.26 (m, 3H, Ar-H) , 7.60 (d, J = 7.3 Hz, 1H, Ar-H) , 7.64–7.72 (m, 3H, alkene-H & Ar-H) ,
7.78-7.88 (m, 3H, alkene-H & Ar-H) , 7.96-8.04 (m, 2H, alkene-H & Ar-H) , 8.40 (s, 1H, Ar-
H) , 8.85 (s, 1H, triazole-H), 12.22 (s, 1H, OH). Anal. Calcd. for C26H17BrN4O4 (529.34):
Calculated/Found: 58.99/59.12 (%C), 3.24/3.47 (%H), 10.58/10.81 (%N).
4.1.7.6. (E)-1-((1-(3-(3-(4-Chlorophenyl)acryloyl)-4-hydroxyphenyl)-1H-1,2,3-triazol-4-
yl)methyl)indoline-2,3-dione (8f)
Yield 66.6%; brown solid; mp 228-230 ºC; IR (ύ cm-1, KBr): 3444 (O-H), 3142 (Ar-H), 2943
(C-H aliphatic), 1752, 1735, 1644 (C=O), 1579, 1503 (C=C), 682 (Ar-H). 1H NMR (400 MHz,
δ ppm DMSO-d6): 5.09 (s, 2H, CH2) , 7.14–7.24 (m, 6H, Ar-H), 7.59–7.68 (m, 3H, alkene-H
23

& Ar-H) , 7.75–7.90 (m, 2H, alkene-H & Ar-H) , 7.98 (d, J = 8.7 Hz, 1H, Ar-H) , 8.35 (s, 1H,
Ar-H) , 8.85 (s, 1H, triazole-H), 12.28 (s, 1H, OH). Anal. Calcd. for C26H17ClN4O4 (484.89):
Calculated/Found: 64.40/64.73 (%C), 3.53/3.69 (%H), 11.55/11.86 (%N).
4.1.7.7. (E)-1-((1-(4-Hydroxy-3-(3-(4-hydroxyphenyl)acryloyl)phenyl)-1H-1,2,3-triazol-
4-yl)methyl)indoline-2,3-dione (8g)
Yield 71.9%; reddish brown solid; mp 244-246 ºC; IR (ύ cm-1, KBr): 3458 (O-H), 3081 (Ar-
H), 2926 (C-H aliphatic), 1735, 1645 (C=O), 1576, 1470 (C=C), 700 (=C-H). 1H NMR (400
MHz, δ ppm DMSO- d6): 5.09 (s, 2H, CH2), 7.13–7.23 (m, 3H, Ar-H), 7.58-7.69 (m, 2H,
alkene-H & Ar-H), 7.87 (d, J = 15.7 Hz, 1H, alkene-H), 7.96–8.01 (dd, J = 8.5, 2.0 Hz, 1H,
Ar-H), 8.08–8.16 (m, 3H, alkene-H & Ar-H), 8.31 (d, J = 8.6 Hz, 2H, Ar-H), 8.37 (d, J = 1.9
Hz, 1H, Ar-H), 8.85 (s, 1H, triazole-H), 12.03 (s, 1H, OH). Anal. Calcd. for C26H18N4O5
(466.44): Calculated/Found: 66.95/66.80 (%C), 3.89/3.97 (%H), 12.01/12.24 (%N).
4.1.7.8. (E)-1-((1-(4-Hydroxy-3-(3-(4-methoxyphenyl)acryloyl)phenyl)-1H-1,2,3-triazol-
4-yl)methyl)indoline-2,3-dione (8h): Yield 64%; red solid; mp 274-276 ºC; IR (ύ cm-1,
KBr): 3400 (O-H), 3159 (Ar-H), 2921 (C-H aliphatic), 1744, 1643 (C=O), 1579, 1469 (C=C),
682 (=C-H). 1H NMR (400 MHz, δ ppm DMSO-d6): 3.85 (s, 3H, O-CH3), 5.09 (s, 2H, CH2),
7.05 (d, J = 8.5 Hz, 2H, Ar-H), 7.13–7.20 (m, 2H, Ar-H), 7.25 (d, J = 8.1 Hz, 1H, Ar-H),
7.58–7.70 (m, 2H, alkene-H & Ar-H), 7.85–7.90 (m, 4H, alkene-H &Ar-H), 7.95–7.99 (dd, J
= 8.7, 1.9 Hz, 1H, Ar-H), 8.44 (d, J = 1.9 Hz, 1H, Ar-H), 8.86 (s, 1H, triazole H), 12.53 (s,
1H, OH). Anal. Calcd. for C27H20N4O5 (480.47): Calculated/Found: 67.49/67.65 (%C),
4.20/4.37 (%H), 11.66/11.49 (%N).
4.1.7.9. (E)-1-((1-(3-(3-(3,4-Dimethoxyphenyl)acryloyl)-4-hydroxyphenyl)-1H-1,2,3-
triazol-4-yl)methyl)indoline-2,3-dione (8i): Yield 67.7 %; yellowish green solid; mp 211-
212 ºC; IR (ύ cm-1, KBr): 3447 (O-H), 3080 (Ar-H), 2936 (C-H aliphatic), 1740, 1640
(C=O), 1571, 1511 (C=C), 711 (=C-H). 1H NMR (400 MHz, δ ppm DMSO-d6): 3.84 (s, 6H, -
OCH3), 5.09 (s, 2H, , CH2) , 7.05 (d, J = 7.6 Hz, 1H, Ar-H) , 7.12–7.28 (m, 4H, Ar-H), 7.42–
7.53 (m, 2H, alkene-H & Ar-H) , 7.58–7.70 (m, 2H, alkene-H & Ar-H) , 7.78–7.90 (m, 2H,
alkene-H & Ar-H) , 7.98 (d, J = 7.9 Hz, 1H, Ar-H) , 8.41 (s, 1H, Ar-H) , 8.86 (s, 1H, triazole-
H) , 12.51 (s, 1H. OH). Anal. calcd. for C28H22N4O6 (510.50): Calculated/Found: 65.88/66.12
(%C), 4.34/4.48 (%H), 10.97/11.18 (%N).
4.1.7.10. (E)-1-((1-(4-Hydroxy-3-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)-1H-1,2,3-
triazol-4-yl)methyl)indoline-2,3-dione (8j): Yield 63.3 %; brown solid; mp 267-269 ºC; IR
(ύ cm-1, KBr): 3420 (O-H), 3071 (Ar-H), 2919 (C-H aliphatic), 1739, 1614 (C=O), 1493
(C=C), 762 (Ar-H). 1H NMR (400 MHz, δ ppm DMSO-d6): 3.55 (s, 3H, 4-(OCH3)), 3.73 (s,
24

6H, 3,5-(OCH3)), 5.06 (s, 2H, CH2), 7.13–7.24 (m, 2H, Ar-H), 7.33–7.48 (m, 3H, Ar-H),
7.55–7.69 (m, 4H, alkene-H & Ar-H), 7.85–7.95 (m, 2H, alkene-H & Ar-H), 8.79 (s, 1H,
triazole-H), 13.22 (s, 1H, OH). Anal. calcd. for C29H24N4O7 (540.52): Calculated/Found:
64.44/64.81 (%C), 4.48/4.59 (%H), 10.37/10.59 (%N).
4.2. Biology
4.2.1. In vitro cyclooxygenase inhibition assay
Cyclooxygenase inhibition studies were carried out at the department of Biochemistry,
Faculty of Medicine, Cairo University, Cairo, Egypt. Synthesized compounds were tested for
their ability to inhibit ovine COX-1 and human recombinant COX-2 using a COX inhibitor
screening assay kit (Catalog No. 560131, Cayman Chemical, Ann Arbor, MI, USA)
according to the manufacturer’s instructions and the deviation from the mean is < 10% of the
mean value.
4.2.2. In vitro lipoxygenase inhibition assay
Lipoxygenase inhibition studies were carried out at the department of Biochemistry, Faculty
of Medicine, Cairo University, Cairo, Egypt. Synthesized compounds tested for their ability
to inhibit LOX enzyme using LOX inhibitor screening assay kit (Catalog No. 760700,
Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions and
the deviation from the mean is < 10% of the mean value
4.2.3. In vivo Anti-inflammatory activity by carrageenan induced paw edema method
The in vivo anti-inflammatory activity of the selected compounds (6e, 6f, 8b, 8e and 8h) were
tested by carrageenan induced paw edema method [31] in rats in comparison to Celecoxib®
as a reference drug. The test is based on the pedal inflammation in rat paws induced by
subplantar injection of carrageenan suspension (0.2 mL of 1% solution in normal saline) into
the right hind paw of the rats. Male adult albino rats (120–150 g) were divided into groups,
each of five animals. The thickness of rat paw was measured by a Vernier calliper (SMIEC,
China) before and 30 min after carrageenan injection to detect the carrageenan induced
inflammation. Each test compound at a dose of 28 µmol/Kg (dissolved in 1% sodium
carboxymethyl cellulose solution in normal saline) was injected i.p. to different groups of
rats. Control group received a vehicle (1% sodium carboxymethyl cellulose solution in
normal saline), while the two reference groups received Celecoxib and Diclofenac sodium
i.p. at the same dose of the tested compounds. The difference between the thicknesses of the
two paws was taken as a measure of edema. The measurement was carried out at 1, 2, 3 and 4
h after injection of the test compounds, reference drugs. The percentages of edema inhibition
were calculated according to the following equation. Two-way ANOVA followed by
25

Dunnett’s multiple comparisons test was performed using GraphPad Prism version 7.00 for
Windows, GraphPad Software, La Jolla California USA.
where VR: Average right paw thickness, VL: Average left paw thickness.
4.2.4. Docking and Molecular Modeling
Molecular modeling studies was carried out at the department of medicinal chemistry, faculty
of pharmacy, Assiut University, Assiut, Egypt. All the molecular modeling calculations and
docking simulation studies were performed on a Processor Intel(R) Pentium (R) CPU
N3510@ 1.99GHz and 4 GB Memory with Microsoft Windows 8.1 pro (64 Bit) operating
system using Molecular Operating Environment (MOE 2014.0901, 2014; Chemical
Computing Group, Canada) as the computational software.
All MOE minimizations were performed until a RMSD gradient of 0.01 Kcal/mol/A with the
forcefield (MMFF94) to calculate the partial charges automatically. The Alpha Triangle
placement was chosen to derive the poses by random superposition of ligand atom triplets
through alpha sphere dummies in the receptor site. The London ∆G scoring function
estimates the free energy of binding of the ligand from a given pose. Refining the results was
completed using the MMFF94 forcefield, and rescoring of the refined results using the
London ∆G scoring function was applied. The output database dock file was created with
different poses for each ligand and arranged according to the final score function (S), which
is the score of the last stage that was not set to zero.
Acknowledgements
This work was supported by Faculty of pharmacy, Assiut University.
Declaration of Competing Interest
The authors have declared no conflict of interest
Supplementary material
Supplementary data to this article can be found online at ……………………………….
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