Design of balanced COX inhibitors based on anti-inflammatory and/or COX-2 inhibitory ascidian metabolites

Article abstract of DOI:10.1016/j.ejmech.2019.07.016

The aim of this study was to design and synthesize COX-1/COX-2 balanced inhibitors incorporating the structural motifs of anti-inflammatory ascidian metabolites. We designed a series of substituted indole analogs that incorporate the key structures of the ascidian metabolites, herdmanines C and D. The synthesized analogs were tested for their inhibitory activity against COX-1 and COX-2, and compound 5m, which displayed balanced inhibition, was further evaluated for in vitro anti-inflammatory activity. Compound 5m suppressed the expression of pro-inflammatory factors, including iNOS, COX-2, TNF-α, and IL-6 in LPS-stimulated murine RAW264.7 macrophages. The reduction of PGE₂, NO, and ROS was also observed, together with the suppression of NF-κB, IKK, and IκBα phosphorylation. Our results characterized 5m as a COX-1/COX-2 balanced inhibitor that subsequently caused ROS inhibition and NF-κB suppression, and culminated in the suppression of iNOS, COX-2, TNF-α, and IL-6 expression.

Full text of DOI:10.1016/j.ejmech.2019.07.016

Accepted Manuscript
Design of balanced COX inhibitors based on anti-inflammatory and/or COX-2
inhibitory ascidian metabolites
Zhiran Ju, Mingzhi Su, Jongki Hong, Eun La Kim, Hyung Ryong Moon, Hae Young
Chung, Suhkmann Kim, Jee H. Jung
PII:
S0223-5234(19)30637-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.016
EJMECH 11513
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 May 2019
Revised Date: 5 July 2019
Accepted Date: 5 July 2019
Please cite this article as: Z. Ju, M. Su, J. Hong, E. La Kim, H.R. Moon, H.Y. Chung, S. Kim, J.H.
Jung, Design of balanced COX inhibitors based on anti-inflammatory and/or COX-2 inhibitory
ascidian metabolites, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.07.016.
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LPS
20 ng/mL)
5m (20 uM)
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Design of balanced COX inhibitors based on anti-inflammatory
and/or COX-2 inhibitory ascidian metabolites
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Zhiran Ju , Mingzhi Su , Jongki Hong , Eun La Kim , Hyung Ryong Moon , Hae Young
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Chung , Suhkmann Kim , Jee H. Jung
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College of Pharmacy, Pusan National University, Busan 46241, Republic of Korea
College of Pharmacy, Kyunghee University, Seoul 02447, Republic of Korea
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Center for Proteome Biophysics, Department of Chemistry, Pusan National University, Busan 46241,
Republic of Korea
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*Correspondence: jhjung@pusan.ac.kr (J.H.J.); +82-51-510-2803 (J.H.J.).
These authors equally contributed to this work
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Abstract: The aim of this study was to design and synthesize COX-1/COX-2 balanced
inhibitors incorporating the structural motifs of anti-inflammatory ascidian metabolites. We
designed a series of substituted indole analogs that incorporate the key structures of the
ascidian metabolites, herdmanines C and D. The synthesized analogs were tested for their
inhibitory activity against COX-1 and COX-2, and compound 5m, which displayed balanced
inhibition, was further evaluated for in vitro anti-inflammatory activity. Compound 5m
suppressed the expression of pro-inflammatory factors, including iNOS, COX-2, TNF-α, and
IL-6 in LPS-stimulated murine RAW264.7 macrophages. The reduction of PGE , NO, and
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ROS was also observed, together with the suppression of NF-κB, IKK, and IκBα
phosphorylation. Our results characterized 5m as a COX-1/COX-2 balanced inhibitor that
subsequently caused ROS inhibition and NF-κB suppression, and culminated in the
suppression of iNOS, COX-2, TNF-α, and IL-6 expression.
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Keywords: COX-1/COX-2, balanced inhibition, anti-inflammatory, NF-κB pathway, indole,
ascidian metabolite
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1. Introduction
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Although non-steroidal anti-inflammatory drugs (NSAIDs) are the most frequent choice
of treatment for inflammatory symptoms, they are often accompanied by undesirable side
effects on the renal, hepatic, and cardiovascular systems [1]. Therefore, the discovery of new
NSAIDs with better safety profiles remains a major challenge. Generally, two different
strategies are employed for the development of new NSAIDs. The first is the development of
inhibitors of the production of prostaglandin (PG) E and arachidonic acid metabolites, which
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are potent mediators of inflammation. Cyclooxygenases (COX-1 and -2) are essential for the
biosynthesis of these mediators [1,2,9]. The other is the development of inhibitors of iNOS
(inducible nitric oxide synthase). iNOS contributes to acute and chronic inflammation through
the production of nitric oxide as a cytotoxic inflammatory mediator [3-4]. Traditional
NSAIDs exhibit pharmacological action through the inhibition of cyclooxygenase (COX)
activity in vivo and the reduction of the biosynthesis of prostaglandins in local tissues. At
present, it is believed that NSAID inhibitory effects of COX is the basis of their
pharmacodynamic effect. Therefore, COX proteins are recognized as indispensable targets for
drug development [1,5-7]. Two isoforms of COX, with distinct physiological roles, are well
defined. COX-1, which is constitutively expressed in various tissues, is described as a
housekeeping enzyme that regulates normal cellular processes, such as gastric cytoprotection,
vascular homeostasis, platelet aggregation, and kidney function [7]. In contrast, COX-2
remains at very low levels in most tissues and is only rapidly upregulated during states of
inflammation, in which it causes the elevated production of prostanoids that occurs at sites of
disease and inflammation [5-7].
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Traditional non-selective COX inhibitors, such as aspirin, phenazone, and indomethacin,
are effective for the treatment of inflammatory diseases, but are also associated with major
drawbacks through the decrease in the cytoprotective action of the constitutive COX-1
isoform in the gastrointestinal tract leading to ulcerogenic, hepatic, and renal toxicity [7-9].
Therefore, COX-2 selective inhibitors, such as celecoxib, rofecoxib, and valdecoxib, were
developed as anti-inflammatory agents with reduced gastrointestinal side effects. However,
these COX-2 selective inhibitors were found to be associated with other side effects, such as

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cardiovascular disorders due to imbalance in the COX pathway [9-11]. These undesirable side
effects often resulted in their withdrawal from the market. Recently, Wallace et al. indicated
that the inhibition of COX-1 led to the upregulation of COX-2, which may counteract the
deleterious effects caused by COX-1 inhibition, such as gastric hypermobility and the
subsequent events caused by PG deficiency [7,8]. Furthermore, the inhibition of COX-1
significantly contributes to the resolution of inflammation [12]. In the process of ulcer healing,
COX-1 specific inhibitors, as well as COX-2 specific inhibitors, delay healing [12]. These
findings proved that although COX-2 indeed plays a pivotal role in the inflammatory process,
COX-1/COX-2 balanced inhibitors appear more favorable, in some respects, when the critical
side effects of either non-selective or selective inhibitors are considered [13-15].
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Herein, motivated by the anti-inflammatory ascidian metabolites, herdmanines (Fig. 1),
we designed and synthesized two series of indole derivatives of balanced inhibitory activity to
COX-1 and COX-2, with the expectation of producing effective anti-inflammatory agents
with an improved safety profile.
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2. Design strategy for COX inhibitors based on natural anti-inflammatory compounds
and clinically used NSAIDs
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In our previous work, new anti-inflammatory amino acid derivatives herdmanines C and
D were isolated from the ascidian Herdmania momus [16,17]. Herdmanines C and D showed
moderate suppressive effects on COX-2, iNOS, IL-6, and NO production [16]. Therefore,
herdmanines C and D were examined in silico for COX LBD (ligand binding domain) binding
by docking simulation, and they showed fair affinity to the ligand binding domain of COX-1
and 2 (see SI). The guanidyl moiety of herdmanine C and the carboxyl moiety of herdmanine
D showed similar hydrogen bondings as the carboxyl moiety of indomethacin. The carboxyl
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group of indomethacin works as a hydrogen bond donor and forms H bonds with Arg and
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Tyr of COX-2 LBD (Fig. 8B). The 2-imino-4-thiazolidinone moiety of darbufelone also
behaved as a polar head and formed hydrogen bondings with the LBD of COX. Classical
COX inhibitors like indomethacin and darbufelone are composed of polar head and

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hydrophobic tail. Therefore, the target molecules were designed to contain the acylhydrazone
moiety which may work as hydrogen bond donor as isosteres of guanidyl moiety of
herdmanine C, the carboxyl group of indomethacin, and the 2-imino-4-thiazolidinone moiety
of darbufelone. The acylhydrazone moiety is widely used as pharmacophore block for the
synthesis of a variety of novel bioactive compounds, and many hydrazone derivatives have
been claimed to possess anti-inflammatory activity, including COX inhibitory activity [20-24].
In addition to the acylhydrazone moiety, the target scaffold includes indole moiety and
halobenzyl or halobenzoyl moieties, in part to mimic the partial structures of herdmanine D
and indomethacin, and also to facilitate hydrophobic interactions with the ligand binding
domain of COX. The halobenzyl or halobenzoyl moieties are important for binding to the
COX active site, and for stabilization of the hydrophobic interactions with the key amino
acids in COX [25]. Therefore, synthetic derivatives were designed to incorporate
acylhydrazone, indole, and halobenzyl/halobenzoyl moieties (Fig. 1).
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Based on our original design, a hydrogen bond donor acylhydrazone moiety was placed
at the C-3 position of the indole and the physicochemical properties of the alkyl and aryl
groups were varied (4a–4u, Scheme 1). A chlorobenzyl moiety was introduced in 5a–5u and
the halobenzoyl moieties were introduced in 6a/6b at the N-1 position of indole.
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Fig. 1. Design of COX inhibitors incorporating the partial structures of herdmanines and traditional
NSAIDs. Dotted circles show tentative hydrogen bond donors.
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3. Results and Discussion
3.1. Chemistry
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The synthesis of the target compounds is depicted in Scheme 1. Starting from
indole-3-carboxylic acid (1), a methyl ester (2) was prepared (77%) by the esterification
reaction (90°C) with anhydrous methyl alcohol in the presence of a few drops of dilute
sulfuric acid. The 1H-indole-3-carbohydrazide (3) was prepared with a 96% yield by the
hydrazinolysis of 2 with 98% hydrazine hydrate in absolute ethanol. The indole
N-arylhydrazone derivatives (4a–4u) were synthesized by the condensation of 3 with various
substituted aldehydes in the presence of ethanol and a few drops of propionic acid [26], and
the reaction was straightforward in most of the cases. The resulting indole N-arylhydrazone
derivatives (4a–4u) were employed as starting compounds for the synthesis of
indolyl-N-substituted benzyl/benzoyl derivatives. The target compounds,
indolyl-N-substituted benzyl/benzoyl derivatives (5a–5u and 6a–6b), were prepared by the
reaction of 4a–4u with benzyl chloride and benzoyl chloride/bromide in the presence of NaH
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in DMF at room temperature for 24 h [9]. The final products were characterized by H-NMR,
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C-NMR, and mass spectrometric analysis.
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Scheme 1. Reagents and conditions: (i) dilute H SO , MeOH, reflux, 24 h; (ii) NH -NH .H O, EtOH,
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reflux, 2 h; (iii) RCHO/EtOH, propionic acid, reflux, 2.5 h; (iv) 4-Cl-PhCH Cl, NaH, DMF, R.T., 24 h;
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(v) 4-Cl-PhCOCl/4-Br-PhCOCl, NaH, DMF, R.T., 24 h.
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The geometry of the carbon-nitrogen double bond in 5a–5u and 6a–6b was determined
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as (E) through the comparison of H NMR data with those reported for N-acylhydrazone
derivatives [27, 28]. The chemical shift of the H-C=N hydrogen of 5a–5h (δ 7.49–7.55), 5i–
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5u and 6a–6b (δ 8.22–8.37) was almost identical to those of N-acylhydrazone derivatives
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[27, 28] with (E) geometry. The sterically stable E-isomer predominated over the sterically
hindered Z-isomer [29].
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3.2. Biological evaluations
3.2.1. Evaluation of COX-1 and COX-2 inhibitory activities
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The synthesized compounds (5a–5u and 6a–6b) were tested for their ability to inhibit
COX-1 and COX-2 by using an enzyme immunoassay (EIA). Of the tested compounds, the

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cyanophenyl derivative 5m showed substantial COX-1 and COX-2 inhibitory activities, with
IC values of 39.20 and 7.59 µM, respectively (Fig. 2, Table 1). Compounds 5f and 5u also
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showed better activity than other analogs, and this result may be expected from the docking
simulation (see SI). Of the branched alkyl, linear alkyl, and cycloalkyl derivatives, linear
alkyl derivative (5f) appears a better option. However, substantially long alkyl chain (5g)
would not be preferable. According to docking simulation, the COX-1 and COX-2 binding
affinities of 5m, 5f, and 5u were no less than others though the difference was not notable.
Compounds 6a and 6b showed diminished activity than 5m even though they share similar
skeleton with 5m except the N-benzyl/benzoyl substructure. This may be due to lower binding
affinities of 6a and 6b than that of 5m (see SI). The distinctively lower inhibitory activities of
similar phenyl substituted analogs (5j - 5s) compared to that of 5m would also be attributed to
lower binding affinities of 5j - 5s.
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The COX-1 inhibitory activity of 5m was milder than the tested NSAIDs (Table 1).
Whilst, the COX-2 inhibitory activity of 5m significantly potent (IC = 7.59 µM) and it was
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comparable with those of the tested NSAIDs. The COX inhibitory potency and COX-2
selectivity index (IC (COX-1)/IC (COX-2) of 5m were compared with COX-2 selective
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(celecoxib and Dup-697), COX-1 selective (SC-560), and commercial non-selective drugs
(Table 1). The COX-2 selectivity of 5m was higher than ibuprofen, aspirin, and mesalazine,
but it was much lower than that of celecoxib and Dup-697. The COX-2 selectivity of 5m was
similar to that of diclofenac. Notably, diclofenac is a widely used drug in inflammatory
diseases, but has been associated with only mild GI side effects in both clinical trials and
epidemiological studies [30-32]. In addition, diclofenac is devoid of cardiovascular side
effects that are characteristic of COX-2 selective inhibitors such as celecoxib. These results
suggested that the balanced COX-2 selectivity of 5m may be favorable for development of
NSAIDs with attenuated gastrointestinal and cardiovascular side effects. Subsequently, the in
vitro anti-inflammatory activity of 5m was examined at the cellular level by using RAW264.7
murine macrophages.
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Fig. 2. In-vitro COX-1 and COX-2 enzyme inhibition assay of compounds 5a–5u and 6a–6b. SC-560
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is a COX-1 selective inhibitor; DUP-697 and celecoxib (Cel.) are COX-2 selective inhibitors; Con:
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Table 1. In vitro COX-1/COX-2 inhibition (IC , µM) and selectivity index for compound 5m
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Note. : The result (IC , µM) is the mean of three determinations acquired using a COX fluorescent
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inhibitor screening assay kit (Cayman Chemical, MI, USA). : COX-2 selectivity index (COX-1
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was employed as a COX-1 inhibitor.
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3.2.2. Determination of in vitro cytotoxicity
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Prior to the cell-based anti-inflammatory assay, compound 5m was evaluated for its
toxicity to murine macrophages (RAW264.7), rat liver cells (Ac2F), and mouse fibroblast
cells (L929) to determine a suitable concentration for the anti-inflammatory assay. Compound
5m was almost non-toxic to all of these cell lines at concentrations of up to 50 µΜ for 24 h.
Therefore, murine macrophage cells (RAW264.7) were treated with 5m at concentrations below
20 µΜ and subjected to an anti-inflammatory assay.
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Fig. 3. Determination of the toxicity of 5m (10, 25, and 50 µM) to RAW264.7 murine macrophages, the mouse
fibroblast cell line L929, and rat liver Ac2F cells after exposure for 24 h.
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3.2.3. Compound 5m inhibited LPS-induced expression of pro-inflammatory cytokines in
RAW264.7 cells
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To gauge the in vitro anti-inflammatory effect of compound 5m, the protein expression
of the pro-inflammatory factors iNOS and COX-2 was examined by Western blotting. As
expected, LPS stimulation markedly increased iNOS and COX-2 protein expression, but this
increase was significantly and concentration-dependently downregulated by pretreatment of
cells with 5m (Fig. 4). Notably, compound 5m (20 µΜ) attenuated the protein expression of
both iNOS and COX-2 more potently than dexamethasone (10 µΜ).

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Fig. 4. Effects of 5m on LPS-induced iNOS (A) and COX-2 (B) protein expression in RAW264.7 cells.
The cells were treated with different concentrations of 5m, and then cultured in the presence or absence
of LPS (1 µg/mL) for 24 h. Dexamethasone (DEX) was employed as the positive control (10 µM). The
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results were shown as the mean ± SD (n=3) of three independent experiments. p < 0.001 compared
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with the control group; p < 0.01, p < 0.001 compared with the LPS-stimulated group.
During inflammation, pro-inflammatory stimuli induce the production of various
cytokines and inflammatory mediators, such as nitric oxide (NO), prostaglandin E (PGE ),
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tumor necrosis factor α (TNF-α), and interleukin-6 (IL-6). As compound 5m inhibited iNOS
expression, it would, therefore, decrease NO production in macrophages. The amount of NO
in RAW264.7 cell supernatants was measured using Griess reagent. As shown in Figure 5A,
the NO production decreased in a concentration-dependent manner by compound 5m. As an
inflammatory mediator, a high production of PGE will result from inflammatory stimuli. As
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production of PGE , as shown in Figure 5D.
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In addition, the production of TNF-α and IL-6 was determined by enzyme-linked
immunosorbent assay (ELISA). TNF-α and IL-6, which the inflammatory cytokines, are part
of the host response to inflammatory situations and maintain normal cellular conditions [33].
As shown in Figures 5B and 5C, the amount of pro-inflammatory mediators was markedly
increased when RAW264.7 murine macrophages were exposed to LPS, but this increase was
prevented by 5m in a dose-dependent manner. These results indicated that 5m clearly
attenuated an excessive immune reaction in LPS-stimulated RAW264.7 cells.

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Fig. 5. Effects of compound 5m on the production of (A) nitric oxide (NO), (B) interleukin-6 (IL-6),
(C) tumor necrosis factor α (TNF-α), and (D) prostaglandin E (PGE ) in LPS-induced RAW264.7
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macrophages. The cells were pretreated with different concentrations of 5m (2.5, 5, or 10 µM) for 1 h
and then treated with LPS. NO concentration (LPS, 25 ng/mL, 24 h) in the medium was determined by
the Griess method, and the concentration of IL-6 (LPS, 100 ng/mL, 24 h), TNF-α (LPS, 100 ng/mL, 3
h), and PGE (LPS, 100 ng/mL, 24 h) in the medium was determined by ELISA. The results shown are
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representative of three independent experiments. p < 0.001 vs untreated controls. p < 0.01, p <
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3.2.4. Compound 5m reduced ROS levels in RAW264.7 cells
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Reactive oxygen species (ROS) are chemically reactive chemical species containing
oxygen. In a biological system, ROS are formed as a natural byproduct of the normal
metabolism of oxygen and have important roles in cell signaling. ROS are also generated as a
byproduct of prostaglandin biosynthesis from arachidonic acid by COX-2 [34].
Meanwhile, it is commonly accepted that the state of inflammation induces ROS
production and then influences transcription through the regulation of the phosphorylation of
transcription factors. Among the transcription factors, nuclear factor-kappa B (NF-κB) is
involved in the regulation of pro-inflammatory genes, which represents a key step in the
production of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α),

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interleukin-6 (IL-6), iNOS, and cyclooxygenase-2 [34,35]. Therefore, we detected and
quantified cellular oxidative stress. As shown in Figures 6A and 6B, compound 5m
significantly and dose-dependently reduced ROS production compared with the
LPS-treatment group. Notably, at a concentration of 20 µΜ, compound 5m significantly
decreased ROS production with a potency comparable with that of 20 µΜ diclofenac.
Therefore, we speculated that 5m inhibited NF-κB activity through the reduction of ROS and
the subsequent blocking of the NF-κB transcriptional activity of the pro-inflammatory
molecules.
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Fig. 6. Reduction of ROS in RAW264.7 cells treated with 5m and diclofenac (Dic). (A) LPS (1
µg/mL) induced intracellular ROS, which is indicated as green fluorescence by the fluorescent probe,
DCFH-DA. Through treatment with 5m or diclofenac (Dic) (10 µM), ROS expression was
significantly decreased. (B) The fluorescence intensity was quantified by using a fluorescence
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microplate reader. p < 0.001 vs untreated controls. p < 0.001 vs LPS-treated cells.
3.2.5. LPS-induced NF-κB signaling was repressed by treatment with 5m
To investigate further the possible anti-inflammatory mechanism of 5m, the effect of 5m
on the NF-κB pathway was examined. In the present study, we first used Western blotting to
investigate the induction of NF-κB p65 phosphorylation by treatment with 5m in
LPS-activated RAW264.7 cells. Cell extracts were collected after LPS stimulation for 30 min

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to detect the early state of NF-κB p65 signaling. As shown in Figure 7B, the levels of
phosphorylated NF-κB p65 were clearly increased by LPS treatment, but the treatment of 5m
repressed NF-κB p65 phosphorylation in a dose-dependent manner (Fig. 7B). The
phosphorylation of IκB (inhibitor of NF-κB) and IKK (IκB-kinase) is essential for the process
of the nuclear translocation and activation of NF-κB. Inflammatory stimuli, such as toxins,
pathogens, or oxidative stress, induce the phosphorylation of IKK. After phosphorylation,
IKK is as an essential element in the promotion of IκBα phosphorylation, and phosphorylated
IκBα is then ubiquitinated and degraded to release active NF-κB. Subsequently, activated
NF-κB translocates to the nucleus to bind to DNA and promotes the expression of
pro-inflammatory factors [36]. As expected, LPS stimulation markedly increased the level of
phosphorylated IKK; however, it was obviously decreased by the treatment of 5m. In
addition, the level of phosphorylated IκBα was also reduced compared with the LPS-treated
group (Fig. 7C and 7D). Furthermore, the immunofluorescence assay showed that the
LPS-stimulated translocation of NF-κB into the nucleus was reduced by treatment with 5m at
20 µΜ (Fig. 7A). These results indicated that 5m may exert its anti-inflammatory activity
through a reduction in ROS level and the suppression of NF-κB activation in RAW264.7 cells
(Fig. 9).

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Fig. 7. Effect of compound 5m on NF-κB activation in RAW264.7 macrophages. (A) NF-κB p65 was
visualized using confocal microscopy as green fluorescence and the cell nucleus was viewed as cyan
fluorescence by DAPI staining. (B) Phosphorylation of NF-κB. (C) Phosphorylation of IKK. (D)
Phosphorylation of IκBα. The cells were pretreated with compound 5m for 1 h and then stimulated
with lipopolysaccharide (LPS, 20 ng/mL) for 15 min; β-actin was used as the internal control. The
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##
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results shown are representative of three independent experiments. p < 0.01 vs untreated controls. p
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< 0.05, p < 0.01, p < 0.001 vs LPS-treated cells.
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3.3. Molecular docking study
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The COX-1 and COX-2 inhibitory effects and the in vitro anti-inflammatory activity of
5m led us to perform molecular docking studies to understand the ligand-protein interactions

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in detail. To gain insight into the interaction between 5m and COX enzymes, a docking
simulation was performed using ovine COX-1 (PDB ID: Ovis aries 1EQH) and murine
COX-2 (PDB ID: Mus musculus 4COX). The calculated docking poses of 5m were compared
with that of the indomethacin crystal-like pose with COX-2 [25].
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Generally, COX-1 and COX-2 share a 60% homology in amino acid sequence. However,
the conformation for the substrate-binding sites and the catalytic regions are slightly different.
The comparison of any COX-1 and any COX-2 enzymes demonstrates that COX-2 has a
larger and more flexible substrate channel than COX-1, and also that COX-2 has a larger
space at the site to which selective inhibitors could bind, which is composed of amino acids
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Val , Arg , and His (Fig. 8A) [12,25,37]. COX-2 selective inhibitors, such as SC-558, are
located deep in the large bioactive cavity and the specific moiety could form hydrogen bonds
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to Arg and His . In contrast, non-selective COX inhibitors, such as indomethacin and
ibuprofen, also bind in the long hydrophobic channel, but do not occupy the additional pocket
present in COX-2 [25].
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In addition, there are two important polar amino acids inside the active site in a bent
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narrow gap. The amino acids Arg and Tyr , which are located deeper in the active site,
play an important role in the stabilization of the carboxyl group of classical non-selective
NSAIDs in the hydrophobic COX channel. The carboxyl group of the drug forms a hydrogen
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bond with the guanidium group of Arg and Tyr in the binding site (Fig. 8B) [9,12,25].
Notably, compound 5m showed identical hydrogen bond poses with NSAIDs in COX-1 and
COX-2 (Fig. 8C and 8D), which indicated that the carboxylate group of the NSAIDs could be
replaced by an aryl-hydrazone moiety. These interactions were almost essential for
COX-1/COX-2 inhibitory activity, as exemplified by the binding interaction of indomethacin
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[25]. Meanwhile, compound 5m did not form hydrogen bonds with Arg and His . This
lack of binding may explain the non-significant selectivity of 5m to COX-2.

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Fig. 8. Docking and crystal structures of ligand/COX binding. (A) Zoomed view of the bioactive
pocket of the COX-2 (4COX) (the hydrophobic and hydrophilic regions of COX-2 are shown in red
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and white, respectively). An additional larger space is composed of amino acids of Val , Arg , and
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His . (B) Zoomed view of the crystal structure of the COX-2 bound to indomethacin (4COX). The
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carboxyl group of indomethacin forms hydrogen bonds with Arg and Tyr of COX-2 LBD. (C)
Zoomed view of the hydrogen bonding interactions between 5m and the surrounding amino acids,
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Arg and Tyr in COX-1 (1EQH). (D) Zoomed view of the hydrogen bonding interactions between
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5m and the surrounding amino acids, Arg and Tyr in COX-2 (4COX). 4COX is a murine COX-2
(PDB ID: Mus musculus 4COX) and 1EQH is an ovine COX-1 (PDB ID: Ovis aries 1EQH).
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4. Conclusion
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We designed and synthesized two series of new indole substituted derivatives 5a–5u and
6a–6b as COX inhibitors. These derivatives were tested for in vitro COX-1 and COX-2
inhibition. Compound 5m showed most the potent inhibition of both enzymes with higher
selectivity to COX-2, close to that of diclofenac. Compound 5m was, thus, selected as a
balanced COX inhibitor and evaluated for its in vitro anti-inflammatory activity. Compound
5m exerted anti-inflammatory activity through the suppression of the expression of
pro-inflammatory factors, including iNOS, NO, COX-2, PGE , TNF-α, and IL-6 in
2
LPS-stimulated murine RAW264.7 macrophages. A reduction in ROS was also observed.
Compound 5m decreased the phosphorylation of IKK, IκBα, and NF-κB. A possible
anti-inflammatory mechanism of 5m was proposed based on the in vitro results. Compound
5m may first inhibit COX-2, leading to a decrease in ROS production. Decreased ROS
production culminates in the suppression of NF-κB activation and endonuclear translocation,
thereby attenuating the expression of pro-inflammatory mediators, such as iNOS, COX-2,
TNF-α, and IL-6 (Fig. 9). These results may have important implications for the therapeutic
potential of 5m, and compound 5m may serve as a potential anti-inflammatory lead. Further
investigation on the comparison of side effects with NSAIDs, especially non-selective and
selective COX inhibitors, may be of value.
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Fig. 9. A diagram of the speculative anti-inflammatory mechanism of the COX inhibitor 5m in

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LPS-induced RAW264.7 cells.
5. Experimental section
5.1. Chemistry
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All reagents used were commercially available; some organic solvents were redistilled
under a positive pressure, if necessary. Reactions were monitored by thin-layer
chromatography (TLC) on glass plates coated with silica gel using a fluorescent indicator
1
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(GF254, Merck, Germany). The H and C NMR spectral data were recorded on Varian Unity
500 MHz and 400 MHz NMR spectrometers, respectively. The high-resolution fast-atom
bombardment mass spectrometry (HRFABMS) data were obtained by using an Agilent 1200
UHPLC accurate-mass Q-TOF MS spectrometer. All chemical reagents were purchased from
Sigma-Aldrich and Alfa Aesar.
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5.1.1. Synthesis of compounds 5a–5u
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General procedure for 2: A solution of indole-3-carboxylic acid (1.0 mmol-equiv.) and the
appropriate anhydrous methyl alcohol (25 mL) were refluxed in the presence of a few drops
of sulfuric acid (97%) for 24 h. The progress of the reaction was monitored by TLC (Silica
gel 60F , Merck, Germany). Precipitated 1H-indole-3-carboxylate methyl ester was filtered
254
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and recrystallized from an ethanol-water (3:2) mixture.
1
5.1.1.1. Methyl H-indole-3-carboxylate 2. Pale yellow solid after recrystallization; 77% yield
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3
3
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H-NMR (500 MHz, DMSO-d , δ = ppm) δ 11.94 (s, 1H), 8.09 (s, 1H), 8.02 (d, J = 7.5
6
1
3
Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.26–7.14 (m, 2H), 3.81 (s, 3H). C-NMR (125 MHz,
DMSO-d , δ = ppm) δ 164.8, 136.4, 132.4, 125.7, 122.4, 121.3, 120.4, 112.4, 106.3, 50.6.
6
+
HRFABMS m/z 176.0673 [M+H] (calcd for C H NO , 176.0706).
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General procedure for 4a–u: 1H-Indole-3-carboxylate methyl ester 2 (1.0 mmol-equiv.) was
refluxed with hydrazine hydrate (12.5 g, 0.25 M) in appropriate ethanol (30 mL) for 2 h. The
progress of the reaction was monitored by TLC. After cooling the reaction mixture to room
temperature, the mixtures were filtered to give white solid crude products without purification.

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Next, indole-hydrazide (3, 1.0 mmol) in ethanol (30 mL) was added dropwise into the
appropriate aldehyde (1.5 mmol-equiv.) and a few drops of propionic acid; the mixture was
stirred and refluxed for 2.5 h. After cooling, the precipitates were filtered and washed several
times by methanol to yield the crystal substances 4a–u.
General procedure for 5a–u/6a–b: A solution of 4a–u (1 mmol) in DMF (10 mL) was added
to the solution of sodium hydride (NaH) (1.5 mmol) in DMF (10 mL) and the reaction
mixture was stirred for 1 h at room temperature. Benzyl chloride/benzoyl chloride (1.5 mmol)
was added, and the reaction mixture was stirred for 24 h at room temperature. The reaction
mixture was poured into ice water, filtered off, and then washed several times with methanol
to yield the target compounds 5a–u/6a–b.
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5.1.1.1. (E)-1-(4-Chlorobenzyl)-N'-(2-methylpropylidene)-1H-indole-3-carbohydrazide 5a.
Pale yellow solid after recrystallization; 35% yield:
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H-NMR (500 MHz, DMSO-d , δ = ppm) δ 10.92 (s, 1H), 8.20 (s, 1H), 7.54 (d, J = 7.9
6
Hz, 1H), 7.41 (m, 2H), 7.28 (d, J = 8.1 Hz, 2H), 7.18 (m, 2H), 7.17 (m, 2H), 5.49 (s, 2H),
1
3
2.53 (m, 1H), 1.06 (d, J = 6.5 Hz, 6H). C-NMR (125 MHz, DMSO-d , δ = ppm) δ 164.1,
6
155.2, 136.0, 132.3, 129.3, 128.7 (×6), 122.4, 121.0 (×2), 110.6 (×2), 48.7, 30.9, 19.7 (×2).
+
HRFABMS m/z 354.1358 [M+H] (calcd for C H ClN O, 354.1368).
2
0
20
3
3
3
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5.1.1.2. (E)-1-(4-Chlorobenzyl)-N'-(2,2-dimethylpropylidene)-1H-indole-3-carbohydrazide
5b. Pale yellow solid after recrystallization; 55% yield:
1
3
3
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95
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H-NMR (500 MHz, DMSO-d , δ = ppm) δ 10.87 (s, 1H), 8.22 (s, 1H), 7.55 (d, J = 7.8
6
Hz, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 7.7 Hz, 2H), 7.19 (m, 2H), 7.17 (m, 2H), 5.48
1
3
(s, 2H), 1.07 (s, 9H). C-NMR (125 MHz, DMSO-d , δ = ppm) δ 165.6, 156.1, 135.9, 132.3,
6
129.3, 128.7 (×6), 122.4, 121.0 (×2), 110.6 (×2), 48.7, 34.4, 19.7 (×3). HRFABMS m/z
+
368.1525 [M+H] (calcd for C H ClN O, 368.1524).
2
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22
3
3
4
99
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5.1.1.3. (E)-1-(4-Chlorobenzyl)-N'-(2-methylbutylidene)-1H-indole-3-carbohydrazide 5c. Pale
yellow solid after recrystallization; 42% yield:
1
4
4
01
02
H-NMR (500 MHz, DMSO-d , δ = ppm) δ 10.95 (s, 1H), 8.20 (s, 1H), 7.54 (d, J = 7.9
6
Hz, 1H), 7.42 (m, 2H), 7.28 (d, J = 8.3 Hz, 2H), 7.20 (m, 2H), 7.17 (m, 2H), 5.50 (s, 2H),

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2.31 (m, 1H), 1.50 (m, 1H), 1.40 (m, 1H), 1.05 (d, J = 6.2 Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H).
13
C-NMR (125 MHz, DMSO-d , δ= ppm) δ 164.3, 153.9, 136.3, 132.3, 129.3, 128.7 (×6),
6
+
122.4, 121.1 (×2), 110.6 (×2), 48.7, 37.6, 27.0, 17.3, 11.4. HRFABMS m/z 368.1525 [M+H]
(calcd for C H ClN O, 368.1524).
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4
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5.1.1.4. (E)-1-(4-Chlorobenzyl)-N'-(cyclopropylmethylene)-1H-indole-3-carbohydrazide 5d.
Pale yellow solid after recrystallization; 60% yield:
1
4
4
4
4
4
4
09
10
11
12
13
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H-NMR (500 MHz, DMSO-d , δ = ppm) δ 10.91 (s, 1H), 8.15 (s, 1H), 7.49 (d, J = 8.0
6
Hz, 1H), 7.39 (m, 2H), 7.24 (d, J = 8.5 Hz, 2H), 7.16 (m, 2H), 7.14 (m, 2H), 5.48 (s, 2H),
1
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1.66 (m, 1H), 0.86 (dd, J = 8.1, 2.2 Hz, 2H), 0.63 (dd, J = 6.6, 4.1 Hz, 2H). C-NMR (125
MHz, DMSO-d , δ = ppm) δ 165.3, 154.1, 136.2, 132.2, 129.1 (×2)
,
128.6 (×4), 122.3, 121.5,
6
+
120.9 (×2), 110.5 (×2), 48.7, 13.4, 5.8 (×2)
C H ClN O, 352.1211).
.
HRFABMS m/z 352.1205 [M+H] (calcd for
20
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4
4
15
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5.1.1.5. (E)-1-(4-Chlorobenzyl)-N'-(cyclohexylmethylene)-1H-indole-3-carbohydrazide 5e.
Pale yellow solid after recrystallization; 45% yield:
1
4
4
4
4
4
4
17
18
19
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21
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H-NMR (500 MHz, DMSO-d , δ = ppm) δ 10.93 (s, 1H), 8.13 (s, 1H), 7.54 (d, J = 7.6
6
Hz, 1H), 7.41 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 7.6 Hz, 2H), 7.18 (m, 2H), 7.16 (m, 2H), 5.49
1
3
(s, 2H), 2.23 (m, 1H), 1.73 (m, 4H), 164 (m, 1H), 1.29 (m, 1H), 1.24 (m, 4H). C-NMR (125
MHz, DMSO-d , δ = ppm) δ 164.1, 152.8, 136.2, 132.3, 129.2, 128.6 (×4), 122.3 (×2), 121.5,
6
+
121.0 (×2), 110.5 (×2), 48.7, 29.8 (×2), 25.6, 25.1 (×3). HRFABMS m/z 394.1668 [M+H]
(calcd for C H ClN O, 394.1681).
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23
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5.1.1.6. (E)-1-(4-Chlorobenzyl)-N'-hexylidene-1H-indole-3-carbohydrazide 5f. Pale yellow
solid after recrystallization; 50% yield:
1
4
4
4
4
4
4
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26
27
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H-NMR (500 MHz, DMSO-d , δ = ppm) δ 10.96 (s, 1H), 8.19 (s, 1H), 7.52 (d, J = 7.9
6
Hz, 1H), 7.40 (m, 2H), 7.25 (m, 2H), 7.18 (m, 2H), 7.15 (m, 2H), 5.50 (s, 2H), 2.24 (m, 2H),
1
3
1.49 (m, 2H), 1.29 (m, 4H), 0.87 (t, J = 5.6 Hz, 3H). C-NMR (125 MHz, DMSO-d , δ =
6
ppm) δ 166.3, 153.8, 136.2 (×2), 132.2, 129.0 (×2), 128.6 (×2), 122.3 (×2), 121.5, 120.9 (×2),
+
110.5 (×2), 48.7, 31.7, 30.7, 25.7, 21.8, 13.7. HRFABMS m/z 382.1695 [M+H] (calcd for
C H ClN O, 382.1681).
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5.1.1.7. (E)-1-(4-Chlorobenzyl)-N'-nonylidene-1H-indole-3-carbohydrazide 5g. Pale yellow
solid after recrystallization; 55% yield:
1
4
4
4
4
4
4
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34
35
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H-NMR (500 MHz, DMSO-d , δ = ppm) δ 10.95 (s, 1H), 8.17 (s, 1H), 7.50 (d, J = 8.0
6
Hz, 1H), 7.38 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 7.16 (m, 2H), 7.15 (m, 2H), 5.48
(s, 2H), 2.23 (dd, J = 12.9, 7.3 Hz, 2H), 1.47 (m, 2H), 1.24 (m, 10H), 0.83 (t, J = 6.4 Hz, 3H).
13
C-NMR (125 MHz, DMSO-d , δ = ppm) δ 165.5, 154.5, 136.2 (×2), 132.2, 128.9 (×2),
6
128.5 (×2), 122.3×2, 121.5, 120.9×2, 110.5 (×2), 48.7, 31.7, 31.1, 28.7, 28.5, 26.0, 23.2, 21.9,
+
13.8. HRFABMS m/z 424.2143 [M+H] (calcd for C H ClN O, 424.2150).
2
5
30
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4
4
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40
5.1.1.8. (E)-1-(4-Chlorobenzyl)-N'-(3-phenylpropylidene)-1H-indole-3-carbohydrazide 5h.
Pale yellow solid after recrystallization; 65% yield:
1
4
4
4
4
4
4
41
42
43
44
45
46
H-NMR (500 MHz, DMSO-d , δ = ppm) δ 11.04 (s, 1H), 8.17 (s, 1H), 7.59 (s, 1H), 7.49
6
(d, J = 8.0 Hz, 1H), 7.37 (d, J = 7.7 Hz, 2H), 7.26 (m, 6H), 7.17 (m, 2H), 7.14 (m, 2H), 5.47
1
3
(s, 2H), 2.82 (t, J = 7.7 Hz, 2H), 2.57 (dt, J = 12.7, 6.3 Hz, 2H). C-NMR (125 MHz,
DMSO-d , δ= ppm) δ 163.8, 153.3, 141.1, 136.2 (×2), 135.9, 132.3, 129.1, 128.7 (×2), 128.4
6
(×2), 128.3 (×2), 125.9, 122.4 (×2), 121.5, 121.1 (×2), 110.6 (×2), 48.8, 33.6, 32.2.
+
HRFABMS m/z 416.1517 [M+H] (calcd for C H ClN O, 416.1524).
2
5
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3
4
4
47
48
5.1.1.9. (E)-N'-benzylidene-1-(4-chlorobenzyl)-1H-indole-3-carbohydrazide 5i. Pale yellow
solid after recrystallization; 45% yield:
1
4
4
4
4
4
49
50
51
52
53
H-NMR (500 MHz, DMSO-d , δ = ppm) δ 11.48 (s, 1H), 8.36 (s, 1H), 8.25 (s, 1H), 7.68
6
(s, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.44 (m, 5H), 7.34 (s, 2H), 7.22 (m, 2H), 7.21 (m, 2H), 5.56
13
(s, 2H). C-NMR (125 MHz, DMSO-d , δ = ppm) δ 164.4, 154.3, 136.2 (×2), 135.9, 134.6
6
(×2), 132.3, 129.5, 129.3, 128.8 (×2), 128.7 (×2), 126.7, 122.5 (×2), 121.5, 121.2 (×2), 110.6
+
(×2), 48.8. HRFABMS m/z 388.1198 [M+H] (calcd for C H ClN O, 388.1211).
2
3
18
3
4
4
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5.1.1.10. (E)-1-(4-Chlorobenzyl)-N'-(4-hydroxybenzylidene)-1H-indole-3-carbohydrazide 5j.
Pale yellow solid after recrystallization; 80% yield:
1
4
4
56
57
H-NMR (500 MHz, DMSO-d , δ = ppm) δ 11.70 (s, 1H), 11.26 (s, 1H), 8.23 (s, 1H),
6
8.20 (s, 1H), 7.66 (d, J = 8.6 Hz, 1H), 7.50 (m, 5H), 7.18 (m, 2H), 7.16 (m, 2H), 7.10 (d, J =

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8.7 Hz, 2H), 5.17 (s, 2H). C-NMR (125 MHz, DMSO-d , δ = ppm) δ 168.9, 159.2, 156.4,
6
135.8, 132.4, 129.4 (×3), 128.4 (×3), 128.2 (×2), 127.7, 122.1 (×2), 121.1, 120.6 (×2), 115.2
+
(×2), 111.8, 68.5. HRFABMS m/z 404.1165 [M+H] (calcd for C H ClN O , 404.1160).
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18
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4
4
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5.1.1.11. (E)-1-(4-Chlorobenzyl)-N'-(4-chlorobenzylidene)-1H-indole-3-carbohydrazide 5k.
Pale yellow solid after recrystallization; 85% yield:
1
4
4
4
4
4
4
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64
65
66
67
68
H-NMR (500 MHz, DMSO-d , δ = ppm) δ 11.46 (s, 1H), 8.30 (s, 1H), 8.22 (s, 1H), 7.67
6
(s, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.3 Hz, 2H), 7.42 (d, J = 8.3 Hz, 2H), 7.30 (d, J
1
3
= 7.3 Hz, 2H), 7.21 (m, 2H), 7.18 (m, 2H), 5.53 (s, 2H). C-NMR (125 MHz, DMSO-d , δ =
6
ppm) δ 166.1, 154.3, 136.1, 135.8, 133.8, 133.5, 132.3, 129.2 (×2), 128.8 (×2), 128.6 (×2),
128.2 (×2), 122.5 (×2), 121.4, 121.2 (×2), 110.6 (×2), 48.7. HRFABMS m/z 422.0828 [M+H]
(calcd for C H Cl N O, 422.0821).
+
2
3
17
2
3
4
4
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70
5.1.1.12. (E)-1-(4-Chlorobenzyl)-N'-(4-methoxybenzylidene)-1H-indole-3-carbohydrazide 5l.
Pale yellow solid after recrystallization; 85% yield:
1
4
4
4
4
4
4
71
72
73
74
75
76
H-NMR (500 MHz, DMSO-d , δ = ppm) δ 11.31 (s, 1H), 8.24 (s, 2H), 7.60 (s, 1H), 7.57
6
(d, J = 7.9 Hz, 1H), 7.45 (m, 2H), 7.33 (m, 2H), 7.22 (m, 2H), 7.20 (m, 2H), 7.01 (d, J = 8.3
1
3
Hz, 2H), 5.54 (s, 2H), 3.81 (s, 3H). C-NMR (125 MHz, DMSO-d , δ = ppm) δ 163.9, 160.4,
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154.3, 136.2 (×2), 135.2, 132.4, 129.3, 128.6 (×2), 128.2 (×2), 127.2, 122.4 (×2), 121.5, 121.1
+
(×2), 114.3 (×2), 110.5 (×2), 55.2, 48.7. HRFABMS m/z 418.1322 [M+H] (calcd for
C H ClN O , 418.1317).
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77
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5.1.1.13. (E)-1-(4-Chlorobenzyl)-N'-(4-cyanobenzylidene)-1H-indole-3-carbohydrazide 5m.
Pale yellow solid after recrystallization; 58% yield:
1
4
4
4
4
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4
79
80
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H-NMR (500 MHz, DMSO-d , δ = ppm) δ 11.49 (s, 1H), 8.32 (s, 1H), 8.23 (s, 1H), 7.64
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(m, 3H), 7.57 (d, J = 8.0 Hz, 1H), 7.44 (d, J = 8.2 Hz, 2H), 7.32 (d, J = 7.2 Hz, 2H), 7.22 (m,
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3
2H), 7.21 (m, 2H), 5.55 (s, 2H). C-NMR (125 MHz, DMSO-d , δ = ppm) δ 168.2, 157.4,
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139.8, 136.8, 136.6, 133.3 (×2), 133.1, 130.0, 129.4 (×4), 129.2, 127.9 (×2), 123.3, 122.1,
+
122.0 (×2), 119.3, 111.9, 111.4, 49.4. HRFABMS m/z 413.1168 [M+H] (calcd for
C H ClN O, 413.1164).
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5.1.1.14. (E)-N'-(4-bromobenzylidene)-1-(4-chlorobenzyl)-1H-indole-3-carbohydrazide 5n.
Pale yellow solid after recrystallization; 65% yield:
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H-NMR (500 MHz, DMSO-d , δ = ppm) δ 11.66 (s, 1H), 8.35 (s, 1H), 8.23 (s, 1H), 7.90
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(m, 1H), 7.86 (s, 2H), 7.58 (d, J = 8.2 Hz, 1H), 7.45 (d, J = 7.2 Hz, 2H), 7.32 (J = 7.7 Hz,
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2H), 7.23 (m, 2H), 7.21 (m, 2H), 5.56 (s, 2H). C-NMR (125 MHz, DMSO-d , δ = ppm) δ
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164.8, 155.7, 136.1 (×2), 135.8, 133.9, 132.3 (×2), 131.7, 129.2 (×2), 128.6 (×2), 128.5 (×2),
+
122.5 (×2), 121.5, 121.2 (×2), 110.6 (×2), 48.7. HRFABMS m/z 466.0314 [M+H] (calcd for
C H BrClN O, 466.0316).
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4
93
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5.1.1.15. (E)-4-((2-(1-(4-Chlorobenzyl)-1H-indole-3-carbonyl)hydrazono)methyl)benzoic acid
5o. Pale yellow solid after recrystallization; 78% yield:
1
4
4
4
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H-NMR (500 MHz, DMSO-d , δ = ppm) δ 11.61 (s, 1H), 8.36 (s, 1H), 8.25 (s, 1H), 7.82
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(s, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.49 (m, 2H), 7.43 (d, J = 8.4 Hz,
1
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1H), 7.32 (d, J = 7.8 Hz, 2H), 7.24 (m, 2H), 7.22 (m, 2H), 5.37 (s, 2H). C-NMR (125 MHz,
DMSO-d , δ = ppm) δ 166.3, 165.1, 155.2, 139.2, 136.1, 135.0, 132.3, 129.8 (×2), 129.7 (×2),
6
129.2, 128.6 (×2), 128.4 (×2), 126.8, 122.5 (×2), 121.4, 121.2 (×2), 110.6, 48.7. HRFABMS
+
m/z 432.0966 [M+H] (calcd for C H ClN O , 432.0964).
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5.1.1.16. (E)-1-(4-Chlorobenzyl)-N'-(4-fluorobenzylidene)-1H-indole-3-carbohydrazide 5p.
Pale yellow solid after recrystallization; 85% yield:
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5
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H-NMR (500 MHz, DMSO-d , δ = ppm) δ 11.43 (s, 1H), 8.31 (s, 1H), 8.23 (s, 1H), 7.72
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(s, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.44 (d, J = 8.2 Hz, 2H), 7.30 (m, 2H), 7.27 (d, J = 8.7 Hz,
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2H), 7.21 (m, 2H), 7.17 (m, 2H), 5.54 (s, 2H). C-NMR (125 MHz, DMSO-d , δ= ppm) δ
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165.6, 161.5, 156.9, 136.1, 132.3, 131.2, 129.2 (×2), 128.8 (×2), 128.7 (×2), 128.6 (×2),
+
122.4, 121.4, 121.1 (×2), 115.8, 115.6, 110.6 (×2), 48.7. HRFABMS m/z 406.1112 [M+H]
(calcd for C H ClFN O , 406.1117).
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5.1.1.17. (E)-1-(4-Chlorobenzyl)-N'-(3,5-dichlorobenzylidene)-1H-indole-3-carbohydrazide
5q. Pale yellow solid after recrystallization; 86% yield:
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H-NMR (500 MHz, DMSO-d , δ = ppm) δ 11.71 (s, 1H), 8.37 (s, 1H), 8.21 (s, 1H), 7.74
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(s, 2H), 7.58 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 7.7 Hz, 2H), 7.22 (m,
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2H), 7.20 (m, 2H), 5.55 (s, 2H). C-NMR (125 MHz, DMSO-d , δ = ppm) δ 164.5, 155.2,
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136.2 (×2), 134.5 (×2), 129.0 (×2), 128.9 (×2), 128.6 (×2), 128.4, 124.9 (×2), 122.5 (×2),
+
121.8, 121.2 (×2), 110.6 (×2), 48.7. HRFABMS m/z 456.0426 [M+H] (calcd for
C H Cl N O, 456.0286).
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5.1.1.18. (E)-1-(4-Chlorobenzyl)-N'-(3,5-dibromobenzylidene)-1H-indole-3-carbohydrazide
5r. Pale yellow solid after recrystallization; 75% yield:
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H-NMR (500 MHz, DMSO-d , δ = ppm) δ 11.71 (s, 1H), 8.36 (s, 1H), 8.21 (s, 1H), 7.88
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(s, 2H), 7.57 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 7.4 Hz, 2H), 7.22 (m,
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2H), 7.20 (m, 2H), 5.54 (s, 2H). C-NMR (125 MHz, DMSO-d , δ = ppm) δ 162.8, 157.8,
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140.8, 138.8, 136.2, 133.6, 132.3, 129.0 (×3), 128.6 (×3), 128.1 (×2), 122.8 (×2), 122.5, 121.2
+
(×2), 110.6 (×2), 48.7. HRFABMS m/z 543.9271 [M+H] (calcd for C H Br ClN O,
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3
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543.9276).
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5.1.1. 19.
(E)-N'-(3-chloro-5-methoxybenzylidene)-1-(4-chlorobenzyl)-1H-indole-3-Carbohydrazide 5s.
Pale yellow solid after recrystallization; 75% yield:
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H-NMR (500 MHz, DMSO-d , δ = ppm) δ 11.45 (s, 1H), 8.30 (s, 1H), 8.23 (s, 1H), 7.94 (d,
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J = 10.2 Hz, 1H), 7.65 (s, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 8.3 Hz, 2H), 7.32 (s, 2H),
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7.21 (m, 2H), 7.19 (m, 2H), 5.53 (s, 2H), 3.90 (s, 3H). C-NMR (125 MHz, DMSO-d , δ =
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ppm) δ 166.0, 156.2, 136.2 (×2), 132.3, 130.6, 129.1 (×2), 128.7, 128.6 (×4), 127.6, 122.4
+
(×2), 121.4, 121.1 (×2), 112.7, 111.1, 110.6, 56.4, 48.7. HRFABMS m/z 496.0259 [M+H]
(calcd for C H BrClN O , 496.0276).
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5.1.1.20.
(E)-1-(4-Chlorobenzyl)-N'-((4-methylthiazol-5-yl)methylene)-1H-indole-3-carbohydrazide 5t.
Pale yellow solid after recrystallization; 88% yield:
1
5
5
5
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H-NMR (500 MHz, DMSO-d , δ = ppm) δ 11.49 (s, 1H), 9.00 (s, 1H), 8.59 (s, 1H), 8.26
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(s, 1H), 8.20 (s, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.40 (d, J = 8.2 Hz, 2H), 7.29 (s, 2H), 7.19 (m,
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2H), 5.49 (s, 2H), 2.45 (s, 3H). C-NMR (125 MHz, DMSO-d , δ = ppm) δ 164.5, 154.1,
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153.0, 136.2, 132.4, 129.3, 128.8 (×4), 128.1 (×2), 122.6 (×2), 121.4, 121.3 (×2), 110.7 (×2),
+
48.8, 15.3. HRFABMS m/z 409.0873 [M+H] (calcd for C H ClN OS, 409.0884).
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5.1.1.21. (E)-1-(4-Chlorobenzyl)-N'-(furan-2-ylmethylene)-1H-indole-3-carbohydrazide 5u.
Pale yellow solid after recrystallization; 67% yield:
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49
H-NMR (500 MHz, DMSO-d , δ = ppm) δ 11.50 (s, 1H), 8.36 (s, 1H), 8.25 (s, 1H), 7.55
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(d, J = 7.7 Hz, 1H), 7.42 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 7.1 Hz, 2H), 7.21 (m, 2H), 7.20 (m,
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3
2H), 6.86 (d, J = 3.0 Hz, 1H), 6.63 (dd, J = 3.1, 1.6 Hz, 1H), 5.53 (s, 2H). C-NMR (125
MHz, DMSO-d , δ = ppm) δ 166.8, 149.8, 144.5, 136.1 (×2), 135.8, 132.3, 129.2 (×2), 128.6
6
+
(×4), 122.5 (×2), 121.5, 121.2 (×2), 112.0, 110.6, 48.8. HRFABMS m/z 378.0840 [M+H]
(calcd for C H ClN O , 378.0858).
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16
3
2
5
5
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5.1.1.22. (E)-1-(4-Chlorobenzoyl)-N'-(4-cyanobenzylidene)-1H-indole-3-carbohydrazide 6a.
Pale yellow solid after recrystallization; 88% yield:
1
5
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5
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H-NMR (500 MHz, DMSO-d , δ = ppm) δ 11.84 (s, 1H), 8.32 (s, 1H), 8.26 (s, 1H), 7.89
6
(s, 1H), 7.87 (m, 2H), 7.85 (m, 2H), 7.70 (m, 2H), 7.66 (s, 1H), 7.41 (m, 2H), 7.39 (m, 2H).
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C-NMR (125 MHz, DMSO-d , δ= ppm) δ 167.7, 163.5, 159.6, 132.5 (×3), 131.3, 128.9 (×6),
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127.4, 125.4 (×2), 124.7 (×2), 122.0, 118.5, 115.6 (×2), 111.7 (×2). HRFABMS m/z 427.0961
+
[M+H] (calcd for C H ClN O , 427.0956).
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2
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5
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5.1.1.23. (E)-1-(4-Bromobenzoyl)-N'-(4-cyanobenzylidene)-1H-indole-3-carbohydrazide 6b.
Pale yellow solid after recrystallization; 85% yield:
1
5
5
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5
5
59
60
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H-NMR (500 MHz, DMSO-d , δ = ppm) δ 11.88 (s, 1H), 8.36 (s, 1H), 8.29 (s, 1H), 7.90
6
(s, 1H), 7.88 (m, 2H), 7.87 (m, 2H), 7.83 (m, 2H), 7.72 (s, 1H), 7.46 (m, 2H), 7.43 (m, 2H).
13
C-NMR (125 MHz, DMSO-d , δ = ppm) δ 168.4, 160.2, 157.4, 145.3, 142.9, 139.3, 133.3
6
(×2), 132.6 (×5), 132.1, 128.0, 126.1, 125.4 (×2), 122.4, 119.2, 116.3 (×2), 112.4 (×2).
+
HRFABMS m/z 471.0441 [M+H] (calcd for C H BrN O , 471.0451).
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15
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5.2. Biological evaluation
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5.2.1.1. Cell culture

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RAW264.7 murine macrophages were purchased from the Korean Cell Line Bank
(KCLB®, Seoul, Korea) and rat liver Ac2F cells and mouse fibroblast cells (L929) were
obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells
were cultured at 37°C in a 5% CO humidified incubator and maintained in high glucose
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Dulbecco’s Modified Eagle Medium (DMEM, Nissui, Tokyo, Japan) supplemented with 100
mg/mL streptomycin, 2.5 mg/L amphotericin B, and 10% heat-inactivated fetal bovine serum
(FBS).
5.2.1.2. In vitro cytotoxicity assay
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Suspensions of tested cell lines ( 1.0 × 10 cells/well) were seeded in a 96-well culture
plates, cultured for 12 h, then treated with various diluted concentrations of 5m for 24 h. The
control cells were treated with culture medium alone. The test compounds were evaluated at
three dilutions and the highest concentration was 50 µM. Cell viabilities were evaluated by
using water soluble tetrazolium (WST) reagent (EZ-CyTox, Daeil Lab Service Co., Ltd.,
Seoul, Korea), which was added to each well (10 µL) and incubated at 37°C for 1 h. The
absorbance at 450 nm was read by using an iMark Microplate Absorbance Reader (Bio-Rad
Laboratories, Hercules, CA, USA). In all experiments, cells in the exponential phase were
used.
5.2.1. 3. Cyclooxygenase (COX) inhibition assays
The assay was performed by using a COX fluorescent inhibitor screening assay kit
(Cayman Chemical, MI, USA) in accordance with the manufacturer’s protocol, COX-1 (ovine)
and COX-2 (human recombinant) were utilized to catalyze arachidonic acid into PGG , which
2
is reacted with ADHP (10-acetyl-3,7-dihydroxyphenoxazine) to produce the highly
fluorescent compound resorufin. Resorufin fluorescence can be analyzed using an excitation
wavelength of 510 nm and an emission wavelength of 580 nm. In brief, 150 µL of assay
buffer, 10 µL heme, 10 µL enzyme (COX-1 or COX-2), and 10 µL vehicle were mixed in a
well to serve as 100% initial activity wells; 160 µL of assay buffer, 10 µL heme and 10 µL
vehicle were mixed in a well and served as background wells; 150 µL of assay buffer, 10 µL
heme, and 10 µL enzyme (COX-1 or COX-2) and 10 µL compounds were mixed in a well and

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served as inhibitor wells. The plate was incubated for 5 min at room temperature, and then 10
µL of ADHP was added to each well. To initiate the reaction, 10 µL of arachidonic acid
solution was quickly added to each well, incubated for 2 min at room temperature, and then
the fluorescence spectrum was measured by using an excitation wavelength of 510 nm and an
emission wavelength of 580 nm by TriStar LB 941 Multimode Microplate Reader (Bad
Wildbad, Germany).
5.2.1.4. Production of NO, TNF-α, IL-6, and PGE2
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RAW264.7 macrophages (cal. 1 × 10 cells/well) were seeded in a 96-well culture plate
and cultured for 12 h. The cells were pre-treated with various concentrations of drug for 1 h
and then co-incubated with 25 ng/mL LPS for 24 h. The NO concentrations in medium were
determined by using the Griess assay. Griess reagent (80 µL) was added to the medium
supernatants (80 µL) and then incubated at 37°C for 15 min in the dark. The absorbance at
520 nm was measured by using an iMark Microplate Absorbance Reader (Bio-Rad
Laboratories, Hercules, CA, USA). NO concentrations were calculated by using 0–100 µM
sodium nitrite standards. TNF-α and IL-6 expression in culture medium was quantified by
using a sandwich-type ELISA kit (Biolegend, San Diego, CA, USA) and the absorbance at
450 nm was measured. The production of prostaglandin E (PGE ) in medium was determined
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2
by using the PGE express ELISA kit (Cayman Chemical, MI, USA) in accordance with the
2
protocol and the absorbance at 415 nm was measured.
5.2.1.5. Reactive oxygen species (ROS) measurement
RAW264.7 macrophages were grown on a confocal dish and treated with the test
compound treatment for 1 h. LPS solution was added into the dish at a final concentration of 1
µg/mL and continually cultured for 24 h. The medium was removed and washed with PBS;
DCFH-DA diluted in FBS-free medium to a final concentration of 100 µM was added and
cultured at 37°C for 30 min. The medium was removed and the cells were washed once with
PBS. The fluorescence was viewed using a confocal microscope (FluoView FV10i; Olympus,
Australia) with an excitation wavelength of 485 nm and an emission wavelength of 520 nm.
The quantification of cellular oxidative stress was performed by using a slightly

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modified previously reported method [38]. RAW264.7 macrophages (cal. 1 × 10 cells/well)
were seeded into black 96-well cell culture plates and cultured for 12 h. The cells were
pre-treated with test compound for 1 h and then co-incubated with 1 µg/mL LPS for 24 h. The
medium was removed and washed with PBS; 100 µL DCFH-DA diluted in FBS-free medium
to a final concentration of 100 µM was added and cultured at 37°C for 30 min. The medium
was removed, 100 µL PBS was added to each well, and the fluorescence was detected at an
excitation wavelength of 485 nm and an emission wavelength of 520 nm by TriStar LB 941
Multimode Microplate Reader (Bad Wildbad, Germany).
5.2.1.6. Immunofluorescence staining of NF-κB p65 in RAW264.7 cells
The cells were grown on a confocal dish and treated with the test compounds for 24 h.
Subsequently, the cells were fixed in 10% formalin solution for 15 min, washed three times
with PBS, treated with 0.5% (v/v) Triton X-100/PBS for 15 min, washed three times with
PBS again, and then blocked at room temperature for 30 min in 10% FBS/PBS. The cells
were then incubated with rabbit anti-NFκB-p65 antibody (Cell Signaling Technology, USA)
at 4°C overnight, washed three times with PBS, incubated for 30 min at room temperature
with secondary antibody anti-rabbit Alexa 488 (Cell Signaling Technology, USA) as a
molecular probe, washed three times with PBS again, and then incubated with DAPI (5
µg/mL) at room temperature for 20 min. The localization of NFκB-p65 was observed by
using a confocal microscope (FluoView FV10i, Olympus, Australia) with an excitation
wavelength of 499 nm and an emission wavelength of 520 nm.
5.2.1.7. Western blotting
RAW264.7 cells were harvested and suspended in lysis buffer containing protease and
phosphatase inhibitor cocktails. The protein concentration was determined by using a BCA
protein assay (Thermo Scientific, Rockford, IL, USA). Equal amounts of proteins were
resolved by 10% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred
to polyvinylidene difluoride (PVDF) membranes. Non-specific binding to the membranes was
blocked by incubation of the membrane in Tris-buffered saline containing 0.1% Tween 20
(TBS-T) and 5% skimmed milk for 1 h at room temperature, and then incubated with specific