A competent synthesis and efficient anti-inflammatory responses of isatinimino acridinedione moiety via suppression of in vivo NF-κB, COX-2 and iNOS signaling

Article abstract of DOI:10.1016/j.bioorg.2019.103047

A potent Nonsterodial Anti-inflammatory Drug (NSAID) candidates has been conceived and built by an assembly of a hydrophilic, fluorescent and COX-2 inhibiting units in the same molecule. The isatinimino-acridinedione core (TM-7) was achieved in a simple t

Full text of DOI:10.1016/j.bioorg.2019.103047

Accepted Manuscript
A Competent Synthesis and Efficient Anti-inflammatory responses of Isatini-
mino Acridinedione moiety via Suppression of in vivo NF-κB, COX-2 and iNOS
Signaling
Govindasami Periyasami, Paulrayer Antonisamy, Karthikeyan Perumal, Stalin
Antony, Mostafizur Rahaman, Asma A Alothman
PII:
S0045-2068(19)30416-X
https://doi.org/10.1016/j.bioorg.2019.103047
103047
DOI:
Article Number:
Reference:
YBIOO 103047
Bioorganic Chemistry
To appear in:
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Revised Date:
Accepted Date:
17 March 2019
28 May 2019
5 June 2019
Please cite this article as: G. Periyasami, P. Antonisamy, K. Perumal, S. Antony, M. Rahaman, A.A. Alothman, A
Competent Synthesis and Efficient Anti-inflammatory responses of Isatinimino Acridinedione moiety via
Suppression of in vivo NF-κB, COX-2 and iNOS Signaling, Bioorganic Chemistry (2019), doi: https://doi.org/
10.1016/j.bioorg.2019.103047
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1
A Competent Synthesis and Efficient Anti-inflammatory responses of Isatinimino
Acridinedione moiety via Suppression of in vivo NF-κB, COX-2 and iNOS
Signaling
Govindasami Periyasamia,*, Paulrayer Antonisamyb, Karthikeyan Perumalc, Stalin Antonyd,
Mostafizur Rahamana, Asma AAlothmana
a Department of Chemistry, College of Science, King Saud University, Riyadh-11451, SaudiArabia.
b
Department of Korean Physiology, Wonkwang University School of Korean Medicine, 460 Iksan-
daero, Iksan City, Jeonbuk, 570-749, Republic of Korea.
c Department of Chemistry and Biochemistry, Ohio State University, 170A CBEC, 151 Woodruff
Avenue, Columbus, Ohio 43210.
d Department of Traditional Chinese Medicine, Zhejiang A&F University, Hangzhou 311300, China
*Corresponding author:
Govindasami Periyasami (email: pkandhan@ksu.edu.sa)

2
Abstract
A potent Nonsterodial Anti-inflammatory Drug (NSAID) candidates has been conceived and
built by an assembly of a hydrophilic, fluorescent and COX-2 inhibiting units in the same
molecule. The isatinimino-acridione core (TM-7) was achieved in a simple three step
synthetic procedure viz (i) a multicomponent reaction between dimidone, aldehyde and amine
to furnish the nitroacridinedione (4), (ii) reduction step and (iii) schiffs-base condensation
with isatin. The excellent anti-inflammatory pharmacological efficiency of the drug was
established by in vivo biological experiments. Accordingly, it was found that the treatment
with the synthesized isatimino analogues (dosage: 30 mg/kg) inhibited protein expression of
cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nuclear factor kappa
B (NF-κB) as well as production of prostaglandin E2 (PGE2), nitric oxide (NO), tumor
necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), and interleukin-6 (IL-6) levels
induced by carrageenan. Further, a comparative molecular modeling analysis of TM-7 carried
out with the crystal structure of aspirin acetylated human COX-2 suggested effectively
binding and efficient accommodation inside the active site’s gorge.
Key words: Anti-inflammatory, Paw oedema, Carrageenan, Acridinedione, COX-2, iNOS,
NF-kB, in silico docking, 5F19 receptor.

3
Introduction
Inflammation is a normal physiological reaction of the body against invading pathogens,
which is normally identified by redness, swelling, pain, and heat. A lot of scientific studies
have delivered proof that inflammation is involved in the pathogenesis of several diseases
including cancer [1], aging [2], cardiovascular dysfunction [3], and other life-threatening and
devastating disorders. Acute inflammation is a process that involves a significant production
of several inflammatory and pro-inflammatory mediators, free radicals, and activation of
complex enzymes. Carrageenan-induced paw oedema study is a well-known acute
inflammation model commonly used for testing novel anti-inflammatory drugs. Following
this procedure, biphasic oedema is induced in the sub-plantar surface of rat hind paw by
injecting Carrageenan. The early phase is associated with the release of histamine, bradykinin,
serotonin, and prostaglandins (PGs) produced by cyclooxygenase enzymes (COX), while the
delayed phase contributes to neutrophil infiltration, and the ongoing production of PGs [4].
The delayed phase of carrageenan-induced acute inflammation also releases neutrophil-
derived reactive oxygen species (ROS), nitric oxide (NO), and pro-inflammatory cytokines
including interleukin-1 β (IL-1 β) and tumor necrosis factor (TNF-α) [5]. Diverse scientific
reports have suggested that drugs targeting the COX enzyme, pro-inflammatory protein
expression (e.g. inducible nitric oxide synthase (iNOS)), and free radical formation might
deliver significant anti-inflammatory effects than the currently available therapeutic agents
[6]. In this regard, the present study aims to provide the evaluation of anti-inflammatory
activity of a newly synthesized isatin-imine acridiendione molecule, along with a brief
discussion on its synthetic chemistry.

4
Acridinedione motifs continue to receive substantial attention from various biological and
biomedical fronts including photodynamic therapy. [7] This can be attributed to their broad
spectrum of pharmaceutical properties such that anti-tumor [8], antimalarial [9], antiprotozoal
[10], antileukemic [11], anti-nociceptive [12], and anti-inflammatory properties.[13] Owing
to the structural similarity of acridinedione core unit with 1,4-dihydropyridine, and
biologically important co-enzyme Nicotinamide adenine dinucleotide (NADH), it has
recently acquired rejuvenated importance in the field of biomedical research. Moreover, due
to the bi-chromophoric property of acridinedione dyes, it has been demonstrated that by
judicious variation of substitution groups at the 9th and/or 10th positions, they can be
modified as ideal fluorescent probe for proteins [14], hydrogen-bonding assemblies [15, 16]
and also fluorescent sensors [17, 18]. Also, 9-aryl substituted acridinedione analogues have
been used as an effective vascular potassium channel openers. Apart from this, acridine-1,8-
dione analogues have been reported as novel GCN5 inhibitors that are involved in metabolic
disorders and cancer progression, particularly, in colon cancer and lung cancer. Collectively,
the substituted acridinedione motif has evidence in the literature as an attractive and efficient
target for therapeutic applications. However, the reported therapeutic applications of
acridinedione against inflammation activity are limited and it remains an essential goal in
medicinal chemistry.
Inspiringly, hybrid small molecules composed of NSAIDs are more efficient, in terms of
potency, time consumption, and has less side effects compared with the commercially
available anti-inflammatory drugs [19]. Herein, we report the design and synthesis of a low
molecular weight acridine-1,8-dione hybrid containing a broad spectrum of enzyme
inhibiting substitutions such as the isatin analogue, linked as 3-(phenylimino)indolin-2-one at

5
the 10th position and phenolic substitution at the 9th position as a hydrophilic unit. Obtaining
amide functionality in isatin has shown a broad range of biological properties [20], including
anti-HIV [21], anti-cancer [22], and anti-inflammatory properties [23]. Interestingly, the
amide group is a great alternative for the carboxylic groups in NSAID drugs, for example,
indomethacin, meclofenamic acid, and ketoprofen showed greater selectivity for COX-2
enzyme [24]. We anticipated that, the ability of the designed compound will have a broad
spectrum of fluorescent emission due to the bichromophoric nature of the central acridine-
1,8-dione unit and also it can inhibit the COX-2 enzyme as well as it can easily pass-through
protein channels due to the presence of the hydrophilic phenolic unit.
Results and discussion
Chemistry
Acridine-1,8-dione bridged NSAID-COX inhibitor synthesis
The current research endeavor envisions the synthesis and an in vivo evaluation of anti-
inflammatory nature of a fluorescent dye unit spacer containing a COX-2 inhibitor decorated
with a lipophilic substitution obtained by an easily accessible and efficient procedure. Hence,
the drug target was proposedly designed as small molecular hybrids containing a selective
NSAID (COX inhibitor) inhibitor amide group, and a lipophilic phenolic group linked
through an acridine-1,8-dione dye center unit in a 1:1 ratio (Figure 1). We hypothesized that
the isatin amide functionality may compete as good as the COOH moiety for efficient COX
inhibition and will therefore usher good anti-inflammatory activity. In Scheme 1, the retro
synthetic analysis, which directed us to the possible synthetic route to achieve the target
molecule is described.

6
Figure 1. Structure of the acridine-1,8-dione dye core NSAID-COXs inhibitor
Scheme 1. The retrosynthetic approach for the synthesis of acridine-1,8-dione dye bridged
NSAID-COX inhibitor.

7
In path A, the most prominent direct Hantzsch reaction may produce dimeric acridiendione
due to the presence of two aromatic amine groups present in m-phenylenediamine, and it will
not be suitable for further Schiff condensation with an isatin moiety. Path B offers the
advantage of being an easy one step method; however, a report from Wang et al. [25]
suggests that the imine group of the 2-indolinone imino-amine might also involve in the
cyclization and therefore it may not retain in the final product. Hence, path C was perceived
to be an appropriate synthetic route to obtain the desired product.
Initially, the required precursor tetraketone was synthesized through a Knoevenagel
condensation followed by a Michael addition reaction between 4-hydroxy benzaldehyde (1)
and dimidone (2). Further the generated tetraketone underwent cyclization with 3-nitro
aniline (3) at room temperature in EtOH/H2O (1:1) with a catalytic amount of acetic acid as a
solvent. The yielded crude nitroacridienedione (4) was purified by a filter column, and the
product was further treated with a reducing agent such as Fe powder with ammonium
chloride to furnish the corresponding aminoacridine-1,8-dione (5) in good yield. The
synthetic route of amine functionalized acridinedione is illustrated in Scheme 2.
Scheme 2. Synthesis of acridine-1,8-dione amine (5)
As depicted in Scheme 3, the target molecule (TM) isatin-imineacridindione (TM-7) was
synthesized through Schiff base condensation methodology by refluxing equimolar quantities

8
of aminoacridinedione (5) and isatin (6) in ethanol with a catalytic amount of acetic acid. The
reaction completion was monitored by TLC, and the crude product was purified through flash
silica gel column chromatography.
Scheme 3. Synthesis of target molecule TM-7 through Schiff base condensation of amine (5)
and isatin (6)
The structure of the product TM-7 was confirmed by their spectral data. In the FT-IR
spectrum of TM-7, two broad stretching vibrational absorption bands were found to appear at
-1
-1
3427 cm and 3382 cm corresponding to aromatic OH and iminooxindole amide NH
respectively. A sharp absorption band observed at 1736 cm-1 corresponds to carbonyl groups
of acridinedione and the amide carbonyl of iminooxindole exhibited a sharp band at 1716 cm-
-1
1. Aromatic and aliphatic C-H stretching bands were observed at 3121 cm-1 and 2955 cm
respectively. A band which appeared at 1618 cm-1 was assigned to imine C=N stretching
1
frequency. The H-NMR spectrum showed one proton singlet at  4.88 ppm corresponding to
benzylic CH and the aromatic protons resonated as multiplets in the region between  6.51-
7.36. In the 13C DEPT-135 spectrum, two intense signals appeared in the negative region
corresponding to the methylene carbons present in the compound. Further, the 13C NMR

9
spectrum of TM-7, displayed oxindole carbonyl carbon resonances at 163.82 ppm and that of
the acridinedione carbonyl carbon at 195.62 ppm. The assignment of carbon signal at 163.8
ppm support the presence of imine carbon in the target molecule. The NMR spectra of
synthesized compounds are presented in Electronic Supplementary Information (ESI)†. In
addition, the mass spectrum (ESI-MS) of TM-7 exhibited the molecular ion peak at m/z
585.28 corresponding to the molecular mass of TM-7. The compound also gave satisfactory
results from elemental analysis.
In vivo anti-inflammatory studies
The Carrageenan-induced hind paw edema is a well-known model of acute inflammation
involving a variety of inflammatory mediators and has widely been used to assess the anti-
inflammatory effect of new drugs. The macroscopic appearance and paw oedema inhibition
of indomethacin and the test TM-7 against carrageenan-induced hind paw oedema are shown
in Figure 2. Indomethacin and the tested TM-7 showed 52.02% and 53.13% inhibition of
inflammation at the 5th hour when compared to the control (Figure 3).
The TM-7 significantly inhibited TNF-α (1.62 fold), IL-1β (2.94 fold), and IL-6 (2.38 fold)
levels similar to indomethacin TNF-α (1.58 fold), IL-1β (2.60 fold), and IL-6 (2.45 fold)
(Figure 4 A-C). PGE2 (1.57 fold) and nitrite levels (1.88 fold) were significantly decreased
by the treatment of TM-7 compared to the control group (Figure. 5A and B). In order to
identify whether the anti-inflammatory activity of TM-7 was reasonable due to its antioxidant
activity, the hepatoprotective properties of TM-7 were evaluated by malondialdehyde (MDA)
level measurement as an index of lipid peroxidation. As shown in Figure 5c, the MDA level
significantly increased (2.39 fold) in liver tissue of rats at the 5th hour after carrageenan

10
injection. On the other hand, the MDA level was declined significantly (2.17 fold) by
treatment with TM-7. Indomethacin produced significant inhibition on the MDA level similar
to compound 7. MDA is a metabolic product of lipid peroxidation and its level is expected to
rise during oxidative stress. The current results displayed that TM-7 (30 mg/kg) reduce the
severity of liver damage by decreasing the MDA levels. A growing body of evidence
proposes the anti-oxidative effect of TM-7, which is consistent with previous reports. [26]
Expression of COX-2, iNOS, and pro-inflammatory cytokines are known to be regulated by
NF-κB at the transcriptional level (Lee et al., 2015). In the present study, treatment with TM-
7 (30 mg/kg) resulted in a significant reduction of iNOS (1.71 fold), COX-2 (2.15 fold), and
NF-κB (2.14 fold) protein expressions in carrageenan-induced hind paw oedema indicating
that TM-7 could inhibit iNOS, COX-2 expression through the suppression of NF-κB (Figure
6A-D). In order to evaluate the transudative, exudative, and proliferative components of
subacute inflammation, we have applied the cotton pellet induced granuloma method. The
results are shown in Figure 7. TM-7 (30 mg/kg) and standard drug (indomethacin 10 mg/kg)
elicited a significant inhibitory activity on the wet weight of granuloma.
Docking stimulation
To validate the ligand-receptor associations, TM-7 was selected for docking studies and the
commercially available anti-inflammatory drug Indometacin as the reference compound for
comparative study. We anticipated that it may provide insights on binding possibilities with
lower binding energy through docking with receptor COX-2 (5F19). The synthesized COX-2
inhibiting TM-7 molecule was subjected to docking experiments in order to elucidate its anti-
inflammatory response. We expected that it can activate the molecules due to their presence

11
for hydrophilic unit. The results of the docking experiment of the selected ligand TM-7 with
5F19 showed efficient interactions with the active amino acids in the active site pocket. The
binding interaction with the amino acid residues THR`60/O, ARG`61/HE, SER`121/O,
LYS`532/HZ2 were identified in the least energy value (-8.82 kcal/mol). Notably, the values
closely matched with the binding energy of Indomethacin (-8.98 kcal/mol). having interaction
with amino acids receptors CYS`47/HN, TYR`130/HH, LYS`137/HZ1, GLN`461/2HE2 in
the active pocket. Further, the inhibition constant and ligand efficiency of TM-7 (341.7 nM)
is significantly better than with reference drug indomethacin (263.12 nM). Since TM-7
showed the binding interaction with the different amino acids but in the same active pocket
like that of Indomethacin, and comparable to the crystal structure of aspirin acetylated human
cyclooxygenase-2 [27]. Furthermore, the in vivo study of our synthesized target showed the
significant inhibition against COX-2. Additionally, the TM-7 has been well packed in the
active pocket of COX-2 structure and it represents the strong binding interaction with the
active amino acids [28]. Hence, it is apparent that the TM-7 might be develop a new
inhibition pathway and act as a novel inhibitor for COX-2 and its associated enzymes. The
docking results are shown in Table 1 also Figure 8 & 9 represented the 3D picture and 2D
interaction diagram of the corresponding binding conformations of TM-7 and Indomethacin
has been presented respectively.

12
Table 1. in silico molecular docking of TM-7 and Indomethacin with aspirin acetylated
human cyclooxygenase-2 (5F19)
Protein Binding amino
Binding
Energy
Inhibition
VDW_HB
Ligand
Ligand
(PDB
acid residues
Constant desolv_energy efficiency
ID)
(kcal/mol)
(nM)
(kcal/mol)
Thr`60/O,
TM-7
5F19
5F19
Arg`61/He,
Ser`121/O,
-8.82
-8.98
341.7
-10.07
-8.87
0.20
0.36
Lys`532/Hz2
Cys`47/Hn,
Tyr`130/Hh,
Lys`137/Hz1,
Gln`461/2He2
Indo
methacin
263.12
Experimental
Synthesis of nitro substituted acridinedione 4
5,5-Dimethylcyclohexane-1,3-dione (2 equiv.) was dissolved in 20 mL EtOH/H2O (1:1) and
then 4-hydroxybenzaldehyde (1 equiv.) was added and the mixture was stirred at room
temperature. The reaction mixture turns to be cloudy and stirring was continued for another
30 min for complete generation of tetraketone. After complete conversion of the starting
materials (as monitored by TLC), 4-nitroaniline and a catalytic amount of acetic acid were
added. The reaction mixture was stirred at room temperature for another 24 h. The white
tetraketone underwent cyclization reaction with aromatic amine and turns to bright yellow
solid. The crude product was transferred to cold water and the solid was washed thoroughly
with distilled water and dried under vacuum. The crude nitro functionalized acridinedione (4)
was carried over to the next step without further purification. Yield: (Rf = 0.73, 1.28 g, 86%);
-1
mp 132 C; FT-IR (KBr, cm ): 3422 (O-H stretch), 1726 (C=O stretch), 1530 and 1350 (N-O
stretch); 1H NMR (400 MHz, DMSO-d6, ppm) H 0.72 -0.86 (m, 12H, Me proton), 1.97-2.21
(m, 8H, CH2 proton), 4.93 (s,1H, CH proton), 6.60-6.62 (ABq(d), 2H, Ar, J= 8 MHz), 7.09-

13
7.11 (ABq (d), 2H, Ar, J= 8 MHz), 7.85-7.88 (m, 3H, Ar), 8.39-8.40 (s, 1H, Ar), 9.09 (s, 1H,
phenolic OH); 13C NMR (100 MHz, DMSO-d6) C 26.1, 29.2, 32.1, 40.8, 49.6, 113.7, 114.68,
124.3, 128.5, 136.7, 139.4, 148.6, 149.5, 155.3, 162.5, 195.2; LS-MS (ESI): 486.38 (M+).
Anal. Calcd C29H30N2O5: C, 71.59; H, 6.21; N, 5.76. Found: C, 71.71; H, 6.18; N, 5.90%.
Synthesis of amino acridinedione 5
To a solution of nitroacridiendione (4) (1 equiv) in ethanol (7 mL, 3.5 equi) and H2O (2 mL,
1 equiv), iron powder (325 mesh, 3 equiv) and NH4Cl (0.6 equiv) were added at room
temperature. The reaction mixture was stirred at 85 °C for 1 h, cooled to room temperature,
and filtered through celite. The solid was washed thoroughly with ethyl acetate, and then the
filtrate was concentrated under reduced pressure and extracted with ethyl acetate. The organic
phase was dried over anhydrous MgSO4, filtered, and concentrated. The crude product was
purified by flash column chromatography with EtOAc: hexane (1:1, v/v) as eluent affording
amino functionalized acridiendione 5. Yield: (Rf = 0.65, 1.10 g, 94 %); mp 76 C; FT-IR
-1
(KBr, cm ): 3438 (N-H stretch), 3420 (O-H stretch), 3362 (N-H stretch), 1728 (C=O stretch),
1
1615 (N-H bending); H NMR (400 MHz, DMSO-d6, ppm) H 0.71-0.87 (m, 12H, Me
proton), 1.85-2.21 (m, 8H, CH2 proton), 4.90 (s,1H, CH proton), 5.51 (br s, 2H, NH2), 6.45-
6.68 (m, 3H, Ar), 6.58-6.60 (ABq(d), 2H, Ar, J= 8 MHz), 7.03-7.05 (ABq (d), 2H, Ar, J= 8
MHz), 7.18 (s, 1H, Ar), 9.10 (s, 1H, phenolic OH); 13C NMR (100 MHz, DMSO-d6) C 26.1,
29.5, 31.9, 40.1, 49.7, 113.1, 114.5, 126.2, 128.3, 130.1, 137.1, 139.2, 148.9, 150.2, 155.2,
160.6, 195.2; LS-MS (ESI): 456.31 (M+). Anal. Calcd C29H32N2O3: C, 76.29; H, 7.06; N,
6.14. Found: C, 76.33; H, 7.15; N, 6.32%.

14
Synthesis of isatinimino acridindione TM-7
An equimolar mixture of amino acridinedione 5 and isatin 6 in 20 mL of EtOH in the
presence of catalytic amount of acetic acid was heated with constant stirring at 90 C for 1 h.
After completing the reaction, monitored by TLC, the reaction mixture was transferred to
cold water and the solid was washed thoroughly. The crude product was further purified by
flash column chromatography using EtOAc:hexane (8:2 v/v) as eluent yielding isatinimino
acridindione (TM-7) as yellow solid. Yield: (Rf = 0.50 in 100% ethyl acetate, 0.95 g, 86 %);
-1
mp 168 C; FT-IR (KBr, cm ): 3427 (O-H stretch), 3382 (amide N-H stretch), 3121 (Ar C-
H), 2955 (Aliphatic C-H), 1736 (C=O stretch), 1716 (amide C=O), 1618 (C=N Stretch); 1H
NMR (500 MHz, DMSO-d6, ppm) H 0.64 -0.88 (m, 12H, Me proton), 1.84-2.24 (m, 8H,
CH2 proton), 4.88 (s,1H, CH proton), 6.51-7.36 (m, 12H, Ar), 9.07 (s, OH), 11.03 (s, NH);
13C NMR (125 MHz, DMSO-d6) C 26.6, 29.9, 32.5, 41.7, 50.1, 111.4, 112.4, 113.7, 115.1,
118.6, 122.4, 128.9, 129.1, 132.3, 135.5, 137.5, 139.0, 140.3, 146.6, 147.8, 149.9, 150.7,
155.7, 163.8, 195.6; LS-MS (ESI): 585.28 (M+). Anal. Calcd C37H35N3O4: C, 75.88; H, 6.02;
N, 7.17. Found: C, 75.97; H, 6.30; N, 7.29%.
Materials and Methods
Animals
Male Wistar albino rats (200–210 g) were used for the current scientific investigation.
Animals were maintained on a humidity of 60–70% at nearly 25 ± 1 °C with the 12 h
light/dark rotation and have free access to water and diet. All the animal studies were
conducted agreeing to the ethical norms permitted by Ministry of Social Justice and
Empowerment, Government of India and Institutional Animal Ethics Committee guidelines.

15
Carrageenan-induced hind paw oedema in rats
Test drug TM-7 (30 mg/kg) and indomethacin (10 mg/kg) were dissolved in 0.5% CMC and
treated orally 1 h earlier carrageenan induction. After that, carrageenan was injected
subcutaneously into the right hind paw of rats. Rat hind paw thickness was measured initially
and then at 1, 2, 3, 4, and 5 h after the carrageenan injection with digital Vernier calipers [29].
After 5 h, rats were sacrificed and the carrageenan-induced hind paw oedema feet’s were
dissected and stored at – 80 °C until further use.
Measurement of the Tumor Necrosis Factor α, Interleukin 1 β, and IL-6 Level in Paw
Tissue
Rat hind paw tissue was homogenized in 500 µl of buffer containing protease inhibitors, and
IL-1β, TNF-α, and IL-6 levels were determined using a commercially available ELISA kit
(eBioscience, USA) according to the manufacturer instructions.
Hepatic MDA Assay
The MAD level was identified at 532 nm by using the previous method. 1,1,3,3-
Tetramethoxypropane was used as standard [30].
Western Blot Analysis of iNOS, COX-2 and NFkB
Rat hind paw soft tissues were removed and homogenized by RIPA buffer. The homogenates
were centrifuged at 13,000 rpm for 10 min, and 10 µg of total proteins from the supernatants
was separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF)
membranes. The membranes were incubated with anti-iNOS, anti-COX-2, anti-NFkB or anti-
β-actin antibodies in 5% skim milk in TBST overnight at 4 °C with 6 rpm. The PVDF

16
membranes were then incubated with horseradish peroxidase-conjugated secondary antibody.
The immunoreactive protein bands were detected by the enhanced chemiluminescence (ECL)
reagent. ImageJ 1.47 v software (National Institute of Health) was used to quantify the
chemiluminescent intensities of protein signals.
Cotton pellet-induced granuloma in rats
Cotton pellets (35 ± 1 mg) induced granuloma was generated on the axilla region of the rats.
Rats were treated by TM-7 (30 mg/kg) or indomethacin (10 mg/kg) once a day for seven
successive days from the day of cotton pellet grafting. On the 8th day, cotton pellets were
removed from animals and dried in a hot air oven at 60 °C. The granuloma level was assessed
by subtracting the cotton pellet weight on 0 day from eighth day [31].
Statistical analysis
The results were expressed as the Mean ± SD. Results obtained from the present study were
analyzed using One way ANOVA followed by Tukey’s multiple comparison tests.
Differences between the data were considered significant at P < 0.05.
In silico molecular docking study
Ligands: The structure of TM-7 and the standard control drug Indomethacin was drawn in
ChemDraw Ultra version 12.0 and the connection errors have been checked. Both compounds
were optimized and energy minimized using PRODRG server [32] and the low energy
confirmation of the selected ligands was docked using AutoDock Tools v 1.5.6. (autodock
and autogrid) 4.2 software (http://www.scripps.edu/mb/olson/doc/autodock) and followed by
the reported methodology by Stalin et al. [33].

17
Receptor: The three dimensional X-ray crystal structure of target protein, aspirin acetylated
human cyclooxygenase-2 (PDB ID: 5F19) was obtained from the protein data bank
(https://www.rcsb.org/structure/5F19). The active sites of the target protein were identified
using the CASTp (Computed Atlas of Surface Topography of proteins) server. Based on the
target molecules preparation protocol from Autodock, water molecules and hetero groups
were deleted from the target enzyme molecules. The target enzyme molecules preparation
wizard has been used to assign bond orders, formal charges (Kollman and Gasteiger-type
charges), and missing information on connectivity of enzyme structures in PDB.
Docking: Molecular interaction and simulation was performed on the ligand TM-7 and
Indomethacin as reference molecule in order to generate superlative pose with highest least
score, to reveal its complete accommodation with 5F19 protein active site pocket.
Conclusion
In conclusion, the isatinimino acridienedion motif was synthesized successfully with high
yield and purity. The results of the present study suggested that the synthesized isatinimino
acridiendione showed considerable anti-inflammatory effects in carrageenan-induced hind rat
paw oedema, which is equivalent to standard drug indomethacin. The molecular mechanisms
of target molecule might be related to the decline of NF-kB, TNF-α, IL-6, and IL-1β that may
possibly result in inhibition of COX-2 and iNOS expression and its product (PGE2 and NO).
Our results deliver new perspectives on the therapeutic use of isatinimine acridiendione TM-7
in the management of inflammatory diseases.

18
Acknowledgements
The authors express their appreciation to the Deanship of Scientific Research at King
Saud University for funding this work through research group no. RG-1440-111.
Electronic Supplementary data
† Experiment details and NMR spectra for synthesized compounds were available in
Electronic supplementary information.
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Figure 2. Macroscopic images of carrageenan-induced hind paw oedema and the anti-
inflammatory effect of TM-7 (30 mg/kg) and indomethacin (10 mg/kg). (A) Normal control;
(B) control; (C) TM-7 (30 mg/kg); and (D) indomethacin (10mg/kg).

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TM -7 (30 mg/kg)
Figure 3. Effect of TM-7 (30 mg/kg) and indomethacin (10 mg/kg) on carrageenan induced hind paw oedema in rats

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TM -7
TM -7
(30 mg/kg)
(30 mg/kg)
A
B
TM -7
(30 mg/kg)
C
Figure 4. Effect of TM-7 (30 mg/kg) and indomethacin (10 mg/kg) on TNF-α, IL-1β, and IL-6 levels

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Figure 5. Effect of TM-7 (30 mg/kg) and indomethacin (10 mg/kg) on PGE2, nitrite, and MDA levels

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Figure 6. Effect of TM-7 (30 mg/kg) and indomethacin (10 mg/kg) on COX-2, iNOS, and NF-κB protein expression levels.

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Figure 7. Anti-inflammatory activity of TM-7 (30 mg/kg) and indomethacin (10 mg/kg) against cotton pellet-induced granuloma model in rats.

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Figure 8. (a) 3D interaction diagram and (b) 2D interaction diagram of TM-7 with active site of target human cyclooxygenase-2 (5F19). The
hydrogen bond interactions are shown as dotted yellow and black lines and the hydrophobic regions are shown in green coloured lines.

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Figure 9. (a) 3D interaction diagram and (b) 2D interaction diagram of reference drug Indomethacin with active site of target receptor human
cyclooxygenase-2 (5F19). The hydrogen bond interactions are shown as dotted yellow and black lines and the hydrophobic regions are shown in
green coloured lines.

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Graphical abstract