Design, synthesis, biological evaluation and docking study of novel indole-2-amide as anti-inflammatory agents with dual inhibition of COX and 5-LOX

Article abstract of DOI:10.1016/j.ejmech.2019.07.004

In this work, a series of novel indole-2-amide compounds were designed, synthesized, characterized and the anti-inflammatory activity in vivo were evaluated. Compounds 8a, 10b, 12h, and 12l exhibited marked anti-inflammatory activity in 2,4-Dinitrofluorob

Full text of DOI:10.1016/j.ejmech.2019.07.004

Accepted Manuscript
Design, synthesis, biological evaluation and docking study of novel indole-2-amide as
anti-inflammatory agents with dual inhibition of COX and 5-LOX
Yuanzheng Huang, Bin Zhang, Jiaming Li, Huicai Liu, Yanchun Zhang, Zhang Yang,
Wandong Liu
PII:
S0223-5234(19)30625-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.004
EJMECH 11501
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 January 2019
Revised Date: 1 July 2019
Accepted Date: 1 July 2019
Please cite this article as: Y. Huang, B. Zhang, J. Li, H. Liu, Y. Zhang, Z. Yang, W. Liu, Design,
synthesis, biological evaluation and docking study of novel indole-2-amide as anti-inflammatory agents
with dual inhibition of COX and 5-LOX, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.07.004.
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ACCEPTED MANUSCRIPT
Design, Synthesis, biological evaluation
and docking study of novel
Indole-2-amide as anti-inflammatory
agents with dual inhibition of COX and
5-LOX
Yuanzheng Huang, Bin Zhang, Jiaming Li,
Huicai Liu, Yanchun Zhang, Zhang Yang,
Wandong Liu
Abstract: A series of novel indole-2-amide
compounds were designed, synthesized and
the
dual
inhibitory
activity
of
COX/5-LOX were demonstrated.

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Design, Synthesis, biological evaluation and docking study of novel
Indole-2-amide as anti-inflammatory agents with dual inhibition of
COX and 5-LOX
Yuanzheng Huang a, Bin Zhang a, Jiaming Li a, b, *, Huicai Liu a, Yanchun Zhang a, b
,
Zhang Yang a, Wandong Liu a
(a School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230031, China;
b Department of Medicinal Chemistry, Anhui Academy of Chinese Medicine, Hefei
230031, China)
*Corresponding author: Jiaming Li
E-mail:lijiaming2017@sina.com
Abstract
In this work, a series of novel indole-2-amide compounds were designed, synthesized,
characterized and the anti-inflammatory activity in vivo were evaluated. Compounds
8a, 10b, 12h, and 12l exhibited marked anti-inflammatory activity in
2,4-Dinitrofluorobenzenethe (DNFB) - induced mice auricle edema model. Further,
compounds 8a 10b and 12h exhibited potential in vitro COX-2 inhibitory activity
(IC50 = 21.86, 23.3 and 23.21 nM, respectively), while the reference drug celecoxib
was 11.20 nM. The most promising compound 10b was exhibited the highest
selectivity for COX-2 (selectivity index (COX-1/COX-2) = 17.45) and moderate
5-LOX inhibitory activity (IC50 = 66 nM), which comparable to positive controlled
zileuton (IC50 = 38.91 nM). In addition, the test results showed compounds 10b and
12h no significant cytotoxic activity on normal cells (RAW264.7). Further, at the
active sites of the COX-1, COX-2 co-crystals, 3b and 4l showed higher binding forces
in the molecular docking study, which consistent with the results of in vitro
experiments. These results demonstrated that these compounds had dual inhibitory
activity of COX/5-LOX, providing clues for further searching for safer and more
effective anti-inflammatory drugs.
Keywords: Indole; Anti-inflammatory; COXs; 5-LOX; Docking
1. Introduction

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As one of the most frequently used drugs in the world, traditional non-steroidal
anti-inflammatory drugs (NSAIDs) achieve antipyretic, analgesic and
anti-inflammatory effects by inhibiting COX enzyme-associated inflammatory
cytokines synthesis [1]. COX is one of the key enzymes in the arachidonic acid (AA)
metabolic pathway. It include two isoenzymes (COX-1 and COX-2), which mediates
the production of prostaglandins and thromboxanes from arachidonic acid [2].
According to current research, COX-1 is an intrinsic enzyme, and mainly distributed
in the stomach, kidney and platelets, which can maintain the gastric and renal
homeostasis; COX-2 is an inducible enzyme, and its expression is enhanced in the
case of tissue damage, inflammation, etc [3]. Therefore, long-term use of traditional
NSAIDs such as aspirin may cause serious gastrointestinal side effects, which are
very unfavorable for the treatment of some chronic inflammation [4]. Compared with
traditional NSAIDs, selective COX-2 inhibitors significantly reduce gastrointestinal
side effects, but at the same time increase the likelihood of cardiovascular disease
[5,6].
5-lipoxygenase (5-LOX) is one of the three major isozymes of liquid oxygen
(LOX), involved in the metabolic process of AA, and there is a compensatory
mechanism with COX [7]. The Leukotrienes (LTs) ultimately produced by the LOX
pathway are considered as important mediators of inflammatory and allergic diseases,
and are believed to be closely related to cancer and cardiovascular disease [7,8].
Therefore, it is possible to decrease the potential cardiovascular disease risk of
NSAIDs by inhibiting 5-LOX and reducing the synthesis of LTs. At the same time,
studies have shown that LTs are involved in the gastrointestinal reactions caused by
NSAIDs [9]. It is worth noting that although 5-LOX plays an important role in
various physiological processes such as inflammation, the 5-LOX inhibitor - Zileuton
don’t show an obvious anti-inflammatory effect in related experiments [10].
Therefore, it is possible to cut down side effects such as gastrointestinal tract and
cardiovascular while maintaining the basic activity of NSAIDs through strategy of
COX/5-LOX co-inhibition, thereby obtaining safer and more effective

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anti-inflammatory drugs.
As a dual inhibitor of COX/5-LOX, licofelone and darbufelone were first
designed and entered the clinic as an analgesic and anti-inflammatory drug, but failed
to be marketed due to efficacy or toxicity. Therefore, for the time being, the design
and research of dual inhibitors is still urgently needed in the market [11]. And in
recent years, researchers have also discovered some excellent compounds with dual
inhibitory potential. among which, Mostafa etc designed and synthesized a compound
as COX/5-LOX double inhibition by using quinoline as a backbone and a 2-amide
structure as a linker, Its IC50 values of COX-1 = 8.6 µM, COX-2 = 1.13 µM and
5-LOX = 7.61 µM, both comparable to positive drugs [12]. In addition, indole ring is
an important fragment with anti-inflammatory effects, and indomethacin has been
used as a non-steroidal anti-inflammatory drug [13,14]. Palwinder and co-workers
combined three heterocyclic structures of indole-chrysin-barbiturate to synthesize
compound 2 and exhibited extremely high double inhibitory activity with IC50 values
of COX-2 = 1 nM and 5-LOX = 1.5 nM, respectively, better than celecoxib and
zileuton [15]. And Reddy synthesized a series of compounds by modifying the
structure of N-benzyl indole, in which compound 3 showed good COX/LOX dual
inhibitory activity with IC50 values of COX-2 = 3.9 µM and 5-LOX = 94 µM [16].
(Fig. 1)
Encouraged by these findings, 5-chloroindole-2-carboxylic acid was used as the
fundamental structure to construct amide bond by ethylamine, pyrrolidine and
piperazine, and simultaneously combined with the tryptamine and ligustrazine
fragments which have a wide range of biological activities, in order to obtain
compounds which have good anti-inflammatory activity (Fig. 2). The first six
compounds were synthesized and evaluated for their in vivo anti-inflammatory
activity, and the most active compound 8a was selected for further modification to
confer different electronic and lipophilic properties to the molecules. Similarly, the
newly synthesized fourteen compounds were also evaluated for their in vivo
anti-inflammatory activity. Further, the most active compounds 8a, 10b, 12h, 12l

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were investigated for their in vitro COX/5-LOX inhibition. In addition, we also
conducted molecular docking studies on compounds 10b and 12h to explore feasible
combinations of compounds with the two enzymes.
Fig. 1 Chemical structure of indole-based COX or LOX inhibitor compound and dual COX/LOX copound of
contain amide
Fig. 2 Idea of newly designed indole-2-carboxamide
2. Result and discussion
2.1. Chemical synthesis

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Schemes 1 - 4 describes the synthetic route for intermediates and final
compounds.
The preparation of the carboxylic acid fragments 3a-b are described in Scheme 1.
Since there were two active sites in the 5-Chloroindole-2-carboxylic acid, the
carboxylic acid group was first protected by methyl esterification. The obtained
methyl ester was reacted with 4-chlorobenzyl chloride or 4-fluorobenzyl chloride, and
NaH was added to increase the reactivity to obtain 3a-b [17]. The two steps had good
yields of 91.5% and 86.4%, respectively. The structures of the compounds 3a-b were
1
confirmed by H NMR and 13C NMR spectroscopic analyses as well as MS
spectrometry, and the absence of indole NH peak of the compound 2 and presence of
1
a peak of two protons integration due to benzyl group at 5.87 ppm in H NMR
spectrum of 3a-b confirmed the reaction. Finally, they were hydrolyzed in a hot
alkaline solution to obtain 3a-b.
The preparation of amino fragments 5 and 7 were described in Scheme 2.
Dichloromethane solution of 4 with an excess of anhydrous piperazine afforded amine
5 in 77% yield. On the other hand, compound 4 was subjected to substitution using 1 -
Boc - 3 - aminopyrrolidine in dichloromethane to yield intermediate 6. And
compound 6 was hydrolyzed to the amine 7 using trifluoroacetic acid (TFA) under an
alkaline environment [18].
The formation of compounds’ amide bond are described in Schemes 3 and 4.
The HOBT and EDCI were first combined with the carboxylic acid fragment to form
an active lipid, which increaseed the reactivity, and then added the above-mentioned
amine fragment to form an amide under an alkaline environment [19]. All the
1
structures of indole-2-carboxamides were confirmed by H NMR and 13C NMR
spectroscopic analyses as well as MS spectrometry. Taking compound 8a as an
example, 1H NMR spectra showed the presence of a peak assigned to ArCH2N at 5.5
ppm and a peak assigned to Piperazine-CH2 at 3.5 ppm. At the same time, the mass
spectrometry data further confirmed the structure of 8a, which gave a molecular ion
m/z 521.17 (M+H)+.

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Reagents and conditions: (a) SOCl2, Methanol, 75 ; (b) Appropriate benzyl, NaH, DMF, rt; (c) NaOH, alcohol,
75
.
Scheme 1 Synthetic pathways for compounds 1-3
Reagents and conditions: (a) piperazine, CH2Cl2, rt; (b) 1-Boc-3-aminopyrrolidine, THF, reflux; (c) TFA, CH2Cl2,
rt
Scheme 2 Synthetic pathways for compounds 5-7
Reagents and conditions: (a) EDCI, HOBT, TEA, DMF, rt
Scheme 3 Synthetic pathway for compounds 8a-b, 9a-b, 10a-b

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Reagents and conditions: (a) EDCI, HOBT, TEA, DMF, rt
Scheme 4 Synthetic pathway for compounds 12a-n
2.2. Biological evaluation
2.2.1. Anti-inflammatory activity
The anti-inflammatory activity of the synthesized compounds was evaluated by
utilizing the mice auricle edema model using dexamethasone as a positive drug [20].
The experimental results were shown in Table 1 and Table 2 and expressed as means
± standard deviation (SD). And one-way analysis of variance was performed on the
control group and the test group using SPSS 23.0 statistical software. The difference
was significant at p < 0.05.
In the inflammatory model, the compounds were divided into two batches to
evaluate the activity of inhibition of mouse ear swelling. Each test compound was

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administered by intragastric administration (positive group was given dexamethasone
0.5 mg/kg, and compound group was given 5 mg/kg, respectively). After 6 days of
adaptive feeding, DNFB was used to stimulate inflammation, and the mice were
sacrificed 24 hours later. The left ear and the right ear were weighed and the swelling
degree and inhibition rate were calculated [20].
Among the first activity results, 8a (64.9%) and 9b (52.5%) had significant
inhibitory activity compared with the control group (p < 0.01). There was no
significant difference between 8a and the positive drug dexamethasone. 12h (43.1%)
and 12l (40.5%) had significant inhibitory activity compared with the model group (p
< 0.01), which 12h was equivalent to the positive drug.
Table 1
The percentages of edema inhibition of compounds 8a-b, 9a-b, 10a-b and dexamethasone
Dose levels
left ear
(mg)
Right ear
(mg)
swelling degree
(mg)
inhibition
(%)
Compound
(mg/kg)
Blank
-
-
14.3±1.0
13.5±1.2
13.8±1.0
12.9±0.8
12.5±1.1
12.5±0.9
12.9±0.6
13.8±0.9
13.8±1.5
13.9±1.2
30.9±3.5
20.1±4.8
19.0±3.2
21.5±3.7
22.5±3.7
21.7±3.4
23.5±3.2
22.5±4.6
-
-
Control
17.4±3.6
6.3±4.7aa
6.1±2.5aa
9.0±4.3a
10.0±3.1
8.8±4.1a
9.7±4.1a
8.7±4.7aa
-
Dexamethasone
0.5
5
63.8
64.9
48.3
42.5
49.4
44.3
50.0
8a
8b
5
9a
5
9b
5
10a
10b
5
5
Ap < 0.05，aap < 0.01 vs control
swelling degree (mg) = WR - WL
inhibition (%) = ((WR - WL) control - (WR - WL) treated) / (WR - WL) control * 100%
WR = Average weight of the right ear; WL = Average weight of the left ear
Table 2
The percentages of edema inhibition of compounds 12a-n and dexamethasone

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Dose levels
left ear
(mg)
Right ear
(mg)
swelling degree
(mg)
inhibition
(%)
Compound
(mg/kg)
Blank
-
-
11.8±0.7
12.5±1.1
12.3±0.8
11.3±1.0
11.5±1.0
12.3±1.0
13.8±1.0
12.1±0.7
12±0.9
12.2±0.7
25.7±2.2
19.3±1.2
21.3±3.2
21±2.8
-
-
Control
13.2±2.3
7±1.9aa
-
Dexamethasone
0.5
5
46.8
24.0
27.8
36.7
25.4
32.9
34.2
35.4
43.1
29.8
39.2
29.1
40.5
32.9
30.4
12a
12b
12c
12d
12e
12f
12g
12h
12i
10±3.7
5
9.5±3.8
8.3±3.2a
9.9±3.4
8.8±2.3a
8.7±3.9a
8.5±2.9a
7.5±2.1aa
9.0±2.9
8.0±2.8a
9.3±3.6
7.8±2.9aa
8.8±3.4a
9.2±2.4a
5
20.7±3.4
23.7±3.6
21±2.7
5
5
5
20.7±3.8
21±2.4
5
12.5±1.1
11.7±0.8
13.9±1.2
12.5±1.4
12.2±0.7
12±0.9
5
19.2±1.9
22.9±3.1
20.5±3.0
21.5±3.7
19.8±2.1
21±2.9
5
12j
5
12k
12l
5
5
12m
12n
5
12.2±0.7
12±0.9
5
21.2±3.2
Ap < 0.05，aap < 0.01 vs control
swelling degree (mg) = WR - WL
inhibition (%) = ((WR - WL) control - (WR - WL) treated) / (WR - WL) control * 100%
WR = Average weight of the right ear; WL = Average weight of the left ear
2.2.2. In vitro COX-1/COX-2 inhibition assay
Further, compounds with high anti-inflammatory activity such as 8a, 10b, 12h,
and 12l were evaluated for their COX inhibitory activity in vitro by
immunofluorescence analysis, and indomethacin and celecoxib were used as positive
controls [21]. The mean fluorescence values were calculated at an emission
wavelength of 587 nm and the potency of the testing compounds was determined as

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the concentration causing 50% enzyme inhibition (IC50). In addition, the selectivity
index (IS) of COX-2 was calculated.
As shown in Table 3, all compounds exhibited good inhibitory ability to COX-2
and selectivity to COX-2 (IC50 = 31.91-21.86 nm) was higher than that of COX-1
(IC50 = 406.6-195.0 nm). The SI ranges from 8.4 to 17.45. All the tested compounds
had higher COX inhibition than indomethacin but slightly less inhibition of COX-2
than celecoxib. These results indicated that compounds with good anti-inflammatory
activity exhibited high inhibitory activity against COX, and inhibition COX may be
one of the pathways by which these compounds exert anti-inflammatory effects.
Table 3
In vitro COX-1, COX-2 and 5-LOX enzymatic inhibition assay of the compounds 8a,10b,12h and 12l.
IC50a (nM)
Compound
COX-1/COX-2
COX-1
312.4±28.75
406.6±35.65
195.0±10.34
361.4±21.35
489.9±18.73
>500
COX-2
21.86±1.37
23.3±1.39
23.21±1.51
31.91±1.78
1466±86.59
11.20±0.97
ND
5-LOX
339.0±30.62
66.14±6.26
83.54±6.28
209.5±9.57
ND
selectivity index(SI)b
8a
10b
14.29
17.45
8.4
12h
12l
11.33
0.33
>40
Indomethacin
Celecoxib
Zileuton
ND
ND
ND
38.91±2.16
a IC50: Represents the concentration of the test compound that is required for 50% inhibition in vitro.
b SI: IC50 (COX-1) / IC50 (COX-2)
ND: not determined
2.2.3 In vitro 5-LOX inhibition assay
Similarly, the evaluation of 5-LOX inhibitory activity was performed on 8a, 10b,
12h and 12l, and the obtained data is listed in Table 3 [22]. Among them,compounds

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10b and 12h had moderate inhibitory activities (IC50 = 66.14 and 83.54 nM,
respectively) while the positive control zileuton was 38.91 nM. However, 8a and 12l
showed no significant inhibitory activity against 5-LOX. By analyzing the structure of
the compounds, we had found that the electron-donating group of methoxy group may
increase the inhibitory activity of the compound for 5-LOX compared to the electron
withdrawing group.
2.2.4 In vitro cytotoxic activity
Further, compounds 10b and 12h were evaluated for their cytotoxicity on normal
macrophage cell line (RAW264.7). The results showed that compounds 10b and 12h
had no significant effect on the survival of RAW264.7 cells below 50 µM (including
50 µM). The cell survival rate of each concentration group was not significant
difference from that of the blank group (Fig. 3).
Fig. 3 Cell viability experiment was performed at 5-50 µM concentration and there was no significantly cytotoxic
of compounds 10b and 12h at the concentration up to 50 µM.
2.3 Molecular docking study
In order to investigate the possible binding conformations of compounds 10b and
12h with either COX-1 (PDB code 1PGF) [23] or COX-2 (PDB code 1CX2) [24]
receptors, molecular docking study was carried out using AUTODOCK 4.2.6
modeling software [25]. The docking results were processed with PYMOL to obtain
the molecular chimeric maps, and the positions at which the compounds formed

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hydrogen bonds and binding pockets were analyzed [26].
To ensure the feasibility of the docking results, we extracted the eutectic ligands
IMM and SC-558 from the 3D protein structure of COX-1 and COX-2. In Fig. 4, the
docking method of IMM (A) and SC-558 (B) was compared with the eutectic ligand
to verify the accuracy of the docking study. The same docking parameters were used
in subsequent docking of 10b and 12h.
Fig. 5 (A) showed the combination of 12h and COX-1. 12h enter the active
pockets of HIS90, ARG120, SER530 and TYR385 [27], and combined HIS90 with
hydrogen bonds. In Fig. 5 (B), the combination of 12h and IMM was similar, and the
docking scores for 12h and IMM were -11.74 and -10.62, respectively. Be different
from Ile523 in COX-1, COX-2 binding site was present an additional side pocket
because of the amino acid residue Val523, which increases the active area of COX-2
by 20% [28]. The results of the docking simulation of 12h into COX-1 active site
showed that this compound was forced to adopt a longitudinal binding pattern due to
presence of Ile523, which closed the side pocket and pushed the compound to the
bottom of the active site. On the contrary, in Fig. 6 (B and D), the orientation of the
5-chloroindole moieties in 10b and 12h, respectively within the additional side pocket
of COX-2 was similar to fitting of sulfonamide moiety of SC-558 into the same side
pocket. Due to the different chemical nature of the sulfonamides in the chlorine atom,
figure 5 illustrated that the compounds 10b and 12h have certain COX-2 selectivity,
and may also explain the difference in activity between the compounds and celecoxib
[29]. Simultaneously, 12h forms a hydrogen bond with the important residue ARG120,
which is the same as AA (Fig 6 (B)), while 10b forms a hydrogen bond with the
active pocket apical residue TYR385. These two hits exhibited similar binding pattern
and interactions to that of the cocrystallized SC-558 ligand (Fig 6 (D)) [30].

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A
B
Fig 4 (A) Comparison between the docked pose of IMM (yellow) as produced by docking experiment and the
co-crystallized ligand of this inhibitor within COX-1 (gray, PDB code: 1PGF). (B) Comparison between the
docked pose of SC-558 (yellow) as produced by docking experiment and the co-crystallized ligand of this inhibitor
within COX-2 (gray, PDB code: 1CX2).
B
A
Fig 5 (A) Docking and binding pattern of compound 12h (orange) into COX-1 active site (PDB code: 1PGF); (B)
The superimposition of the docked pose 12h (yellow) and the co-crystallized IMM (magenta) within active site of
COX-1. Dashed lines represent hydrogen bonds. The hydrophobic area of active site was represented by green; and
the hydrophilic area was indicated by yellow.
A
B

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C
D
Fig 6 (A) Docking and binding pattern of compound 12h (orange) into COX-2 active site (PDB code: 1CX2). (B)
The superimposition of the docked pose 12h (yellow) and the co-crystallized SC-558 (magenta) within active site
of COX-2. (C) Docking and binding pattern of compound 10b (orange) into COX-2 active site (PDB code: 1CX2).
(D) The superimposition of the docked pose 10b (yellow) and the co-crystallized SC-558 (magenta) within active
site of COX-2. Dashed lines represent hydrogen bonds. The hydrophobic area of active site was represented by
green; and the hydrophilic area was indicated by yellow.
3 Conclusion
In the present study, a series of 2-indole-amides derivative was synthesized as
anti-inflammatory agents. Compounds 8a, 10b, 12h and 12l exhibited good
anti-inflammatory activity in the mice auricle edema model. There was no significant
cytotoxic activity on RAW264.7. Further, the experiments conducted in-depth studies on
the anti-inflammatory mechanisms of there compounds. In the enzyme
immunosuppression experiment, the compounds were found to have a good inhibitory
activity against the COX/5-LOX enzyme. Among them, 13i had the best inhibitory
activity against COX-1 (IC50 = 195 nM); 9a, 11b, 13i had potent inhibitory activities
against COX-2, which were 21.86 nM, 23.3 nM and 23.21 nM, respectively; while
11b and 13i exhibited 5-LOX inhibitory activity (IC50 = 66.14 nM, 83.54 nM,
respectively). Therefore, were it was confirmed that the compounds exert its
anti-inflammatory activity by inhibiting the COX/5-LOX pathway. According to Guo
Zongru's strategy of moderate inhibition[31], due to the mutual restriction of COX-1
and COX-2 in the body, excessive inhibition of COX-2 will break this kind of balance,
which will lead to some side effects that are not expected [31]. Compounds 10b and
12h exhibited moderate inhibition of the COX/5-LOX and temperate selectivity for

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COX-2, which may be more safer than celecoxib. In addition, docking studies have
explained and supported the results of in vitro enzyme activity experiments. By
analyzing the binding pattern of the compounds to the active site of the enzyme, it
was find that the 5-chloroindole structure in the compounds 10b and 12h entered the
COX-2 side pocket, which was similar to the sulfonamide group in SC-558. In
conclusion, combined with the results of the mice auricle edema and the enzyme
immunosuppression experiments, this study led to discovery of two promising
compounds with high potency against COX and 5-LOX, providing clues for further
discovery of safe and effective anti-inflammatory drugs.
4. Experimental
4.1. Chemistry
The reagents and solvents for reaction were purchased from common commercial suppliers. If
necessary, purification processes were carried out prior to their use. Melting points are
determined on melting point apparatus (RDCSY-I) and are uncorrected. 1H NMR and 13C NMR
spectra were recorded on 400 MHz and 100 MHz instruments (Bruker, Fallanden, Switzerland),
respectively, with tetramethylsilane (TMS) as internal standard. MS spectra were measured with a
Hewlett-Packard 1100 LC/MSD spectrometer (Agilent, Waldbronn, Germany).
4.1.1. methyl 5-chloro-1H-indole-2-carboxylate (2)
To a solution of 5-Chloroindole-2-carboxylic acid (5g, 25.6 mmol, 1 eq) in 30 mL of MeOH
was added SOCl2 (3.7g, 30.8mmol, 1.2 eq) dropwise within 30 min at 0 . Subsequently, the
reaction mixture was stirred at 35
for 2 h. Then, the solvent was evaporated under reduced
pressure, and the residue was purified by column chromatography using ethylacetate/petroleum,
1
giving intermediate 2 as a yellow solid, yield 93%. m.p. 212.2-214.7 . H NMR (400 MHz,
CDCl3) δ 8.95 (s, 1H, NH), 7.67 (s, 1H, ArH), 7.35 (d, J = 8.7 Hz, 1H, ArH), 7.31-7.25 (m, 2H,
ArH, ArCH), 3.95 (s, 3H, OCH3); 13C NMR (100 MHz, CDCl3) δ 162.1, 135.1, 128.4, 126.6,
126.0, 121.8, 112.98, 112.93, 108.1, 52.2.

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4.1.2. 1-benzyl-5-chloro-1H-indole-2-carboxylic acid (3a-b)
To a stirred suspension of NaH (60 percent dispersion inmineral oil; 0.7 g, 17.3 mmol, 1.2 eq)
in DMF (10 mL) was added dropwise solution of 1 (3g, 14.4 mmol, 1 eq) in DMF (30 mL) at 0
,
and the mixture was stirred at room temperature for 30 min. To the mixture was added dropwise
4-Chlorobenzyl chloride or 4-Fluorobenzyl chloride (14.4 mmol, 1 eq), and the resulting
mixturewas stirred at room temperature for 30 min. The reaction was quenched with water and
acidified with acetic acid. The formed precipitate was filtered, washed with water, and dried under
vacuum to obtain the gray solid.
To a solution of the above compounds (7.8 mmol, 1 eq) in ethanol (30 ml), 10 % NaOH
solution (15 ml) was added with stirring for 15 min at 75 . The residue after removal of ethanol
under reduced pressure was then taken into water and acidified with concentrated hydrochloric
acid. The formed precipitate was filtered, washed with water, and dried under vacuum to give 3a-b
as white solid.
4.1.2.1. 5-chloro-1-(4-chlorobenzyl)-1H-indole-2-carboxylic acid (3a)
Compound 3a was synthesized according to the synthetic procedure given above as white
solid in yield of 85%, 1H-NMR (400 MHz, DMSO-d6) δ 7.80 (d, J = 2.0 Hz, 1H, ArH), 7.61 (d, J
= 8.9 Hz, 1H, ArH), 7.37 – 7.29 (m, 4H, ArH, ArCH=), 7.05 – 7.01 (m, 2H, ArH), 5.87 (s, 2H,
NCH2Ar), COOH (none); ESI-Mass for C16H11Cl2NO2: m/z (M+H)+ 317.7
4.1.2.1. 5-chloro-1-(4-fluorobenzyl)-1H-indole-2-carboxylic acid (3b)
Compound 3b was synthesized according to the synthetic procedure given above in yield of
86%, m.p. 202.8-205.2; 1H-NMR (400 MHz, DMSO-d6) δ: 7.79 (d, J = 2.1 Hz, 1H, ArH), 7.62 (d,
J = 8.7 Hz, 1H, ArH), 7.30 (s, 1H, ArCH=), 7.29 (dd, J = 2.0, 8.7 Hz, 1H, ArH), 7.01 - 7.09 (m,
4H, ArH), 5.87 (s, 2H, NCH2Ar), COOH (none); ESI-Mass for C16H11ClFNO2: m/z (M+H)+ 301.8
4.1.3. 2,3,5-trimethyl-6-(piperazin-1-ylmethyl)pyrazine (5)
Anhydrous piperazine (3.0 g, 34.9 mmol, 6 eq) was dissolved in dichloromethane (60 mL).
2-(chloromethyl)-3,5,6-trimethylpyrazine (0.99g, 5.8 mmol, 1 eq) in dichloromethane (30 mL)
was added dropwise to the solution at room temperature . The reaction mixture was stirred for

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about 4 h. Then, the mixture was washed with saturated K2CO3 solution (30 mL × 3) and water
(30 mL × 3). The organic layer was dried over anhydrous Na2SO4. The filtrate was evaporated
under reduced pressure and desired products 5. ESI-Mass for C16H11ClFNO2: m/z (M+H)+ 221.11.
4.1.4. tert-butyl3-(((3,5,6-trimethylpyrazin-2-yl)methyl)amino)pyrrolidine-1-carboxylate (6)
1-Boc-3-aminopyrrolidine (1.08 g, 5.8 mmol, 1 eq) was dissolved in THF (60 mL).
2-(chloromethyl)-3,5,6-trimethylpyrazine (1g, 5.8 mmol, 1 eq) in THF (30 mL) was added
dropwise to the solution at room temperature . The reaction mixture was stirred at reflux for 4
hours. Then, the mixture was evaporated under reduced pressure after filtered. Obtaining oil 6 for
the next reaction
4.1.5. N-((3,5,6-trimethylpyrazin-2-yl)methyl)pyrrolidin-3-amine (7)
To a stirred solution of 6 (1g, 3.1 mmol, 1 eq) in dichloromethane (20 mL) was added
dropwise trifluoroacetic acid (3.5 g, 31 mmol, 10 eq), and the mixture was stirred at room
temperature for 12 h. The progress of the reaction was monitored by TLC. After concentration at
reduced pressure, the residue was purified by flash column chromatography
(methanol/dichloromethane = 1:20 - 1:10) afforded the title compound 7.
4.1.6. (1-benzyl-5-chloro-1H-indol-2-yl)(4-((3,5,6-trimethylpyrazin-2-yl)methyl)piperazin-1-yl)
methanone (8a-b)
To a stirred solution of 3a-b (1.65 mmol,1 eq.) in DMF (25 ml ), HOBT (3.3 mmol,2 eq.) and
EDCI ( 4.12 mmol 2.5 eq.) was added at 25 . After 15 min, 6 (0.36 g 1.65 mmol,1 eq.) dissolved
in DMF (10 mL) was added. The reaction mixture was continued to keep at 25 with stirring for
4 h. A saturated solution of citric acid was slowly added with stirring for an additional 30 min
followed by addition of H2O and EtOAc. The organic layer was washed with brine, dried over
anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by flash
column chromatography (PE/EA = 5:1 - 2:1 ) afforded the title compounds (8a-b).
Compounds 8a-b were synthesized according to the synthetic procedure given above (See
details in Supplemental Data).

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4.1.7. (1-benzyl-5-chloro-1H-indol-2-yl)(3-(((3,5,6-trimethylpyrazin-2-yl)methyl)ami-no)
pyrrolidin-1-yl)methanone (9a-b)
To a stirred solution of 3a-b (1.65 mmol,1 eq.) in DMF (25 ml ), HOBT (3.3 mmol,2 eq.) and
EDCI (4.12 mmol, 2.5 eq.) was added at 25 . After 15 min, 8 (0.36 g, 1.65 mmol, 1 eq) dissolved
in DMF (10 mL) was added. The reaction mixture was continued to keep at 25 with stirring for
4 h. A saturated solution of citric acid was slowly added with stirring for an additional 30 min
followed by addition of H2O and EtOAc. The organic layer was washed with brine, dried over
anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by flash
column chromatography (PE/EA= 5:1 - 2:1 ) afforded the title compounds (9a-b).
Compounds 9a-b were synthesized according to the synthetic procedure given above (See
details in Supplemental Data).
4.1.8. 1-benzyl-5-chloro-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-1H-indole-2-carboxa-mide (10a-
b)
To a stirred solution of 3a-b (1.65 mmol,1 eq) in DMF (25 ml), HOBT (3.3 mmol,2 eq) and
EDCI ( 4.12 mmol, 2.5 eq ) was added at 25 . After 15 min, 5-Methoxytryptamine (0.31 g, 1.65
mmol, 1 eq) dissolved in DMF (10 mL) was added. The reaction mixture was continued to keep at
25 with stirring for 4 h. A saturated solution of citric acid was slowly added with stirring for an
additional 30 min followed by addition of H2O and EtOAc. The organic layer was washed with
brine, dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was
purified by flash column chromatography (PE/EA = 5:1-2:1) afforded the title compound (10a-b).
Compounds 10a-b were synthesized according to the synthetic procedure given above (See
details in Supplemental Data).
4.1.9. (1-benzyl-5-chloro-1H-indol-2-yl)(4-benzylpiperazin-1-yl)methanone (12a-n)
To a stirred solution of 3a-b ( 1.65mmol,1 eq ) in DMF (25 ml ), HOBT ( 3.3 mmol,2 eq )
and EDCI (4.12 mmol 2.5 eq) was added at 25 . After 15 min, monosubstituted benzyl
piperazine (11a-n) (1.65 mmol,1 eq) dissolved in DMF (10 mL) was added. The reaction mixture
was continued to keep at 25 with stirring for 4 h. A saturated solution of citric acid was slowly
added with stirring for an additional 30 min followed by addition of H2O and EtOAc. The organic

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layer was washed with brine, dried over anhydrous Na2SO4 and evaporated under reduced
pressure. The residue was purified by flash column chromatography (PE/EA = 5:1-2:1) afforded
the title compound (12a-n).
Compounds 12a-n were synthesized according to the synthetic procedure given above (See
details in Supplemental Data).
4.2. Biological activity
4.2.1. DNFB-induced mice auricle edema bioassay
Materials: male ICR mice weighing (18 - 22 g); 1% DNFB solution (composed by 0.1 mL
DNFB solution,
8
mL acetone and
2
mL olive oil); Dexamethasone; sodium
carboxymethylcellulose solution (CMC-NA) were provided by Hefei Yigong Medicine Co., Ltd.
Experimentation: animals were randomly divided into 9 groups according to weights, 6 in
each group. They were were normal group, model group, positive group and other 6 compound
groups. The normal group and the model group were given the same dose of the normal saline.
The positive group was given dexamethasone 0.5 mg/kg, and the compound groups were given
compound 5 mg/kg, respectively. All drugs were adiministered by intragastric administration. The
drug was administered once a day for 6 days. After 7 days of adaptive feeding, the normal group
did not do any treatment. In the other groups, the right ear of the mice were uniformly coated with
10 uL of 1 % DNFB solution, and the left ear was coated with the same amount of solution of
acetone and olive oil for comparison. After 24 hours, the animals were sacrificed, and round ear
slices were cut off from the same sites of left and right ears by 8 mm hole puncher, and weighed to
calculate the swelling degree and inhibition rate. Calculation was carried out according to the
following equations.
swelling degree (mg) = VR - VL
Inhibitory rate of ear swelling (%) = ((VR - VL) control - (VR - VL) treated) / (VR - VL)
control * 100%
VR = Average weight of the right ear; VL = Average weight of the left ear
4.2.2. In vitro COX-1/COX-2 inhibition assay
The inhibitory COX activity of the tested compounds and the reference was assayed using

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COX-1 Inhibitor Screening Kit (Fluorometric) (BioVision, Inc., Mountain View, CA, USA) ；
COX-2 Inhibitor Screening Kit (Beyotime Biotech Co., Ltd, China) according to the
manufacturer’s instructions. COX-1/COX-2 Assay Buffer (398 µL), COX-1 Cofactor (2 µL) and
Arachidonic acid solution were placed in a 96-well plate. The test compounds were added to
above solution. The plate incubated for 5 min at 37 ℃ and fluorescence was measured. The
excitation wavelength was 535 nm and the emission wavelength was 587 nm.
The recording groups were divided into RFU (Relative Fluorescence Unit) blank control,
RFU 100% enzyme activity control, RFU positive drug control, and RFU test compound.
Calculation of the inhibition rate was carried out according to the following equations
Inhibition rate (%) = (RFU (enzyme) - RFU (test compound / positive)) / (RFU (enzyme) -
RFU (blank) * 100%
RFU (enzyme) = RFU 100% enzyme activity control; RFU (test compound) = RFU test
compound; RFU (blank) = RFU blank control; RFU (positive) = RFU positive drug control.
The assays were performed in triplicate and the IC50 values were calculated from the
concentration curves.
4.2.3. In vitro 5-LOX inhibition assay
The inhibitory activity of the test compounds against soya bean 5-LOX was evaluated by
using Abnova lipoxygenase assay kit (catalog No. KA1329, Cayman Chemicals). IC50 values of
the tested compounds were carried out according to procedures and instructions given with the
assay kit and in accordance to previously reported methods
4.2.4. In vitro cytotoxicity
RAW264.7 cell line was purchased from the American Tissue Culture Collection (ATCC,
Manassas, VA, USA).
Suspensions of tested cell lines (ca. 1.0 × 105 cells/ml) were placed in 96-well microtiter
plates and incubated with serum-free medium for 2 h at 37℃ and 5% CO2. Subsequently, the
supernatant was discarded and incubate with RPMI 1640 medium containing different
concentrations of compounds (concentration: 0 µM, 5 µM, 10 µM, 20 µM, 30 µM, 40 µM, 50 µM)
for 24 h. Four hours before the end of incubation, add 20 µL of MTT solution (5 mg/mL) to each

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well. After the incubation, the 96-well plate was centrifuged at 1500 rpm for 3 minutes, the
supernatant of each well was discarded, 150 µL of DMSO was added to each well, and the cell
shaker was shaken for 10 min. After the crystals were sufficiently dissolved, the OD570 was
measured by a microplate reader.
4.2.5. Docking studies
The molecular docking simulation was performed using ANTODOCK 4.2.6 imbedded into
AutoDockTools - 1.5.6. The active sites were generated from the co-crystallized ligands (IMM
and SC-558)[23, 24] within COX-1 and COX-2 protein structures (PDB codes: 1PGF and 1CX2),
respectively. Polar hydrogens were added to all ligands and proteins with the AutoDock Tools
(ADT) program prior to docking with Autodock 4.2.6 program[25]. A grid box 40 Å in size was
centered on the active site of protein. This docking approach was validated by successful
pose-retrieval of the co-crystallized ligands of COX-2 crystal structures. All graphical
representations in Figs. 4 - 6 were rendered using PyMOL[26]
.
5. Conflict of interest
There are no conflicts of interest.
Acknowledgment
The authors are grateful to Hefei Yigong Medicine Co., Ltd for carrying out the in vivo
anti-inflammatory assay.

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●
A series of novel indole-2-amide compounds were designed, synthesized.
● Construct amide bond by ethylamine, pyrrolidine and piperazine.
● 8a, 10b, 12h, and 12l showed most potent in vivo anti-inflammatory activity.
● 10b and 12h showed significant suppressive activity for COX/5-LOX.
● Conducted molecular docking studies on compounds 10b and 12h.