Inhibition of nitric oxide and prostaglandin E2 production by pyrrolylated-chalcones: Synthesis, biological activity, crystal structure analysis, and molecular docking studies

Article abstract of DOI:10.1016/j.bioorg.2019.103376

In search of potent anti-inflammatory agents, twenty-four chalcone derivatives including seven new compounds (13 – 17, 21 and 23) containing pyrrole moiety were designed, synthesized, and assessed for their nitric oxide (NO) and prostaglandin E₂ (PGE₂) suppression ability on IFN-γ/LPS-induced RAW 264.7 macrophage cells. Results showed that none of the synthesized compounds were PAINS-associated molecules, with 3-(2,5-dimethoxyphenyl)-1-(1H-pyrrol-2-yl)-prop-2-en-1-one (compound 16) exhibiting remarkable inhibition activity towards PGE₂ and NO production with IC₅₀ values of 0.5 ± 1.5 μM and 12.1 ± 1.5 μM, respectively. Physicochemical and ADMET studies showed that majority of the compounds obey to Lipinski's rule of five (RO5) having high blood brain barrier (BBB) penetration, human intestinal absorption (HIA), P- glycoprotein (PgP) inhibition and plasma binding protein (PPB) inhibition. The obtained atomic coordinates for the single-crystal XRD of 16 were then applied in a molecular docking simulation, and compound 16 was found to participate in a number of important binding interactions in the binding sites of ERK and mPGES-1. Based on these results, we have observed the potential of compound 16 as a new hit anti-inflammatory agent, and these findings could serve as a basis for further studies on its mechanism of action.

Full text of DOI:10.1016/j.bioorg.2019.103376

Journal Pre-proofs
Inhibition of nitric oxide and prostaglandin E2 production by pyrrolylated-
chalcones: Synthesis, biological activity, crystal structure analysis, and molec-
ular docking studies
Siti Munirah Mohd Faudzi, Maryam Aisyam Abdullah, Mohd Rashidi Abdull
Manap, Ahmad Zaidi Ismail, Kamal Rullah, Mohd Fadhlizil Fasihi Mohd
Aluwi, Aizi Nor Mazila Ramli, Faridah Abas, Nordin H. Lajis
PII:
S0045-2068(19)31521-4
DOI:
https://doi.org/10.1016/j.bioorg.2019.103376
YBIOO 103376
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
13 September 2019
7 October 2019
19 October 2019
Please cite this article as: S. Munirah Mohd Faudzi, M. Aisyam Abdullah, M. Rashidi Abdull Manap, A. Zaidi
Ismail, K. Rullah, M. Fadhlizil Fasihi Mohd Aluwi, A. Nor Mazila Ramli, F. Abas, N.H. Lajis, Inhibition of nitric
oxide and prostaglandin E2 production by pyrrolylated-chalcones: Synthesis, biological activity, crystal structure
analysis, and molecular docking studies, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.103376
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Inhibition of nitric oxide and prostaglandin E2 production by
pyrrolylated-chalcones: Synthesis, biological activity,
crystal structure analysis, and molecular docking studies
Siti Munirah Mohd Faudzia,b,*, Maryam Aisyam Abdullaha, Mohd Rashidi Abdull Manapa,
Ahmad Zaidi Ismaila, Kamal Rullahc, Mohd Fadhlizil Fasihi Mohd Aluwid, Aizi Nor Mazila
Ramlid, Faridah Abasb,e, Nordin H. Lajisb
a Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia
b Laboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia
c
Department of Pharmaceutical Chemistry, Kuliyyah of Pharmacy, International Islamic University Malaysia, Bandar Indera Mahkota, 25200,
Kuantan, Pahang, Malaysia
d Faculty of Industrial Sciences & Technology, Universiti Malaysia Pahang, Lebuhraya Tun Razak, Gambang, Kuantan,26300, Pahang, Malaysia
e Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia
*Corresponding author’s email: sitimunirah@upm.edu.my
Postal address: Department of Chemistry, Faculty of Science, Universiti Putra Malaysia,
43400, Serdang, Selangor, Malaysia.
ABSTRACT
In search of potent anti-inflammatory agents, twenty-four chalcone derivatives including seven new
compounds (13 – 17, 21 and 23) containing pyrrole moiety were designed, synthesized, and assessed
for their nitric oxide (NO) and prostaglandin E2 (PGE2) suppression ability on IFN-γ/LPS-induced RAW
264.7 macrophage cells. Results showed that none of the synthesized compounds were PAINS-
associated molecules, with 3-(2,5-dimethoxyphenyl)-1-(1H-pyrrol-2-yl)-prop-2-en-1-one (compound 16)
exhibiting remarkable inhibition activity towards PGE2 and NO production with IC50 values of 0.5 ± 1.5
µM and 12.1 ± 1.5 µM, respectively. Physicochemical and ADMET studies showed that majority of the
compounds obey to Lipinski's rule of five (RO5) having high blood brain barrier (BBB) penetration,
human intestinal absorption (HIA), P- glycoprotein (PgP) inhibition and plasma binding protein (PPB)
inhibition. The obtained atomic coordinates for the single-crystal XRD of 16 were then applied in a
molecular docking simulation, and compound 16 was found to participate in a number of important
binding interactions in the binding sites of ERK and mPGES-1. Based on these results, we have
observed the potential of compound 16 as a new hit anti-inflammatory agent, and these findings could
serve as a basis for further studies on its mechanism of action.
1. Introduction
Inflammation is a complex host’s defensive reaction which involves the activation and release of
inflammatory mediators, fluids, and leukocytes from the blood into extravascular tissues in response to
infections with the purpose of healing wounds and promoting recovery [1]. Inflammation can be
classified into two types, namely acute and chronic inflammations. Acute inflammation is a short-term
process involving the removal of pathogens by constituting a form of innate immunity, which may
progress to chronic inflammation when they persist for a long time. This results in various deleterious
effects on the host cells and tissues, and potentially leading to the development of cancer and
cardiovascular diseases [2]. Upon inflammation stimulation, macrophages produce a variety of
inflammatory mediators including nitric oxide (NO) and prostaglandin E2 (PGE2) [3]. NO is an important
free radical molecule that is involved in the regulation of physiological and pathophysiological
mechanisms in nervous, cardiovascular, and immunological systems [4,5]. However, the
overproduction of NO might contribute to the immune-pathology of macrophage-dependent
inflammation and degenerative diseases, including cancer [6,7]. Meanwhile, PGE2 is a product of the
COX pathway, and is well established as a lipid mediator that is responsible for inflammatory-related
disorders [8]. Therefore, inhibitory effects towards NO and PGE2 production is a promising therapeutic
target in the development of potential anti-inflammatory agents.
A chalcone is a small bioactive molecule that belongs to the flavonoid family and possesses a wide
range of pharmacological properties including anti-inflammatory, anti-microbial, and anti-cancer [9,10].

Meanwhile, pyrrole-containing small molecules have been widely used clinically as analgesic and anti-
inflammatory agents, particularly as a cyclooxygenase- 2 (COX-2) inhibitor with high potency and better
gastrointestinal safety [11,12]. Besides that, this compound of interest also has been reported as anti-
cancer, antioxidant and antimicrobial agents [13, 14, 15, 16]. Based on this information, we incorporated
the pyrrole scaffold into a chalcone structure by replacing one of the aromatic rings and evaluated its
potential as anti-inflammatory molecules.
In the present study, we screened the anti-inflammatory activity of the 24 molecules of pyrrolylated-
chalcones specifically on NO and PGE2 using in vitro IFN-γ/LPS-induced RAW 264.7 macrophage
cellular assay. We then performed the in-silico prediction studies including a pan-assay interference
compounds (PAINS) filtering and physicochemical properties determination using the FAF-Drugs4, as
well as ADMET analysis via PreADMET server. The crystals of the most bioactive molecule were grown
and analysed to allow comparison and prediction of the optimal structure of the compound, prior to the
execution of molecular docking to understand the molecular mechanism of action in the most potent
compound that exhibits prominent PGE2 inhibition.
2. Results and Discussion
2.1. Chemistry
Twenty-four pyrrolylated-chalcone analogues were prepared via Claisen-Schmidt condensation by
reacting 2-acetylpyrrole with their appropriate substituted benzaldehydes in ethanolic sodium hydroxide
for 24 hours in room temperature (Scheme 1) [17]. A detailed analysis of the aliphatic region in the 1H-
and 13C-NMR spectra of compounds 1-24 revealed the presence of methine groups, α and β (δH 7.18 -
7.65 ppm and 7.37 - 8.17 ppm; δC 116.3 - 130.1 ppm and 115.4 - 160.7 ppm) with J value of 15.0 Hz,
in which the signals indicate the presence of trans α,β-unsaturated-ketone moiety. The structure of the
synthesized compounds was then subjected to full characterisation by a complete analysis of 1D-NMR
spectroscopy and mass spectrometry (MS) (Spectroscopic data of new analogues are compiled in the
Supporting Information). The presence of electron-donating groups (-OCH3, -CH3, and –OH),
particularly the methoxy groups on the benzene ring gave a higher yield in the range of 40.2 to 97.6%,
while relatively lower yields (<63.0%) were observed with the presence of an electron-withdrawing
group as exhibited by the halogenated compounds (10-12, and 15).
O
O
O
a
H
CH3
+
R
R
N
N
H
H
Scheme 1. Synthesis of the pyrrolylated-chalcones. Reagents and conditions: (a) NaOH, ethanol, RT (overnight)

Table 1. NO and PGE2 suppression activities and cytotoxicity of compounds 1-24 in LPS/IFN-γ-stimulated RAW264.7 cells.
Ar
N
H
O
Ring A
Compounds
Ar (ring A)
NO inhibition (%)
± S.E.M at 50 µM
NO inhibition
IC50 (µM) ±
S.E.M at 50 µM
PGE2 inhibition
(%) ± S.E.M at
50 µM
PGE2
Cell viability
(%) ± S.E.M
at 50 µM
inhibition IC50
(µM) ± S.E.M
at 50 µM
Curcumin
99.3 ± 0.2
14.7 ± 0.2
>100
>100
80
NS-398
1
97.0 ± 1.3
82.0 ± 0.3
52.8 ± 1.5
66.6 ± 0.8
86.7 ± 0.6
74.0 ± 2.7
69.1 ± 2.2
61.6 ± 0.5
80.8 ± 1.4
79.1 ± 2.0
77.4 ± 1.0
63.0 ± 0.5
83.8 ± 0.7
17.0 ± 1.3
74.7 ± 1.8
86.5 ± 0.6
88.7 ± 2.6
89.6 ± 0.7
85.5 ± 0.6
68.3 ± 1.3
71.9 ± 1.8
62.8 ± 2.6
73.5 ± 0.6
83.4 ± 3.7
41.4 ± 0.9
0.1 ± 0.2
NDb
Phenyl
42.4 ± 0.8
87.3 ± 5.1
32.8 ± 4.8
72.4 ± 1.6
62.4 ± 2.6
36.8 ± 3.7
76.4 ± 2.2
59.0 ± 0.5
9.2 ± 5.9
NDa
6.0 ± 1.7
NDa
2
3-methylphenyl
NDb
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3
4-methylphenyl
NDb
4
2-methoxyphenyl
3-methoxyphenyl
4-methoxyphenyl
3-hydroxyphenyl
4-hydroxyphenyl
2-fluorophenyl
13.1 ± 0.6
46.4 ± 2.0
NDa
4.5 ± 2.2
NDb
5
6
NDb
7
13.6 ± 1.3
42.7 ± 1.4
NDa
NDb
8
3.3 ± 0.7
NDb
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
3-fluorophenyl
19.9 ± 4.7
28.8 ± 3.4
51.6 ± 2.4
64.5 ± 4.6
41.6 ± 2.1
71.2 ± 1.2
98.9 ± 0.5
64.8 ± 4.8
96.9 ± 1.1
8.6 ± 2.7
NDa
NDb
4-fluorophenyl
NDa
NDb
2-bromophenyl
NDa
NDb
3,4-dimethoxyphenyl
3-hydroxy,4-methoxyphenyl
3-chloro,4-hydroxyphenyl
2,5-dimethoxyphenyl
2,3,4-trimethoxyphenyl
3,4,5-trimethoxyphenyl
1H-pyrrol-2-yl
35.1 ± 2.1
NDa
NDb
NDb
34.0 ± 0.4
12.1 ± 1.5
39.1 ± 2.5
23.2 ± 0.7
NDa
4.1 ± 1.1
0.5 ± 1.5
12.1 ± 2.5
6.9 ± 1.6
NDb
Thiophenyl
21.5 ± 1.8
89.2 ± 2.9
8.9 ± 3.6
NDa
NDb
3-methyl-thiophenyl
Furan-2-yl
43.2 ± 2.2
NDa
NDb
NDb
5-methylfuran-2-yl
4-(benzyloxy)-phenyl
12.8 ± 3.1
5.4 ± 4.4
NDa
NDb
NDa
NDb
NDa = The IC50 of the selected molecules were not determined as the NO inhibition was less than 55%
NDb = The IC50 of the selected molecules were not determined as the PGE2 inhibition was less than 84%, except for compound 8 (with 51.6% of
NO inhibition)
2.2. NO and PGE2 inhibition in LPS/IFN-γ-induced macrophages
The synthesized pyrrolylated-chalcone series including seven new compounds (13 – 17, 21 and 23) were first screened for
their inhibitory activity towards nitric oxide (NO) in IFN-γ/LPS-induced RAW 264.7 macrophage cells [17,18]. Based on the
preliminary evaluation, twelve compounds were shown to significantly inhibit the LPS/IFN-γ-stimulated NO production with
the percentages of inhibition greater than 50.0%, signifying their potential to serve as anti-inflammatory agents (Figure 1).
The twelve compounds (2, 4, 5, 7, 8, 12, 13, 15, 16, 17, 18, and 21) were then subjected to IC50 determination and compared
to the positive control, curcumin [19]. Cytotoxicity of the tested compounds were determined using MTT assay, where the
results indicated that no compounds were cytotoxic to the RAW264.7 macrophage cells at the concentration of 50 µM. The
inhibitory effects of the pyrrolylated-chalcones on the NO production is shown in Table 1.
The result of the NO inhibitory screening showed that compounds 2, 4, 7, and 16 displayed significant NO inhibitory activity
with IC50 values less than 15.0 µM, signifying an enhanced potency in comparison to curcumin. Compound 2 exhibited the
highest NO inhibition effect with an IC50 value of 6.0 ± 1.7 µM, which is 2-fold stronger than curcumin (14.7 ± 0.2 µM) in a
dose-dependent manner (Figure 2A and Figure 2B). Following that, the MTT assay result has proven that the potent activity
of compound 2 was not due to its cytotoxic effect (Figure 2C). Based on these results, it appears that the meta-methylphenyl

group is an important factor in NO inhibitory activity. However, the actual impact of methyl group on this specific bioactivity is
still in question.
Figure 1. NO inhibitory effects of compounds 1–24 at a testing concentration of 50 µM
(A)
(B)
150
100
50
*
0
.5
25
50
rol
Cont 0.7
.25
6
12
8125
.5625 3.125
1
Concentrations of compound 2 (M)
(C)
Figure 2. (A) Dose-response curve of compound 2 on NO inhibition in LPS/IFN-γ-stimulated RAW264.7 cells, (B) NO secretion of LPS/IFN-
γ-stimulated RAW264.7 macrophage cells upon treatment with analogue 2 at different concentrations, (C) Cell viability of LPS/IFN-γ-
stimulated RAW264.7 macrophage cells upon treatment with analogue 2 at different concentrations via MTT assay.
Further comparisons on the variation of substituents on the phenyl ring revealed that the substitution of the electron donating
group (hydroxyl and methyl, respectively) at the meta-position is preferable as indicated by a relatively stronger NO
suppression of 2 over 3. A similar observation was also seen in the comparisons of compounds 7 and 8, in which compound
7, a meta-hydroxylated chalcone, displayed stronger activities by three folds compared to the other analogue. This finding
agrees with a previous work, which reported that meta-hydroxyl-containing chalcones enhances anti-inflammatory and anti-
cancer properties [20].
While meta-substituted (methyl and hydroxyl)-bearing compounds showed significant activity, the analogues containing either
meta-methoxyl or para-oxygenated moiety (compounds 6, 8, 14, 15, 17, and 18) exhibited mild to poor NO inhibition activity.
This result was in close agreement with Kim et al., which reported that para-substitution on chalcone analogues did not
improve the suppression activity on NO production [21].
The introduction of the electron-withdrawing group (halogen) regardless of their position on the phenyl ring revealed poor NO
inhibitory properties, as displayed by compounds 9-12 in comparison to its unsubstituted compound 1. In contrast, the
analogue containing para-hydroxyl-bearing chlorinated ring, 15 showed mild NO inhibition. This result suggested that the
presence of a hydroxyl group in the phenyl ring may have obscured the negative impacts of the halogen moiety.
We also investigated the replacement of phenyl functionality with a heterocyclic system towards anti-inflammatory activity,
as showed by compounds 19-23. It is worth noting that only the 3-methylthiophenyl analogue (compound 21) displayed mild
NO inhibition with an IC50 value of 43.2 ± 2.2 µM, while other analogues are relatively inactive. This observation was in
contrast with the findings of Qiu et al., which stated that heteroaromatic-bearing compounds reduces anti-inflammation and
anti-cancer efficacy [22]. However, the mild inhibition can be rationalised by the importance of the 3-methyl group in the
thiophenyl system, which mitigates the negative contribution of the heterocyclic group, resulting in the alteration of its
interaction with the mediators.
All pyrrolylated-chalcones were also assessed for their PGE2 inhibitory activity on LPS/IFN-γ-induced RAW264.7 cells at the
concentration of 50.0 µM [18]. The results obtained were analysed and presented in Table 1. The production of PGE2 is
activated in the event of an infection or inflammation. The accumulation of excessive PGE2 in the body may elicit powerful
immunosuppressive properties that may contribute to chronic inflammation, potentially leading to various diseases such as
cancer, rheumatoid arthritis, atherosclerosis, and pain [19b, 23]. Six compounds (4, 8, 15, 16, 17, and 18) were found to
exhibit significant inhibition of PGE2 production, giving IC50 values ranging between 0.5 ± 1.5 µM to 12.1 ± 2.5 µM. Compound
16, composed of 2,5-dimethoxyphenyl, demonstrated the highest PGE2 suppression activity in a dose-dependent manner
(Figure 3) with IC50 of 0.5 ± 1.5 µM showed a comparable suppression activity to the standard drug, NS-398 (IC50 = 0.1 ± 0.2
µM). Most of the active compounds that inhibit PGE2 contains an ortho- and para-methoxyl group on the phenyl ring, except
for compounds 8 and 15 which possess a hydroxyl group at the para-position.

1000
800
600
400
200
0
****
)
****
****
****
****
25
50
.25
6
12.5
8125 5625
3.125
S-398
1.
N
0.7
Concentrations of compound 16 (M)
Figure 3. Effects of compound 16 on PGE2 in /IFN-γ-stimulated RAW264.7 macrophage cells upon treatment with different concentrations.
This observation is consistent with previous studies, which stated that the presence of ortho-methoxyl moiety in chalcones
enhances anti-inflammatory activity significantly [21]. Furthermore, compounds comprised of methoxyl groups at the C-2 and
C-5 positions of the phenyl ring on the chalcone scaffold were reported to potently attenuate TNF-α induced NF-kβ [24].
Following this data, compound 16 also exhibited stronger PGE2 suppression by nine-folds than that observed in 2-methoxyl
containing compound 4. Hence, the presence of 2,5-dimethoxyl moiety might contribute to a higher electron density, as well
as a higher hydrogen bond potential of the phenyl functionality, which might be involved in π-π interactions or hydrogen
bonding in the active site, an important factor in the PGE2 inhibitory property. Likewise, introducing a strong electron
withdrawal (F and Br) group or heteroatom system to the phenyl ring of pyrrolylated-chalcones also decreases the effects of
PGE2 inhibition, as well as the NO suppression activity.
2.3. Pan-assay interference compounds (PAINS) and aggregator identification
PAINS is defined as a common structural motif with an ability to interfere with biochemical assays through a number of
mechanisms, including protein binding interactions [25] and the formation of colloidal aggregates [26], which may result in
misleading assay readings. The increasing number of PAINS reported in research publications in recent years has caught
the attention of the scientific community, specifically medicinal chemists, as the occurrence of PAINS may cause the spread
of misinformation in the drug discovery field. Hence, noting or identifying any PAINS-containing molecules has become a
crucial step to validate that the function of a compound is as expected prior to removing it in the early stages of a drug
discovery project. In view of this, we input all 24 pyrrolylated-chalcones into publicly available filter servers, FAF-Drugs4 [27]
and Aggregator Advisor [28] for PAINS and aggregator identification, respectively.
Based on the PAINS identification analysis, none of the pyrrolylated-chalcones were PAINS-associated compounds (see
Supporting Information). Likewise, through the aggregator identification analysis, none of the compounds were similar to any
known aggregator. However, several molecules (compounds 2, 3, 12, and 24) with a fairly high calculated LogP (3.18 to
4.28) may be potential robust macromolecular aggregates, in which the respective LogP range was previously reported for
many other aggregators.
The physicochemical and ADMET properties of twenty-four pyrrolylated-chalcones were also analysed in-silico through FAF-
Drugs4 server. The tabulated results in Table 2 show that all compound did not violate the Lipinski’s rule of five (RO5). The
famous RO5 is a rule in pharmaceutical science to evaluate the ‘drug-like’ characteristics and their oral bioavailability in
humans based on several factors, including molecular weight less than 500 (MW ≤ 500), no more than 5 hydrogen bond
donor groups (HBD ≤ 5), no more than 10 hydrogen bond acceptor groups (HBA ≤ 10), and a calculated partition coefficient
between n-octanol and water less than 5 (log P ≤ 5, log P measure lipophilicity). In addition to assess for their drug-likeness,
all molecules were followed the rule set by Veber et al (2002), which stated that compound with a total surface area less than
140 Å2 (TPSA ≤ 140) and no more than 10 rotatable bonds (NRB ≤ 10) confer to play a major role in oral
bioavailability [29]. Furthermore, the FAF-drug4 results estimated all the compounds to possess good
bioavailability confirming to both VEBER (good oral bioavailability if NRB ≤ 10 and TPSA ≤ 140 Å2) and EGAN
(good oral bioavailability if -1 ≤ log P ≤ 5.8 and TPSA ≤ 130 Å2) rules [30]. All molecules are non-inducer of
phospholipidosis and have a very low Fsp3 values which may correlate to their low solubility (Table 3).

Table 2. Phytochemical properties of compounds calculated via FAF-Drugs4
Compounds
MW
Log P
2.81
3.18
3.18
3.20
2.78
2.78
2.46
2.46
3.12
2.91
2.91
3.50
2.76
2.43
3.09
2.76
2.73
2.73
1.64
2.53
2.89
2.06
2.31
4.28
HBD
1
1
1
1
1
1
2
2
1
1
1
1
1
2
2
1
1
1
2
1
1
1
1
1
HBA
2
2
2
3
3
3
3
3
2
2
2
2
4
4
3
4
5
5
3
2
2
3
3
3
TPSA (Å2)
32.86
32.86
32.86
42.09
42.09
42.09
53.09
53.09
32.86
32.86
32.86
32.86
51.32
62.32
53.09
51.32
60.55
60.55
48.65
61.10
61.10
46.00
46.00
42.09
NRB
3
3
3
4
4
4
3
3
3
3
3
3
5
4
3
5
6
6
3
3
3
3
3
6
RO5 violations
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
197.23
211.26
211.26
227.26
227.26
227.26
213.23
213.23
215.22
215.22
215.22
276.13
257.28
243.26
247.68
257.28
287.31
287.31
186.21
203.26
217.29
187.19
201.22
303.35
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Table 3. Druglikeness properties of pyrrolylated chalcones calculated by FAF-Drugs4
Compounds
Oral
bioavailability
(VEBER)
Good
Oral
bioavailability
(EGEN)
Good
Phospho-
lipidosis
Fsp3
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
0
0.07
0.07
0.07
0.07
0.07
0
0
0
0
0
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
0
0.13
0.07
0
0.13
0.19
0.19
0
0
0.08
0
0.08
0.05
The ADMET properties prediction are important in early stage of drug discovery and development to improve the efficiency
of therapeutic candidates. The ADMET analysis was performed on five important parameters, such as blood-brain barrier
(BBB) penetration, human intestinal absorption (HIA), CYP2C19 inhibition, P-glycoprotein (PgP) inhibition and plasma protein
binding (PPB) via PreADMET server and the results were compiled in Table 4.
Table 4. ADMET properties of pyrrolylated-chalcones calculated using PreADMET
Compounds
BBB
HIA
CYP_2C19_inhibition
Inhibitor
PgP_inhibition
Non-Inhibitor
Inhibitor
Inhibitor
Non-Inhibitor
Inhibitor
PPB
1
2
3
4
5
6.8838
8.1930
8.0885
5.1507
4.2176
91.9972
92.2454
92.2454
91.2391
91.2391
86.1394
86.2123
85.7490
83.0044
81.5552
Inhibitor
Inhibitor
Inhibitor
Inhibitor

6
7
8
9
2.4055
4.0003
3.7905
7.4443
7.4443
7.4352
8.8674
1.5961
0.7921
5.3137
2.7124
0.8464
0.9443
4.6323
2.6632
3.5918
3.1248
3.4293
8.2620
91.2391
87.6705
87.6705
92.0083
92.0083
92.0083
93.6777
91.1406
86.9387
90.0396
91.1407
91.2580
91.2580
85.9961
90.9639
91.3088
89.0042
89.4765
93.4625
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Non-Inhibitor
Non-Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Inhibitor
Non-Inhibitor
Non-Inhibitor
Inhibitor
Inhibitor
Inhibitor
Non-Inhibitor
Inhibitor
Inhibitor
Non-Inhibitor
Non-Inhibitor
Inhibitor
84.2860
79.5653
82.8919
83.4339
83.1704
84.9441
99.3681
82.3664
80.2829
94.8936
82.6915
82.5769
81.7537
67.1263
87.9581
90.4369
83.6832
84.3267
91.1920
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
BBB is commonly related to the drugs delivery process involving the central nervous system (CNS), in which drugs that target
CNS should possess higher BBB penetration while drugs that target the peripheral organs should have lower BBB penetration
to minimize the adverse side effects on CNS. BBB penetration characteristic was based on (a) high BBB penetration to CNS
> 2.0, (b) medium BBB penetration to CNS 0.1< BBB <2.0, and (c) weak BBB absorption to CNS < 0.1 [30]. Our results
demonstrated that all compounds predicted to possess good BBB absorption except for compounds 13, 14, 17 and 18 with
medium BBB penetration, signifying their potential as effective molecules in the treatment of CNS-related disorders.
HIA impart as one of the most important issues in drug discovery, as many negative effects can be intensified by lower-
solubility drugs including poor absorption and bioavailability after oral dosing [17]. The prediction of HIA of pyrrolylated-
chalcones was evaluated based on (a) High absorption 70% <HIA< 100%, (b) medium absorption 20% <HIA< 70%, and (c)
poor absorption 0% <HIA< 20% [30]. All compounds were displayed good human intestinal absorption, reflecting their
potency as good oral drugs.
CYP2C19, a Cytochrome P450 enzyme is responsible for the metabolism of drugs in the liver. It was found that all compounds
(1-24) were CYP2C19 inhibitors, indicating their intolerance towards this CYP enzyme. Meanwhile, the results indicated that
most of the chalcone series except for compounds 1, 4, 7, 8, 14, 15, 19, 22 and 23 were PgP inhibitor, where PgP is a type
of ATPase of membrane transport proteins responsible for efflux of drugs and is a major component of BBB [30].
Plasma proteins may adsorb a significant amount of drug molecules once they have entered the bloodstream and to be
distributed to their specific therapeutic targets. PPB can affect the pharmacokinetics of drug substances because the bound
molecules are temporarily shielded from metabolism. Hence, only unbound drugs exhibit noteworthy pharmacological effects
[17]. PPB analysis was performed based on (a) Strong bound compound PPB > 90%, and (b) weakly bound compound PPB
< 90% [30]. As presented, most of the compounds displayed a poor binding between the drug molecules and plasma protein,
while compounds 12, 15, 21 and 24 showed strong binding interaction between the drugs with protein plasma.
Based on the in-silico studies comprising PAINS and aggregator filters, as well as physicochemical and ADMET profile, we
can concluded that compound 16 was not classified as both PAINS and potential aggregator. Besides that, compound 16
was predicted with good physicochemical and ADMET properties, except for their lower solubility (Fsp3 = 019) and slightly
weaker protein plasma binding. However, these properties can be improved via structural modification through SAR
approach.
2.4. Single crystal XRD compound 16
Crystals of the most active compound 16, (3-(2,5-dimethoxyphenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one), which is suitable for
X-ray diffraction analysis, were grown from slow evaporation of ethanol forming ‘plate-like’ crystals. Compound 16 was
crystallized in a triclinic system with P-1 space group, where one asymmetric unit contains three molecules of 16. The final
refinement led to a final R value of 7.71% with the structure of compound 16 shown in Figure 4. The crystal structure of 16
confirms the expected molecular structure, as suggested in the earlier NMR, MS, and FTIR analyses.
Bond lengths and selected angles of compound 16 are respectively shown in Table 5 and Table 6 below. The bond lengths
and angles of compound 16 showed almost similar values with some related molecules [31]. The phenyl ring C-C bond
lengths were in the range of 1.379 (5) – 1.409 (4) Å with an average ring bond angle of 120°, which matches the typical value
for an aromatic phenyl ring. Meanwhile, the pyrrole ring was observed to possess a bond angle range of 106.8 – 110.0°,
which also matches the values commonly found in pyrrole rings. However, it is interesting to note that compound 16 varies
from most substituted chalcones, in that this compound is non-planar with a dihedral angle of 21.47° between the
dimethoxyphenyl and pyrrole ring system. This angle was measure by creating two “planes”, where the first plane is in-line
with the pyrrole ring and another plane is in-line with the dimethoxyphenyl moiety. Angle where these two planes meet and
intersect was then measured to give the dihedral angle mention above. Substitutions on the phenyl ring or the pyrrole of the
compound are commonly found to be the reason for this observation [32,33]. Intramolecular hydrogen bonds were present
in the structure, involving the adjacent hydrogen and carbonyl groups. Atom O1 of the carbonyl was observed to be involved
in the intramolecular hydrogen bond with the hydrogen of C3”(C3”-H3”). This explains the C1”=O1 bond length that was

observed to be longer than the usual carbonyl bonds. The presence of the intermolecular N1-H1····O1 hydrogen bond causes
the arrangement of the molecule into centrosymmetric dimers, in which later will grow into layers that are arranged parallel
to each other (see Supporting Information).
Figure 4. The molecular structure of compound 16, with displacement ellipsoids drawn at 30% probability level.
Table 5. Bond lengths of non-hydrogen atoms (e.s.d.s are given in parentheses) in compound 16
Atom
O2
O2
O1
N1
N1
C3
C3
C3
Atom
C2
C7
C1"
C5'
C2'
C3"
C2
Length/Å
1.373(4)
1.437(3)
1.255(3)
1.354(4)
1.371(4)
1.456(5)
1.409(4)
1.403(5)
1.471(5)
1.440(5)
1.330(5)
Atom
C2'
C1
C46
C3'
C8
C2
C5
C5
C5
Atom
C3'
C6
C55
C4'
O3
C1
C4
C6
O3
Length/Å
1.386(4)
1.384(5)
1.393(5)
1.396(5)
1.422(4)
1.384(5)
1.379(5)
1.392(4)
1.376(4)
1.382(5)
C4
C1"
C1"
C3"
C2"
C2'
C2"
C5'
C4'
Table 6. Selected bond angle of compound 16 (e.s.d.s are given in parentheses).
Atom
C4
C1
C4
C2
Atom
C3
C2
C5
C1
Atom
C2
C3
C6
C6
Angle/˚
117.7(3)
120.0(4)
119.4(4)
121.2(3)
119.6(4)
122.0(3)
Atom
C5'
N1
N1
C2'
C5'
Atom
Atom
Angle/˚
110.0(3)
107.9(4)
106.8(3)
107.9(3)
107.2(3)
N1
C2'
C4'
C3'
C4'
C3'
C5'
C2'
C3'
C4'
C1
C5
C6
C4
C5
C3
2.5. In silico molecular docking studies
The nuclear factor kappa-β (NF-κβ) and mitogen-activated protein kinase (MAPK) signalling pathways are recognised as
crucial transcription factors known to regulate the inducible nitric oxide synthase (iNOS) and COX-2 expression, respectively.
Interruptions of these signalling pathways may down-regulate the iNOS and COX-2 expressions, thus reducing NO and PGE2
production. Since the pyrrolylated-chalcones were found to be more pronounced during the inhibition of PGE2 compared to
NO, we predicted that it might preferably target the MAPK signalling pathway. Therefore, the obtained crystal XRD of the
most active compound 16 was employed in the docking simulation using the protein crystal structures of ERK (PDB ID:
3C9W), JNK (PDB ID: 3O2M), p38 (PDB ID: 2GTN), COX-2 (PDB ID: 6COX), and mPGES-1.
Since the complete crystallized three-dimensional structure of murine mPGES-1 is currently not available, a homology model
was built from the human protein crystal structure mPGES-1 (4YL3) [34]. The conserved region in human mPGES-1 was
selected as a putative binding site for compound 16 based on the fact that the MK-886 inhibitor selectively inhibits human
mPGES-1 activity. The BLASTP analysis revealed that M. musculus mPGES-1 exhibits high similarity with all the solved 3D
structures in the PDB database, with the highest match showing 79% identity and 100% coverage to the crystallographic
structure of human microsomal prostaglandin E synthase-1 (PGES-1) form. To elucidate the possible binding interactions of
compound 16, the protein model of targeted M. musculus proteins were constructed using SWISS-MODEL. Among the
various potential templates for the molecular modelling purposes of M. musculus mPGES-1, a crystal structure of mPGES-1
inhibitor complexes (PDB ID: 4YL3) which had the highest sequence identity and coverage, and satisfied QMEAN Z-scores
and GMQE score, was selected as a template. The resulting model was subjected to energy minimisation to avoid poor
molecular contacts, and to obtain a relaxed conformation. The quality of the refined M. musculus mPGES-1 was evaluated
using PROCHECK and ERRAT programs. The plot of the M. musculus mPGES-1 model indicated that 96.1% of the residues
were located in the most favoured region, and the remaining residues resided in the additional allowed regions. No residues
were found in the generously and disallowed regions, which indicates the good stereochemical quality of the M. musculus
mPGES-1 model. The ERRAT score for the M. musculus mPGES-1 model was 100%, suggesting that the backbone
conformation and non-bonded interactions of the model were all reasonable within a normal range. The Ramachandran and

ERRAT analyses of the M. musculus mPGES-1 model gave a satisfying and acceptable score for further analysis, which can
be viewed in Supporting Information. The M. musculus mPGES-1 homology model built was utilised as a molecular target in
the docking experiment.
The binding energies were used as a measurement to compare the binding affinity of compound 16 with the co-crystallized
ligands and inhibitor, respectively (Table 7). Among the target enzymes, compound 16 showed favourable binding energy of
-5.73 kcal/mol (ERK) and -4.97 kcal/mol (mPGES-1) which closely resembled the binding energy of their co-crystallized
ligands and inhibitor, -6.09 kcal/mol and -5.37 kcal/mol, respectively. Deep in the ERK binding site, the N atom of pyrrolyl
moiety and O atom of the keto group interacted by forming strong hydrogen bonds with Met106 (2.22 Å and 2.27 Å,
respectively). Furthermore, hydrophobic interactions could be observed between 2,5-methoxyphenyl and pyrrolyl rings with
Val37, Ala50, Lys52, and Ile29, correspondingly (Figure 5A). This result was in agreement with the crystal structure of the
ERK2-hypothemycin covalent complex [35].On one hand, compound 16 was found to participate in several important binding
interactions in the binding site of mPGES-1. Strong hydrogen bonds could be observed between the oxygen atom of methoxy
moiety of 16 with Arg127 (equivalent position of Arg126 in human mPGES-1) (1.67 Å) in the mPGES-1 binding site. On the
other hand, the amine of pyrrolyl moiety was found to interact with Glu78 (equivalent position of Glu77 in human mPGES-1)
by forming a hydrogen bond (2.17 Å). Weak π-σ (4.90 Å) and hydrophobic interactions (4.53 Å) between pyrrolyl moiety with
Arg73 (equivalent position of Arg72 in human mPGES-1) could also be observed (Figure 5B). Such interactions are almost
essential for the mPGES-1 inhibitory activity based on the binding interactions displayed by mPGES-1 inhibitor MK886, in
the mPGES-1 binding site. Overall, compound 16 displayed lower binding affinity in other enzymes as compared to their co-
crystallized ligands. Thus, it is suggested that compound 16 could preferably target ERK or mPGES-1, subsequently reducing
PGE2 production.
Table 7. Comparison of binding energy between compound 16 with target enzyme inhibitors
Binding energy (kcal/mol)
Enzyme
Compound 16
Co-crystallized
ligand*
ERK
JNK
p38
COX-2
mPGES-1
-5.73
-5.90
-7.08
-8.10
-4.97
-6.09
-8.29
-9.12
-10.74
-5.37†
(A)
(B)
Figure 5. Predicted binding pose retrieved from the docking simulation of compound 16 in (A) ERK and (B) mPGES-1 putative binding
sites. The atom colouring for the compound is as follows; carbon in grey, oxygen in red, nitrogen in blue, and hydrogen in white. The
green line indicates hydrogen-bonding interaction, orange line indicates π-α interaction, while pink line indicates hydrophobic
interaction. Distance is indicated in angstroms, Å.

3. Conclusions
The present investigation describes the synthesis of twenty-four molecule hybrids combining a pyrrole group into a chalcone
scaffold as a new route to anti-inflammatory agents including seven new compounds (13 – 17, 21 and 23), where none of
molecules are PAINS-associated compounds. Compound 16 exhibited the most significant inhibition on both NO and PGE2
production in LPS/IFN- γ-stimulated RAW264.7 cells with IC50 values of 12.1 ± 1.5 µM and 0.5 ± 1.5 µM respectively and
were non-cytotoxic at the tested concentrations. Since the compound was determined to be more potent in inhibiting PGE2
production compared to NO production, we predicted that it might preferentially target the MAPK signalling pathway.
Therefore, the obtained crystal XRD coordinates of the most active compound 16 was employed in docking simulations and
the results showed that compound 16 participated in a number of important binding interactions in the binding site of mPGES-
1 but possesses lower predicted binding affinities in other enzymes compared to their co-crystallized ligands. Thus, it is
suggested that 16 could preferentially target ERK or mPGES-1, resulting in the subsequent reduction of PGE2 production.
Accordingly, compound 16 could be considered as a structural lead that merits further SAR investigation for the development
of optimised anti-inflammatory agents that possess notable PGE2 inhibition. Further work will address the mechanistic study
of the regulatory protein expression associated with the MAPK signalling pathway for a deeper understanding of its molecular
mechanism.
4. Experimental
4.1. Materials and equipment
All chemicals and reagents were purchased from Merck and Sigma Aldrich. The solvents used in the spectroscopic
measurements were of spectroscopic grade. The reaction mixtures were extracted with suitable organic solvents, dried over
anhydrous magnesium sulphate, and concentrated by a rotatory evaporator. The progress of the reaction was monitored via
thin layer chromatography using aluminium TLC sheets of silica gel 60 f254 (layer thickness 0.2 mm) from Merck.
Subsequently, the purification was performed via gravitational column chromatography using Merck silica gel 60 PF254 No.
7734 (mesh 70-230) and Merck silica gel 60 PF254 No. 9835 (mesh 230-400). Mass spectra were recorded using a GCMS-
1
QP5050A (Shimadzu, Kyoto, Japan) Mass Spectrometer. H and 13C NMR spectra were measured in CDCl3 (unless
otherwise indicated) on a Varian 500 MHz NMR Spectrometer (Varian Inc., California, USA). Chemical shifts were reported
in δ (ppm) values relative to the standard tetramethylsilane (TMS), while the melting points were determined using a Fisher-
Johns melting point apparatus.
4.2. General procedure for the synthesis of pyrrolylated-chalcones (compounds 1-24)
Equimolar quantities of the 2-acetyl pyrrole (2 mmol) and substituted benzaldehydes (2 mmol) were dissolved in 5 ml of
absolute ethanol. The resulting mixture was stirred for 5 minutes before 3 ml of 40% NaOH aqueous solution was added
dropwise. After the addition was complete, the reaction mixture was stirred overnight at room temperature. The adduct
solution was poured into crushed ice and extracted with ethyl acetate (3 x 10 ml). The organic layer was washed with distilled
water (3 x 10 ml), dried over anhydrous MgSO4, and concentrated under reduced pressure. Finally, the crudes were purified
by column chromatography on silica gel using hexane:ethyl acetate as a solvent system [17].
5
4
3'
4'
5'
3
3'
4'
5'
6
4
3
R
R
5
X
N 2'
H
2
N 2'
H
1
2
O
O
Compound 19-23
Compound 1-18, 24
4.3. Compound characterisation
4.3.1. (E)-3-(phenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (1)
Yellow solid; 39.80 %; mp: 130-131 °C; 1H NMR (500 MHz, CDCl3): δ = 6.36 (t, 1H), 7.09 (d, 1H), 7.13 (d, 1H), 7.36 (d, 1H,
3JH-H =15.5 Hz), 7.40-7.44 (m, 3H), 7.64 (dd, 2H), 7.84 (d, 1H, 3JH-H =15.5 Hz), 9.81 ppm (br. s, 1H); 13NMR (126 MHz, CDCl3):
δ = 178.9, 142.3, 135.0, 133.1, 130.2, 128.9, 128.3, 125.4, 121.9, 116.3, 111.0 ppm. HRMS (ESI): m/z calcd for C13H11NO-
H+: 197.0841 [M-H]+; found: 197.0839.
4.3.2. (E)-3-(3-methylphenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (2)
Yellow solid; 80.36 %; mp: 111-112 °C; 1H NMR (500 MHz, acetone-d6): δ = 2.37 (s, 3H), 6.30 (t, 1H), 7.21 (d, 1H), 7.23 (d,
1H, 3JH-H =7.5 Hz), 7.29 (d, 1H), 7.32 (t, 1H), 7.56 (d, 1H, 3JH-H =8.0 Hz), 7.62 (d, 1H), 7.67 (d, 1H, 3JH-H =15.0 Hz), 7.72 (s,
1H), 11.01 ppm (br. s, 1H). 13NMR (126 MHz, acetone-d6): δ = 177.9, 141.1, 138.4, 135.3, 130.7, 128.8, 128.7, 125.6, 125.5,
122.5, 117.6, 110.1, 20.4 ppm (CH3). HRMS (ESI): m/z calcd for C14H13NO-H+: 210.0997[M-H] +; found: 210.0999.
4.3.3. (E)-3-(4-methylphenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (3)

Yellow solid; 46.21 %; mp: 142-143°C; 1H NMR (500 MHz, acetone-d6): δ = 2.36 (s, 3H), 6.30 (t, 1H), 7.21 (d, 1H), 7.26 (d,
2H, 3JH-H =8.5 Hz), 7.28 (d, 1H), 7.56 (d, 1H, 3JH-H =15.5 Hz), 7.67 (d, 2H, J=8.0 Hz), 7.70 (d, 1H, 3JH-H = 15.0 Hz), 7.72 (s,
1H), 11.01 ppm (br. s, 1H). 13NMR (126 MHz, CDCl3); δ = 178.9, 142.5, 134.9, 131.1, 128.9, 128.8, 125.6, 125.3, 121.7,
111.0, 21.4 ppm. HRMS (ESI): m/z calcd for C14H13NO-H+: 210.0997 [M-H]+; found: 210.0998.
4.3.4. (E)-3-(2-methoxyphenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (4)
Yellow solid; 60.41 %; mp: 112-113 °C; 1H NMR (500 MHz, acetone-d6): δ = 3.96 (s, 3H), 6.31 (t, 1H), 7.01 (d, 1H), 7.10 (d,
1H, 3JH-H =8.0 Hz), 7.20 (d, 1H), 7.24 (d, 1H), 7.41 (t, 1H), 7.65 (d, 1H, 3JH-H =15.6 Hz), 7.83 (d, 1H, 3JH-H =7.0 Hz), 8.10 (d,
3
1H, JH-H =15.0 Hz), 11.01 ppm (br. s, 1H). 13NMR (126 MHz, acetone-d6): δ = 178.3, 158.6, 136.0, 133.5, 131.5, 128.2,
125.4, 123.8, 122.7, 120.6, 116.1, 111.4, 110.1, 55.1 ppm. HRMS (ESI): m/z calcd for C14H13NO2-H+: 226.0946 [M-H]+; found:
226.0948.
4.3.5. (E)-3-(3-methoxyphenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (5)
Yellow solid; 11.42 %; mp: 133-134 °C; 1H NMR (500 MHz, acetone-d6): δ = 3.87 (s, 3H), 6.31 (t, 1H), 7.01 (d, 1H, 3JH-H =5.5
Hz), 7.22 (d, 1H), 7.30 (d, 1H), 7.36 (d, 3H, 3JH-H =13.0 Hz), 7.37 (q, 2H, 3JH-H =15.0 Hz), 11.04 ppm (br. s, 1H). 13NMR (126
MHz, CDCl3): δ = 178.8, 159.8, 142.2, 136.4, 133.1, 129.9, 125.4, 122.2, 121.0, 116.4, 115.9, 113.4, 111.0, 55.3 ppm. HRMS
(ESI): m/z calcd for C14H13NO2-H+: 226.0946 [M-H]+; found: 226.0948.
4.3.6. (E)-3-(4-methoxyphenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (6)
Yellow solid; 58.69 %; mp: 121-122 °C; 1H NMR (500 MHz, acetone-d6): δ = 3.86 (s, 3H), 6.30 (t, 1H), 7.00 (d, 2H, 3JH-H =8.0
Hz), 7.19 (d, 1H), 7.26 (d, 1H), 7.53 (d, 1H, 3JH-H =15.7 Hz), 7.68 (d, 2H, 3JH-H =8.8 Hz), 7.75 (d, 1H, 3JH-H =15.0 Hz), 10.99
ppm (br. s, 1H). 13NMR (126 MHz, acetone-d6): δ = 178.1, 161.5, 140.9, 130.0, 127.9, 125.3, 120.3, 116.0, 114.3, 110.1 ppm.
HRMS (ESI): m/z calcd for C14H13NO2-H+: 226.0946 [M-H]+; found: 226.0948.
4.3.7. (E)-3-(3-hydroxyphenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (7)
Yellow solid; 44.60 %; mp: 122-123 °C; 1H NMR (500 MHz, acetone-d6): δ = 3.00 (br. s, 1H), 6.31 (t, 1H), 6.92 (d, 1H), 7.29
3
3
(s, 2H), 7.24 (d, 3H), 7.58 (d, 1H, JH-H =15.0 Hz), 7.67 (d, 1H, JH-H =15.0 Hz), 11.05 ppm (br. s, 1H). 13NMR (126 MHz,
acetone-d6): δ = 178.3, 158.6, 136.0, 133.5, 131.5, 128.2, 125.4, 123.8, 122.7, 120.6, 116.1, 111.4, 110.1, 55.1 ppm. HRMS
(ESI): m/z calcd for C13H11NO2-H+: 212.0790 [M-H]+; found: 212.0788.
4.3.8. (E)-3-(4-hydroxyphenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (8)
Yellow solid; 5.60 %; mp: 78-79 °C; 1H NMR (500 MHz, acetone-d6): δ = 6.30 (t, 1H), 6.91 (d, 2H, 3JH-H =8.1 Hz), 7.18 (d,
1H), 7.23 (d, 1H), 7.48 (d, 1H, 3JH-H =15.7 Hz), 7.66 (d, 2H, 3JH-H =8.0 Hz), 7.69 (d, 1H, 3JH-H =15.0 Hz), 8.90 (br. s, 1H), 10.96
ppm (br. s, 1H). 13NMR (126 MHz, acetone-d6): δ = 178.1, 159.5, 141.1, 133.4, 130.1, 126.7, 125.0, 119.4, 115.7, 115.7,
110.0 ppm. HRMS (ESI): m/z calcd for C13H11NO2-H+: 212.0790 [M-H]+; found: 212.0788.
4.3.9. (E)-3-(2-fluorophenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (9)
Yellow solid; 99.80 %; mp: 135-136 °C; 1H NMR (500 MHz, CDCl3): δ = 6.36 (t, 1H), 7.10 (d, 1H’), 7.14 (d, 1H), 7.20 (t, 1H),
7.38 (d, 1H), 7.47 (d, 1H, 3JH-H =15.6 Hz), 7.64 (t, 1H), 7.65 (t, 1H), 7.93 (d, 1H, 3JH-H =15.0 Hz), 9.83 ppm (br. s, 1H). 13NMR
(126 MHz, CDCl3): δ = 178.8, 162.7, 160.7, 135.0, 131.7, 131.5, 129.6, 125.9, 124.6, 124.4, 116.8, 116.2, 111.0 ppm. HRMS
(ESI): m/z calcd for C13H10FNO-H+: 214.0746 [M-H]+; found: 214.0747.
4.3.10. (E)-3-(3-fluorophenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (10)
Yellow solid; 25.58 %; mp: 119-120 °C; 1H NMR (500 MHz, CDCl3): δ = 6.36 (t, 1H), 7.09 (s, 1H), 7.10 (d, 1H), 7.13 (d, 1H),
7.33 (d, 2H), 7.35 (d, 1H, 3JH-H =15.0 Hz), 7.39 (t, 1H), 7.77 (d, 1H, 3JH-H =15.0 Hz), 9.77 ppm (br. s, 1H). 13NMR (126 MHz,
CDCl3): δ = 178.4, 164.4, 140.8, 137.3, 133.0, 130.5, 125.8, 124.4, 123.2, 116.7, 114.7, 114.4, 111.1 ppm. HRMS (ESI): m/z
calcd for C13H10FNO-H+: 214.0746 [M-H]+; found: 214.0747.
4.3.11. (E)-3-(4-fluorophenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (11)
Yellow solid; 25.60 %; mp: 120-121 °C; 1H NMR (500 MHz, CDCl3): δ = 6.36 (t, 1H), 7.08 (d, 1H), 7.09 (d, 1H), 7.12 (d, 2H,
3JH-H =5.0 Hz), 7.28 (d, 1H, 3JH-H =15.0 Hz), 7.63 (d, 2H, 3JH-H =5.0 Hz), 7.79 (d, 1H, 3JH-H =15.0 Hz), 9.78 ppm (br. s, 1H).
13NMR (126 MHz, CDCl3): δ = 178.7, 164.9, 141.0, 133.1, 131.3, 130.2, 130.1, 125.2, 121.7, 116.2, 116.0 ppm. HRMS (ESI):
m/z calcd for C13H10FNO-H+: 214.0746 [M-H]+; found: 214.0747.
4.3.12. (E)-3-(2-bromophenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (12)
Yellow solid; 63.00 %; mp: 102-103 °C; 1H NMR (500 MHz, CDCl3): δ = 6.36 (t, 1H), 7.08 (d, 1H), 7.14 (d, 1H), 7.24 (t, 1H),
7.28 (d, 1H, 3JH-H =15.0 Hz), 7.36 (t, 1H), 7.64 (d, 1H), 7.73 (d, 1H), 8.17 (d, 1H, 3JH-H = 15.0 Hz), 9.85 ppm (br. s, 1H). 13NMR
(126 MHz, CDCl3): δ = 178.5, 140.7, 135.2, 133.5, 133.0, 131.0, 127.8, 127.6, 125.8, 125.7, 124.9, 116.4, 111.1 ppm. HRMS
(ESI): m/z calcd for C13H10BrNO-H+: 273.1286 [M-H]+; found: 273.1283.
4.3.13. (E)-3-(3,4-dimethoxyphenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (13)
Yellow solid; 66.27 %; mp: 145-146 °C; 1H NMR (500 MHz, acetone-d6): δ = 3.87 (s, 3H), 3.90 (s, 3H), 6.30 (t, 1H), 7.02 (d,
1H, 3JH-H =8.0 Hz), 7.19 (d, 1H), 7.23 (d, 1H), 7.31 (d, 1H, 3JH-H =8.0 Hz), 7.53 (d, 1H, 3JH-H =15.6 Hz), 7.68 (d, 1H, 3JH-H =15.0
Hz), 10.97 ppm (br. s, 1H). 13NMR (126 MHz, acetone-d6): δ = 177.9, 151.2, 149.4, 141.0, 133.2, 127.9, 125.0, 120.1, 115.7,
112.6, 111.3, 110.3, 109.7, 55.0, 54.9 ppm. HRMS (ESI): m/z calcd for C15H15NO3-H+: 256.1052 [M-H]+; found: 256.1053.
4.3.14. (E)-3-(3-hydroxy-4-methoxyphenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (14)
Yellow solid; 40.17 %; mp: 188-189 °C; 1H NMR (500 MHz, acetone-d6): δ = 3.91 (s, 3H), 6.30 (t, 1H), 7.01 (d, 1H, 3JH-H =8.3
Hz), 7.19 (d, 1H), 7.21 (d, 1H), 7.28 (d, 1H), 7.33 (s, 1H), 7.48 (d, 1H, 3JH-H =15.6 Hz), 7.63 (d, 1H, 3JH-H =15.0 Hz), 7.80 (s,

1H), 11.00 ppm (br. s, 1H). 13NMR (126 MHz, acetone-d6): δ = 178.2, 149.7, 146.9, 141.3, 133.5, 128.5, 125.3, 121.8, 120.3,
116.1, 113.8, 111.4, 110.1, 55.4 ppm. HRMS (ESI): m/z calcd for C14H13NO3-H+: 242.0895 [M-H]+; found: 242.0897.
4.3.15. (E)-3-(3-chloro-4-hydroxyphenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (15)
Yellow solid; 7.77 %; mp: 187-188 °C; 1H NMR (500 MHz, acetone-d6): δ = 6.30 (t, 1H), 7.08 (d, 1H, 3JH-H =8.0 Hz), 7.20 (d,
1H), 7.30 (d, 1H), 7.55 (d, 2H, 3JH-H =15.0 Hz), 7.59 (d, 1H, 3JH-H =15.0 Hz), 7.86 (s, 1H), 9.38 (s, 1H), 11.01 ppm (br. s, 1H).
13NMR (126 MHz, acetone-d6): δ = 177.9, 154.7, 139.6, 133.3, 129.5, 128.7, 128.1, 125.4, 121.0, 120.8, 116.8, 116.2, 110.0
ppm. HRMS (ESI): m/z calcd for C13H10ClNO2-H+: 246.0400 [M-H]+; found: 246.0404.
4.3.16. (E)-3-(2,5-dimethoxyphenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (16)
Yellow solid; 65.27 %; mp: 107-108 °C; 1H NMR (500 MHz, acetone-d6): δ = 3.82 (s, 3H), 3.90 (s, 3H), 6.31 (t, 1H), 7.00 (d,
1H), 7.02 (d, 1H), 7.21 (d, 1H), 7.26 (d, 1H), 7.41 (s, 1H), 7.65 (d, 1H, 3JH-H =15.0 Hz), 8.08 (d, 1H, 3JH-H =15.0 Hz), 11.02
ppm (br. s, 1H). 13NMR (126 MHz, acetone-d6): δ = 178.2, 153.7, 152.9, 135.7, 133.4, 125.4, 124.4, 122.8, 116.9, 116.2,
112.7, 112.6, 110.0, 55.5, 55.1 ppm. HRMS (ESI): m/z calcd for C15H15NO3-H+: 256.1052 [M-H]+; found: 256.1053.
4.3.17. (E)-3-(2,3,4-trimethoxyphenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (17)
1
Yellow solid; 59.64 %; mp: 98-99 °C; H NMR (500 MHz, acetone-d6): δ = 3.84 (s, 3H), 3.91 (s, 3H), 3.93 (s, 3H), 6.30 (t,
1H), 6.88 (d, 1H, 3JH-H =8.3 Hz), 7.21 (d, 2H), 7.55 (d, 1H, 3JH-H =15.6 Hz), 7.61 (d, 1H, 3JH-H =8.0 Hz), 7.97 (d, 1H, 3JH-H
=
15.0 Hz), 11.03 ppm (br. s, 1H). 13NMR (126 MHz, acetone-d6): δ = 178.7, 155.7, 153.3, 142.4, 135.8, 133.5, 125.2, 122.9,
121.8, 121.3, 115.8, 110.0, 108.1, 60.9, 60.0, 55.5 ppm. HRMS (ESI): m/z calcd for C16H17NO4-H+: 286.1158 [M-H]+; found:
286.1153.
4.3.18. (E)-3-(3,4,5-trimethoxyphenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (18)
Yellow solid; 97.56 %; mp: 139-140 °C; 1H NMR (500 MHz, CDCl3): δ = 3.90 (s, 3H), 3.93 (s, 6H), 6.36 (t, 1H), 6.86 (s, 2H),
7.11 (d, 2H), 7.28 (d, 1H, 3JH-H =15.0 Hz), 7.75 (d, 1H, 3JH-H =15.0 Hz), 9.71 ppm (br. s, 1H,). 13NMR (126 MHz, CDCl3): δ =
178.7, 153.4, 142.4, 140.1, 133.1, 130.5, 125.3, 121.2, 116.2, 111.0, 105.5, 61.00, 56.2 ppm. HRMS (ESI): m/z calcd for
C16H17NO4-H+: 286.1158 [M-H]+; found: 286.1153.
4.3.19. (E)-1,3-di(1H-pyrrol-2-yl)prop-2-en-1-one (19)
Yellow solid; 5.38 %; mp: 159-160 °C; 1H NMR (500 MHz, acetone-d6): δ = 6.22 (t, 1H), 6.27 (t, 1H), 6.64 (d, 1H), 7.04 (m,
2H), 7.15 (d, 1H), 7.32 (d, 1H, 3JH-H =15.0 Hz), 7.61 (d, 1H, 3JH-H =15.0 Hz), 10.80 (br. s, 1H), 10.92 ppm (br. s, 1H). 13NMR
(126 MHz, acetone-d6): δ = 178.0, 133.7, 131.0, 122.6, 122.5, 124.6, 116.0, 114.8, 114.6, 110.3, 109.8 ppm. HRMS (ESI):
m/z calcd for C11H10N2O-H+: 187.0793 [M-H]+; found: 187.0795.
4.3.20. (E)-1-(1H-pyrrol-2-yl)-3-(thiophen-2-yl)prop-2-en-1-one (20)
Yellow solid; 71.22 %; mp: 123-124 °C; 1H NMR (500 MHz, CDCl3): δ = 6.34 (t, 1H), 7.09 (d, 2H), 7.17 (t, 1H), 7.36 (d, 1H),
7.41 (d, 1H, 3JH-H =15.0 Hz), 7.58 (d, 1H), 7.81 (d, 1H, 3JH-H =15.0 Hz), 9.86 ppm (br. s, 1H). 13NMR (126 MHz, CDCl3): δ =
179.1,138.2, 135.8, 133.1, 128.4, 126.9, 125.3, 125.2, 121.7, 116.1, 111.0 ppm. HRMS (ESI): m/z calcd for C11H9NOS-H+:
202.0405 [M-H]+; found: 202.0403.
4.3.21. (E)-1-(1H-pyrrol-2-yl)-3-(3-methylthiophen-2-yl)prop-2-en-1-one (21)
Yellow solid; 89.90 %; mp: 125-126 °C; 1H NMR (500 MHz, CDCl3): δ = 2.39 (s, 3H), 6.34 (t, 1H), 6.90 (d, 1H), 7.05 (d, 2H),
7.10 (d, 1H), 7.27 (d, 1H, 3JH-H =15.0 Hz), 8.00 (d, 1H, 3JH-H =15.0 Hz), 9.75 ppm (br. s, 1H). 13NMR (126 MHz, CDCl3): δ =
178.6, 142.0, 135.0, 133.2, 131.3, 126.8, 125.1, 124.0, 120.0, 116.0, 110.9, 14.3 ppm. HRMS (ESI): m/z calcd for C12H11NOS-
H+: 216.0561 [M-H]+; found: 216.0564.
4.3.22. (E)-3-(furan-2-yl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (22)
Orange solid; 89.90 %; mp: 125-126 °C; 1H NMR (500 MHz, CDCl3): δ = 6.34 (t, 1H), 6.50 (d, 1H), 6.58 (d, 1H), 7.07 (d, 1H),
7.10 (d, 1H), 7.25 (d, 1H, 3JH-H =15.0 Hz), 7.52 (d, 1H), 7.58 (d, 1H, 3JH-H =15.0 Hz), 9.85 ppm (br. s, 1H). 13NMR (126 MHz,
CDCl3): δ = 178.6, 144.6, 133.2, 128.4, 125.2, 119.6, 115.4, 116.3, 112.5, 111.0 ppm. HRMS (ESI): m/z calcd for C11H9NO2-
H+: 186.0633 [M-H]+; found: 186.0631.
4.3.23. (E)-3-(5-methylfuran-2-yl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (23)
Orange solid; 64.39 %; mp: 155-156 °C; 1H NMR (500 MHz, CDCl3): δ = 2.39 (s, 3H), 6.11 (d, 1H), 6.33 (t, 1H), 6.58 (d, 1H),
7.08 (d, 2H), 7.18 (d, 1H, 3JH-H =15.0 Hz), 7.51 (d, 1H, 3JH-H =15.0 Hz), 9.69 ppm (br. s, 1H). 13NMR (126 MHz, CDCl3): δ =
178.8, 155.5, 150.7, 133.3, 128.6, 124.8, 117.7, 117.4, 115.9, 110.7, 109.1, 14.00 ppm. HRMS (ESI): m/z calcd for
C12H11NO2-H+: 200.0790 [M-H]+; found: 200.0988.
4.3.24. (E)-3-(4-(benzyloxy)phenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (24)
Yellow solid; 4.95 %; mp: 155-156 °C; 1H NMR (500 MHz, CDCl3): δ = 5.12 (s, 2H), 6.34 (t, 1H), 6.35 (d, 1H), 7.01 (m, 3H),
7.06 (d, 2H), 7.23 (d, 1H), 7.40 (d, 2H), 7.42 (d, 1H, 3JH-H =15.0 Hz), 7.44 (d, 2H), 7.79 (d, 1H, 3JH-H =15.0 Hz), 9.64 ppm (br.
s, 1H). 13NMR (126 MHz, CDCl3): δ = 177.0, 145.1, 136.7, 132.4, 130.7, 128.9), 127.6, 127.5, 127.1, 126.0, 121.3, 114.2,
111.7, 70.8 ppm. HRMS (ESI): m/z calcd for C20H17NO2-H+: 302.1259 [M-H]+; found: 302.1261.
4.4. Biological evaluation
4.4.1. Biology reagents
RAW 264.7 murine macrophage cells obtained from American Type Culture Collection (ATCC, Rockville, MD), Dulbecco’s
modified Eagle’s medium (DMEM), Fetal bovine serum (FBS), Penicilin/Streptomycin, Lipopolisaccharide (LPS: E. coli,

serotype 0111:B4), Interferon gamma (IFN-γ), Trypan blue, Triple express (detach media), 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), and other chemicals were purchased from Sigma Aldrich and Life Technologies [17].
The prostaglandin E2 express EIA kit (Cayman Chemical, MI, USA) contained monoclonal antibody, acetylcholinesterase
enzyme (AChE) tracer, Polysorbate 20, and antiserum dye [18,19b].
4.4.2. Cell culture
The RAW 264.7 murine macrophage cells obtained from ATCC were grown in fresh DMEM with phenol red in a culture flask,
supplemented with 10% FBS and 1% of penicillin/streptomycin before incubation under 5% CO2 and 95% air at 37°C [17].
4.4.3. Cell stimulation and treatment
The attached ATCC RAW 264.7 cells at confluence of 90-95% were detached using Triple Express, prior to being seeded
into a 96-well culture plate at 5 x 104/ 50 µl per well and incubated for 24 hours. The cells were then induced with recombinant
5 mg/ml of LPS and 1 ng/ml of IFN-γ in the presence or absence of test samples for 17-20 hours. The level of NO was
determined with a Griess assay [17], while the concentration of PGE2 was measured via PGE2 EIA kit (Code No. 500141)
[18, 19b].
4.4.4. NO determination (Griess assay)
The NO concentration was then quantified with a Griess assay by reacting 50 µl of Griess reagent (1% sulfanamide and 0.1%
N-(1-naphthyl)ethylenediaminedihydrochloride in 2.5% phosphoric acid) and 50 µl of cell culture supernatant at room
temperature for 10 minutes. The optical density was measured at 550 nm with a microplate reader after 5 minutes of
incubation at room temperature [17].
4.4.5. Determination of PGE2
50 µl of cell culture supernatants were collected and evaluated for PGE2 secretion using a PGE2 EIA kit, following the
protocols provided by the manufacturer. The concentrations of PGE2 in each well was quantified by a microplate reader and
calculated from their respective standard curves graph [18, 19b].
4.4.6. Cell viability (MTT assay)
Each well was added with 100 µl DMEM and 20 µl of MTT (5 mg/ml in PBS) after the supernatant was removed. The plate
was then incubated under 5% CO2 and 95% air at 37°C for 4 hours. The mixture of culture media and MTT in all wells were
discarded and the purplish formazan crystals formed were dissolved in 100% dimethylsulfoxide (DMSO) and further
incubated at room temperature for 15-20 minutes. The absorbance was taken at 570 nm using a microplate reader, with the
absorbance of the formazan formed in the control (untreated) cells taken as 100% viability [17].
4.5. In silico studies (PAINS and aggregator filtration, physicochemical and ADMET properties)
4.5.1. In-silico PAINS identification and physicochemical properties determination
All chemical structures of pyrrolylated-chalcones were drawn using ChemDraw Ultra 12.0 and converted to SMILES
(Simplified Molecular Input Line Entry System) format. The SMILES formulas were then converted into Symyx SDF (Spatial
Data File) file format as recommended by the FAF-Drugs4 Bank Formatter (http://mobyle.rpbs.univ-paris-diderot.fr/cgi-
bin/portal.py#forms::Bank-Formatter), before automatically redirected as the input file into the FAF-Drugs4 server
(http://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal.py#forms::FAF-Drugs4) for the filtration of PAINS using PAINS filter A,
B, and C [27]. The generated results is available at the following address (http://mobyle.rpbs.univ-paris-diderot.fr/cgi-
bin/portal.py?form=FAF-Drugs4#jobs::FAF-Drugs4.T04085527502060).
4.5.2. In-silico aggregator identification
The SMILES file containing the synthesized molecules generated from the in-silico PAINS analysis was input into the
Aggregator Advisor server (http://advisor.bkslab.org/) for the identification of potential aggregators [28].
4.5.3 In-silico prediction of ADMET
All chemical structures of pyrrolylated-chalcones were drawn using available drawing platform at
https://preadmet.bmdrc.kr/adme/ for the determination of ADMET properties.
4.6. Crystal growth and X-ray
The accuracy of molecular docking is known to be highly dependent on the initial conformation of the input ligand. Therefore,
crystals of the most active compound 16, 3-(2,5-dimethoxyphenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one, which is suitable for X-
ray diffraction analysis, were grown from the slow evaporation of ethanol. The ‘plate-like’ crystals of compound 16 were then
mounted on a fibre loop using perfluoropolyether oil before rapid cooling to 100K using an Oxford Cryosystems Cobra unit
[36]. Then, the crystal and refinement data of compound 16 were measured using an Oxford Diffraction Gemini ECCD
diffractometer. All measurements were made with graphite monochromatic Mo Kα radiation (λ = 0.71073 Å). Data collected
were then integrated and processed using the CrysAlis Pro software suite (Agilent Technologies UK Ltd, Yarnton, England).
All non-hydrogen atoms were refined anisotropically. All other hydrogen atoms were included in calculated positions but not
refined. The structure was solved by direct methods and full-matrix least-squares refinement on F2 using SHELXS [37] in an
Olex2 interface [36]. The cif file is available in Mendeley Data (http://dx.doi.org/10.17632/tzbf4tnw4n.1).

4.7. Molecular docking methodology
4.7.1. Homology modelling
Amino acid sequences of microsomal prostaglandin E synthase 1 (mPGES-1) and inhibitor of nuclear factor kappa-B kinase
subunit beta (IKKβ) from M. musculus were retrieved from the UniProt protein database. The resulting amino acid sequences
Q9JM51 and O88351, were subjected to NCBI BLASTP sequence analyses for Sequence similarity search against protein
databank sequences. These amino acid sequences were used as the input for the development of 3D models using SWISS-
MODEL [38], a fully automated protein structure-modelling server which is reachable through the ExPASy web server. The
model with the highest sequence identity and coverage with the template was selected and evaluated. In addition, the best
model was also selected based on the satisfied QMEAN Z-scores and GMQE score. QMEAN Z-scores around zero indicates
a good agreement between the model structure and experimental structures of similar size, while higher GMQE numbers
indicates a higher reliability to reflect the expected accuracy of a model built with that alignment and template, and the
coverage of the target. Energy minimisation of the resulting models was performed using the steepest descent algorithm
implemented in GROMOS from Deepview to obtain a relaxed conformation. The minimised models were assessed using
PROCHECK and ERRAT.
4.7.2. Molecular docking
AutoDock 4.2 [39] was used to dock the compounds into protein active sites. Lamarckian-Genetic Algorithm (LGA) was
employed in this docking study. Murine ERK (3C9W), JNK (3O2M), p38 (2GTN), and COX-2 (6COX) crystal structures were
retrieved from the Brookhaven Protein Data Bank. Homology model for murine mPGES-1 and IKKβ built were used to dock
the compounds. All the proteins were pre-treated before the docking. Hydrogen atoms were added to the protein structure.
Protein-ligand interactions were analysed using Discovery Studio Visualizer 3.5 [40].
Acknowledgement
This study was financially supported by Universiti Putra Malaysia under the Research University Grant Scheme (RUGS)
(9301700 and 9480700) and Malaysia Toray Science Foundation (MTSF). The first author also acknowledges the support
from the Ministry of Education Malaysia under the SLAB scholarship.
Declaration and Competing Interest
The authors declared that there is of conflict of interest.
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GRAPHICAL ABSTRACT

Highlights
 Compound 16 displayed prominent PGE2 over NO inhibition with
IC50 of 0.5 ± 1.5 µM
 Compound 16 is non-PAINS associated molecule with high blood
brain barrier (BBB) penetration, human intestinal absorption
(HIA), P- glycoprotein (PgP) inhibition and plasma binding protein
(PPB) inhibition
 Compound 16 was found to participate in a number of important
binding interactions in the binding sites of ERK and mPGES-1

CONFLICT OF INTEREST
The authors declared that there is of conflict of interest.