Design, synthesis and evaluation of a series of non-steroidal anti-inflammatory drug conjugates as novel neuroinflammatory inhibitors

Article abstract of DOI:10.1016/j.intimp.2015.02.033

Neuroinflammation is involved in the process of several central nervous system (CNS) diseases such as Parkinson's disease, Alzheimer's disease, ischemia and multiple sclerosis. As the macrophages in the central nervous system, microglial cell function in

Full text of DOI:10.1016/j.intimp.2015.02.033

International Immunopharmacology 25 (2015) 528–537
Contents lists available at ScienceDirect
International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp
Design, synthesis and evaluation of a series of non-steroidal
anti-inflammatory drug conjugates as novel
neuroinflammatory inhibitors
1
1
Zhixiang Xu , Jing Wu , Jiyue Zheng, Haikuo Ma, Hongjian Zhang, Xuechu Zhen,
Long Tai Zheng ⁎, Xiaohu Zhang ⁎
Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Su Zhou,
Jiangsu 215021, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Neuroinflammation is involved in the process of several central nervous system (CNS) diseases such as
Parkinson's disease, Alzheimer's disease, ischemia and multiple sclerosis. As the macrophages in the central
nervous system, microglial cell function in the innate immunity of the brain and are largely responsible for the
inflammation-mediated neurotoxicity. Prevention of microglia activation might alleviate neuronal damage and
degeneration under the inflammatory conditions, and therefore, represents a possible therapeutic approach to
the aforementioned CNS diseases. Here we report the synthesis of a number of non-steroidal anti-inflammatory
drug (NSAID) conjugates, and the evaluation of their anti-inflammatory effects in lipopolysaccharide
(LPS)-stimulated BV-2 microglial cells and primary mouse microglial cells. Among the tested analogues,
compounds 8 and 11 demonstrated potent inhibition of nitric oxide production with no or weak cell toxicity.
Compound 8 also significantly suppressed the expression of tumor necrosis factor (TNF)-α, interleukin
Received 6 December 2014
Received in revised form 14 February 2015
Accepted 20 February 2015
Available online 10 March 2015
Keywords:
Neuroinflammation
Neurodegenerative diseases
Microglia activation
Drug conjugates
Neuroprotection
(
IL)-6, cyclooxygenase (COX)-2 as well as inducible nitric oxide synthase (iNOS) in LPS-stimulated BV-2
microglial cells. Further mechanistic studies indicated that compound 8 significantly suppressed phosphoryla-
tion of mitogen-activated protein kinases (MAPKs) and subsequent activation of activator of transcription 1
(AP-1). Furthermore, in a co-culture system, compound 8 inhibited the cytotoxicity generated by LPS-activated
microglia toward HT-22 neuroblastoma cells. Collectively, these experimental results demonstrated that
compound 8 possessed potent anti-neuroinflammatory activity via inhibition of microglia activation, and
might serve as a potential lead for the therapeutic treatment of neuroinflammatory diseases.
©
2015 Elsevier B.V. All rights reserved.
1
. Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs, representatives,
diclofenac and 5-ASA, are shown in Fig. 1) are the treatment of choice
for peripheral acute and chronic inflammation and pain [5,6]. They
have also been shown to reduce the risk of developing Parkinson's
disease and Alzheimer's disease [7,8]. The beneficial effects have been
attributed mainly to the anti-inflammatory functions elicited by these
drugs. However, the application of NSAIDs in CNS was circumvented
by their limited exposure in the brain [9], resulting in the need for
higher doses, and therefore, higher risk of side effects. Many NSAID
analogues were developed in the past decades, aiming at enhancing
efficacy, reducing side effects, or both [10,11]. One example was
the NSAID–ADT–OH conjugate (Fig. 1, ACS-15, compound 6; ATB-429,
compound 7) [12,13]. ADT–OH (Fig. 1, compound 3) is a slow-releasing
hydrogen sulfide donor. Hydrogen sulfide has been demonstrated to
show multiple physiological functions including neuronal protection
and anti-inflammatory effects [14–16]. The conjugate ACS-15 indeed
exhibited enhanced potency compared with diclofenac (compound 1)
in the inhibition of neuroinflammation induced by microglial and
astrocytic activation [17].
Neuroinflammation is a self-defense reaction in the central nervous sys-
tem (CNS) to protect the human body from injurious stimuli and to initiate
the healing process. However, chronic neuroinflammation could become
the causative factor in the pathogenesis of a broad range of diseases such
as Parkinson's disease, Alzheimer's disease, ischemia, and multiple sclerosis
[1,2]. As the macrophages in the central nervous system, microglial cell
function in the innate immunity of the brain and are largely responsible
for the inflammation-mediated neurotoxicity [3,4]. Prevention of microglia
activation might alleviate neuronal damage and degeneration under the
causative inflammatory conditions, and therefore, represents a possible
therapeutic approach to the aforementioned CNS diseases.
⁎
Corresponding authors. Tel./fax: +86 512 65880380.
E-mail addresses: zhenglongtai@suda.edu.cn (L.T. Zheng), xiaohuzhang@suda.edu.cn
(X. Zhang).
1
These authors contributed equally to this paper.
http://dx.doi.org/10.1016/j.intimp.2015.02.033
567-5769/© 2015 Elsevier B.V. All rights reserved.
1

Z. Xu et al. / International Immunopharmacology 25 (2015) 528–537
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Fig. 1. Examples of NSAIDs, multi-functional ligands, known conjugates (6, 7), and new conjugates (8–11).
Encouraged by these results, we decided to explore other NSAID
conjugates as potential drug leads to attenuate neuroinflammation.
Cysteamine (Fig. 1, compound 4), an anti-oxidant aminothiol, was
approved by FDA for the treatment of nephropathic cystinosis, a rare
lysosomal storage disease [18]. Cysteamine exerts its biological func-
tions through multiple mechanisms, including anti-oxidation, metal
chelation, among others [19]. Oxidative neuronal damage and abnormal
metal homeostasis are both involved in the development of Alzheimer's
disease and Parkinson's disease [20–22], thus making cysteamine an
attractive candidate to conjugate with NSAIDs. Because the conjugates
are intended for CNS indications, and therefore need to cross the blood
brain barrier, certain physical–chemical properties, such as low mo-
lecular weight, low polar surface area are especially important [23].
The fact that cysteamine has an extremely small molecular weight,
along with an excellent safety profiles, further strengthens its candi-
dacy as a NSAID conjugate; therefore, compound 8 was synthesized.
An alternative way to conjugate cysteamine to diclofenac via an amide
bond was developed [24]; however, the free thiol may suffer from
spontaneous oxidation, which could complicate biological analysis.
Resveratrol, a natural stilbene which is a constituent of red wine, has
been shown to prevent or slow a wide range of ailments such as cardio-
vascular disease, cancer and ischemic injuries [25,26]. Like cysteamine,
resveratrol also exerts its biological functions through multiple mecha-
nisms. Well studied molecular targets of resveratrol include quinone
reductase 1 and 2 (anti-oxidation) [27], COX (anti-inflammation) [28],
and SIRT1 (longevity) [29–31], a member of the sirtuin deacylase family.
Despite overwhelmingly favorable results, however, the therapeutic
application of resveratrol was hampered by its fast metabolism in circu-
lation [32]. Major metabolites are sulfate and glucuronide on the
hydroxyl groups of resveratrol. Here we propose using diclofenac to
block the metabolic soft spots of resveratrol; therefore, compounds
9–11 were synthesized. The conjugates may bring the multifunctional
effects from diclofenac and resveratrol into synergy.
In the primary screening experiment, NO release was used as a
marker during LPS-stimulated BV-2 microglial cell activation. We have
included compounds 1–7 as reference compounds to evaluate the
anti-neuroinflammatory activities of compounds 8–11.
2. Materials and methods
2.1. Chemistry
The synthetic approach to compounds 8–11 is outlined in Fig. 2.
Treatment of diclofenac with tert-butyl 2-mercaptoethylcarbamate

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Z. Xu et al. / International Immunopharmacology 25 (2015) 528–537
Fig. 2. Reagents and conditions: (a) tert-butyl 2-mercaptoethylcarbamate, DCC, DMAP, DCM, r. t., 16 h. (b) HCl/EtOAc, r. t., 8 h. (c) DCC, DMAP, DCM, dioxane, r. t., 24 h. (d) ClCH
2 2
OSO Cl,
Bu HSO , DCM, H O, r. t., 1 h. (e) NaI, acetone, r. t., 24 h. (f) TBSCl, imidazole, DMF, r. t., 24 h. (g) Ag CO , MeCN, r. t., 10 h. (h) TBAF, AcOH, r. t., 0.5 h.
4
4
2
2
3
and N,N-dicyclohexylcarbodiimide (DCC) provided intermediate 12,
which was deprotected with HCl/EtOH to give the desired compound
mouse IFN-γ were obtained from R&D Systems (Minneapolis, MN).
Anti-iNOS and anti-COX-2 were obtained from Abcam (Cambridge,
MA). Anti-IκBα and anti-phospho-IκBα were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). Anti-ERK1/2, p38, JNK and c-jun
were obtained from Cell Signaling Technology (Beverly, MA). DMEM,
DMEM/F-12 and FBS were obtained from Invitrogen (Carlsbad, CA).
BV-2 murine microglia cell line, RAW 264.7 macrophage cell and
HT-22 mouse neuroblastoma cell lines were cultured in DMEM con-
8
as an HCl salt. Compound 9 was obtained via direct coupling of
diclofenac with resveratrol using standard DCC coupling conditions. To
synthesize compounds 10 and 11, chloromethyl ester 13 was prepared
by alkylation of diclofenac using chloromethyl chlorosulfate, and then
reacted with NaI in acetone to yield iodomethyl ester 14. Resveratrol
was treated with TBSCl and imidazole to give a mixture of compounds
1
5 and 16, which were alkylated with iodomethyl ester 14 and subse-
2
taining 10% FBS, 1% penicillin/streptomycin at 37 °C, 5% CO .
quently deprotected with TBAF to afford compounds 10 and 11 (Fig. 2).
2.2.2. Primary culture of microglia and astrocytes
2
2
.2. Biological assay
All of the animal studies were approved by the Institutional Review
Board of Soochow University. The care and use of animals were per-
formed in accordance with the guidelines of National Institute of Health.
Primary culture of microglia and astrocytes were isolated from cerebral
cortices of newborn of Institute of Cancer Research (ICR) mice as
described previously [33]. In brief, stripped meninges were incubated
in serum free DMEM/F12 containing 0.2% of trypsin for 10 min at
.2.1. Reagents and cell culture
The reagents used in this study are following: Anti-α-tubulin, horse-
radish peroxidase-conjugated secondary antibodies and bacterial lipo-
polysaccharide (LPS) (Escherichia coli serotype 055:B5) were obtained
from Sigma-Aldrich (St. Louis, MO). ELISA kits and recombinant

Z. Xu et al. / International Immunopharmacology 25 (2015) 528–537
531
3
7 °C and then repeatedly suspended using different pore size of tips in
2.2.8. NF-κB and AP-1 reporter assay
DMEM/F12 containing 10% FBS. After centrifugation and resuspension,
the cells were plated on poly-D-lysine-coated 75 mm flasks and cultured
for 14 days at 37 °C, 5% CO . The microglia cells were separated from
2
mixed glial cultures by shaking at 150 rpm for 2 h. The astrocyte
cultures were obtained from mixed glial cell by shaking at 280 rpm for
The establishment of stable BV-2 cell line expressing NF-κB or AP-1
reporter construct was performed as previously described [37]. In
brief, the BV-2 microglia cells stably expressing NF-κB or AP-1
constructs (Cignal™ Lenti Reporters, Qiagen) were seeded on 24 well
5
plates (1 × 10 cells/well). The cells were pretreated with compounds
1
2 h. The purity of cultured microglia or astrocytes ( 95%) were con-
or vehicle for 30 min prior to LPS (0.1 μg/mL) treatment. After 16 h of
LPS stimulation, luciferase activity was measured using the luciferase
assay kit (Promega) according to the instruction of the manufacturer
and the level of promoter activity was expressed as relative units.
firmed by immunostaining with CD11b or GFAP respectively (data not
shown).
2
.2.3. Cytotoxicity assay
The measurement of the cell viability was determined by 3-(4,5-
2.2.9. Microglia/neuron co-culture
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay as previously described [34]. In brief, the cells were pretreated
with different concentration of compound prior to LPS challenge for
For the co-culture of microglia/neuron, microglia condition media
system was used as described previously [37]. In brief, BV-2 microglia
cells were seeded in 6 well cell culture plates. The BV-2 microglia cells
were pretreated with compound or vehicle for 30 min prior to LPS
(0.1 μg/mL) treatment. After stimulation by LPS for 24 h, BV-2 cells
condition medium (CM) was collected. The HT-22 neuroblastoma cells
were incubated with CM from compound or vehicle treated BV-2 cells
for 36 h, and then the cell viability was accessed by MTT assay.
3
0 min. After incubation with LPS for 24 h, the cell culture media
were removed and 30 μL of MTT (0.5 mg/mL) solution was added
to each well. After incubation for 4 h at 37 °C, the cell culture super-
natants were aspirated and 100 μL of dimethyl sulfoxide (DMSO)
was added into each well. The absorbance of solubilized formazan
was measured by microplate reader at 540 nm of absorbance.
2.2.10. Statistical analysis
2
.2.4. Nitrite quantification
For the statistical analysis, SPSS software (version 16) was used. The
The measurement of NO levels in condition medium was performed
data are presented as the mean ± standard deviation of 3 independent
experiments. For the multiple comparisons, the data were analyzed
using One-way ANOVA followed by the Student Newman Keuls post
hoc analysis. A value of p b 0.05 was considered significant.
with Griess reagent as described previously [35]. In brief, after treat-
ment of the cells with different concentration of compound or stimulat-
ing agents, the cell culture media were collected. 50 μL of the cell culture
medium was added to 96 well plates containing 50 μL of Griess reagent
[36] and incubated for 10 min at room temperature. The optical density
3. Results
was obtained by measuring absorbance at 550 nm with microplate
−
reader. To calculate concentration of NO
2
, serially diluted sodium
3.1. Inhibition of NO production in activated microglial cells
nitrite solution was used as the standard reagent.
Excessive activation of microglia is a signature of neuroinflammation
and is related to the neurodegenerative progress by releasing various
neurotoxic mediators [1]. The inhibition of microglial activation might
be an effective therapeutic option for the treatment of neurodegenera-
tive diseases. NO, a major pro-inflammatory factor, is a hallmark of
microglial activation and plays an important role in the neuro-
inflammatory process and neuronal cell death [1]. Therefore, we evalu-
ated the inhibitory effects of the novel NSAID conjugates on the NO
generation in LPS activated microglia cells. In parallel, we evaluated
cell viability by MTT assay to mitigate the possibility that the decrease
of NO level was due to cell growth inhibition or cytotoxicity. The results
were summarized in Table 1. The two NSAIDs, diclofenac and 5-ASA,
2
.2.5. Measurement of level of TNF-α, IL-6 and PGE
The amounts of TNF-α, IL-6 and PGE in the cell culture medium
2
2
were measured by specific ELISA kits according to the instruction of
the manufacturer.
2
.2.6. Isolation of total RNA and quantitative real-time PCR
Cells were cultured in 6 well plates at a density of 2 × 10 cells/well.
5
After incubation for overnight, the cells were pretreated with different
concentration of compound or vehicle for 30 min prior to stimulation
of LPS (0.1 μg/mL) for 6 h. The extraction of total RNA and preparation
of cDNA were performed using TRIzol reagent (Invitrogen, CA) and
reverse transcription kit (Takara, Dalian, China) respectively. The exper-
iment was performed in accordance with the instruction of the manu-
facturer. The specific primers used for PCR are the following: iNOS
forward, TAG GCA GAG ATT GGA GGC CTT G; iNOS reverse, GGG TTG
CTG AAC TTC CAG TC; COX-2 forward, CAG GCT GAA CTT CGA AAC A;
COX-2 reverse, GCT CAC GAG GCC ACT GAT ACC TA; TNF-α forward,
CAG GAG GGA GAA CAG AAA CTC CA; TNF-α reverse, CCT GGT TGG
CTG CTT GCT T; IL-6 forward, TCC AGG ATG AGG ACA TGA GCA C; IL-6
reverse, GAA CGT CAC ACA CCA GCA GGT TA; and GAPDH forward,
TGT GTC CGT CGT GGA TCT GA, GAPDH reverse, TTGCTG TTG AAG TCG
CAG GAG. Quantitative real-time PCR was conducted with CFX96 PCR
instrument (BIORAD, USA) using specific primers and SYBR Premix II
kit (Takara, Dalian, China). The CT values of each target gene were
normalized to that of GAPDH. For calculation of relative quantification,
the delta CT method was used [34].
Table 1
Compound activity on inhibition of NO production in LPS-activated BV-2 microglia and
cell viability.
IC50 (μM)a
Cell viability (%)b
Compound
(10 μM)
(5 μM)
1
2
3
4
5
6
7
8
9
10
246.64 ± 2.39
59.87 ± 1.77
16.24 ± 1.21
124.25 ± 2.09
22.86 ± 1.35
16.00 ± 1.20
19.56 ± 1.29
4.80 ± 0.68
107.35
95.92
96.08
97.86
103.37
96.98
98.96
104.46
103.14
93.91
93.83
102.79
97.06
97.62
99.78
106.81
94.56
100.37
102.26
96.67
93.91
93.98
24.14 ± 1.38
18.95 ± 1.27
14.43 ± 1.15
11
a
2
.2.7. Western blot analysis
IC50: the concentration that produces 50% inhibitory effect on NO production. Values
The cells were pre-incubated with different concentration of com-
obtained from three independent experiments and the mean inhibition of NO production
relative to the LPS control for respective compound. The data are presented as mean
pound or vehicle for 30 min prior to treatment of LPS (0.1 μg/mL).
After LPS treatment, the cell lysates were obtained and the equal
amounts of proteins were subjected to Western Blot analysis as described
previously [36].
±
standard deviation.
b
Cell viability after treatment with (5–10 μM) of each compound was expressed as
a percentage (%) of the value obtained from LPS alone group. The data are presented
as mean ± standard deviation.

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Z. Xu et al. / International Immunopharmacology 25 (2015) 528–537
were virtually inactive (247 and 60 μM, respectively) under the current
experimental conditions. Cysteamine was also inactive (124 μM).
However, both ADT–OH and resveratrol demonstrated moderate inhib-
itory activities (16 and 23 μM, respectively), consistent with those of the
previous reports [13,17,39]. ACS-15 and ATB-429 both showed
improvement of the inhibitory potency (16 and 20 μM, respectively)
compared with their corresponding NSAIDs, diclofenac, and 5-ASA,
respectively. However, this improvement could be attributed to the
inhibitory activity from the other half of the conjugate, ADT–OH
(
16 μM). The three resveratrol-diclofenac conjugates, compounds 9,
1
0, and 11, all demonstrated moderate inhibitory effects (24, 19 and
1
4 μM, respectively) comparable to or better than that of resveratrol
(
23 μM). Among them, compound 11 exhibited a decent improve-
ment compared with both parent molecules, indicating that a synergetic
effect was indeed possible. This was validated in the case of cysteamine–
diclofenac conjugate, as compound 8 (4.8 μM) was much more potent
compared with diclofenac and cysteamine. All of the 11 compounds
were well tolerated by the BV-2 microglial cells in the two concentra-
tions (5 and 10 μM) tested as demonstrated by the cell viability assay
(
Table 1). Compounds 1, 4, 5, 8 and 11 were selected for a detailed
dose responses assessment (Fig. 3A–E). As shown in Fig. 3, compounds
and 11 significantly reduced NO production in a dose dependent
8
Fig. 4. Effect of compound 8 on NO production in LPS-stimulated BV-2 microglia, primary
microglia, RAW 264.7 macrophage cells, and LPS/IFN-γ-stimulated primary astrocytes.
The cells were pretreated with compound 8 (1.25–5 μM) for 30 min, followed by LPS
manner in the LPS-stimulated BV-2 microglial cells. Consistent with
the IC50 determination experiments, compound 8 is much more potent
than the parent compounds 1 and 4 on inhibiting NO production in
LPS-stimulated BV-2 microglial cells. Similarly, compound 11 was also
more effective than the parent compounds 1 and 5. Compound 8 repre-
sented the most potent compound that we evaluated in this assay. The
generality of the inhibitory effect of compound 8 on NO production
was confirmed in LPS or LPS/IFN-γ stimulated BV-2 microglial cells,
primary microglia, RAW 264.7 murine macrophage cells and primary
astrocytes (Fig. 4). In addition, cell viability assay demonstrated that
(0.1 μg/mL) or LPS/IFN-γ (50 unit/mL) treatment for 24 h. The nitrite in the cell culture
medium was quantified using Griess reaction (A, BV-2 cells; B, primary microglia; C,
RAW 264.7 cells; D, primary astrocytes). The results shown were obtained from 3
independent experiments. Asterisk indicates significant difference from LPS or LPS/IFN-γ
treatment alone group (*p b 0.05, **p b 0.01).
compound 8 at the indicated concentrations (1.25–5 μM) did not de-
crease the viability of the aforementioned cells in the presence or ab-
sence of LPS or LPS/IFN-γ (data not shown).
Fig. 3. Effect of compounds (A, 1; B, 4; C, 5; D, 8; E, 11) on NO production in LPS-induced BV-2 microglial cells. BV-2 microglial cells were pretreated with indicated concentrations of
compounds for 30 min, followed by LPS (0.1 μg/mL) treatment for 24 h. The contents of nitrite in the cell culture medium were quantified using Griess reaction. Comparison of the effect
of compounds 1, 4, 8 and equal molar mixture of compounds 1 and 4 on NO production in LPS-induced BV-2 microglia cells (F). Data represent the mean ± standard deviation of 3
independent experiments carried out in triplicate. Asterisk indicates significant difference from LPS treatment alone group (*p b 0.05, **p b 0.01). #p b 0.01 indicates significant difference
from compound 8 treatment group.

Z. Xu et al. / International Immunopharmacology 25 (2015) 528–537
533
3
.2. Compound 8 inhibited the expression of pro-inflammatory genes in
kinases (MAPKs), including ERK, p38 and c-Jun N-terminal kinase
(JNK) [47]. Therefore, the effect of compound 8 on the MAPK pathways
was investigated by Western blot. The results revealed that compound
8 suppressed the phosphorylation of ERK and JNK in LPS-activated
BV-2 microglial cells, whereas p38 phosphorylation was not affected
(Fig. 7C). Since activated ERK and JNK directly bind and phosphorylate
c-jun which is a major component of AP-1 transcriptional factor in
LPS-activated microglia [47,48], the effect of compound 8 on c-jun phos-
phorylation was determined by Western blot. As shown in Fig. 7D,
compound 8 markedly suppressed LPS induced phosphorylation of
c-jun in LPS-activated BV-2 microglia cells. To confirm that compound
8 suppressed LPS-induced microglial activation through the inhibition
of AP-1 activity, we next examined the effect of compound 8 on LPS-
induced AP-1 luciferase activity. As shown in Fig. 7E, compound 8 signif-
icantly inhibited LPS induced AP-1 luciferase activity in BV-2 microglia
cells. This is consistent with reports that diclofenac inhibited AP-1
activity in various cell lines [13,49]. These results suggest that MAPK/
AP-1 pathway might be implicated in the inhibitory effects of compound
8 on the LPS triggered inflammatory responses in microglia cells.
LPS-stimulated BV-2 microglial cells
Inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2
expression are induced in activated microglia and responsible for syn-
thesis of NO and PGE , respectively [40]. Likewise, pro-inflammatory
2
cytokines such as TNF-α, IL-6 are over produced in activated microglia
cells and actively contribute to neurodegenerative process [41]. The
effect of compound 8 on iNOS, COX-2, TNF-α and IL-6 gene expression
at mRNA levels was determined by quantitative real-time PCR. As
shown in Fig. 5, compound 8 suppressed the gene expression of iNOS,
COX-2, TNF-α and IL-6 in a dose dependent manner in LPS-activated
BV-2 microglial cells. The inhibitory effect of compound 8 on the expres-
sion of iNOS, COX-2, TNF-α, IL-6 and PGE
confirmed by immunoblotting analysis. As shown in Fig. 6, compound 8
significantly inhibited LPS-induced iNOS, COX-2, TNF-α, IL-6 and PGE
expression at protein level.
2
at protein levels was further
2
3
.3. Compound 8 inhibited MAPK phosphorylation and AP-1 activation in
LPS-activated BV-2 microglial cells
NF-κB is a key transcriptional factor that controls the expression of
inflammatory cytokines and enzymes in activated microglia [42]. The
inhibition of NF-κB activity has been implicated in the suppressive effect
of numerous chemicals on the gene expression of iNOS, COX-2, TNF-α,
and IL-6 in microglial cells [43,44]. We therefore investigated the effect
of compound 8 on NF-κB activation in LPS-activated microglial cells. As
shown in Fig. 7A and B, compound 8 did not suppress LPS-induced IκB
phosphorylation, IκB degradation and NF-κB luciferase activity, suggest-
ing that the NF-κB pathway was not involved in the anti-inflammatory
mechanism of compound 8 in BV-2 cells. Previously, it was demonstrated
3.4. Compound 8 reduced microglial neurotoxicity in microglia/neuron
co-culture model
To investigate the potential neuroprotective effect of compound 8
in vitro, we took advantage of a microglia/neuron co-culture model.
The neuronal toxicity of conditioned media (CM) of LPS-activated
BV-2 microglial cells was evaluated with HT-22 hippocampal cells. A
significant decrease of HT-22 cell viability was observed after the
HT-22 cells were treated with CM harvested from LPS-activated BV-2
microglial cells. However, the viability of HT-22 cells being treated
with CM harvested from compound 8 pre-incubated BV-2 cells was
significantly improved (Fig. 8A). To investigate whether compound 8
could protect against oxidative stress-induced HT-22 cell death, we
2
that inhibition of NF-κB activity by S-diclofenac was dependant on H S
releasing property [45]. It seemed that compound 8 elicited its anti-
inflammatory effect through an alternative mechanism. AP-1 activation
is also known to play a critical role in the signal transduction that controls
pro-inflammatory gene expression in activated glial cells [46]. AP-1
activation in glial cells is mainly mediated by mitogen-activated protein
determined the viability of HT-22 cells after H
in the presence or absence of compound 8. The results showed that
compound 8 also ameliorate H induced HT-22 cell death (Fig. 8B).
2 2
O treatment for 24 h
2 2
O
Fig. 5. Effect of compound 8 on iNOS, TNF-α, IL-6 and COX-2 expression at mRNA level in LPS-stimulated BV-2 microglial cells. The BV-2 microglial cells were pretreated with compound 8
1.25–5 μM) for 30 min, followed by LPS treatment (0.1 μg/mL). The iNOS (A), TNF-α (B), COX-2 (C) and IL-6 (D) mRNA levels were determined by SYBR green quantitative-RT-PCR. Data
represent the mean ± standard deviation of 3 independent experiments carried out in triplicate. Asterisk indicates significant difference from LPS or LPS/IFN-γ treatment alone group
*p b 0.05, **p b 0.01).
(
(

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Z. Xu et al. / International Immunopharmacology 25 (2015) 528–537
Fig. 6. Effect of compound 8 on iNOS, TNF-α, IL-6 and COX-2 expression at protein level in LPS-stimulated BV-2 microglial cells. The BV-2 microglial cells were pretreated with compound 8
1.25–5 μM) for 30 min, followed by LPS treatment (0.1 μg/mL). The iNOS (A, upper), COX-2 (A, lower) levels were determined by Western blot. The amounts of PGE2 (B), TNF-α (C) and
(
IL-6 (D) in the supernatants were measured using ELISA kit. Data represent the mean ± standard deviation of 3 independent experiments carried out in triplicate. Asterisk indicates
significant difference from LPS or LPS/IFN-γ treatment alone group (*p b 0.05, **p b 0.01).
Fig. 7. Effect of compound 8 on NF-κB, MAPKs and AP-1 activation in LPS-activated BV-2 microglial cells. BV-2 microglial cells were pretreated with compound 8 (1.25–5 μM) for 30 min,
followed by LPS treatment (0.1 μg/mL) for indicated time point. The phosphorylated–IκB-α (10 min) and IκB-α (20 min) levels were determined by Western blot using respective
antibodies (A). BV-2 cells stably expressing NF-κB (B) or AP-1 (E) reporter construct was pretreated with compound 8 (5 μM) for 30 min, followed by LPS treatment (0.1 μg/mL) for
1
6 h. Luciferase activity was measured by luminometry. The luciferase activity is expressed as relative values and the values of control are set to a relative value of 1. Data represent
the mean ± standard deviation of 3 independent experiments carried out in triplicate. Asterisk indicates significant difference from LPS or LPS/IFN-γ treatment alone group (*p b 0.05,
*p b 0.01). The activation of MAPKs (ERK1/2, upper; p38, middle; JNK, lower) was determined by Western blot using respective antibodies (C). The phosphorylated-c-jun (1 h) level
was determined by Western blot (D).
*

Z. Xu et al. / International Immunopharmacology 25 (2015) 528–537
535
Fig. 8. Effect of compound 8 on microglial neurotoxicity. The BV-2 microglial cells were pretreated with compound 8 (1.25–5 μM) for 30 min, followed by LPS treatment (0.1 μg/mL). After
treatment of LPS for 24 h, CM from control, compound 8 alone, LPS alone and LPS/compound 8 treated BV-2 microglial cells was added to HT-22 cells in 96 well plates. HT-22 cell viability
was assessed by MTT assay after 36 h (A). HT-22 cells were pretreated with indicated concentration of compound 8 prior to 500 μM of H O for 30 min. The HT-22 cell viability was
2 2
measured by MTT assay (B). Data represent the mean ± standard deviation of 3 independent experiments carried out in triplicate. Asterisk indicates significant difference from LPS or
LPS/IFN-γ treatment alone group (*p b 0.05, **p b 0.01).
3
.5. Stability of compound 8, in vitro and in vivo transition
diclofenac-L-cysteine conjugate exhibited enhanced anti-inflammatory
activity in vivo [50]. In line with this, we observed that diclofenac–cyste-
amine conjugate inhibited NO production more effectively than that of
the combination of diclofenac and cysteamine, suggesting that the syner-
gistic effect of the conjugate was unlikely related to the hydrolysis of the
thioester bond. Additional literature reports showed that amide prodrug
of ibuprofen and naproxen were metabolized very slowly in vivo [51],
and amide derivatives of indomethacin were not readily hydrolyzed
into indomethacin in plasma. Taken together, we suspect that the syner-
gistic effect of diclofenac–cysteamine conjugate on NO inhibition in LPS-
activated microglia cells might involve different mechanisms: antioxi-
dant cysteamine enhanced anti-inflammatory action of diclofenac, such
as by increasing cellular glutathione levels; or compound 8 or its metab-
olites per se through alternative mechanisms. It is well known that iNOS
and COX-2 catalyze the production of NO and PGE2 from L-arginine and
arachidonic acid, respectively, and are shown to be induced by various
inflammatory stimuli including pro-inflammatory cytokine and LPS. Al-
though diclofenac is a selective COX inhibitor, many studies have demon-
strated that diclofenac and its derivates exhibited inhibitory effects on
expression of iNOS, COX-2 and pro-inflammatory cytokines including
TNF-a, IL-6 [13,17]. In agreement with this study, we found that com-
pound 8 markedly suppressed expression of iNOS, COX-2, TNF-a and
IL-6 at mRNA or protein levels, suggesting that this compound may be
a potential lead for the therapeutic treatment of microglial activation-
associated neuroinflammatory diseases.
Transduction of LPS signaling within microglia was mainly mediated
by Toll-like receptor 4 (TLR4). TLR4 activation leads to activation of
many intracellular signaling pathways including IκB kinase (IKK) com-
plex, MAPKs and PI3K/Akt [48]. These intracellular signal molecules ul-
timately lead to the activation of transcriptional factors NF-κB and AP-1,
which control gene expression of many pro-inflammatory cytokines
and enzymes. It is well established that LPS induced NF-κB activation
is mediated by IκB phosphorylation, IκB degradation and nuclear trans-
location of NF-κB. In the nucleus, the NF-κB binds to a κB site in the pro-
moter region of target genes, thereby regulating transcriptional gene
expression [52]. In our work, it was observed that compound 8 inhibited
neither the IκB degradation nor the NF-κB promoter activity in LPS-
activated microglia cells. This was consistent with previous observation
that diclofenac alone did not suppress the activation of NF-κB [13]. In
addition to NF-κB, AP-1 is another main transcriptional factor that
plays a critical role in the expression of pro-inflammatory genes such
as iNOS, COX-2, IL-6 and ROS [48]. Transcriptional activation of AP-1 is
mediated by the phosphorylation of MAPKs which has been implicated
in the signal transduction pathways for inflammatory responses of mi-
croglia cells. It was demonstrated that diclofenac and its derivates sig-
nificantly attenuated LPS triggered AP-1 DNA-binding activity [13]. In
agreement with these results, we also found that diclofenac–cysteamine
conjugate significantly inhibited LPS-induced phosphorylation of ERK1/
2 and JNK, ultimately led to the blockage of transactivation of AP-1.
Since compound 8 is a thioester conjugate, we did preliminary sta-
bility test on it. Compound 8 as an HCl salt was stable at room temper-
ature for days. Compound 8 was also evaluated in water and DMSO and
remained unchanged after 24 h at room temperature. However, when
we evaluated compound 8 in rat pharmacokinetics (P.K.) study, com-
pound 8 demonstrated a large clearance (N100 mL/min/kg), indicating
a high hurdle to translate the in vitro results into in vivo efficacy (data
not shown).
4
. Discussion
In the present study, we synthesized a number of non-steroidal
anti-inflammatory drug (NSAID) conjugates and evaluated their
anti-inflammatory effects in microglia cells. Among the tested analogues,
compounds 8 (diclofenac–cysteamine conjugate) and 11 (diclofenac–
resveratrol conjugate) exhibited potent inhibitory activities on nitric
oxide production with no or weak cell toxicity. Compound 8 significantly
suppressed the expression of tumor necrosis factor (TNF)-α, interleukin
(
(
IL)-6, cyclooxygenase (COX)-2 as well as inducible nitric oxide synthase
iNOS) in LPS-stimulated BV-2 microglia cells. Further mechanistic
studies indicated that compound 8 significantly suppressed phosphory-
lation of mitogen-activated protein kinases (MAPKs) and subsequent
activation of activator of transcription 1 (AP-1). Furthermore, in a co-
culture system, compound 8 inhibited the cytotoxicity generated by
LPS-activated microglia toward HT-22 neuroblastoma cells.
Over activation of microglia is a signature of neuroinflammation and
contributes to the neurodegenerative progress by releasing various
neurotoxic mediators [1]. The inhibition of microglial activation repre-
sented effective potential therapeutic approach for the treatment of
neurodegenerative diseases. NO, a major pro-inflammatory factor, is a
hallmark of microglial activation and plays an important role in the
neuroinflammatory process and neuronal cell death [1]. Therefore, we
evaluated the inhibitory effects of the novel NSAID conjugates on the
NO generation in LPS activated microglia cells. We found that (i) the
parent drugs (diclofenac and cysteamine) alone showed weak inhibitory
activity on NO production; (ii) co-administration of diclofenac and cys-
teamine with equimolar doses resulted in enhanced inhibition of
NO production; (iii) diclofenac–cysteamine conjugate (compound 8)
demonstrated the most potent inhibitory effect on NO production,
much more than that of the combined diclofenac and cysteamine
(
Table 1 and Fig. 3). Previously, it was reported that S-diclofenac
displayed enhanced anti-inflammatory activity over the individual
parent compound in an additive fashion because the conjugate broke
down into diclofenac and H S [13]. On the other hand, it was demon-
2
strated that co-administration of diclofenac and L-cysteine displayed
less anti-inflammatory activity than individual parent compound, while

536
Z. Xu et al. / International Immunopharmacology 25 (2015) 528–537
Previous studies indicated that ERK is an upstream signal molecule of
NF-κB in peripheral cells [53]. On the contrary, it was suggested that
NF-κB and ERK are two unrelated signal pathways in inflammatory
responses [54]. We have shown that an ERK specific inhibitor, U0126,
did not block NF-κB activation, suggesting that NF-κB activation is
not induced by ERK phosphorylation [37]. We therefore suggest
that diclofenac–cysteamine conjugate reduces the production of pro-
inflammatory mediators though inhibition of the intracellular signal
transduction pathway MAPKs/AP-1.
Pro-inflammatory cytokines and free radicals released by activated
microglial cells are the major neurotoxic factors and are actively in-
volved in the neuronal cell apoptosis and progression of neuronal
degeneration associated diseases [38,55]. Thus, the inhibition of
microglial activation may mitigate neurotoxicity [33]. In fact, a number
of epidemiological studies have demonstrated that the use of NSAID is
associated with lower risk of neurodegenerative diseases. In addition
to inflammation, oxidative stress is also implicated in the pathogenesis
of neurodegenerative diseases. Cysteamine is an organic compound
generated in mammals as product of the coenzyme A metabolism
GAPDH glyceraldehydes 3-phosphate dehydrogenase;
GFAP
HBSS
ICR
glial fibrillary acidic protein;
Hank's balanced salt solution;
institute of cancer research;
interferon;
IFN
IκB
inhibitor of kappa B;
IL
interleukin;
iNOS
JNK
LPS
inducible nitric oxide synthase;
c-Jun N-terminal kinase;
lipopolysaccharide;
MAPKs mitogen-activated protein kinases;
MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide;
NF-κB
nuclear factor-kappa B;
NSAID
non-steroidal anti-inflammatory drug;
prostaglandin E2;
PGE
2
RT-PCR real-time polymerase chain reaction;
S.D.
SDS
standard deviation;
sodium dodecyl sulfate;
[18]. Like cysteamine, cysteine can also be oxidized into taurine, which
Tris–HCl tris-(2-carboxyethyl)-phosphine hydrochloride.
is one of the most abundant amino acids in the brain and possesses
anti-oxidant activity [56]. Increasing evidence suggested that cyste-
amine and cysteine sustain neuroprotective and anti-oxidative proper-
ties in vitro as well as in vivo [57]. In the current study, we found
that diclofenac–cysteamine not only reduce microglial cell activation
Acknowledgments
This work was supported by National Natural Science Foundation
of China (Grant No. 81273372, 81372688,81473090, 81130023,
31373382); National Basic Research Plan (973) of the Ministry of Science
and Technology of China (2011CB5C4403); China Postdoctoral Science
Foundation (2013M541729); Natural Science Foundation of Jiangsu
Province (BK20140313); BM2013003; and PAPD (A Project Funded by
the Priority Academic Program Development of Jiangsu Higher Education
Institutions).
2 2
induced neurotoxicity, but also inhibit H O induced neuronal cells
death. These result suggested that two potential mechanisms are in-
volved in the neuroprotection of compound 8: i) diclofenac–cysteamine
conjugate suppress microglial neurotoxicity by inhibiting microglial
2 2
activation; ii) diclofenac–cysteamine could directly scavenge H O
induced free radicals in neurons. In fact, it was demonstrated that
another NSAIDs–cysteamine conjugates significantly inhibit lipid per-
oxidation of rat hepatic microsomal lipids and directly interacted
with free radical DPPH [50]. Although the condition media of LPS-
stimulated microglia may not reflect microenvironments of neurode-
generative diseases, it partially mimics the neuroinflammatory condi-
tions where secreted pro-inflammatory factors by microglia cells
induce neuronal cell apoptosis. Ultimately, the neuroprotective effects
of these compounds need to be validated in proper neuroinflammatory
disease animal models. Collectively, these experimental results demon-
strated that compound 8 possessed potent anti-neuroinflammatory
activity via inhibition of microglia activation, and might serve as a
potential lead for the therapeutic treatment of neuroinflammatory
diseases.
Appendix A. Supplementary data
Supplementary data (Experimental data and NMR spectra) associated
with this article can be found, in the online version. Supplementary data
to this article can be found online at doi: http://dx.doi.org/10.1016/j.
intimp.2015.02.033
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