Indolyl-isoxazolidines attenuate LPS-stimulated pro-inflammatory cytokines and increase survival in a mouse model of sepsis: Identification of potent lead

Article abstract of DOI:10.1016/j.ejmech.2018.04.004

A library of indolyl-isoxazolidines (6–9) has been synthesized by regio- and stereoselective microwave irradiated 1,3-dipolar cycloadditions of C-(3-indolyl)-N-phenylnitrone (2′) with variedly substituted dipolarophiles (3′-5′) and screened for their anti-inflammatory activities through inhibition of pro-inflammatory cytokines such as TNF-α and IL-6. Amongst the evaluated compounds (6–9), bicyclic isoxazolidine (9a) was found to exhibit significant inhibitory potential against LPS induced human IL-6 and TNF-α in THP-1 cells. Compound 9a was further assessed for in vivo analgesic and anti-inflammatory activities via acetic acid induced writhing and carrageenan induced paw edema models in mice, respectively. The results showed that compound possesses potent anti-inflammatory-analgesic activity comparable to indomethacin and did not show toxicity up to a 2000 mg kg^?1 dose as evidenced by histopathological studies. Consequently, the most active compound 9a was also evaluated against LPS-induced septic death and exhibited a significant protection in in vivo mouse model. Taken all together, the results suggest that the compound 9a is able to attenuate pro-inflammatory cytokines such as IL-6 and TNF-α; accelerate resolution of inflammation, and also increased survival rate of septic mice. Therefore, these “lead” isoxazolidines can be used as promising candidate for further analgesic/anti-inflammatory drug design and development.

Full text of DOI:10.1016/j.ejmech.2018.04.004

Accepted Manuscript
Indolyl-isoxazolidines attenuates LPS-stimulated pro-inflammatory cytokines and
increases survival in a mouse model of sepsis: Identification of potent lead
Gagandeep Singh, Gurjit Singh, Rajbir Bhatti, Mehak Gupta, Ajay Kumar, Ankita
Sharma, Mohan Paul Singh Ishar
PII:
S0223-5234(18)30331-3
10.1016/j.ejmech.2018.04.004
EJMECH 10353
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 September 2017
Revised Date: 24 March 2018
Accepted Date: 2 April 2018
Please cite this article as: G. Singh, G. Singh, R. Bhatti, M. Gupta, A. Kumar, A. Sharma, M.P. Singh
Ishar, Indolyl-isoxazolidines attenuates LPS-stimulated pro-inflammatory cytokines and increases
survival in a mouse model of sepsis: Identification of potent lead, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.04.004.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT
Leave for space
Indolyl-Isoxazolidines attenuates LPS-stimulated
pro-inflammatory cytokines and increases survival in a mouse model of sepsis:
Identification of potent lead
Gagandeep Singh,a Gurjit Singh,a Rajbir Bhatti,a Mehak Gupta,c Ajay Kumar,c Ankita Sharma,c
Mohan Paul Singh Ishar a,b
*

ACCEPTED MANUSCRIPT
Indolyl-Isoxazolidines attenuates LPS-stimulated pro-inflammatory cytokines
and increases survival in a mouse model of sepsis: Identification of potent lead
Gagandeep Singh,a Gurjit Singh,a Rajbir Bhatti,a Mehak Gupta,c Ajay Kumar,c Ankita
Sharma,c Mohan Paul Singh Ishar a,b
*
a
Bio-Organic and Photochemistry Laboratory, Department of Pharmaceutical Sciences, Guru Nanak Dev
University, Amritsar-143 005, Punjab, India.
Vice Chancellor, Maharaja Ranjit Singh Punjab Technical University, Badal Road, Bathinda.
Cancer Pharmacology Division, Indian Institute of Integrative Medicine (CSIR), Jammu-180001, India.
b
c
Corresponding author. E-mail address: mpsishar@yahoo.com
Abstract: A library of indolyl-isoxazolidines (6-9) has been synthesized by regio- and
stereoselective microwave irradiated 1,3-dipolar cycloadditions of C-(3-indolyl)-N-phenylnitrone
(2′) with variedly substituted dipolarophiles (3′-5′) and screened for their anti-inflammatory
activities through inhibition of pro-inflammatory cytokines such as TNF-α and IL-6. Amongst
the evaluated compounds (6-9), bicyclic isoxazolidine (9a) was found to exhibit significant
inhibitory potential against LPS induced human IL-6 and TNF-α in THP-1 cells. Compound 9a
was further assessed for in vivo analgesic and anti-inflammatory activities via acetic acid induced
writhing and carrageenan induced paw edema models in mice, respectively. The results showed
that compound possesses potent anti-inflammatory-analgesic activity comparable to
indomethacin and did not show toxicity up to a 2000 mg Kg−1 dose as evidenced by
histopathological studies. Consequently, the most active compound 9a was also evaluated
against LPS-induced septic death and exhibited a significant protection in in vivo mouse model.
Taken all together, the results suggest that the compound 9a is able to attenuate pro-
inflammatory cytokines such as IL-6 and TNF-α; accelerate resolution of inflammation, and also
increased survival rate of septic mice. Therefore, these “lead” isoxazolidines can be used as
promising candidate for further analgesic/anti-inflammatory drug design and development.
Keywords: 1,3-dipolar cycloaddition; indolyl-isoxazolidines; pro-inflammatory cytokines; IL-6;
TNF- α; anti-inflammatory-analgesic activity; sepsis.
Inflammation is an important mechanism contributing to the host’s defense against pathogenic
challenge and the restoration of normal tissue structure. Activated macrophages, the primary pro-
inflammatory cells, mediate most cellular and molecular inflammation networks by producing
NO, prostaglandin E2 (PGE2), and cytokines such as tumor necrosis factor-a (TNF-α) and
interleukins (ILs) [1]. The dysregulated and elevated expression of cell adhesion molecules, such
as ICAM-1, VCAM-1 and E-selectin, in response to pro-inflammatory cytokines such as TNF-α,
IL-6, IL-1β and a bacterial polysaccharide, LPS on the luminal surface of vascular endothelial
cells, are critical early event in inflammatory processes [2]. Therefore, reducing the expression
1

ACCEPTED MANUSCRIPT
levels of LPS-inducible inflammatory mediators is considered as an effective way to attenuate a
variety of disorders derived from the inflammation triggered by activated macrophages. It has
also been demonstrated that IL-6 plays a vital role in synoviocytes proliferation and osteoclast
differentiation through NF-kB expression [3]. The selective targeting of IL-6 over TNF-α has
been considered beneficial, as TNF-α inhibitors (monoclonal antibodies) have a history of
adverse side effects [4]. Therefore, inhibiting IL-6 by small molecules presents new
opportunities to treat rheumatoid arthritis.
Consequently, enormous efforts are directed towards the structural design leading to the
discovery of novel anti-inflammatory agents. Therapeutic importance of 3-heterosubstituted
indoles has also been well established (Figure-1) [5]. The involvement of C3 functionalized
indole nucleus has been used extensively to obtain biologically active compounds [6]. Even
though it has varied actions, the role of indole in anti-inflammatory therapy is of prime interest
which is prominently highlighted in the drug molecule indomethacin.
O
OH
O
N
N
O
EtOOC
S
Cl
N
O
R
n
O
N
O
N
O
O
N
H
N
O
H
H2N
Cl
Tenidap
2
1
Indomethacine
O
N
O
H
O
N
HN
R
.HCl
N
R
H
NH
O
R1
O
N
R2
N
O
O
N
H
O
H
H
OH
3
5
4
Figure 1: C-3 functionalized indoles as anti-inflammatory-analgesic agents
Isoxazolidines belong to an important class of heterocyclic compounds having a wide range of
biological activities such as anti-inflammatory, anti-viral and anti-microbial [7]. We have
recently reported pyridyl-isoxazolidines as nicotinic analogs for their nootropic activities [8] and
chromanyl–isoxazolidines as apoptosis inducers through the mitochondrial-dependent pathway
in HL-60 cells [9]. Therefore, taking cognizance of therapeutic potential of C3 functionalized
indoles as anti-inflammatory agents and extensive pharmacological activities of isoxazolidines; it
2

ACCEPTED MANUSCRIPT
was decided to evaluate previously synthesized indolyl-isoxazolidines [10] along with new
derivatives, as inhibitors of pro-inflammatory cytokines through in vitro and in vivo expression
of TNF-α, and IL-6, along with in vivo analgesic-anti-inflammatory activities.
Material and Methods
:
Animals: Swiss albino mice (2-3 months old) of either sex, weighing 25-35 g (Animal House Facility,
Guru Nanak Dev University, Amritsar, India) were habituated to the temperature, humidity and lighting
(12 h light/dark cycle) controlled housing facility and group housed for at least 3 days before behavioral
studies began. Animals had free access to food (standard laboratory rodent’s chow) and water throughout
the study. Before beginning the experiments, animals were acclimated to the laboratory environment for
at least 30 min. All animal experimental protocols were approved by the Institutional Animal Ethical
Committee (IAEC) of CPCSEA. All efforts were made to minimize animal suffering and to decrease the
number of animals used.
Drugs and chemicals: All the chemicals and drugs used such as carboxymethylcellulose,
lipopolysaccharide (LPS), MTT and indomethacin were purchased from Sigma Aldrich, USA; whereas,
evaluated indolyl-isoxazolidines (6-9) were obtained by 1,3-dipolar cycloaddition reactions of C-(3-
indolyl)-N-phenylnitrone (2
′) with monosubstituted (3a-f), di-substituted (4′a-b) and cyclic
dipolarophiles (5 a-d) as described earlier (Scheme-1) [10]; authenticated using NMR spectroscopy and
′
mass spectrometry. A suspension of various doses of the compounds was prepared in 0.1% CMC
solution. 0.9% saline was used as vehicle control. All the drugs were administered intraperitoneally (i.p.).
Figure-2: Variedly substituted indolyl-isoxazolidine analogs (6-9).
In vitro effect on cytokines level in THP-1 cells: Human leukemia THP-1 cells were seeded in a 24-well
plate at a density of 3.5 x 105 cells/ml of RPMI medium per well. THP-1 cells were then treated with
PMA (20 ng/ml) for differentiation into macrophages for 18 h followed by rest period of 48 h. Cells were
3

ACCEPTED MANUSCRIPT
further co-treated with 5 µM of each test compound and 1 µg/ml of lipopolysaccharide (LPS) in a serum-
free medium. Culture supernatant was harvested after 24 h for analysis of different cytokines by using
OptEIA ELISA Kits from BD Biosciences. Total protein content for all the samples was calculated by
using Bradford reagent from Bio-Rad Laboratories. All the samples were normalized by dividing cytokine
concentration with total quantity of protein.
Cell proliferation assay using MTT: THP-1 cells were plated in 96-well plate in 100 µL of medium and
treated on the same day with various concentrations of test compounds 8, 9a, and 9b, and incubated for
24−48 h. The MTT dye, at a concentration of 2.5 mg/mL, was added for 4 h at 37 °C. The supernatant
was aspirated, and MTT−formazon crystals were dissolved in 150
at 540 on an ELISA microplate reader. Cell growth was calculated by comparing the absorbance of
treated versus untreated cells.
Effect of compounds on the production of LPS-induced pro-inflammatory cytokines TNF-
6 in vivo model: The effect of compounds 9a and 9b was investigated on the secretion of cytokines
(TNF- / IL-6) in Swiss albino mice of either sex weighing 25-35g. Mice were then randomly divided into
µL of DMSO. The OD was measured
λ
α
and IL-
α
8 groups (n = 4 per group). The treatment to each group was given in following format: group I (vehicle),
group II (LPS, 1 mg/kg), groups III, IV and V (compound 9a at doses 5, 10 and 20 mg/kg, respectively),
groups VI, VII and VIII (compound 9b at doses 5, 10 and 20 mg/kg, respectively). Blood sample was
collected by cardiac puncture using heparin as anticoagulant and immediately centrifuged at 5000 rpm for
10 minutes. Blood plasma was collected and frozen at -20°C until activity. Cytokines (TNF-α/ IL-6)
concentration was determined by using enzyme linked immunosorbent assay (ELISA), using commercial
kits from Krishgen Biosystems, Mumbai-India, as per manufacturer’s instructions. Blood plasma cytokine
concentrations are expressed as picogram per milliliter (pg/mL).
Acetic acid-induced abdominal writhing: The acetic acid-induced abdominal writhing test was used as
described previously by Koster et al. Four experimental groups of mice were treated (I.P) with vehicle
(0.1% carboxy methyl cellulose), compounds 9a and 9b (12.5 mg/kg), indomethacin (10 mg/kg) 30 min,
before the administration of acetic acid solution (0.6 % v/v, 10 ml/kg, i.p.). The number of writhing was
calculated for each mice, completed a period of 30 min after acetic acid injection, and the results were
expressed as the means ± SEM of number of writhing.
Carrageenan-induced paw edema assay: For anti-inflammatory activity, carrageenan induced paw
edema model was used as previously reported [5b]. Experimental groups of animals were treated with
vehicle (0.1% carboxy methyl cellulose), indomethacin (10 mg/kg, p.o.), and compound 9a, 30 minutes
before the injection of 50 µL of carrageenan (1% w/v) in the right hind paw. Then the paw edema was
measured by vernier caliper (Precise) at 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h and 6 h after injection of
carrageenan.
4

ACCEPTED MANUSCRIPT
Involvement of cyclooxygenase or opioid receptor pathways: Six groups of mice with 5 animals in
each group were taken for exploring the mechanism of action of compound 9a, the animals were
pretreated with misoprostol, for studying the involvement of the cyclooxygenase pathway and to check
the involvement of the opioid receptors during the inhibition of algesia, the animals were pretreated with
naloxon. Details of the protocols are shown in Figure-3.
30 min
30 min
Acetic acid
30 min
Comp.9a (12.5 mg/Kg)
Misoprostol (100 µg kg-1)
Observation
30 min
30 min
30 min
Acetic acid
Comp.9a (12.5 mg/Kg)
Naloxone (80 µg kg-1)
Observation
Figure-3: Schematic representation of experimental protocol for exploring the involvement of
cyclooxygenase pathway or opioid receptor in the observed analgesic effect of compound 9a.
Compound 9a attenuated LPS-induced inflammatory mortality in mouse model: Mice weighing 25-
35 g were treated with compound 9a (12.5 mg/kg) by i.v. injection either 15 min before or 15 min after
the i.v. injection of LPS (20 mg/kg). Control animals received a similar volume of vehicle. Body weight
change and mortality were recorded for 10 days.
Statistical analysis: Statistical analysis was performed by one way analysis of variance (ANOVA)
followed by Tukey’s multiple comparison test as the post hoc test. All results are expressed as mean ±
standard error of mean (SEM). The IC50 calculations (in vivo) were carried out using Design-Expert
software, Stat-EasInc., Minneapolis, USA.
Acute toxicity studies: Four groups of animals with three animals per group were taken. The first group
was administered the vehicle and served as the control group; the second, third, and fourth groups were
treated with compound 9a at doses of 50, 300, and 2000 mg kg−1, respectively. All of the treatments were
administered after 4 h of fasting. Thereafter, the animals were observed continuously for the first 4 h and
periodically thereafter for 24 h. After 14 days, one animal each in the control and highest dose (2000 mg
kg−1) groups was sacrificed, and histological studies were conducted using H and E staining.
Results and Discussion
Initially, the 1,3-dipolar cycloaddition reactions of C-(3-indolyl)-N-phenylnitrone (2′) with
monosubstituted (3′a-f) and di-substituted (4′a-b) dipolarophiles were carried out by irradiating
equimolar amount of the addends (neat) in a focused monomode microwave reactor, leading to
the synthesis of variedly substituted isoxazolidines (6-8). A perusal of literature reveals that
5

ACCEPTED MANUSCRIPT
bicyclic isoxazolidines are widely distributed in natural products and valuable pharmaceutical
agents as well as synthetic intermediates. A number of desirable biologically properties [10d-f]
have been reported for molecules containing this bicyclic scaffold. Therefore, keeping in view
the high therapeutic importance of bicyclic isoxazolidines, the reaction of C-(3-indolyl)-N-
phenylnitrone (2′) with various cyclic dipolarophiles (5′a-d) were carried out under similar set of
conditions to obtain bicyclic isoxazolidines (9a-d). After completion of the reaction (TLC) the
residues obtained were resolved by column chromatography over silica gel to obtain the
cycloadducts (6-9, Scheme 1) [10]. We had earlier reported characterization of the regio- and
stereoisomeric indolyl-isoxazolidines on the basis of NMR and X-ray crystallographic studies
[10a-c].
Scheme-1: Reaction of nitrone (2′) with variedly substituted dipolarophiles (3-5)
6

ACCEPTED MANUSCRIPT
In vitro effect on cytokines level: The release of pro-inflammatory cytokines is an important
mechanism by which the immune cells regulate the inflammatory responses and contribute to
various inflammatory and autoimmune disorders. All the synthesized compounds (6-9) were
analyzed for their ability to decrease lipopolysaccharide (LPS) induced tumor necrosis factor-α
(TNF-α) and interleukin-6 (IL-6) production in THP-1 cells (Table-1).
Table 1: Inhibitory effect of compounds 6-9 on LPS induced pro-inflammatory cytokines IL-6
and TNF-α.
Compound No.
Concentration
IL-6 inhibition
0.51 ± 0.43
TNF-α inhibition
1.40 ± 0.66
UT Control
LPS
6a
6b
6c
1 µg/ml
5µM
5µM
5µM
5µM
5µM
5µM
5µM
5µM
5µM
5µM
5µM
5µM
5µM
5µM
5µM
6.06 ± 0.55
5.40 ± 0.44
1.12 ± 0.62
2.77 ± 0.51
4.08 ± 0.44
3.20 ± 0.32
2.06 ± 0.46
1.67 ± 0.57
1.15 ± 0.51
3.03 ± 0.42
3.01 ± 1.18
1.13 ± 0.20
1.34 ± 0.91
1.43 ± 0.53
3.09 ± 0.39
1.65 ± 0.32
6.04 ± 0.54
14.93 ± 1.70
4.58 ± 0.72
3.92 ± 1.11
7.27 ± 0.15
7.77 ± 0.43
3.98 ± 0.44
3.00 ± 0.43
4.37 ± 1.12
2.59 ± 0.31
3.98 ± 0.11
2.74 ± 1.71
2.37 ± 0.76
2.21 ± 0.40
2.42 ± 0.29
4.28 ± 0.12
6d
6e
6f
6′d
6
′e
6
′f
7
8
9a
9b
9c
9d
Notes: Human leukemia THP-1 cells after differentiation with PMA, were subjected to co-treatment
with the test compounds (6-9; 5 µM) and LPS (1 µg/ml) for 24 h. Supernatant was used for the
analysis of cytokines by OptEIA ELISA kits from BD Biosciences, according to the manufacturer’s
protocol. All the samples were normalized by dividing cytokine’s concentration with total protein
content. Data shown here are average of three similar experiments ± SD.
7

ACCEPTED MANUSCRIPT
Several compounds from this series of molecules exhibited significant inhibitory activity against
LPS induced human IL-6 and TNF-α in THP-1 cells. However, compounds 6b-c bearing p-tolyl
and pyridyl groups at C5- of isoxazolidine moiety, respectively, 6′d-6′e (methyl- and ethyl-ester
functionality at C4- position of isoxazolidine moiety), 8 (di-substituted isoxazolidine), 9a-b, and
9d (bicyclic isoxazolidines) at 5µM, showed particularly high activity against the induction of
IL-6, where, the induction was reduced by 4-5 folds. Some of the compounds also reduced the
induction of TNF-α; though the inhibitory effect was not as strong as against IL-6. Before the in
vivo evaluation, the most potent compounds (8, 9a and 9b) were checked for their cytotoxic
effect on THP-1 cells, to confirm that the suppressive effect of the these active compounds from
in vitro assay is not because of cytotoxicity and the IC50 of these compounds 8, 9a and 9b against
THP-1 cell line was found to be >100 µM (24 h) and 74, 87, and 78 µM (48 h), respectively.
This indicates that these compounds possess excellent therapeutic window for IL-6 and TNF-α
inhibition with respect to THP-1 cell cytotoxicity. Therefore, the most active compounds were
further chosen for in vivo evaluation, to determine whether, the lead compounds identified from
in vitro studies were able to replicate the activity in animal models.
Effect of compounds on the production of LPS-induced pro-inflammatory cytokines TNF-α
and IL-6 in vivo model: Based on the results of preliminary cytokine inhibition screening, two
most active derivatives 9a and 9b were chosen for in vivo evaluation (dose-dependent inhibitory
effects) against LPS-induced TNF-α and IL-6 release. The mice were fed with compounds at
three doses (5, 10 and 20 mg/kg), followed by stimulation with LPS. Blood samples were
collected and cytokine levels were measured in plasma (Figure 4 and 5).
8

ACCEPTED MANUSCRIPT
Figure-4: Effect of compound 9a on production of TNF-
α
(A) and IL-6 (C) in Swiss albino mice; All
values are expressed as mean ± S.E.M. Statistical differences were determined by one way analysis of
variance (ANOVA) followed by Tukey’s test *p<.005, versus control group and #p<0.01versus LPS
treated group. (B) Second order fitted regression equation for IC50 (TNF-α) by varying the dose of comp.
9a; (D) Second order fitted regression equation for IC50 (IL-6) by varying the dose of comp. 9a
It was observed that the treatment with compound 9a at the dose of 10 and 20 mg/kg,
significantly reduced the plasma level of TNF-α upto 53.8 % and 86.06 % respectively, (Figure-
4A) and IL-6 upto 73.71 % and 89.92 % respectively (Figure-4C). In order to determine the IC50
values for compound 9a against TNF-α and IL-6, respectively, the experiment were run by
varying the dose of investigational compound 9a from 5 mg kg-1 to 20 mg kg-1 in triplicates. The
dose response data of compound 9a showing % inhibition of TNF-α, which was modeled using
analysis of variance (ANOVA) with best fitted quadratic model. The model was highly
significant at 99.99% (p-value <0.001) with non significant lack of fit and the IC50 value for
compound 9a against TNF-
α inhibition is achieved at 9.4 mg/kg (Figure 4B). The fitted
regression equation to find percentage inhibition against TNF-α (X1) is given in equation 1.
% inhibition (X1) = - 37.640 + 12.064 × A – 0.294 A2
(1)
Where, A = Dose (compound 9a) is in mg/kg of body weight.
9

ACCEPTED MANUSCRIPT
Whereas, the dose response data of compound 9a showing percent inhibition for IL-6 was best
fitted using cubic model; the IC50 value against IL-6 for compound 9a is achieved at 3.5 mg/kg
(Figure 4D). Regression equation for percent inhibition of IL-6 (X2) is given as equation 2.
X2 (% inhibition of IL-6) = - 4.145 + 19.546 × A – 1.673 A2 + 0.046 × A3
(2)
where, A= Dose of compound 9a (mg/kg of body weight).
Figure-5: Effect of compound 9b on production of TNF-α (A) and IL-6 (C) in serum in vivo LPS model;
All values are expressed as mean ± S.E.M. Statistical differences were determined by one way analysis of
variance (ANOVA) followed by Tukey’s test *p<.005, versus control group and #p<0.01 versus LPS
treated group; (B) Second order fitted regression equation for IC50 (TNF-α) by varying the dose of comp.
9b; (D) Second order fitted regression equation for IC50 (IL-6) by varying the dose of comp. 9b
However, at the same doses, compound 9b inhibit the LPS induced plasma level of TNF-α (52.9
% and 62.1 %; Figure-5A) and IL-6 (60.46 % and 65.48 %; Figure-5C). Further, the IC50 values
for compound 9b (against TNF-α = 8.7 mg/kg and IL-6 = 7.4 mg/kg) were also determined in a
similar set of experiment by varying the dose of investigational compound 9b from 5 mg kg-1 to
20 mg kg-1 in triplicate and the dose response data of compound 9b showing percent inhibition of
10

ACCEPTED MANUSCRIPT
TNF-α (Figure 5B) and IL-6 (Figure 5D) was best fitted using quadratic model. The model was
highly significant at 99.99% (p-value<0.001) with non significant lack of fit indicated model
fitting. The fitted regression equations for percent inhibition of TNF-
α
(Y1) and IL-6 (Y2) are
given in equation 3-4, respectively.
Y1 (% inhibition of TNF-
where, A= Dose of compound 9b (mg/kg) of body weight.
α
) = 24.781 + 3.645 × A – 0.088 A2
(3)
(4)
Y2 (% inhibition of IL-6) = - 0.152 + 8.858 x A – 0.278 A2
where, A= Dose of compound 9b (mg/kg) of body weight.
On the basis of the promising efficacy of compound 9a (IC50 value against TNF-α = 9.4 mg/kg
and IL-6 = 3.5 mg/kg) and compound 9b (IC50 value against TNF-α = 8.7 mg/kg and IL-6 = 7.4
mg/kg) in in vivo LPS models, revealed that both the compounds 9a and 9b showed prominent
decrease in the levels of IL-6 in comparison to TNF-α; and further taken for studying in vivo
analgesic/anti-inflammatory potential in animal models.
In vivo analgesic activity: Acetic acid induced writhing test is used for detecting both central
and peripheral analgesia. Intraperitoneal administration of acetic acid releases prostaglandins and
sympathomimetic mediators such as PGE2 and PGF2α and their levels are increased in the
peritoneal fluid of the acetic acid administered mice [11]. The compounds such as 9a and 9b
showing significant inhibition of LPS induced IL-6 and TNF-α were evaluated for their
antinociceptive activity by the writhing test and results of writhing test (Figure-6A) indicate that
compound 9a exhibited most potent antinociceptive activity by reducing the number of abnormal
muscle constrictions to 80.74% inhibition at 12.5 mg/kg dose, as compared to the standard drug
indomethacin which cause 58.15% inhibition at 10 mg/kg.
In vivo anti-inflammatory activity: Because, compound 9a was found to inhibit the inductions
of LPS induced IL-6 and TNF-α (both in vitro and in vivo) and possess significant analgesic
activity as compared to indomethacin, it was decided to determine whether compound 9a affects
acute-phase inflammation in animal models. In this study, we used the carrageenan induced paw
edema model as it is suitable experimental model for evaluating the anti-edematous and
hyperalgesia effect and is believed to be biphasic; the first phase (1 h) involves the release of
serotonin and histamines, and the second phase (>1 h) is mediated by prostaglandin, a
cyclooxygenase product [12]. Carrageenan induces the release of TNF-α, which subsequently
promotes IL-1β and IL-6 production in tissues, and in vivo anti-inflammatory results revealed
11

ACCEPTED MANUSCRIPT
that compound 9a (12.5 mg/kg) reduced edema by 36.92% at 6 hours, whereas, the positive
control, indomethacin (10 mg/kg), reduced edema by 34.51% (Figure-6B). It was observed that
the compound 9a inhibited the carrageenan-induced paw oedema at all-time points (1–6 h from
the carrageenan treatment, Figure-6B). These results suggest that the compound 9a inhibited the
biphasic inflammation induced by carrageenan.
Involvement of cyclooxygenase or opioid receptor pathways: To further understand the
mechanism of action of compound 9a, the animals were pretreated with misoprostol, for
studying the involvement of the cyclooxygenase pathway and to check the involvement of the
opioid receptors during the inhibition of algesia, the animals were pretreated with naloxone. The
schematic representation of the experimental protocol is given in Figure-3.
The pretreatment of the animal with naloxone, an opioid receptor antagonist did not reverse the
analgesic effect of compound 9a, whereas, pretreatment with misoprostol, a prostaglandin
analogue was found to decreased the analgesic effect of compound 9a, although the difference
was not statistically significant (Figure-6C).
Figure-6: (A) Effect of compounds 9a and 9b on acetic acid induced writhing in mice; (B) Effect of
compound 9a on carrageenan induced rat paw inflammation in mice; (C) Effect of misoprostol and
naloxone on analgesic effect of compound 9a on acetic acid induced writhing in mice. All values are
expressed as mean ±SEM. * p<0.05 Vs control.
12

ACCEPTED MANUSCRIPT
It is well known that severe pain especially of neuropathic origin involves a complicated
interplay of neuromodulators and cytokines. Although, the precise pathogenic pathway is not
well understood, there is growing body of evidence that postulates the involvement of mediators
such as eicosanoids, interleukins IL-6, bradykinin and TNF-α as the major culprits [13]. It has
been postulated that in painful inflammatory conditions such as osteoarthritis, there is formation
of pro-inflammatory cytokines including IL-1, IL-6, IL-15 and TNF-α which trigger a cascade of
inflammatory reactions including activation of proteases, other chemokines as well as auto-
stimulation, lipooxygenases, cyclooxygenases and metalloproteases (MMPs) etc. which lead to
an exaggerated inflammatory reaction and pain [14]. The compound 9a was found to decrease
the lipopolysaccharide induced expression of IL-6 and TNF-α in human leukemia THP cell lines.
Thus, it was essential also to check the effect of compound 9a on the expression of COX-2 and
iNOS (Figure 7). Results of western blotting experiment reveals that the expression of iNOS
displayed a concentration dependent decline in the expression which confirms the negative
regulatory effect of compound 9a on NF-κB pathway, whereas, COX-2 expression was also
reduced in a concentration dependent manner, indicating the broad anti-inflammatory effect of
compound 9a.
Figure 7: THP-1 cells were co-treated with LPS and different concentrations of compound 9a for 24 h.
Cells were lysed with RIPA and lysates were analyzed for the expression of COX-2 and iNOS by western
blotting; beta actin was used as internal control.
In addition, the in vivo studies in mice revealed compound 9a has a potent analgesic and anti-
inflammatory activity, which was comparable to the standard drug indomethacin. Along with
this, partial reversal of the observed analgesic response in acetic acid induced writhing was
observed on pretreatment with misoprostol, a PGE2 analogue. Therefore, it may tentatively be
13

ACCEPTED MANUSCRIPT
suggested that compound 9a is acting through interplay of pathways including inhibition of
inflammatory cytokines as well as decreased formation of prostaglandins (Figure-8A).
Compound 9a enhances the survival rate in septic mouse model: As a major endotoxin, LPS
from gram-negative bacteria has been implicated as a major cause of sepsis, thus, based on
significant analgesic and anti-inflammatory effects of compound 9a and for the potential clinical
application, we have further determined whether compound 9a attenuates endotoxin shock
through inhibiting LPS-induced inflammatory response. Mice were injected with LPS at the
dosage of 20 mg/kg intravenously in the presence or absence of compound 9a, and their survival
rates were monitored for 10 days, respectively, as shown in Figure-8B. All animals treated with
LPS alone died as a result of the septic shock, whereas, animals treated with compound 9a (12.5
mg/kg) 15 min after LPS injection (for therapeutic effect), the survival rates were significantly
increased as compared to that of the positive control group (LPS treated, Figure-8B).
Figure-8: (A) Tentative mechanism of action of compound 9a; (B) Compound 9a improves survival of
mice subjected to a lethal dose of LPS. Mice were treated with compound 9a (12.5 mg/kg) 15 min after
the injection of 20 mg/kg of LPS. Survival was recorded for 10 days after the LPS injection at the interval
of 1 day.
Acute Toxicity Studies: OECD guidelines (OECD, 2001) [15] were followed for acute toxicity
studies of compound 9a. Four groups of animals with three animals per group were taken, the
first group was administered the vehicle and served as the control group; the second, third, and
fourth groups were treated with compound 9a at doses of 50, 300, and 2000 mg kg−1,
respectively. All of the treatments were administered after 4 h of fasting. Thereafter, the animals
were observed continuously for the first 4 h and periodically for 24 h. After 14 days, one animal
14

ACCEPTED MANUSCRIPT
each from the control and the highest dose (2000 mg kg−1) group was sacrificed, and histological
studies were conducted using H and E staining. Results revealed that gross behavioral
abnormality was not observed in any of the four groups of animals and even no significant
changes were evident in the myocardium, liver or kidney of the animals treated with the highest
dose of compound 9a (2000 mg kg−1) as compared to those of the control group (Figure-9).
Figure-9: Histology of control (A) and compound 9a treated heart (B), control (C) and compound 9a
treated liver (D), and control (E) and compound 9a treated kidney (F).
Conclusions:
15

ACCEPTED MANUSCRIPT
A focused library of substituted indolyl-isoxazolidines (6-9) has been synthesized by regio- and
stereoselective 1,3-dipolar cycloaddition reactions and evaluated for their activity against pro-
inflammatory cytokines IL-6 and TNF-α in LPS stimulated model. The in vitro studies showed
that all the compounds possess significant IL-6 and TNF-α inhibition. In particular, compound
9a, is most potent inhibitor of IL-6 and TNF-α. Testing of this compound on albino mice showed
reversal of algesia and inflammation with much more efficacy as compared to the standard drug
indomethacin. The mechanistic studies indicated that the anti-inflammatory and analgesic effects
of compound 9a share a common molecular pathway probably involving the inhibition of pro-
inflammatory mediators. Thus, our findings clearly demonstrated that compound 9a exhibited
significant inhibitory effects on LPS-induced inflammation and had potential in the prevention
and treatment for LPS-induced sepsis, and can be exploited as ‘lead’ compounds for the design
of new analgesic/anti-inflammatory agents.
Acknowledgments
We express special thanks to Dr. Ram A. Vishwakarma (Director), CSIR-IIIM Jammu, India, for
providing necessary research facilities. University Grants Commission, New Delhi is
acknowledged for grant under University with potential for Excellence to Guru Nanak Dev
University.
Additional Information
Competing Interests: The authors declare no competing financial interests.
References
[1] J.J. Watters, J.A. Sommer, Z.A. Pfeiffer, U. Prabhu, A.N. Guerra, P.J. Bertics, A
Differential Role for the Mitogen-activated Protein Kinases in Lipopolysaccharide Signaling
The MEK/ERK pathway is not essential for nitric oxide and interleukin 1β production, J.
Bio. Chem. 277 (2002), 9077-9087.
[2] T.A. Springer, Traffic signals for lymphocyte recirculation and leukocyte emigration: the
multistep paradigm, Cell 76 (1994), 301-314.
[3] P. Palmqvist, E. Persson, H.H. Conaway, U.H. Lerner, IL-6, leukemia inhibitory factor,
and oncostatin M stimulate bone resorption and regulate the expression of receptor activator
of NF-κB ligand, osteoprotegerin, and receptor activator of NF-κB in mouse calvariae, J.
Immunol. 169 (2002), 3353-3362.
16

ACCEPTED MANUSCRIPT
[4] (a) O. Addimanda, N. Possemato, P. Macchioni, C. Salvarani, Efficacy and safety of
tocilizumab in refractory rheumatoid arthritis: a real life cohort from a single centre, Clin.
Exp. Rheumatol. 32 (2014), 460-464; (b) T.W. Huizinga, R.M. Fleischmann, M. Jasson,
A.R. Radin, J. van Adelsberg, S. Fiore, X. Huang, G.D. Yancopoulos, N. Stahl, M.C.
Genovese, Sarilumab, a fully human monoclonal antibody against IL-6Rα in patients with
rheumatoid arthritis and an inadequate response to methotrexate: efficacy and safety results
from the randomised SARIL-RA-MOBILITY Part A trial, Ann. Rheum. Dis. 73 (2014),
1626-1634.
[5] (a) J. Tois, R. Franzén, A. Koskinen, Synthetic approaches towards indoles on solid phase
recent advances and future directions, Tetrahedron 59 (2003), 5395-5405; (b) P. Singh, P.
Prasher, P. Dhillon, R. Bhatti, Indole based peptidomimetics as anti-inflammatory and anti-
hyperalgesic agents: dual inhibition of 5-LOX and COX-2 enzymes, Eur. J. Med. Chem. 97
(2015), 104-123.
[6] (a) N.K. Kaushik, N. Kaushik, P. Attri, N. Kumar, C.H. Kim, A.K. Verma, E.H. Choi,
Biomedical importance of indoles, Molecules 18 (2013), 6620-6662; (b) S.R. Pedada, N.S.
Yarla, P.J. Tambade, B.L. Dhananjaya, A. Bishayee, K.M. Arunasree, G.H. Philip, G.
Dharmapuri, G. Aliev, S. Putta, Synthesis of new secretory phospholipase A 2-inhibitory
indole containing isoxazole derivatives as anti-inflammatory and anticancer agents, Eur. J.
Med. Chem. 112 (2016), 289-297; (c) A. Singh, M. Kaur, S. Sharma, R. Bhatti, P. Singh,
Rational design, synthesis and evaluation of chromone-indole and chromone-pyrazole based
conjugates: Identification of a lead for anti-inflammatory drug, Eur. J. Med. Chem. 77
(2014), 185-192.
[7] (a) S. Malhotra, S. Balwani, A. Dhawan, Y. Kumar, B.K. Singh, C.E. Olsen, A.K.
Prasad, V.S. Parmar, B. Ghosh, Design, synthesis and biological activity evaluation of
regioisomeric spiro-(indoline-isoxazolidines) in the inhibition of TNF-α-induced ICAM-1
expression on human endothelial cells, MedChemComm 3 (2012), 1536-1547; (b) Y. Xiang,
H.-J. Gi, D. Niu, R.F. Schinazi, K. Zhao, An Efficient Route to β-d-Isoxazolidinyl
Nucleosides via Diastereoselective Michael Addition of Hydroxylamine to Unsaturated
Esters, J. Org. Chem. 62 (1997), 7430-7434; (c) V.S. Georgiev, G.B. Mullen, 3, 5-diphenyl-
3-[(1H-imidazol-1-ylmethyl) or (1H-1, 2, 4-triazol-1-ylmethyl)]-2-methyl-4-isoxazolines,
US Pat., Chem. Abstr. 12 (1988), 35841; (d) G. Singh, A. Sharma, H. Kaur, M.P.S. Ishar,
17

ACCEPTED MANUSCRIPT
Chromanyl–isoxazolidines as Antibacterial agents: Synthesis, Biological Evaluation,
Quantitative Structure Activity Relationship, and Molecular Docking Studies, Chem. Bio.
Drug. Des. 87 (2016), 213-223.
[8] N. Sethi, R. Bhatti, M. Ishar, In vivo pharmacological profile of substituted (3-pyridyl)-2-
phenylisoxazolidine analogues of nicotine as novel antinociceptives, Res. Pharm. Sci. 9
(2014), 59.
[9] G. Singh, A. Kaur, V. Sharma, N. Suri, P.R. Sharma, A.K. Saxena, M.P.S. Ishar,
Synthesis and cytotoxicity evaluation of regioisomeric substituted N-phenyl-3′-(chrom-4-
one-3-yl)-isoxazolidines: induction of apoptosis through a mitochondrial-dependent
pathway, MedChemComm 4 (2013), 972-978.
[10] (a) G. Singh, S. Sharma, V. Gupta, T. Raj, P. Singh, M.P.S. Ishar, Formation and
reductive ring opening reactions of indolyl-isoxazolidines: access to novel natural product
analogs and precursors, Tetrahedron 72 (2016), 900-911; (b) G. Singh, T. Raj, V. Gupta,
M.P.S. Ishar, Formation and reductive ring opening reactions of indolyl bicyclic-
isoxazolidines-II: Access to novel natural product analogs, Tetrahedron Lett. 57 (2016),
4688-4692; (c) S.S. Bhella, A.P.S. Pannu, M. Elango, A. Kapoor, M.S. Hundal, M.P.S.
Ishar, Investigations on synthesis of indole based constrained mimetic scaffolds through 1,
3-dipolar cycloadditions of the C-(3-indolyl)-N-phenylnitrone with a variety of olefinic and
allenic dipolarophiles under microwave irradiation, Tetrahedron 65 (2009), 5928-5935; (d)
B. Chakraborty, A. Samanta, P. Sharma, M. Chhetri, S. Kafley, A. Banerjee, C. Sinha, One
pot high yield synthesis of some novel isoxazolidine derivatives using N-methyl-α-chloro
nitrone in water and their antibacterial activity. J. Chem. Pharm. Res. 2 (2010) 727-739; (e)
H.J. Kim, C.H. Chae, K.Y. Yi, K.L. Park, S.E. Yoo, Computational studies of COX-2
inhibitors: 3D-QSAR and docking. Bioorg. Med. Chem. 12 (2004) 1629-1641; (f) R. Badru,
P. Anand, B. Singh, Synthesis and evaluation of hexahydropyrrolo [3,4-d] isoxazole-4, 6-
diones as anti-stress agents. Eur. J. Med. Chem. 48 (2012) 81-91.
[11] R. Deraedt, S. Jouquey, F. Delevallée, M. Flahaut, Release of prostaglandins E and F in an
algogenic reaction and its inhibition, Eur. J. Pharmacology 61 (1980), 17-24.
[12] C.A. Winter, E.A. Risley, G.W. Nuss, Carrageenin-induced edema in hind paw of the rat as
an assay for antiinflammatory drugs, Exper. Bio. Med. 111 (1962), 544-547.
18

ACCEPTED MANUSCRIPT
[13] (a) C. Sommer, Animal studies on neuropathic pain: the role of cytokines and cytokine
receptors in pathogenesis and therapy, Schmerz (Berlin, Germany) 13 (1999), 315-323; (b)
L. Tao, Q. Ding, C. Gao, X. Sun, Resveratrol attenuates neuropathic pain through
balancing pro-inflammatory and anti-inflammatory cytokines release in mice, Int.
Immunopharmacol. 34 (2016), 165-172.
[14] M. Rahmati, A. Mobasheri, M. Mozafari, Inflammatory mediators in osteoarthritis: A
critical review of the state-of-the-art, current prospects, and future challenges, Bone 85
(2016), 81-90.
[15] Acute Oral Toxicity-Acute Toxic Class Method. OECD Guideline for Testing of
Chemicals; Guideline OECD: Paris 423 (2001).
19

ACCEPTED MANUSCRIPT
Highlights of Manuscript
‹ Variedly substituted Indolyl-isoxazolidines (6-9) have been synthesized by regio- and
stereoselective microwave irradiated 1,3-dipolar cycloaddition reactions.
‹ All the synthesized compounds were evaluated for inhibitory effect against LPS induced
pro-inflammatory cytokines (TNF- α and IL-6) in vitro and in vivo.
‹ The most active compound 9a (bicyclic isoxazolidine analog) possesses significant in vivo
anti-inflammatory/analgesic activity comparable to indomethacin.
‹ Moreover, compound 9a, also showed remarkable protective effect against septic shock in
in vivo mouse model.